{'fraction_non_alphanumeric': 0.03483163187566662, 'fraction_numerical': 0.028066433033673625, 'mean_word_length': 4.5885558583106265, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 1, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Mechanism and Properties of Inhibition of Purified Rat Brain Adenylate Cyclase by G Protein βγ-Subunits

The mode of the inhibition of purified rat brain adenylate cyclase by the beta gamma-subunits of G protein (beta gamma) was studied. These subunits inhibited the catalytic activity of the cyclase with the maximal inhibition of 85% and the half-maximal inhibition at about 0.7 nM beta gamma. The complex of beta gamma and adenylate cyclase isolated by density gradient centrifugation contained 1.8-2.0 mol beta gamma per mol of the cyclase when beta gamma was assayed by immunoblotting and by its inhibitory activity on adenylate cyclase. However, the beta gamma concentration-inhibition curves suggest that one of the two beta gamma molecules bound may be essential for the inhibition. The role for the second beta gamma molecule is unknown. As a tentative estimate, 70% of the adenylate cyclase activity remained inhibited by beta gamma when the complex was isolated. The inhibition was not dependent on G alpha s or calmodulin. Although purified adenylate cyclase contained a protein (0.06-0.08 mol/mol of adenylate cyclase) that reacted with anti-G alpha s antibody, this protein was not liberated from the cyclase when it formed a complex with beta gamma. In addition, guanine nucleotide analogs little affected the cyclase activity or the inhibition by beta gamma. The inhibition by beta gamma was reversed by the dilution of the complex, and the following re-addition of beta gamma suppressed the enzyme activity to about 15% of the initial activity again. These findings provide strong evidence that beta gamma inhibits adenylate cyclase directly and reversibly through the formation of the complex.

Adenylate cyclase (ATP pyrophosphate lyase [cyclizing], EC 4.6.1.1) is a component of the signal transduction system in plasma membranes. The enzyme is under dual regulation by the stimulatory and inhibitory receptors and G proteins. The activation of a G protein through the receptor promotes the binding of GTP to the G protein and accelerates the dissociation of G protein subunits into the GTP-binding a-subunit and Pr. The asubunit is unique for each G protein. The GTP-activated as-subunit of Gs directly activates adenylate cyclase. The dissociated ai and Pr of Gi have been considered to inhibit the cyclase activity directly or indirectly (1,2).
The inhibition of adenylate cyclase activity by the iso-lated Pr has been shown in both plasma membranes (3,4) and the solubilized and purified cyclase preparations (5)(6)(7)(8)(9)(10). Although Pr was considered in early studies to inhibit the cyclase activity indirectly by deactivating as, we (5) and Katada et al. (6) proposed that the inhibition of brain adenylate cyclase by Rr was direct. In recent cloning studies of cDNAs for several types of adenylate cyclase, Gilman and colleagues showed that Pr moderately inhibited purified recombinant type I adenylate cyclase activated with as (10). The other five types of cloned adenylate cyclase are not inhibited by Pr (11,12).
In the present study, we present evidence that Pr binds to and reversibly inhibits highly purified brain adenylate cyclase independent of as or calmodulin.
Purification of adenylate cyclase Adenylate cyclase was purified by affinity chromatography on forskolin-agarose according to the method of Pfeuffer et al. (13). The procedure was modified as follows: The membrane homogenate (13-15 mg protein/ml) prepared from 90g of fresh rat brains was mixed with 1/19 volume of 20% (wt./vol.) Lubrol PX, stirred for 60 min and centrifuged for 60 min at 105,000 x g. The supernatant obtained was diluted with 4 volumes of buffer 1 and applied to the forskolin-agarose column (25 ml) equilibrated with the same buffer. The column was washed as described by Pfeuffer et al. (13). The cyclase was eluted with a gradient (150 ml) of 0 to 100 pM forskolin in buffer 2. The cyclase eluted was concentrated in an Amicon concentrator equipped with a PM-30 membrane (Amicon, Lexington, MA, USA). To reduce the NaCl concentration, the concentrated sample was diluted with 3 volumes of buffer 3 lacking Tween and applied to the DEAE-5PW column (0.8 x 7.5 cm) equilibrated with buffer 3. The cyclase was eluted with a gradient (30 ml) of 0-500 mM NaCl.
