text:
Molecular oxygen modulates cytochrome c oxidase function.

This study sought to determine whether molecular oxygen interacts with cytochrome c oxidase to modify its catalytic activity. Such an interaction could explain the observation that mitochondria incubated under low O2 concentrations exhibit a reversible suppression of State 3 respiration. Oxidized bovine heart cytochrome c oxidase was incubated in oxygen concentrations of <50 μM for 4 h. The enzyme exhibited a reversible decrease in Vmax after incubation, compared with control enzyme incubated at higher oxygen concentrations. This change was accompanied by a small increase in the apparent Km of the enzyme for both cytochrome c and oxygen, although the optical absorption spectra of oxidized, cycling, or reduced enzyme were not affected. Spectroscopy studies after 4 h of incubation revealed that heme a3 was 33% reduced during cycling at [O2] = 25 μM whereas enzyme at [O2] = 135 μM was only 18% reduced, suggesting that the site of inhibition occurred at the electron transfer step between heme a3 and O2. These results provide a mechanistic explanation for the observation that intact cells or mitochondria exhibit a reversible inhibition of respiration during prolonged exposure to [O2] <25 mM, by demonstrating that the catalytic activity of cytochrome c oxidase function is similarly inhibited, possibly through an allosteric effect of molecular O2 on the enzyme.

This study sought to determine whether molecular oxygen interacts with cytochrome c oxidase to modify its catalytic activity. Such an interaction could explain the observation that mitochondria incubated under low O 2 concentrations exhibit a reversible suppression of State 3 respiration. Oxidized bovine heart cytochrome c oxidase was incubated in oxygen concentrations of <50 M for 4 h. The enzyme exhibited a reversible decrease in V max after incubation, compared with control enzyme incubated at higher oxygen concentrations. This change was accompanied by a small increase in the apparent K m of the enzyme for both cytochrome c and oxygen, although the optical absorption spectra of oxidized, cycling, or reduced enzyme were not affected. Spectroscopy studies after 4 h of incubation revealed that heme a 3 was 33% reduced during cycling at [O 2 ] ؍ 25 M whereas enzyme at [O 2 ] ؍ 135 M was only 18% reduced, suggesting that the site of inhibition occurred at the electron transfer step between heme a 3

and O 2 . These results provide a mechanistic explanation for the observation that intact cells or mitochondria exhibit a reversible inhibition of respiration during prolonged exposure to [O 2 ] <25 mM, by demonstrating that the catalytic activity of cytochrome c oxidase function is similarly inhibited, possibly through an allosteric effect of molecular O 2 on the enzyme.
Cytochrome c oxidase is the terminal oxidase of cellular respiration and catalyzes the transfer of electrons from ferrocytochrome c to molecular oxygen (1,2). The free energy release associated with this electron transfer is coupled to the translocation of protons from the mitochondrial matrix to the cytosol, generating a proton electrochemical gradient across the inner mitochondrial membrane. This potential is subsequently used to sustain a number of mitochondrial functions including the synthesis of ATP. The enzyme contains four redox-active metal centers, two hemes (a and a 3 ), and two redox-active coppers, Cu A and Cu B (3). Cu A is thought to reside in subunit II of the enzyme and to consist of a binuclear center that accepts electrons from reduced cytochrome c. Electrons are transferred to heme a (4) and then to the binuclear oxygen binding site, heme a 3 and Cu B , in subunit I. Electrons are finally transferred to O 2 , although the details of this process are incompletely understood.
The kinetic behavior of the oxidase has been studied exten-sively (5) Chemicals-All biochemicals were obtained from Sigma. The Pdmeso-tetra-(4-carboxyphyenyl) porphine dye used for phosphorescencequenching measurements of oxygen concentration was obtained from Medical Systems, Inc., Greenvale, NY. The isolated cytochrome c oxidase was a gift from Dr. Martin Horvath.
Preparations-Bovine heart cytochrome c oxidase concentrations were based on a value of ⑀ ϭ 1.41 ϫ 10 5 M Ϫ1 ⅐cm Ϫ1 at 421 nm for the oxidized enzyme. The enzyme was prepared by the method of Hartzell and Beinert (8) and was stored at a concentration of 0.6 mM at Ϫ80°C until immediately before use. Cytochrome c was reduced as described previously (9). Briefly, bovine cytochrome c (⑀ ϭ 2.12 ϫ 10 4 M Ϫ1 ⅐cm Ϫ1 at 550 minus 540 nm) (10) was dissolved in a mixture containing equal amounts of 20 mM boric acid and 0.2 M sodium borohydride (pH ϭ 10.2) and incubated for 1 h. Cytochrome c reduction was stopped using sodium phosphate (pH ϭ 6.5).
Cytochrome c Oxidase Incubation-Oxidized bovine cytochrome c oxidase (1-10 nM) (11) was incubated in spinner flasks in 100 mM KH 2 PO 4 containing 0.5% dodecyl ␤-D-maltoside at pH ϭ 7.4, 25°C (7). The head space of the flask was continuously flushed with gas containing oxygen ranging from 2.8 to 14.2%, balance nitrogen. The solution was continuously mixed with a magnetic stirrer (60 rpm), and the dissolved oxygen tension was monitored using a polarographic electrode placed in a side port of the flask (12). The flasks were also equipped with glass tubing extending into the media to allow the anaerobic removal of aliquots for analysis.
Measurements of Oxygen Consumption-The apparent K m of cytochrome c oxidase with respect to cytochrome c was assessed in stirred solutions by measuring the decrease in oxygen partial pressure in a water-jacketed respirometer (1.5 ml) equipped with a calibrated polarographic O 2 electrode. Bovine cytochrome c (5 M), TMPD, 1 (500 M), and ascorbate (5 mM) were used as electron donors, and the decrease in oxygen tension over 5-10 min was recorded using a 12-bit analog-todigital converter. However, this method was not suitable for assessing the apparent K m of cytochrome c oxidase with respect to oxygen because the response speed of polarographic electrodes declines at [O 2 ] of less than 10 M. Accordingly, for those studies the oxygen concentrations in solution were measured using the oxygen-dependent phosphorescence quenching of a porphine dye (13). Accuracy of the system was confirmed by comparison of test solutions assessed simultaneously by polaro-* This work was supported by NHLBI Grants HL32646 and HL35440. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. graphic and optical systems under quasi-steady-state conditions. The time constant for phosphorescence decay under anoxia was confirmed in solutions using the glucose oxidase reaction to scavenge O 2 . In both O 2 measurement systems, the turnover number (s Ϫ1 ) for cytochrome c oxidase was calculated by dividing O 2 consumption (nmol O 2 /s) by the enzyme concentration (nM). In studies requiring the use of TMPD and ascorbate, the contribution of autooxidation of ascorbate (14) was small but was nevertheless measured and subtracted from the overall rate of oxygen consumption.
Spectrophotometric Determination of the K m of Cytochrome Oxidase for Cytochrome c-The apparent K m and V max of the enzyme with respect to cytochrome c were determined spectrophotometrically by measuring the oxidation of reduced cytochrome c over time from the absorbance at 550 minus 540 nm. First-order rate constants were calculated from the slopes of the linear plots of ln(C t t Ϫ C oxd ) against time, where C t is the concentration of ferrocytochrome c at time t and C oxd is the concentration of ferrocytochrome c after the addition of ferricyanide, which completes the oxidation of cytochrome c (15). Reaction velocities were calculated from the product of the first-order rate constants and the concentrations of ferrocytochrome c. These were divided by four to obtain the equivalent rates of O 2 uptake. Apparent K m and V max were calculated from the negative reciprocal slope and the intercept with the abscissa, respectively, of plots /S against (Eadie-Hofstee plots), where ϭ turnover number (s Ϫ1 ) and S ϭ ferrocytochrome c concentration (in M).
Absorbance Spectra-Optical spectra (Hewlett-Packard 8453A diode array spectrophotometer) were recorded in stirred solutions at 25°C in a cuvette that was filled via a capillary chimney (1.5 mm diameter, 4 cm length). The spectrophotometer was interfaced to a computer that was used to control data acquisition and to store results. Spectra of the enzyme were recorded at 10-s intervals with an integration time of 1 s in 1-nm wavelength steps. Steady-state turnover of cytochrome c oxidase (1-2 M) was initiated by adding cytochrome c (5 M), TMPD (400 M), and ascorbate (5 mM). As the cuvette solution reached anaerobicity, the cytochrome c oxidase became fully reduced. The anaerobicity of the solution in the cuvette was verified using the phosphorescence quenching method. ] of less than 50 M produced an inhibition of the enzymatic turnover number that was rapidly reversed if the enzyme was subsequently exposed even briefly to oxygen concentrations greater than 50 M.
Conceivably, the oxygen dependence of the cytochrome c oxidase turnover number could have been a consequence of an increase in the apparent K m for cytochrome c and/or an increase in the apparent K m for O 2 . The apparent K m with respect to cytochrome c was therefore determined from Eadie-Hofstee plots using polarographic measurements of O 2 consumption in the presence of different concentrations of ferrocytochrome c, with TMPD and ascorbate added to reduce ferricytochrome c (Fig. 2, A and B). nM) was measured spectrophotometrically from the change in ferrocytochrome c concentration over time. In these studies, TMPD and ascorbate were not added, so the concentration of ferrocytochrome c decreased with time. As shown in Fig. 3, the oxidase exhibited first-order kinetics, and the first-order rate constant decreased as the initial cytochrome c concentration was increased from 0.8 to 20 M after 4 h incubation, regardless of whether the [O 2 ] was 135 M (Fig. 3A) or 25 M (Fig. 3B). Enzyme incubated at [O 2 ] ϭ 135 M yielded a V max and apparent K m for cytochrome c of 57 s Ϫ1 and 1.53 M, respectively (Fig. 3C), under the conditions of this assay. The V max and the apparent K m for cytochrome c of enzyme incubated at [O 2 ] ϭ 25 M were 38.5 s Ϫ1 and 2.8 M, respectively (Fig. 3D). As expected, these values differed from the corresponding measurements made in the presence of TMPD plus ascorbate (see Fig.  2) due to the ability of TMPD to reduce ferricytochrome c that is still bound to cytochrome oxidase (1). Collectively, these results demonstrate that prolonged exposure to [O 2 ] Ͻ50 M results in a reversible transition of the enzyme from its native state to a relatively inhibited (conformed) state associated with a decrease in V max and an increase in the apparent K m for oxygen and cytochrome c.
To determine whether the redox state of the enzyme during cycling was altered after prolonged exposure to [O 2 ] ϭ 25 M, absorbance at 444 nm was measured in the oxidized state, during cycling in the presence of excess cytochrome c, and in the reduced state after reaching anaerobicity. As shown in Cytochrome c, TMPD, and ascorbate then were added to initiate cycling of the enzyme, and spectra were again recorded (18). After the solution reached anaerobicity, the enzyme became fully reduced and spectra were again recorded.