The cyclase was purified by 4,000-to 5,000-fold from the Lubrol-solubilized supernatant. The yield was 25 -30 pg protein from 180 g of rat brain. The recovery of the cyclase activity from the supernatant was 3 % -4010. The activity of the purified enzyme was 5 -6 pmol cyclic AMP/min/mg protein in the presence of 100 pM forskolin, which is comparable to the enzyme activity reported by Pfeuffer et al. (13).

Assay of adenylate cyclase
Adenylate cyclase was assayed for 15 min at 30'C in 100 p1 of the medium containing 20 mM HEPES, pH 7.0, 20 mM KCI, 3.2 mM MgC12, 0.2 mM ATP, 0.2 mg/ml BSA, 0.2 mM dithiothreitol and 0.015% Lubrol PX unless otherwise mentioned. The reaction was terminated by immersing the tubes in boiling water for 90 sec. The amount of cyclic AMP produced was determined by the cyclic AMP-binding protein assay (14).

Inhibition of adenylate cyclase activity by Pr
Purified adenylate cyclase and Rr purified as previously described (5) were preincubated together for 10 min at 231C in 30 p1 of buffer containing 20 mM HEPES, pH 7.4, 0.7 mg/ml BSA, 0.7 mM dithiothreitol and 0.05% Lubrol. The assay medium (65 p1) containing no ATP was added to the preincubated cyclase and Pr, and the mixture was further preincubated for 40 min at 6C. °ATP (5 pl) was added to the mixture and then adenylate cyclase activity was assayed as above in the presence of 3.2 mM MgC12.

Glycerol-density gradient centrifugation
Purified adenylate cyclase and Ar in 0.1 ml were preincubated for 10 min at 231C and then layered on a 8%-30% (wt./vol.) glycerol-density gradient (4.4 ml) in 25 mM Tris-HCI, pH 8.0, buffer containing 1 mM dithiothreitol and 0.05% Lubrol PX. The gradient was centrifuged for 14 hr at 43,000 rpm (222,000 x g) at 4°C in a Beckman SW50.1 rotor (Beckman Instruments, Palo Alto, CA, USA), and fractions were collected from the bottom of the tubes.

Quantification of Pr
In Fig. 3, Pr were assayed by the Pr-stimulated ADPribosylation of ai/ao with pertussis toxin as previously reported (5,15). An aliquot of the fraction containing Pr or the control buffer was added to the reaction mixture containing ai/ao, pertussis toxin and [32P]NAD. After the reaction, protein was precipitated and washed with trichloroacetic acid. The radioactivity incorporated into the protein was measured in a scintillation counter. Purified AT was used for a standard curve to calculate the Pr concentration in a sample. Upon SDS-PAGE of the precipitated protein, radioactivity was incorporated only into the ai-and a,,-subunits.
The low concentration of Pr seen in Fig. 3 was quantified by the inhibition of adenylate cyclase activity. A 50-p1 aliquot of the fraction containing Pr was added to 5 p1 of the purified cyclase (final concentration of 0.77 nM during the assay), and the mixture was preincubated for 5 min at 23C. Then 40 p1 of the assay medium omitting ATP was added to the mixture, and the preincubation was further continued for 40 min at 6C. After the preincubation, the adenylate cyclase activity was assayed in the presence of ATP and 3.2 mM MgC12. Each assay tube contained 0.77 nM of the added purified adenylate cyclase and the cyclase from the fraction. The enzyme concentration of the latter was separately determined by the enzyme assay in the presence of 50 mM MgC12, which abolished the inhibition by Pr contained in the fraction (see the text). The 010 inhibition of adenylate cyclase activity by Pr was calculated by dividing the enzyme activity determined as above by the activity of the same concentration of adenylate cyclase in the absence of Pr. The latter enzyme activity was calculated from the activity of the purified cyclase (0.25 pmol cyclic AMP/min /nmol enzyme in the absence of Pr) determined under the same conditions. Finally, the Pr concentration during the assay was determined, using the Pr concentration-Olo inhibition curve (the inserted figure of Fig. 3).

Immunoblotting
Immunoblotting of G protein subunits was conducted as described by Towbin et al. (16). Samples were subjected to SDS-PAGE (12% gel) and were transferred to an Immobilon PVDF membrane (Nihon Millipore, Tokyo). Antibodies indicated in the figure legends and 125I-labeled secondary antibody (F(ab")2 fragment) were used. The measurement of radioactivity on the Immobilon PVDF membrane was conducted in a Fujix bioimaging analyzer BAS2000 (Fuji Photo Film, Tokyo) which gave the radioactivity in a PSL unit (Photo-Stimulated Luminescence value). The unit was proportional to the total disintegration of 125I during the exposure. In Figs. 4 and 7, the fractions containing adenylate cyclase and associated Pr from density gradient centrifugation were combined and concentrated in a Millipore Molcut concentrator (nominal cut-off molecular weight of 30,000, Nihon Millipore) before SDS-PAGE. The amount of the concentrated cyclase was estimated by the density of silver-stained cyclase on a polyacrylamide gel, using the purified cyclase as standard, because the concentration procedure resulted in a decrease (about 40%) in the cyclase activity probably due to adsorption of the enzyme to the filtration membrane and the partial inactivation of the enzyme.

Protein determination
The method of Schaffner and Weissmann (17) was used for microassay of protein. Briefly, protein precipitated on a filter with trichloroacetic acid was dyed with amido black. The dye adsorbed to the protein was solubilized with 100 pl of ethanol solution, and the optical density of the solubilized dye was measured at 630 nm in a microcuvette. A linear standard curve was obtained, using up to 2 pg of BSA. With 1 pg of BSA, the optical density at 630 nm was 0.23 -0.25 and the standard deviation was 3% of the mean.

Miscellaneous procedures
SDS-PAGE was carried out by the method of Laemmli (18). The densities of silver-stained protein bands on the polyacrylamide gel were measured in a Shimadzu flyingspot scanner CS9000 (Shimadzu, Kyoto).

Properties of adenylate cyclase inhibition by Pr
Adenylate cyclase was purified from rat brain by successive chromatography on forskolin-agarose and DEAE-5PW columns as described in the Materials and Methods. Figure 1 shows that major proteins of Mr 180,000 and 135,000 were coeluted with adenylate cyclase activity from the forskolin-agarose column. Both the proteins are considered to be adenylate cyclase as discussed later. Densitometric measurement of silver-stained bands in SDS-PAGE of the purified cyclase preparation showed that the proteins of Mr 180,000 and 135,000 accounted for 77% of the total protein in the final enzyme preparation, and these two major proteins were present in a ratio of 1 : 2.0. Based on this ratio, the weight-average molecular weight of 150,000 for adenylate cyclase was calculated. We used the molecular weight of 150,000 and the purity of 77% to calculate the molar concentration of the cyclase. Proteins of Mr 66,000, 38,000, 36,000 and 28,000 (shown by the arrows in Fig. 1) and other minor proteins of Mr 55,000 and 43,000 were also coeluted with the enzyme activity. The purified Pr derived from G; and Go strongly inhibited the catalytic activity of the purified cyclase as shown in Fig. 2. Cyclic AMP accumulating during the assay increased linearly up to at least 20 min both in the presence and absence of Pr. The inhibition was not observed when Pr were denatured by heating. In the presence of 3 mM MgC12 in an excess of Mg-ATP, the Rr concentration for half-maximal inhibition (EC50) was about 0.7 nM and the maximum inhibition was 85%. In the presence of 50 mM excess MgC12, the Pr-dependent inhibition decreased. Particularly, the inhibition was almost abolished when the Pr concentration was less than 1 nM. This property was used to determine the concentration of adenylate cyclase in the fraction containing Pr as described later.