DISCUSSION
Collectively, our data suggest that molecular oxygen directly modifies the V max and the apparent K m of cytochrome c oxidase, leading to a reversible inhibition of the enzyme after it is exposed to low oxygen concentrations (Ͻ50 M) for several hours. To interpret our data we employed Minnaert's mechanism IV, which applies first-order kinetics to explain cytochrome c oxidase kinetics (19). Typically, the first-order rate constants of a simple bimolecular collisional mechanism remain constant with increasing substrate concentrations until V max is reached at finite substrate concentration (20). However, although cytochrome c oxidase kinetics show that the enzyme remains strictly first-order even at high cytochrome c concentrations, the rate constant decreases with increasing cytochrome c concentration (17). Minnaert explained this phenomenon by proposing that ferro-and ferricytochromes have equal binding capacity, with ferrocytochrome forming a productive complex while ferricytochrome can act as a competitive inhibitor. Subsequently, Yonetani and Ray (21) demonstrated that under conditions where first-order kinetics are observed, the K m for ferrocytochrome c was the same as the dissociation constant (K i ) for ferricytochrome c (21).
Minnaert mechanism IV: where [S] ϭ ferrocytochrome c, [P] ϭ ferricytochrome c; (K m ϭ K i ), and k 1 , k 2 , k 3 ϭ electron transfer rates. To apply this model to the interpretation of kinetic data obtained after incubation at low oxygen concentrations, it is necessary to demonstrate that these conditions hold true during both the native and the conformed states of the enzyme. Fig. 3 shows that cytochrome c oxidase exhibits first-order kinetics after incubation at [O 2 ] ϭ 135 M (Fig. 3A) or at [O 2 ] ϭ 25 M (Fig. 3B), thus confirming this assumption. Furthermore, the first-order rate constant was observed to decrease with increasing cytochrome c concen- tration under both incubation conditions. Thus, the assumption for mechanism IV regarding the cytochrome c dependence of enzyme kinetics described by Minnaert remain valid for the enzyme in its conformed and native states.
How might the oxidase V max decrease and the apparent K m for cytochrome c and oxygen increase during prolonged exposure to [O 2 ] Ͻ50 M? Based on Minnaert's mechanism IV, the observed changes could have resulted either from changes in k 1 , k 2 , or k 3 . The rate constant k 3 represents the electron transfer step from a 3 . k 3 is decreased).
These observations are consistent with the hypothesis that molecular oxygen interacts with cytochrome c oxidase to modify its kinetic behavior. The need for prolonged (Ͼ1 h) incubation at low oxygen concentrations to induce conformance, and the rapid reversibility of the inhibition when the enzyme is exposed even briefly to oxygen concentrations greater than 50 M, suggests that a second binding site for oxygen exists on the enzyme, which is distinct from the catalytic binuclear site. When oxygen is bound to this regulatory site the enzyme would assume its normal catalytic activity. However, when incubated at lower oxygen concentrations, O 2 would gradually dissociate from this putative site, causing the enzyme to revert to its inhibited (conformed) state. Exposure to higher oxygen concentrations would result in the rapid reassociation of O 2 , returning the enzyme to its native state. While supportive of this hypothesis, the data of our study do not directly demonstrate the existence of a regulatory site capable of interacting with oxygen. However, the enzyme did appear to revert to its conformed state more quickly when incubated at lower O 2 concentrations, consistent with the notion that oxygen must dissociate from the enzyme to revert to the conformed state. Cytochrome c oxidase has been the focus of extensive analysis over the past few decades, and a large number of different factors are known to influence its kinetic behavior (2,3,5). However, a regulatory effect of oxygen on the catalytic function of the enzyme has not been identified previously. The reason for this may relate to the necessity for prolonged exposure to low oxygen concentrations, and the rapid reversibility of the conformed state upon exposure to higher O 2 concentrations. Classical studies have shown that cellular respiration remains independent of oxygen concentration above a critically low [O 2 ] of 3-5 M (13,22,23). However, because of technical considerations those studies were carried out using hypoxia of short duration, which would not be expected to reveal effects that require more prolonged exposure to elicit. Indeed, the present study represents an extension of our previous studies of prolonged hypoxia in isolated hepatocytes (12) and cultured cardiomyocytes (24). Those studies demonstrated that prolonged exposure to lowered oxygen concentration was associated with a reversible inhibition of cellular respiration, an effect that was similarly evident during TMPD respiration in isolated mitochondria incubated at low oxygen concentrations (6). The previous observation that intact cells of diverse phenotype demonstrate an O 2 -dependent regulation of respiration, combined with the present observation that an inhibition of cytochrome c oxidase activity occurs during prolonged exposure to lowered oxygen concentrations, suggests that this response may represent a component of an adaptive response to lowered oxygen availability. A regulatory effect of molecular oxygen on cytochrome c oxidase would provide a mechanism allowing cells to adjust their respiratory rates in accordance with the ambient oxygen concentrations and could explain how specialized cells within intact tissues are able to respond to changing oxygen concentrations over the physiological range (25). Further elucidation of the significance of this response will require additional studies.


s2fieldsofstudy: Biology Medicine

text:
Regulation of obese (ob) mRNA and plasma leptin levels in rhesus monkeys. Effects of insulin, body weight, and non-insulin-dependent diabetes mellitus.

We have cloned the rhesus monkey obese cDNA and have analyzed its expression in monkeys with a wide range of body weights (lean to very obese) and with or without non-insulin-dependent diabetes mellitus to examine the relationship of ob gene expression to obesity and non-insulin-dependent diabetes mellitus. The sequence of monkey ob protein, excluding the signal peptide, showed 91% identity with the human protein. We observed a significant correlation between the level of ob mRNA and body weight. We also found a significant relationship between ob mRNA and fasting plasma insulin concentration; however, insulin stimulation during a 100-140-min euglycemic/hyperinsulinemic clamp did not result in any changes in ob mRNA levels. Circulating levels of the ob gene product leptin were also significantly correlated with body weight. These results show that ob gene expression is related to body weight and is not acutely regulated by insulin.