Association of Pr with adenylate cyclase
From the pr-induced inhibition described above, it is considered that Pr interacted with adenylate cyclase. This was examined by density gradient centrifugation of Pr and the cyclase.
Purified adenylate cyclase was preincubated with a sufficient amount of Pr to inhibit the cyclase activity maximally and then subjected to the centrifugation in a glycerol-gradient. During the centrifugation, free Pr, assayed by the Pr-stimulated ADP-ribosylation of ai/ao with pertussis toxin as previously reported (5,15), was separated from the cyclase and remained near the top of the gradient (Fig. 3). On the other hand, adenylate cyclase, assayed in the presence of 50 mM or 3 mM Mg2+, sedimented toward the bottom of the gradient (Fig. 3).
As shown in Fig. 2, assay of adenylate cyclase in the presence of 50 mM MgC12 nearly abolished the inhibition by a low concentration of Ay (< 1 nM). This abolition was observed between the examined cyclase concentrations of 0.025 and 0.2 nM, which suggested that the concentration of adenylate cyclase in the fraction containing Pr shown in Fig. 3 could be determined by the assay in the presence of 50 mM MgC12 under these conditions. Actually, when adenylate cyclase centrifuged in a density gradient in the absence of Rr as a control in Fig. 3 was diluted 20-fold (final concentration) in the assay, the cyclase concentrations in the peak fractions of the enzyme determined by the standard assay in the presence of 3.2 mM MgC12 were within the range of 0.025-0.2 nM. Therefore, we assayed adenylate cyclase in the fraction (Fig. 3) in the presence of 50 mM MgC12 by using a final 20-fold dilution, assuming that the cyclase concentration in this fraction was similar to that of the control cyclase centrifuged in the absence of Pr. The determined concentrations of adenylate cyclase from fractions 1-5 in Fig. 3 were 0.05 -0.09 nM during the assay, which were within the range of adenylate cyclase concentration (0.025 -0.2 nM) allowed for the estimation. In addition, the concentrations of Pr from fractions 1-5 determined as described below were 0.07 -0.22 nM during the assay. Therefore, the quantification of adenylate cyclase was performed under proper conditions. The enzyme activity in the presence of 3 mM excess Mgt+, as shown in Fig. 3, was lower than that assayed in the presence of 50 mM Mgt+, although the enzyme assay in the presence of 50 mM and 3 mM Mg2+ gave similar activities in the absence of Pr (see Fig. 2). This indicates that the enzyme activity with 3 mM Mgt+ was under inhibition by Pr contained in the adenylate cyclase fractions. So we estimated the concentration of Ay in the fraction. However, correct quantification of (3r in these fractions by the ADP-ribosylation assay was difficult due to the low ~r concentration. Then, we tried to use the inhibitory activity of i3r on adenylate cyclase to estimate the Rr concentration (Fig. 3). Briefly, an aliquot of each adenylate cyclase fraction was added to the purified cyclase (0.77 nM), and the ° inhibition of the cyclase activity by 1T contained in the fraction added was obtained from the cyclase assay with 3.2 mM MgC12 as described in the Materials and Methods. Then, the 1r concentration was determined from the Pr concentration-076 inhibition curve shown in the inserted figure of Fig . 3.