We have cloned the rhesus monkey obese cDNA and have analyzed its expression in monkeys with a wide range of body weights (lean to very obese) and with or without non-insulin-dependent diabetes mellitus to examine the relationship of ob gene expression to obesity and non-insulin-dependent diabetes mellitus. The sequence of monkey ob protein, excluding the signal peptide, showed 91% identity with the human protein. We observed a significant correlation between the level of ob mRNA and body weight. We also found a significant relationship between ob mRNA and fasting plasma insulin concentration; however, insulin stimulation during a 100 -140-min euglycemic/hyperinsulinemic clamp did not result in any changes in ob mRNA levels. Circulating levels of the ob gene product leptin were also significantly correlated with body weight. These results show that ob gene expression is related to body weight and is not acutely regulated by insulin.
Obesity is the most common nutritional disorder in developed countries and is a major risk factor for hypertension, cardiovascular disease, and non-insulin-dependent diabetes mellitus (NIDDM) 1 (1). Recently, several genes responsible for obesity, including the obese (ob) (2), fat (3), and agouti genes (4) have been identified in genetically obese rodent models. The human and rat ob cDNAs and human and mouse ob genes have also been cloned (2,(5)(6)(7)(8)(9)(10)(11). The ob gene encodes the circulating protein termed leptin (2,12), which may be a helical cytokine (13). Recent experiments utilizing recombinant protein have suggested that leptin modulates appetite, body weight, oxygen consumption, and body temperature (14 -19). However, if leptin is truly a satiety hormone, it is not yet clear how humans or rodents become obese in the presence of high levels of leptin (20,21). The recent identification of a leptin receptor (22) may lead to a better understanding of these discordant findings.
Regulation of the ob gene is an area of intensive investiga-tion. In one study, induction of ob gene expression by corticosterone in normal rats has been shown to reduce body weight and food intake (23). Increased ob mRNA expression has been reported in adipose tissue in ob/ob mice (2) as well as in obese humans, genetically fat rodents (db/db mouse, Zucker fatty rat, Wistar fatty rat, and obese JCR:LA corpulent), ventromedial hypothalamus lesioned obese rodents (6 -9, 24 -27), and dietary-induced obese rodents (28). There have been some reports suggesting that ob mRNA levels are reduced in fasting normal rodents and streptozotocin-induced diabetic rodents and that these levels are restored to normal after refeeding and partially toward normal with insulin treatment of diabetic animals (29 -33). However, this regulation by insulin is still controversial (30,32,34). Many but not all rhesus monkeys (Macaca mulatta) become obese after sexual maturation (35). Obesity in rhesus monkeys, as in humans, is usually associated with insulin resistance and hyperinsulinemia and frequently leads to NIDDM, supporting the rhesus monkey as an excellent model for the study of human obesity and NIDDM (36).
In this paper, we report the cloning of the rhesus monkey ob cDNA. We examine the expression of this gene and its protein in relation to body weight and plasma insulin levels in three groups of monkeys: normal, normoglycemic-hyperinsulinemic, and those that have overt NIDDM. Results showed that the ob mRNA and plasma leptin levels were significantly related to body weight and that ob mRNA was not acutely regulated by insulin.
Procedures-After a consistent 16-h fast, plasma samples for fasting glucose, insulin, and leptin were obtained under light anesthesia (ketamine hydrochloride (10 mg/kg body weight), with dose repeated as necessary). Plasma was frozen for later assays. Abdominal subcutaneous adipose tissue samples were obtained either under ketamine hydrochloride, as noted above, or immediately after anesthesia by intravenous sodium pentobarbital. Tissue samples were immediately frozen in liquid nitrogen and stored at Ϫ165°C. The euglycemic clamp procedure was carried out in five of the lean young adult monkeys from the * These studies were supported by National Institutes of Health Grants DK37717, AG10612, and AG42100 and the International Health Foundation and also by National Institutes of Health Grants DK 44093 and DK50602 (to T. A. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) U58492.
normal group described above. The experimental procedure has been described previously (38) and used an insulin infusion rate of 2400 pmol/m 2 body surface area/min, with glucose maintained at approximately 4.7 mM. Abdominal subcutaneous and omental adipose tissue biopsies were obtained for RNA preparation before initiation of the procedure and again at steady state (approximately 100 -140 min).
Construction and Screening of the Monkey Adipose cDNA Library-Total RNA was extracted from subcutaneous adipose tissue using the TRIzol reagent (Life Technologies, Inc.). Poly(A) ϩ RNA was prepared as described (39). A monkey adipose tissue cDNA library was constructed using a cDNA synthesis kit (SuperScript TM , Life Technologies, Inc.). The resulting cDNA was ligated into the ZAPII vector (Stratagene, La Jolla, CA) and screened as described (40). To obtain a probe to screen the library, the oligonucleotides (5ЈCGCGGAATTCCAAGATGACAC-CAAAACCCT3Ј and 5ЈCGCGCTCGAGGCCACCACCTCTGTGGAG-TA3Ј) were synthesized according to the human ob mRNA sequence and included EcoRI or XhoI restriction enzyme sites (2). ob monkey mRNA was amplified by reverse transcription-polymerase chain reaction using the monkey adipose tissue RNA. The DNA fragment was cloned into pBluescriptII KS(Ϫ) (Stratagene) and sequenced to confirm that this polymerase chain reaction fragment was homologous to the human and mouse ob genes. This fragment, which corresponds to 82-437 of the monkey ob cDNA, was used as a probe to screen the library.
Northern and Slot-blot Hybridization Analysis-Total RNA was extracted from adipose tissues using TRIzol reagent (Life Technologies, Inc.), according to the manufacturer's protocol. Total RNA (20 g) was separated by 0.8% agarose gel containing 2.2 M of formaldehyde. The RNA was transferred to a nylon membrane (Hybond TM -N; Amersham Corp.). For the slot-blot hybridization, 3 g of total RNA was applied to a nitrocellulose filter (Bio-Rad). The membranes and filters were prehybridized at 42°C for 4 h in 50% formamide, 5 ϫ Denhardt's solution, 6 ϫ SSC, 20 mM sodium phosphate (pH 6.5), 1% SDS, and 100 g/ml denatured salmon sperm DNA. The hybridization was carried out at 42°C for 24 h in the same solution, containing 1 ϫ 10 6 cpm/ml 32 Plabeled probe. Filters were washed with 0.1 ϫ SSC and 0.1% SDS twice at room temperature and three times at 65°C (41). The PstI fragment (0.5 kilobase pair) of the monkey ob cDNA (Ϫ47 to 452) was used as a probe. The 440-base pair BglI-KpnI DNA fragment of the human HSP83 cDNA was used as an internal control (42). The relative amounts of ob and HSP83 probes bound to the immobilized mRNAs were quantitated by PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Assay of Plasma Leptin-Plasma leptin levels were measured in a solid-phase sandwich enzyme immunoassay, utilizing an affinity-purified polyvalent antibody immobilized in microtiter wells. Bound leptin was detected with affinity-purified antibody conjugated to horseradish peroxidase and quantitated with a chromogenic substrate. Leptin concentrations were calculated from standard curves generated in the same assay with recombinant leptin.
Other Assays-Plasma glucose was assayed using a Glucose Autoanalyzer II (Beckman Instruments). Plasma insulin was determined by radioimmunoassay (43). Body fat was determined using tritiated water (4 Ci/kg), with dilution determined on plasma samples obtained at 1 h (44).
Statistical Analysis-Group differences were compared by one-way analysis of variance. Data are expressed as the mean Ϯ S.E.

RESULTS AND DISCUSSION
To identify the monkey ob cDNA, we first screened a monkey adipose tissue cDNA library using a polymerase chain reactionderived probe as described under "Experimental Procedures." Twenty-nine clones were identified, and these overlapping clones included the 5Ј-non-coding region and the poly(A) tail. Sequence analysis of these clones revealed that the predicted monkey ob protein is identical in length (167 amino acids) to the human and mouse proteins. The monkey protein (excluding the signal peptide) shows 91% and 84% identity with human and mouse ob proteins, respectively (Fig. 1). In 1 of 8 clones FIG. 1. Rhesus monkey ob cDNA. A, nucleotide and predicted amino acid sequence of the monkey ob cDNA. The glutamine residue that is excluded in one clone is underlined. The putative polyadenylation signal is underlined. B, comparison of the ob amino acid sequences from monkey, human, mouse, and rat. Amino acids that are identical to the monkey sequence are represented by dashed lines. The predicted signal sequence is underlined. ء indicates the glutamine residue that is excluded in one clone. encoding this region, a glutamine at codon 49 was excluded, as has also been reported in the mouse and human ob mRNAs (2,5). This difference has been postulated to be due to splice site "slippage," and the functional consequence of this difference is unclear.
We next examined ob mRNA expression in monkey subcutaneous adipose tissue by Northern blot analysis. Northern blot hybridization revealed that the ob mRNA in monkey is much shorter (0.8 kilobase pair) than that of human, mouse, and rat (4.5 kilobase pairs). Monkey ob mRNA was expressed only in adipose tissue, as has also been shown in humans and rodents (2,5,6,8,9,25,27,29) (Fig. 2). Longer exposures revealed no other bands of other mobilities (data not shown).
Next we determined the mRNA levels in monkey subcutaneous adipose tissue and the plasma leptin levels in three groups of monkeys: (a) normal; (b) hyperinsulinemic; and (c) NIDDM. The average ob mRNA levels were highest in hyperinsulinemic monkeys and lowest in NIDDM monkeys, although the differences between the three groups were not statistically significant by analysis of variance (p ϭ 0.08; Fig. 3A). There were no significant differences in the levels of the control HSP83 mRNA (Fig. 3B). Among the NIDDM monkeys, four monkeys were treated with insulin. The ob mRNA level of insulin-treated monkeys (0.3 Ϯ 0.1 ϫ 10 4 ) was significantly lower than that of the five untreated NIDDM monkeys (1.1 Ϯ 0.2 ϫ 10 4 ). The NIDDM monkeys lose weight before insulin treatment and show insulin levels that are reduced relative to the hyperinsulinemic state. The insulin-treated monkeys were lean (9.9 Ϯ 1.2 kg) and had lower levels of fasting plasma insulin (123 Ϯ 37 pM) compared with untreated NIDDM monkeys (15.1 Ϯ 1.5 kg and 516 Ϯ 107 pM). Thus, this difference may be related to the lower body weight and plasma insulin, not to the insulin treatment.
As shown in Fig. 3 (C and D), ob mRNA levels and body weight were correlated (r ϭ 0.528; p Ͻ 0.01), as were ob mRNA and fasting plasma insulin concentrations (r ϭ 0.514; p Ͻ 0.01; Fig. 3, C and D).
We noted significant variations in the ob mRNA expression levels in six young adult non-obese monkeys that were all of the same age (ϳ7 years old). These monkeys were relatively lean, with body fat less than 22% (body weight, 7.2-11.8 kg; body fat, 5.5-19.3%) and had normal fasting plasma glucose (3.2 Ϯ 0.1 mM) and insulin (354 Ϯ 33 pM) levels. The ob mRNA levels of these young adult monkeys were correlated to body weight (r ϭ 0.866; p Ͻ 0.05, data not shown). This relationship was much stronger in these young adult monkeys than in the other monkeys, including older normal, hyperinsulinemic, and NIDDM monkeys (age, 21.5 Ϯ 1.4, 12.7-30.0 years old). The early increased ob expression in some of the lean young monkeys could relate to the later development of obesity or be a marker of the propensity to develop obesity.
In fasting rats or mice and streptozotocin-diabetic rats, ob mRNA levels were reduced, and levels were increased after refeeding or insulin treatment (29 -33). However, this insulin effect has been controversial. In fasting rats or streptozotocininduced diabetic rats, whether lower ob mRNA levels were due to body weight loss or to decreases in insulin levels could not be determined (32). In vitro experiments using primary adipose cells showed no regulation of ob mRNA by insulin (34), however, another group reported ob up-regulation by insulin (30). To address the issue of whether ob mRNA is acutely regulated by insulin in vivo, we performed the euglycemic/hyperinsulinemic clamp using five of the normal lean young adult monkeys to examine the effect of insulin on ob mRNA levels. There were no significant differences in ob mRNA levels in either subcutaneous or omental adipose tissues (Fig. 4, A and B) between basal and maximally insulin-stimulated clamp conditions measured after 100 -140 min of insulin infusion. Previously, there have been some reports that in rats, ob mRNA levels increased 3-fold after a 6-h insulin infusion clamp or 2-fold after 2 days (30,33). Therefore, we cannot exclude the possibility that ob mRNA might increase after a clamp of longer than 2 h. However, the present results together with previously reported studies involving cultured adipose cells (34) suggest that ob mRNA is not acutely regulated by insulin.
We next analyzed the plasma leptin levels of normal, hyperinsulinemic, and NIDDM monkeys. There were no significant differences in plasma leptin levels between these three groups (data not shown), although the hyperinsulinemic group tended to have higher levels. There was a significant correlation between the plasma leptin level and body weight (Fig. 5) in the total group (r ϭ 0.498; p Ͻ 0.05). This correlation was greater (r ϭ 0.736; p Ͻ 0.001) when the data from one monkey (which was lean and had high leptin levels) was omitted. There were no significant correlations between plasma leptin levels and fasting plasma insulin (r ϭ 0.139) or fasting plasma glucose levels (r ϭ 0.151; data not shown). These results suggest that the expression of the ob gene is related to body weight at both the mRNA and protein levels.


extfieldsofstudy: Biology Medicine

text:
The pleckstrin homology domain mediates transformation by oncogenic dbl through specific intracellular targeting.

The pleckstrin homology (PH) domain is an ~100 amino acid structural motif found in many cellular signaling molecules, including the Dbl oncoprotein and related, putative guanine nucleotide exchange factors (GEFs). Here we have examined the role of the Dbl PH (dPH) domain in the activities of oncogenic Dbl. We report that the dPH domain is not involved in the interaction of Dbl with small GTP-binding proteins and is incapable of transforming NIH 3T3 fibroblasts. On the other hand, co-expression of the dPH domain with oncogenic Dbl inhibits Dbl-induced transformation. A deletion mutant of Dbl that lacks a significant portion of the PH domain retains full GEF activity, but is completely inactive in transformation assays. Replacement of the PH domain by the membrane-targeting sequence of Ras is not sufficient for the recovery of transforming activity. However, subcellular fractionations of Dbl and Dbl mutants revealed that the PH domain is necessary and sufficient for the association of Dbl with the Triton X-100-insoluble cytoskeletal components. Thus, our results suggest that the dPH domain mediates cellular transformation by targeting the Dbl protein to specific cytoskeletal locations to activate Rho-type small GTP-binding proteins.

The pleckstrin homology (PH) domain is an ϳ100 amino acid structural motif found in many cellular signaling molecules, including the Dbl oncoprotein and related, putative guanine nucleotide exchange factors (GEFs). Here we have examined the role of the Dbl PH (dPH) domain in the activities of oncogenic Dbl. We report that the dPH domain is not involved in the interaction of Dbl with small GTP-binding proteins and is incapable of transforming NIH 3T3 fibroblasts. On the other hand, co-expression of the dPH domain with oncogenic Dbl inhibits Dbl-induced transformation. A deletion mutant of Dbl that lacks a significant portion of the PH domain retains full GEF activity, but is completely inactive in transformation assays. Replacement of the PH domain by the membrane-targeting sequence of Ras is not sufficient for the recovery of transforming activity. However, subcellular fractionations of Dbl and Dbl mutants revealed that the PH domain is necessary and sufficient for the association of Dbl with the Triton X-100-insoluble cytoskeletal components. Thus, our results suggest that the dPH domain mediates cellular transformation by targeting the Dbl protein to specific cytoskeletal locations to activate Rho-type small GTPbinding proteins.
The cytoskeletal-associated Dbl oncoprotein transforms NIH 3T3 cells (1) by activation of signaling pathways involving Rho-type GTP-binding proteins (2). Proto-Dbl is a 115-kDa cytoskeletal-associated protein that is found in brain, adrenal glands, and gonads (1). Oncogenic activation occurs as an outcome of an amino-terminal truncation of proto-Dbl, where a recombination event fuses about 10 kDa of an unidentified human gene product (from chromosome 3) on to the carboxylterminal half of Dbl (residues 498 -925). The oncogenic Dbl protein contains at least two putative signaling motifs. The first is a region of 176 amino acids (residues 498 -674) that was originally found to share significant homology with the Saccharomyces cerevisiae cell-division-cycle protein, Cdc24, and the breakpoint cluster region protein, Bcr 1 (3). This region, referred to as the Dbl homology (DH) domain, has been shown to be essential both for the transformation activity of oncogenic Dbl and for its ability to act as a GEF by stimulating the guanine nucleotide exchange activity of Cdc42Hs (4,5). The second putative signaling motif is the pleckstrin homology (PH) domain (6,7) and includes residues 703-812. Although PH domains appear to be relatively poorly conserved, both NMR and x-ray crystallographic studies of the PH domains of pleckstrin, dynamin, and spectrin indicate that they adopt a common three-dimensional structural motif (8 -11).
Over the past few years, a growing family of oncogene products and other growth regulatory proteins have been shown to contain a DH domain in tandem with a PH domain. In addition to Cdc24 and Bcr, these include the Vav, Ost, Ect-2, Lbc, Lfc, and Dbs oncoproteins (12)(13)(14)(15)(16)(17) and the activators of the Ras proteins, Sos (18), and Ras-GRF (19). All indications from previous studies are that the DH domain will form a binding site and in many cases contain GEF activity for Rho-like GTPbinding proteins (8, 9, 13, 14, 20 -22). However, less is known about the roles of the PH domains. In the present study, we have used the Dbl oncoprotein as a model to examine the role of the PH domain in cellular transformation and GEF activity. Cellular Fractionation Studies-Control NIH 3T3 and different NIH 3T3 transfectants were lysed and fractionated into cytosolic (S), Triton X-100-solubilized membrane fractions (T), and Triton X-100-insoluble fractions (I) as described by Graziani et al. (1). Cells were labeled with [ 35 S]methionine and [ 35 S]cysteine for 3 h at 37°C. Specific Dbl products were detected by immunoprecipitation using anti-Dbl-2 antibodies (3), electrophoresed through a 12% polyacrylamide gel and autoradiographed. For the detection of the PH domains (e.g. Figs. 2B and 4D, below), cells were immunoprecipitated with anti-Flag M5 antibodies and electrophoresed through a 15% polyacrylamide gel followed by immunoblotting with anti-Flag M5 antibodies.
Measurements of GDP Dissociation from Cdc42Hs-The [ 3 H]GDP dissociation assays were carried out as described previously (4,5). In Fig. 3A, the amounts of GST-Dbl and GST-DH (see Fig. 1) purified from Sf9 insect cells were estimated by Coomassie Blue staining after SDSpolyacrylamide gel electrophoresis. ϳ200 nM of GST-Dbl or GST-DH were incubated with 1 g of RhoA protein preloaded with [ 3 H]GDP in 100 l of reaction buffer at room temperature, and 16-l aliquots were diluted into 5 ml of ice-cold termination buffer (20 mM Tris-HCl, pH 7.4, 10 mM MgCl 2 , and 100 mM NaCl) at various time points. In