The ,3r concentration could be determined independently of the cyclase concentration in the fraction, because the total cyclase concentration during the assay was 0.77 -1.54 nM including 0.77 nM purified enzyme, and no difference of the curve was observed with 0.77, 1.00 or 1.54 nM cyclase (the inserted figure of Fig. 3). Fig. 4. Quantification of 13r-subunits (Pr) in the adenylate cyclase fraction from density gradient centrifugation by immunoblotting of the p-subunit. The adenylate cyclase fraction was obtained as shown in Fig. 3 and concentrated as described in the Materials and Methods. Immunoblotting was conducted as described in the Materials and Methods. The p-subunit was immunoblotted with an antibody (SW/1) recognizing the carboxyl-terminus of the R-subunit. Radioactivity was measured in a Fujix bioimaging analyzer. Lanes 1, 2 and 5, purified ;3r as standard (0.45, 1.36 and 0.14 pmol, respectively); lane 3, adenylate cyclase fraction (corresponding to the fractions 2-5 in Fig. 3 In fractions 1-5, the Pr concentration was proportional to the cyclase concentration determined by the enzyme assay with 50 mM MgC12 (Fig. 3). The average molar ratio of Pr to the cyclase in fractions 1-5 was 2.0 mol Pr per mol of the cyclase. These results indicate that Pr in the cyclase fraction actually sedimented as a complex with adenylate cyclase. In the fraction 6 and later, the (3r concentration increased markedly due to the sedimentation of free Pr. When adenylate cyclase was omitted from the centrifugation, the amount of Pr in the bottom fractions 1-5 was under the limit of detection. The amount of Pr in the cyclase fraction was also quantified by immunoblotting with anti-p-subunit antibody (SW/1) (Fig. 4). Using purified Pr as the standard, we estimated the content of j3-subunit in the cyclase fraction to be 1.8 mol p per mol of adenylate cyclase.
From the experiments described above, it was found that 1.8-2.0 mol of AT was associated with 1 mol of adenylate cyclase. So, we tried to distinguish by kinetic analysis whether only one or two Pr molecules were required for the inhibition of adenylate cyclase activity. Figure 5A shows the plots of the reciprocal of adenylate cyclase activity (1/v) against Pr concentration (i). In the plots of 11v against i, the plots will be linear if only one inhibitor molecule can bind to the enzyme. The plots will curve upward (parabolically) if more than one inhibitor molecule can bind to the same form of the enzyme (19). We obtained a line that curved downward (line A), similar to that in a case of partial inhibition (19). It is not known whether this downward curve is due to either the property of the enzyme itself or the presence of another type of adenylate cyclase which was refractory to Pr. Then, we tentatively attempted, as the case of the latter, to subtract the enzyme activity remaining in the presence of an excess of Pr from the enzyme activities measured at various Pr concentrations. Re-plotting of the reciprocal of the subtracted activity gave an approximately straight line (line B), but not a line that curved upward.
The data was also analyzed by a Hill plot of the (3r concentration and the inhibition (the reduction in adenyl-ate cyclase activity by Pr) (Fig. 5B). The slope of the line gave a Hill coefficient of 0.85. Therefore, no evidence for the requirement of two Pr molecules in the inhibition was obtained from the analysis above.

as in adenylate cyclase preparation
It is important to examine whether the purified adenylate cyclase contained G protein a-subunits, because asubunits may serve as the binding site for Pr. Figure 6 shows the immunoblots of the purified cyclase with the antibodies (AS/7, GO/1, RM/1, SW/1 and MS/1) raised against the subunits of G proteins. The properties of these antibodies have been well-studied (20). Neither a; nor ao was detected by immunoblotting (Fig.  6A, lanes 1 and 2). In some experiments, a protein of Mr 29,000 in the cyclase preparation reacted with anti-a0 antibody (GO/1) but this was not studied further. The psubunit was not detected in the purified cyclase (Fig. 6B,  lanes 2 and 3).