RESULTS AND DISCUSSION
To investigate the role of the PH domain in cellular transformation mediated by the oncogenic Dbl protein, we analyzed several Dbl mutants for transforming activity in NIH 3T3 cells ( Fig. 1). We found that while the transforming capability of a deletion mutant containing primarily the DH and PH domains (designated as pMA4 in Fig. 1) was similar to that of the Dbl oncogene product, neither the DH domain nor the PH domain (pdDH and pdPH, respectively) alone showed any detectable effects on the growth of 3T3 fibroblasts. However, when Dbl and pdPH were co-expressed in NIH 3T3 cells (dblϩpFlag/ PHdbl, Fig. 2C), we observed a significant reduction of the transforming activity by Dbl. Co-expression of Dbl with a Flagtagged PH domain of the Dbl-related Vav oncoprotein (12), on the other hand, showed effects comparable with those obtained with the pFlag/neo vector control (Fig. 2C), even though it appeared to be more highly expressed (Fig. 2B, lane 2) than the PH domain of Dbl (Fig. 2B, lane 1). The level of expression of the Dbl oncoprotein was essentially equivalent in all cases ( Fig. 2A).
To further confirm the selective inhibition of Dbl-induced transformation by the dPH domain, we used a second mammalian expression vector, pKH3 (23), to express the PH domain from either Dbl, Vav, or from the related yeast Cdc24 protein (20), together with the Dbl oncogene product. As shown in Fig.  2C, the expression of the dPH domain inhibited the focusforming activity of oncogenic Dbl by ϳ40%, whereas co-expression of Dbl with the PH domain of Vav (pKH3/PHvav) had little effect. Mass populations of these transfected cells also were examined for their ability to display anchorage-independent growth. We observed that cells co-expressing Dbl and the dPH domain lost the ability to grow in soft agar (data not shown). In some cases, we found that expression of a Flag-tagged PH domain of Vav caused some inhibition of Dbl-induced growth in soft agar, suggesting that the Vav PH domain (perhaps when expressed at sufficient levels) was capable of competing with the PH domain of Dbl for a cellular target. However, it is likely that the Vav PH domain is a weak competitor, since we often observed no detectable effects with either the Flag-tagged protein or when expressing the PH domain of Vav from the pKH3 vector. We also have found no detectable effects on Dbl transformation when expressing the PH domain from Cdc24 (data not shown). Mass cultures of Dbl transfectants expressing the dPH domain also displayed a less transformed phenotype compared with Dbl transfectants alone or compared with cells co-expressing Dbl and the PH domain of Vav (data not shown). Taken together, these results suggest that the PH domain of Dbl behaves as a selective antagonist of Dbl-induced transformation, possibly by binding to a saturable and specific ligand in cells.
Previously we have shown that the DH domain alone is sufficient for the GEF activity for Cdc42Hs (5). Since oncogenic Dbl also stimulates the guanine nucleotide exchange activity of Rho, we examined whether the Dbl domain is sufficient for stimulating the activation of Rho. To do this, we compared Rho-GEF activities of approximately equal amounts (ϳ200 nM) of insect cell-expressed, purified GST-Dbl and GST-DH domain. No significant differences were observed for the abilities of the GST-Dbl and GST-DH to stimulate [ 3 H]GDP dissociation from RhoA (Fig. 3A). These results suggest that the PH domain does not contribute to the GEF function of Dbl. This is further reinforced by the results in The membrane association of ␤ARK and spectrin has been attributed to their PH domains (24,25). The PH domains of ␤ARK, BTK, PLC␥, IRS-1, and Ras-GRF have been shown to bind to plasma membrane-associated ␤␥ subunits of the heterotrimeric G-proteins (26,27), and they all behave as antagonists of G␤␥-mediated signaling (28). Recent evidence also suggests that PH domains from many signaling molecules including ␤ARK and Ras-GAP can bind to specific phospholipids, namely phosphatidylinositol 4,5-bisphosphate (PIP 2 ) (29). These findings raised the possibility that the PH domain mediates the membrane targeting of oncogenic Dbl. It has been shown that the introduction of a membrane-targeting sequence into the Ras GEFs, Cdc25 and Sos (30,31), was sufficient to activate Ras, and more recently, that the addition of a membrane-targeting sequence in place of the PH domain of the Lfc oncoprotein was able to restore full transformation activity (32). Thus, we examined whether the substitution of the dPH domain with a membrane-targeting sequence enabled the DH domain of Dbl to induce transformation. A chimeric molecule containing the DH domain (residues 498 -756) fused to the Ras membrane-targeting farnesylation signal sequence (designated pdHF in Fig. 1) was constructed and assayed for focus-forming activity in NIH 3T3 cells. This chimera was expressed at a comparable level to oncogenic Dbl and a percentage (10 -20%) of the total chimeric molecules was targeted to the membrane The abilities of these constructs to serve as GEFs for Rho and Cdc42Hs and to transform NIH 3T3 cells are summarized. pdDH represents the Dbl homology domain and pdPH is the pleckstrin homology domain. The abbreviation ffu represents focus forming units. 100% is 3 ϫ 10 5 foci/pmol DNA. pDHF is a construct in which the PH domain sequences downstream from residue 750 are replaced with the carboxyl-terminal 16 amino acids of Ha-Ras, which include both the palmitylation and farnesylation sites (black rectangle). pdHF* is a construct encoding the DH domain of Dbl and the carboxyl-terminal 16 amino acids of Ha-Ras, except that the cysteine, which is normally farnesylated, has been changed to serine (cross-hatched rectangle). The GEF activity for these constructs has not been determined (N/A). The pdDH, pdDHF, and pdDHF* were subcloned from dbl by the polymerase chain reaction and inserted into the mammalian expression vector pZipneo. The GEF activities were measured as described (5) by the in vitro nitrocellulose filter binding assay either using the anti-Dbl immunoprecipitates from the NIH 3T3 transfectants (proto-Dbl, Dbl, and pMA4) or using the insect cell expressed peptides (DH and PH). surface (i.e. the Triton X-100 solubilized fraction (T) in Fig. 4A). However, this did not restore transforming activity to the DH domain (Fig. 1). Although, one possible explanation is that the amount of the chimera expressed at the membrane surface was not sufficient to stimulate a transforming signal, this does not seem likely based on what we have observed regarding the range of expression of oncogenic Dbl that will yield cellular transformation (34).
We have reported previously that significant portions of both proto-and oncogenic Dbl are localized to the Triton X-100insoluble fractions of transfected NIH 3T3 cells, suggesting an association with the cytoskeletal matrix (1). To address the possible role of the dPH domain in mediating this pattern of localization for the Dbl protein, stable transfectants of Dbl and Dbl deletion mutants (Fig. 1) were subjected to subcellular fractionation. The crude membrane fractions (P100) of the cells were solubilized either by 1% Triton X-100 or by treatment with 0.1% SDS and 0.25% sodium deoxycholate. Anti-Dbl immunoprecipitates revealed that a percentage of both the intact oncogenic Dbl protein and a truncation mutant pMA4 associated with the Triton X-100-insoluble fractions of cells (designated by I in Fig. 4, B and C). The amounts of oncogenic Dbl and pMA4 that were present in the Triton X-100-insoluble fraction typically varied between 50 and 70% of the total detectable protein, although in some cases (Fig. 4B) the percentage of oncogenic Dbl in this fraction was less than 50%. However, the DH domain of Dbl, which lacks transforming ability, was localized exclusively to the cytosolic fraction (designated S in Fig. 4C). When cells expressing the Flag-tagged PH domains were subjected to similar fractionation, the PH domains were found associated with the Triton X-100-insoluble fractions (Fig.  4D). These results suggest that the dPH domain is directly responsible for the association of oncogenic Dbl with the Triton X-100-insoluble cytoskeletal fraction and thus may serve to target the catalytic DH domain to the cytoskeleton.
We have reported previously that the DH domain is responsible for Dbl GEF function and is required for Dbl transforming activity (3,5). Here, we demonstrate that while the dPH domain does not seem to be involved in the interactions of Rhotype small GTP-binding proteins with Dbl, it is essential for Dbl transforming activity. Thus, our present findings establish that both the DH and PH domains are required for the cellular function of Dbl. Indeed, the minimum structural unit (pMA4) of oncogenic Dbl conferring complete transforming activity just encompasses the DH domain and the PH domain. The finding that plasma membrane-targeting of Dbl is not sufficient to confer transforming activity, coupled with the requirement of the dPH domain as the necessary and sufficient element for association of the Dbl protein with the Triton X-100-insoluble component, suggests that the function of the PH domain resides in its ability to target the catalytic DH domain to the cytoskeletal matrix. Whether this targeting function holds for other members of Dbl-related GEF family proteins remains to be seen. However, based on the observation that the PH domains of Dbl-related molecules Vav and Cdc24 do not act effectively as inhibitors of Dbl-induced transformation, it is an attractive possibility that different members of the family of Dbl-related proteins may be targeted by their PH domains to distinct cellular locations to activate various Rho-type GTPbinding proteins, in response to different extracellular stimuli such as epidermal growth factor, platelet-derived growth factor, lysophosphatidic acid, and bradykinin. This may also explain the finding that substitution of a membrane-targeting (i.e. Ras-farnesylation) sequence for the PH domain of Lfc restored its transformation capability (32), whereas this substitution did not restore transforming activity to a Dbl protein that just contains the DH domain. It may be that Lfc needs to be targeted to the plasma membrane to optimally couple to other protein components in its signaling pathway while Dbl needs to be targeted to a cytoskeletal location.
The identity of the ligand(s) that binds to the PH domain of oncogenic Dbl will represent an important focus of future studies. It seems likely, that given the hypervariable nature of the putative ligand-binding cleft in the PH domains that have thus far been identified (33), a complex diversity of ligands may exist that are responsible for mediating the actions of various PH domain-containing signaling molecules, including Dbl and related regulatory molecules of small GTP-binding proteins.


extfieldsofstudy: Medicine Chemistry

text:
Apolipoprotein A-I structural modification and the functionality of reconstituted high density lipoprotein particles in cellular cholesterol efflux.

The role of HDL and its major protein constituent, apolipoprotein (apo) A-I, in promoting the removal of excess cholesterol from cultured cells has been well established; however, the mechanisms by which this occurs are not completely understood. To address the effects of apoA-I modification on cellular unesterified (free) cholesterol (FC) efflux, three recombinant human apoA-I deletion mutants and plasma apoA-I were combined with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and FC to make reconstituted high density lipoprotein (rHDL) discoidal complexes. These particles were characterized structurally and for their efficiency as acceptors of mouse L-cell fibroblast cholesterol. The deletion mutant proteins lacked NH2-terminal (apoA-I (Delta44-126)), central (apoA-I (Delta139-170)), or COOH-terminal (apoA-I (Delta190-243)) domains of apoA-I. The three deletion mutants all displayed lipid-binding abilities and formed discoidal complexes that were similar in major diameter (13.2 +/- 1.5 nm) to those formed by human apoA-I when reconstituted at a 100:5:1 (POPC:FC:protein) mole ratio. Gel filtration profiles indicated unreacted protein in the preparation made with apoA-I (Delta190-243), which is consistent with the COOH terminus portion of apoA-I being an important determinant of lipid binding. Measurements of the percent alpha-helix content of the proteins, as well as the number of protein molecules per rHDL particle, gave an indication of the arrangement of the deletion mutant proteins in the discoidal complexes. The rHDL particles containing the deletion mutants had more molecules of protein present than particles containing intact apoA-I, to the extent that a similar number of helical segments was incorporated into each of the discoidal species. Comparison of the experimentally determined number of helical segments with an estimate of the available space indicated that the deletion mutant proteins are probably more loosely arranged than apoA-I around the edge of the rHDL. The abilities of the complexes to remove radiolabeled FC were compared in experiments using cultured mouse L-cell fibroblasts. All four discoidal complexes displayed similar abilities to remove FC from the plasma membrane of L-cells when compared at an acceptor concentration of 50 microg of phospholipid/ml. Thus, none of the deletions imposed in this study notably altered the ability of the rHDL particles to participate in cellular FC efflux. These results suggest that efficient apoA-I-mediated FC efflux requires the presence of amphipathic alpha-helical segments but is not dependent on specific helical segments.

High density lipoproteins (HDL) 1 are a heterogeneous class of particles thought to mediate the flux of unesterified (free) cholesterol (FC) from peripheral cells to the liver in the process of reverse cholesterol transport (1). However, the mechanism directing the incorporation of cell cholesterol into the HDL and the role of HDL's major protein component, apolipoprotein (apo) A-I, are subjects of controversy. Epidemiological studies (2) as well as experiments involving transgenic animal models (3) have suggested that apoA-I is the major determinant of the ability of HDL species to participate in cholesterol efflux, thus much effort has been expended to elucidate the functional domains of this protein.
It has been demonstrated that the presence of amphipathic helical segments in acceptor particles is necessary for efficient efflux of cholesterol from cells (4). The arrangement or conformation of these segments may be a factor in the ability of acceptor particles to sequester cellular cholesterol. Several groups have utilized epitope-specific apoA-I monoclonal antibodies to define segment(s) of apoA-I that may be crucial for FC efflux (5)(6)(7)(8). Banka et al. (5) utilized eight such antibodies and found a region spanning amino acid residues 74 -110 to be important for efficient cholesterol efflux. A similar approach was taken by Luchoomun et al. (6) whose experiments indicated that the domain around amino acid 165 of apoA-I is involved in the efflux of cellular cholesterol. Experiments in which genetic apoA-I variants were combined with dimyristoylphosphatidylcholine have further suggested that substitution of a proline residue at amino acid 165 interferes with a conformation that is essential for cholesterol efflux (7). In contrast, a study by Fielding et al. (8) has demonstrated that the amino acid region 137-144 of apoA-I is adjacent to or part of a structural site in the HDL subspecies, pre-␤ 1 -HDL, which is effective in promoting the efflux of cellular cholesterol. Another study has attempted to distinguish whether monoclonal antibodies specific to apoA-I are able to inhibit cellular cholesterol efflux from intracellular or plasma membrane pools of cholesterol (9). Two Fab fragments spanning the amino acid region 140 -150 were found to inhibit the efflux of intracellular cholesterol but not the cholesterol flux from the plasma membrane. It is apparent that there is marked disagreement about which, if any, domains of apoA-I are critical for cell cholesterol efflux.
Recently, Holvoet et al. (10) have expressed and purified recombinant mature human apoA-I and various deletion mutants for investigating structure-function relationships with regard to activation of lecithin-cholesterol acyltransferase (LCAT). The three deletion mutants: apoA-I (⌬Leu 44 -Leu 126 ), apoA-I (⌬Glu 139 -Leu 170 ), and apoA-I (⌬Ala 190 -Gln 243 ) lack domains thought to be crucial for either phospholipid (PL) binding or LCAT activation. In an attempt to define the domain of apoA-I that is crucial for cholesterol efflux from cells, the present study uses acceptor particles containing these engineered apoA-I molecules reconstituted with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) into homogeneous and highly defined discoidal complexes. The variant proteins were successfully combined with POPC and unesterified cholesterol to form reconstituted HDL (rHDL) particles of similar size, which were subsequently characterized with respect to their structure. Cholesterol efflux studies indicate that when rHDL particles are utilized as acceptors of FC, there are no significant differences in the rates of efflux to the discoidal HDL containing either intact apoA-I or the deletion mutant proteins. These data indicate that the deletion of large segments from either the NH 2 -terminal, central or COOH-terminal regions of the apoA-I molecule does not significantly impair the functionality of the rHDL complexes as acceptors of cellular cholesterol.

Methods
Purification of Apolipoprotein A-I-Human HDL isolated from the fresh plasma of normolipidemic subjects was delipidated in ethanol/ diethyl ether as described by Scanu and Edelstein (11) and purified apoA-I was isolated by anion exchange chromatography on Q-Sepharose and stored in lyophilized form at Ϫ70°C (12). Prior to use the purified A-I was resolubilized in 6 M guanidine HCl and dialyzed extensively against Tris buffer (10 mM Tris, 150 mM NaCl, 1.0 mM EDTA; pH 8.2).