The immunoblotting in Fig. 6A (lane 3) showed that a protein of Mr 43,000 reacted with anti-as antibody, although we have no further evidence that this immunoreactive protein is as. The immunoreactive protein was still found in the adenylate cyclase fraction after the centrifugation in a density gradient. The preincubation of adenylate cyclase with (3r before the centrifugation did not promote the liberation of the immunoreactive protein from the cyclase (Fig. 7). Therefore, the inhibition by Pr is not due to the liberation of the immunoreactive protein from the cyclase. The content of the immunoreactive protein of 43 kDa was estimated by densitometric measurement of silverstained proteins in a Shimadzu flying spot scanner after SDS-PAGE of purified adenylate cyclase. The amount of silver-stained protein of 43 kDa (probably the immunoreactive protein) was 1.8% of that of silver-stained adenylate cyclase on the same gel, that is, 0.06 mol of the immunoreactive protein per mol of the cyclase.
We also attempted to estimate the content from the experiment in Fig. 7, in which the radioactivity on a protein band reacting with anti-as antibody was proportional to the amount of the purified cyclase (0.21-0.88 pmol) loaded on the gel. To calculate the amount of the 43-kDa immunoreactive protein from its radioactivity, we used the radioactivity on the immunoblots of a known amount of s-subunit in Fig. 4. The experiments in Figs. 7 and 4 were conducted simultaneously under the same conditions, and we assumed for the calculation that anti-as and anti-p antibodies used in these experiments reacted with their antigen to the same extent and the second antibody recognized these antibodies with the same efficiency. Then the estimated content of the immunoreactive protein was 0.08 mol per mol of adenylate cyclase.
From the estimation as described above, it is evident that the content of a, if it exists, is too low to serve as the site for the Pr binding in the inhibition.
In the adenylate cyclase assay conducted here, we did not use GTP, a physiological activator of as, or an ATPregenerating system that may phosphorylate the GDP associated with as to GTP. To determine whether as-activated cyclase activity was involved in the pr-dependent inhibition observed here, we examined the effects of guanine nucleotide analogs on adenylate cyclase activity.
Adenylate cyclase was preincubated for 10 min at 23 C in the presence of 5 MM Mg2+ and 100 yM Gpp(NH)p, a hydrolysis-resistant analog of GTP and a strong activator of as, and then the cyclase was assayed for 15 min at 301C (final concentrations of 3.2 mM Mg2+ and 33 pM Gpp(NH)p). Although our previous report (21) on the Gpp(NH)p-dependent activation of partially purified adenylate cyclase with a, showed that preincubation for 30 min with Gpp(NH)p doubled the enzyme activity, we had to limit the preincubation time to 10 min due to instability of the highly purified adenylate cyclase used in the present study. The amounts of cyclic AMP formed in the presence and absence of Gpp(NH)p were 32.9±0.3 and 32.4± 1.0 pmol (mean ±S.E.), respectively. The activation by Gpp(NH)p was not observed. Moreover, the preincubation of the cyclase with 5 MM MgC12 and 30 pM GDPpS, a hydrolysis-resistant analog of GDP, in order to replace the guanine nucleotide bound to as did not cause any reduction of the cyclase activity in the absence of Pr. There was no difference between the Pr concentration (0.11-114 nM)-inhibition curves after preincubation with or without GDPpS (data not shown). These results show that adenylate cyclase activity and the effect of jar observed here were independent of as. Fig. 6. Immunoblotting of purified adenylate cyclase with the antibodies against G protein subunits. Immunoblotting was conducted as described in the Materials and Methods. Panel A: Lanes 1-3, 0.87 pmol of the purified adenylate cyclase ("C"); lanes 4-6, 1.1 pmol of the mixture of ao and a, ("ao/a,"). Antibodies used were AS/7 recognizing the carboxyl-termini of at, a,, and a,2 (lanes 1 and 4), GO/1 recognizing the carboxyl-termini of a,, and a,3 (lanes 2 and 5) and RM/1 recognizing the carboxyl-terminus of a, (lanes 3 and 6). Panel B: Lanes 1 and 4, 1.9 and 2.5 pmol of Pr-subunits respectively; lanes 2 and 3, 0.87 pmol of purified adenylate cyclase ("C"). Antibodies used were SW/l recognizing the carboxyl-terminus common to p,, p2 and p3 (lanes 1 and 2) and MS/l recognizing the amino-terminus of pj (lanes 3 and 4).