Recombinant Human Apolipoprotein A-I and Deletion
Mutants-Preparation of the recombinant proteins was carried out by Holvoet et al. as formerly described in detail (10). Wild-type apoA-I and the apoA-I mutants were expressed in the periplasmic space of Escherichia coli cells and purified to homogeneity to yield the three deletion mutants utilized in this study: apoA-I (⌬Leu 44 -Leu 126 ), apoA-I (⌬Glu 139 -Leu 170 ), and apoA-I (⌬Ala 190 -Gln 243 ).
Preparation of Reconstituted HDL (rHDL)-Particles were prepared using the cholate dispersion/Bio-Bead removal technique as described in detail previously (13). A starting POPC/cholesterol/protein ratio of 100:5:1 (mol:mol:mol) was used and final compositions were determined after concentration (Centriprep 30, Amicon) under low speed centrifugation. The homogeneity and size of the complexes were assessed by gradient gel electrophoresis using precast 8 -25% polyacrylamide gels (Pharmacia Biotech Inc.). Due to indications that the complex preparations contained varying amounts of free protein, the samples were purified by high performance gel filtration (Superdex 200 HR 10/30, Pharmacia Biotech Inc.). The column was calibrated with standard proteins (13) and the hydrodynamic diameters of the particles were calculated from the elution volumes.
Characterization of rHDL-The particles were analyzed chemically using the Markwell modification of the Lowry protein assay (14), while phospholipids were determined as inorganic phosphorus by the method of Sokoloff and Rothblat (15). Unesterified cholesterol was determined after Bligh and Dyer extraction (16) by gas-liquid chromatography analysis (17); cholesteryl methyl ether was utilized as an internal standard in this assay. The number of apoA-I molecules per particle was determined by reaction with dimethyl suberimidate and determining the degree of cross-linking of the apoA-I by SDS-PAGE (18). In addition to hydrodynamic diameter determination by gel filtration chromatography, negative stain electron microscopy (EM) was utilized as described by Forte and Nordhausen (19) to determine the size of the reconstituted particles. Mean particle dimensions of 100 complexes were determined from each negative at ϫ189,000 magnification. The average ␣-helix content of apoA-I when complexed to PL was determined by circular dichroism (CD) spectroscopy using a Jasco J41A spectropolarimeter. Spectra were measured at 25°C in a 0.1-cm path length quartz cuvette as described previously (20); the percent ␣-helix was determined from the molar ellipticities at 222 nm.
Efflux of Plasma Membrane Cholesterol-Mouse L-cell fibroblast monolayers were used to monitor the release of [ 3 H]cholesterol to the extracellular medium as a measure of unesterified (free) cholesterol (FC) efflux, as has been described in detail (4). The cells, present in 12-well cell plates (22 mm), were grown to confluence in minimal essential medium/bicarbonate supplemented with 10% fetal bovine serum in a 37°C humidified incubator in the presence of 95% air and 5% CO 2 . The monolayers were labeled with 2 Ci/ml [1,2-3 H]cholesterol in bicarbonate-buffered minimal essential medium with 2.5% fetal calf serum for 24 h. This was followed by a 12-h incubation in minimal essential medium/bicarbonate containing 1% bovine serum albumin to equilibrate the radioactivity between the various cellular sterol pools. After a brief wash with minimal essential medium/bicarbonate containing HEPES (50 mM), cholesterol efflux measurements were initiated by the application of 1.0 ml/well of the test medium, containing 0.5% bovine serum albumin and the acceptor at the indicated PL concentration. The experiment was conducted in a 37°C incubator with an atmosphere of 95% air and 5% CO 2 . The radioactivity of a 75-l aliquot of the medium was determined at specific time intervals to estimate the fraction of FC released into the medium. Upon completion of the time course, all cell wells were washed with Dulbecco's phosphate-buffered salt solution three times and the cellular lipids were extracted with isopropanol (21). From the extraction, the total amount of radioactive cholesterol per well was determined by liquid scintillation counting.
Data Analysis-The fractional release of FC was determined experimentally and analyzed as described in detail for this system previously (4). The kinetic analysis is based on the assumption that the system is closed and that all FC therefore exists in either the cellular FC pool or in the acceptor FC pool. The equilibration of FC between these two pools was fitted to the single exponential equation Y ϭ Ae ϪBt ϩ C, where Y represents the fraction of radiolabeled FC remaining in the cells, t is the incubation time, A is a pre-exponential term that reflects the fraction of FC that exists in the medium at equilibrium, B is a time constant representing the release of FC, and C is a constant that represents the fraction of labeled FC that remains associated with the cells at equilibrium. A, B, and C were derived by fitting the experimental data to the above equation by nonlinear regression (Graph Pad Prism, Graph Pad Software Inc.). The above equation fit the data more accurately than a double exponential equation. The apparent rate constant for efflux (k e ), which is dependent on the acceptor particle concentration tested, is the product of A and B. The apparent t1 ⁄2 of efflux value in hours was then calculated as follows: t1 ⁄2 ϭ ln2/k e . The t1 ⁄2 values were statistically compared by Student's t test (Graph Pad Prism, Graph Pad Software Inc.).

Structural Characterization of rHDL Particles-Previously,
plasma apoA-I has been shown to form discoidal complexes (rHDL) upon combination with POPC Ϯ cholesterol (22)(23)(24), and the compositional characteristics of these discs have been well defined. Holvoet et al. (10) recently showed that recombinant apoA-I and specific deletion mutants of apoA-I were able to combine in a similar manner with dipalmitoylphosphatidyl-choline to form discoidal complexes. We confirmed that recombinant apoA-I forms reconstituted complexes with POPC and cholesterol and that these complexes are structurally identical to those prepared from plasma apoA-I. In preliminary cell FC efflux studies utilizing rHDL containing either plasma apoA-I or recombinant, wild-type, apoA-I, all measurements of efflux to the two types of rHDL particles were indistinguishable, indicating that the origin of the apoA-I did not affect the ability of the particles to function as acceptors of cellular cholesterol. Therefore, plasma apoA-I was used as the control protein in the studies of recombinant variants.
To study the effects of apoA-I modification on the structure and function of rHDL particles, discoidal complexes were prepared with plasma apoA-I, apoA-I (⌬44 -126), apoA-I (⌬139 -170), or apoA-I (⌬190 -243) at an initial molar ratio of 100:5:1 (POPC:FC:protein). The particles were sized by PAGE, which indicated the presence of free protein in addition to the complexes, particularly with rHDL constructed with apoA-I (⌬44 -126) and apoA-I (⌬190 -243) (approximately 20 and 50% lipidfree protein, respectively). The complexes were isolated from unreacted protein and lipid using gel filtration chromatography on a calibrated column, from which particle hydrodynamic diameters were determined. The gel filtration profiles (Fig. 1) indicated that there was no free protein in the preparations containing plasma apoA-I, apoA-I (⌬44 -126), and apoA-I (⌬139 -170), whereas 26% of apoA-I (⌬190 -243) was found to be unassociated with lipid in its preparation. These results suggest that the extent of free protein detected by PAGE may have been an artifact of the electrophoresis due to stripping of the protein from the complexes as they migrated through the gel. Reduced lipid association of apoA-I (⌬190 -243) was an expected result as this apoA-I mutant is missing the carboxyl terminus of apoA-I, which plays a major role in stabilizing the lipid-bound state of apoA-I (10,(25)(26)(27). The fractions corresponding to the rHDL species were isolated and characterized; the compositions and dimensions of the particles are listed in Table I. Electron microscopy was utilized to confirm that the particles were discoidal in shape, which is apparent from the stacks of discoidal complexes displayed for each preparation in Fig. 2. The composition and size determinations of the plasma apoA-I⅐FC⅐POPC complex are in good agreement with previously published results for a discoidal complex of composition 83:3:1 (molar ratio, POPC:FC:apoA-I) (24). This complex has been measured by PAGE and EM to have a diameter of 10.3 and 11.5 nm, respectively, which is the same as the present measurements of 10.0 and 11.2 nm ( Table I). The EM measurements denote an average major diameter of 13.2 Ϯ 1.5 nm for the four rHDL species described in Table I; the measurements are not significantly different by unpaired t test analysis. The PAGE and gel filtration column measurements of the hydrodynamic diameters indicated that apoA-I (⌬44 -126) formed a slightly larger complex than rHDL prepared with plasma apoA-I, while apoA-I (⌬139 -170) and apoA-I (⌬190 -243) both resulted in slightly smaller complexes. Again, the smaller hydrodynamic diameters of the latter two complexes may be attributed to some protein removal during migration through the gel matrix. Protein cross-linking was used to estimate the number of protein molecules per complex (Fig. 3). The rHDL comprised of intact apoA-I contained a mixture of particles containing 2 (70%) and 3 (30%) molecules of apoA-I per complex. The scans of the gels in Fig. 3 for the complexes containing the mutant proteins indicate that the cross-linking  was incomplete. In these cases, the dominant cross-linked oligomer was assumed to represent the number of protein molecules on the discoidal particle (Table I). The predominant species were four molecules of apoA-I (⌬44 -126), three molecules of apoA-I (⌬139 -170), and three molecules of apoA-I (⌬190 -243). Circular dichroism was used to determine the effect of the various deletions in the apoA-I molecule on the average ␣-helix content of the proteins in the rHDL particles. Table I demonstrates that the ␣-helix content of the intact protein was greater than that of any of the apoA-I deletion mutants. The number of amphipathic helices per protein molecule can be predicted from the number of helical residues in the protein by assuming that there are approximately 22 amino acid residues per helical segment (28). On this basis, there are nine helices in the plasma apoA-I molecules in the rHDL particles; this number is in good agreement with prior work which suggest that with 75% ␣-helix there should be eight helical segments per protein molecule (22,28). The equivalent numbers of helices for apoA-I (⌬44 -126), apoA-I (⌬139 -170), and apoA-I (⌬190 -243) are 4, 7, and 5, respectively.
Efflux of Cellular Cholesterol to Discoidal rHDL Particles-To compare plasma apoA-I/FC/POPC and mutant apoA-I/FC/POPC discoidal complexes as acceptors of cell cholesterol, the rHDL were incubated with radiolabeled mouse L-cell fibro-blasts at a concentration of 50 g of PL/ml for periods of up to 6 h. Measurement of the fraction of radiolabeled FC present in the cells during the time course of incubation with rHDL particles showed that they exhibited similar abilities to remove cellular cholesterol (Fig. 4). Time courses of cholesterol efflux in the systems containing native apoA-I⅐FC⅐POPC and apoA-I (⌬190 -243)⅐FC⅐POPC discoidal complexes are the same. Thus, the deletion of the carboxyl terminus did not impair the ability of the particle to participate in cholesterol efflux. Efflux to apoA-I (⌬44 -126)⅐FC⅐POPC and apoA-I (⌬139 -170)⅐FC⅐POPC rHDL was identical to efflux to particles containing intact apoA-I for the first 2 h of the experiment, after which point they showed some deviation from the control time course. After 6 h of incubation, apoA-I (⌬139 -170)⅐FC⅐POPC rHDL removed slightly less FC and apoA-I (⌬44 -126)⅐FC⅐POPC slightly more than the apoA-I⅐FC⅐POPC particles. All the time courses were fitted to a single exponential decay equation as described under "Methods" to obtain t1 ⁄2 values of efflux for each complex (Table  II). An average t1 ⁄2 of 16.2 Ϯ 1.8 h was measured, with maximum deviations of only 14% from this value. Comparison of the t1 ⁄2 of efflux by unpaired t test analysis indicates that none of the time courses involving incubation with apoA-I mutant⅐FC⅐POPC complexes were statistically different. These data indicate that the apoA-I deletions introduced here do not impair the functionality of the rHDL complexes as acceptors of cellular cholesterol.

DISCUSSION
Formation of rHDL Particles-The experiments summarized in Table I  were able to bind to POPC and cholesterol to form rHDL discoidal complexes. From the helix content and the amino acid residue content of the proteins, the number of ␣-helical segments per protein molecule can be determined through the assumption that each helix is 22 amino acids in length (28). The results indicate that the two molecules of protein present on the apoA-I⅐FC⅐POPC disc are organized such that 18 ␣-helical segments are present (Table III). Despite major structural modification of the apoA-I molecule, the mutant proteins packed onto a disc in such a manner that an equivalent number of ␣-helical segments (18 Ϯ 3 helices) were incorporated. As shown in Table III, with native apoA-I, 20 helices could theoretically be accommodated around the disc perimeter. Since 18 helices are actually present at the disc edge, it follows that the helices in the two protein molecules are packed tightly around the perimeter of the complex. In this arrangement, electrostatic interactions between the antiparallel helices contribute to the stabilization of the protein in this close-packed conformation (29,30). Although the apoA-I helices are tightly packed on the particles described in this study, a "hinge-domain" (28) extending away from the particle surface is probably not present, since there is room to accommodate every helix at the disc edge.
Since the deletion mutant proteins are smaller and have fewer ␣-helical segments, more protein molecules were able to be incorporated onto the disc edge. The limiting number of ␣-helical segments for these rHDL particles was calculated to be 25 for discs containing the NH 2 -terminal and COOH-terminal deletion mutants and 28 for the protein lacking the central domain of apoA-I (Table III). Again, there is sufficient space to accommodate every helix at the disc edge, because the numbers of helices measured experimentally were lower than these limiting values. However, the deletion mutant proteins are not as tightly packed on the disc edge as intact apoA-I, because only 60 -75% of the maximum possible number of helices were incorporated at the disc edge compared with the equivalent value of 90% for the intact apoA-I molecule. It seems that when helical segments are deleted in apoA-I, the interhelix attraction is reduced, resulting in a looser packing in the discoidal particles. In summary, all four types of apoA-I molecule formed similar particles suggesting that no specific amino acid sequence or helix-helix interactions are required for this process. The particles are stable provided that sufficient amphipathic ␣-helices are present to cover the PL acyl chains at the disc edge.
Cholesterol Efflux to rHDL Particles-It is now generally accepted that efflux of plasma membrane cholesterol involves a passive desorption of FC molecules from the membrane followed by diffusion of these molecules through the aqueous phase surrounding the cells and incorporation of the FC into a PL-containing acceptor particle (1,31). Studies have suggested that when FC acceptor particles contain apolipoprotein, there may be an interaction of the protein helices with specific lipid domains in the plasma membrane resulting in the modulation of cholesterol efflux (31)(32)(33)(34), but the details of this association are not yet clear.
This study investigated the possibility that a particular region of apolipoprotein A-I is essential for the efficient release of cholesterol from the plasma membrane. Cholesterol efflux was measured to rHDL containing either wild-type apoA-I or one of the three apoA-I deletion mutants in a system in which other factors that could affect the efflux capability of the particles were eliminated. For example, acceptor particle PL properties and particle size have been shown to be determinants in cholesterol efflux (35,36). In this study, the PL species was constant, and the acceptor particles were incubated with the cell monolayers at equal PL concentrations. In addition, differences due to varying sizes of the acceptor particles were not a factor here as the particles were constructed and isolated in a manner such that they were similar in size. Our study clearly demonstrates the need to isolate the rHDL particles from any free lipid or protein, as the gel filtration profiles indicated heterogeneous preparations. The utilization of homogeneous acceptors ensured that our results were not affected by the presence of these unreacted species. Having taken these factors into account, variations in the ability of the discs to remove cholesterol could be attributed directly to the differences in protein structure.
The results indicate that the deletion of specific segments of apoA-I did not affect the ability of the protein to participate in cholesterol efflux when combined with lipid as a rHDL particle. The t1 ⁄2 values of efflux are the same (Ϯ14%), thus all four particles essentially displayed equivalent abilities to remove cellular FC. Furthermore, none of the deletion mutant discoidal species had slower t1 ⁄2 values of efflux than particles containing the intact plasma apoA-I. These data suggest that specific domains within the apoA-I molecule are not required for efficient efflux. These results are in contrast to studies performed by several groups in which apoA-I-specific mono-  clonal antibodies were used to determine the regions of functional importance. Each of these studies identified a discrete region of apoA-I that was critical for apoA-I-mediated cholesterol efflux. It has been suggested that the region surrounding amino acid residue 165 of apoA-I (6) or the specific amino acid content in this position (7) determines the efficiency of apoA-I in cholesterol efflux. However, this region was deleted in this study in mutant apoA-I (⌬139 -170) without any effect on the participation of the protein in the efflux process. Other regions of apoA-I have been suggested to be essential: amino acids 74 -110 in studies involving rHDL species (5), and 137-144 in the HDL subspecies, pre-␤ HDL (8). These regions were addressed in this study with mutants apoA-I (⌬44 -126) and apoA-I (⌬139 -170), respectively, both of which when combined with lipid did not vary from the ability of intact apoA-I to remove cellular FC. Although small regions of the protein (1-44, 127-138, and 171-189) were not deleted in the mutants studied here, it seems likely that no specific domains of apoA-I are required for efficient apolipoprotein-mediated cholesterol efflux. The reasons for the variation in results between the present study and the monoclonal antibody studies are not entirely clear at this time. However, it is possible that inhibiting antibodies may be positioned in some cases such that they cover a significant portion of the polar phosphatidylcholine head groups, or the "faces," of the discoidal complex. Since the deletion mutant proteins utilized here demonstrate looser helix packing at the disc edge than intact apoA-I, but the discs are equally good FC acceptors, cholesterol molecules probably do not incorporate at the disc edge, but rather by way of the face of the complex. If a monoclonal antibody covers a portion of the face, it follows that cholesterol incorporation will be inhibited.
The results of this study suggest that several structural features of the apoA-I molecule are not determinants of efficient cholesterol efflux. Thus, specific domains of the protein are not essential and, furthermore, interactions between specific helices seem not to be required. The number of protein molecules or helical segments per rHDL complex has no effect on the function of the particle in a cholesterol efflux system. It seems that the essential factor provided by apoA-I is the amphipathic helices required to stabilize the small rHDL particles. This agrees with previous work with synthetic peptides (4), which suggested that amphipathic helix interactions are essential for efflux, but that the number and amino acid sequence of the helices does not affect the process.
Implications for Reverse Cholesterol Transport-In addition to the phenomenon of peripheral cell FC transfer from the plasma membrane to HDL particles that has been described in this study, reverse cholesterol transport in vivo involves the subsequent steps of cholesterol esterification by LCAT and cholesterol ester transfer between lipoprotein particles via cholesterol ester transfer protein (33,37,38). Studies have indicated that discoidal HDL are better substrates for LCAT than spherical HDL particles (39). Although the abilities of the dis-coidal HDL particles containing either wild-type apoA-I or apoA-I deletion mutants to accept cholesterol molecules diffusing from the plasma membrane are similar, experiments of Holvoet et al. (10) indicate that discoidal particles containing these deletion mutants have different reactivities with LCAT. Thus, the conformation of apoA-I probably affects the second step of the reverse cholesterol transport process. The esterification by LCAT is essential to maintain the gradient of cholesterol flux from the cellular membrane (38,40). These results suggest that the structure of apoA-I is critical for proper activation of LCAT but not for formation of HDL particles that can accept FC molecules diffusing from the plasma membrane of a cell.
The current results relate to cholesterol efflux from mouse L-cell fibroblasts where at least 60% of the plasma membrane cholesterol effluxes from a single kinetic pool under the experimental conditions utilized. 2 However, some cell types appear to have several pools of plasma membrane cholesterol which may be accessed preferentially by apoA-I present in a particular conformation (32). Furthermore, it has been proposed that the conformation of apoA-I in pre-␤ HDL species is different to that in spherical HDL (33), and these small discoidal acceptors may access cholesterol in certain human fibroblast plasma membrane domains, such as caveolae, more efficiently (41). Further work is necessary to determine whether particular domains of the apoA-I molecule are important for efflux from these specific plasma membrane pools of cholesterol.  Plasma ApoA-I  194  9  18  20  ApoA-I (⌬44-126)  95  4  16  25  ApoA-I (⌬139-170)  144  7  21  28  ApoA-I (⌬190-243)  119  5  15  25 a Determined from the percent ␣-helix content (Table I) and the total amino acid residues of the protein.
b Number of helices was calculated from the number of helical residues assuming that each helix is 22 amino acids in length. c Calculated by multiplying the number in b by the number of protein molecules per disc (Table I). d The circumference of rHDL particles was calculated from the major diameter measured by EM and corrected for the apolipoprotein contribution by subtracting the diameter of an ␣-helix (1.5 nm). The number of ␣-helices packed around this circumference was estimated by dividing by the ␣-helix diameter. (cf. Ref. 22).