In addition to a, Ca t+/calmodulin was also not involved in the inhibition by (3r. The presence of 1 mM EGTA, to eliminate the effects of Cat+, in the assay medium showed no effect on the inhibition by /3r (data not shown). Adenylate cyclase activity of the complex with (3r and the reversibility of the inhibition by ( r We measured adenylate cyclase activity in the complex fraction obtained in Fig. 3 to determine if the cyclase activity in the complex fraction was inhibited to a similar extent as in the standard assay system (85% inhibition). The complex fraction was added to an equal volume of assay medium so that the dilution of the complex fraction was minimum (0.47 nM cyclase during the assay in a final volume of 100 p1) and also the detergent and glycerol brought in from the complex fraction did not exceed the concentration allowed for the assay. Then, the specific activity (pmol cAMP/min/pmol of the enzyme) of the cyclase in the complex fraction measured was about 60% of that of the control cyclase centrifuged in a density gradient in the absence of pr. Because this result was quite different from that observed in the standard assay system, we considered that ~ r might be dissociated from the cyclase due to the dilution of the complex with the assay medium containing ATP and Mgt' and the elevation of temperature in the assay. Actually, dilution of the complex fraction by 10-fold with the buffer used for density-gradient centrifugation and the following addition of the diluted complex fraction to the assay medium (final concentration, 0.047 nM cyclase) increased the measured specific activity of the cyclase from about 60% to 86% of the control cyclase activity. We also tentatively tried to estimate the cyclase activity in the complex fraction that was not diluted. The % specific activities of the fraction assayed with various extents of dilution were plotted against the cyclase concentration during the assay. Then, the approximately linear line obtained was extrapolated to the cyclase concentration (0.94 nM) in the undiluted complex fraction. As a result, the % specific activity at 0.94 nM cyclase on the plot was estimated to be about 30% of the control (70% inhibition). The estimated % inhibition, which was close to the maximal inhibition in the standard assay system, was tentatively considered to indicate that the adenylate cyclase in the complex stayed nearly fully inhibited by pr under the assay conditions employed here.
If the dissociation of Pr from the cyclase suggested above is the case, the cyclase activity enhanced by dilution must be inhibited again by the re-addition of Pr. In Fig.  8, adenylate cyclase activity of the complex fraction (diluted to 0.08 nM cyclase) assayed under standard conditions was indeed inhibited again by the re-added Pr. Both enzyme activities in the complex fraction and the control adenylate cyclase were inhibited to the maximum (about 15% of the initial activity of the control cyclase) with similar concentrations of (r. These results show that the inhibition by Pr is reversible. The effects of dilution and Pr re-addition described above may be explained if the inhibition is assumed to be a reversible bimolecular interaction between adenylate cyclase and Pr as suggested above, although the formation or dissociation of the complex in a buffer system containing detergent may not be a simple process. Fig. 8. Reversible inhibition of adenylate cyclase from density gradient centrifugation. Adenylate cyclase was preincubated in the presence or absence of pr-subunits (Pr) and centrifuged in a density gradient as described in Fig. 3. The complex of adenylate cyclase and Pr (/) or adenylate cyclase containing no Pr (control adenylate cyclase) (0) was obtained and diluted to a final concentration of 0.08 nM cyclase for the assay. The dilution increased the enzyme activity in the complex fraction from 69% (at 0.4 nM cyclase) to 82% (at 0.08 nM cyclase) of the control cyclase activity. The enzyme activity was assayed with re-addition of the indicated concentration of Pr. The addition of Pr inhibited adenylate cyclase activity in the complex fraction to approximately 15% of the control enzyme activity assayed in the absence of jar. Data shown are representative of the experiments repeated twice.

DISCUSSION
In the present study, we confirmed the previous observations (5,6) that the enzyme activity of brain adenylate cyclase was effectively inhibited by Pr of G proteins. The inhibition shown here was reversible and dependent on the Pr concentration. This suggests that the inhibition by Pr described here may serve as the physiological mechanism for adenylate cyclase regulation in the brain.
Because both p-and r-subunits have subtypes, various forms of Pr exist. Iniguez-Lluhi et al. (22) recently reported that Pr generated by the combination of p1 or P2 with rt, r2 or r3 showed similar inhibitory effects on the activity of type I adenylate cyclase except P1x1 (Pr from transducin) which inhibited the enzyme activity relatively weakly. It is not certain that only particular forms of Pr inhibit adenylate cyclase activity.
In the present study, Pr associated reversibly with adenylate cyclase. Because 1.8-2.0 mol of Pr was found to be associated with one mole of adenylate cyclase, we tried to distinguish whether the inhibition required only one or two (3r molecules. The results suggested that one of the two Pr molecules bound to the cyclase was essential for the inhibition. The property and the function of the other Pr molecule suggested is unknown, but the possibility remains that the binding of the second Pr molecule serves to stabilize the inhibition complex. However, it should be noted that the ratio mentioned above is based on the molecular weights determined by SDS-PAGE and the protein concentrations assayed by using bovine serum albumin as the standard. Our previous (9) and present studies have shown that calmodulin and the as-subunit were not the binding site for Pr in the inhibition observed here. The content of the protein immunoreactive to anti-as antibody (0.06-0.08 mol/mol of the cyclase) was too low to serve as the site of the Pr binding. In addition, the immunoreactive protein was not liberated from the cyclase even in the presence of Pr. Thus it is very unlikely that the inhibition requires a, or calmodulin.
The adenylate cyclase purified here contained major proteins of Mr 180,000 and 135,000 as clearly visible bands on a polyacrylamide gel. Both the proteins appear to be adenylate cyclase on the basis of the reported molecular weights of mammalian adenylate cyclase (105 to 180 kDa) (7,13,(23)(24)(25)(26)(27). Particularly, even Ca t+/calmodulin-sensitive adenylate cyclase purified from the brain by calmodulin-affinity chromatography has a molecular weight of 135,000-140,000 (24)(25)(26). In addition, Orlando et al. (26) found a Ca 2+/calmodulin-sensitive cyclase of Mr 175,000 from the brain whose apparent molecular weight changed to 135,000 in the course of purification. Other forskolin-binding proteins (glucose carriers) have been reported, but they have much smaller molecular weights (Mr 40,000-60,000) than adenylate cyclase (28). Therefore, the two major bands on the gel may be isozymes or they might be generated by a modification such as proteolysis or by a difference in sugar chains covalently attached to the enzyme. The complex of adenylate cyclase and Pr contained both forms of the cyclase (Mr 180,000 and 135,000). However, it is not yet certain whether or not Pr inhibits the two forms of the enzyme to the same extent.
We have no direct evidence that the adenylate cyclase purified here is the type I cyclase that is localized in neural tissues (29). However, the sensitivity of the purified enzyme to Ca 2+/calmodulin (9) or to Pr indicates that the purified cyclase is the type I enzyme or a similar one. Type II adenylate cyclase, which is also found in the brain, is activated by Pr and is not sensitive to Ca 21/calmodulin (11,12). The nature of minor proteins that copurified with the adenylate cyclase is not known. The protein of Mr 38,000 might be the same protein reported by Mollner et al. (30) who found that the proteins of Mr 38,000 associated with adenylate cyclase cross-reacted with the antibody against synaptophysin. The copurified protein of Mr 36,000 was similar in molecular weight to the G protein p-subunit. However, this minor protein did not cross-react with the antibody raised against the carboxyl-or amino-terminal peptide of the p,-subunit, whose carboxyl terminal amino acid sequence is also shared by P2 and N3-subunits (31). Therefore, this protein is not P1 and is unlikely to be h or ~3.