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Preparation and characterization of subfractions of bovine thyroid plasma membranes.

Two subfractions of bovine thyroid plasma membranes, light membranes (L-membranes) and heavy membranes (H-membranes), were obtained by a discontinuous sucrose gradient centrifugation of plasma membranes. Electron microscopy of the plasma membrane and its subfractions showed that the H-membranes were very similar to the plasma membrane fraction, both contained junctional complexes, long membrane sheets, and vesicles. In contrast, the L-membranes consisted mainly of short membrane sheets and vesicles, and only a few junctional complexes. The H-membranes had greater adenylate cyclase activity which responded to thyroid-stimulating hormone (TSH) while this hormone had very little effect on the enzyme activity in the L-membranes. Despite the marked difference in TSH stimulation of adenylate cyclase activity in the H- and L-membrane fractions, specific binding of 125I-TSH was similar in both fractions. The L-membranes had higher specific activities of 5'-nucleotidase and Mg2+ATPase while (Na+ + K+)-ATPase and alkaline phosphatase activities were similar in the two subfractions. Protein kinase activity of H-membranes was not significantly stimulated by exogenous cyclic adenosine 3':5'-monophosphate (cAMP). Plasma membranes and H-membranes contained a substrate capable of being phosphorylated. Such phosphorylation was slightly increased by addition of soluble protein kinase. The phosphorylation of exogenous histone by protein kinase of plasma membranes and H-membranes was augmented by cAMP. In contrast, L-membranes had very little protein kinase activity even when exogenous histone was added. They were not a very good substrate for cytosolic protein kinase.


Two subfractions
of bovine thyroid plasma membranes, light membranes (L-membranes) and heavy membranes (Hmembranes), were obtained by a discontinuous sucrose gradient centrifugation of plasma membranes. Electron microscopy of the plasma membrane and its subfractions showed that the H-membranes were very similar to the plasma membrane fraction, both contained junctional complexes, long membrane sheets, and vesicles. In contrast, the L-membranes consisted mainly of short membrane sheets and vesicles, and only a few junctional complexes. The Hmembranes had greater adenylate cyclase activity which responded to thyroid-stimulating hormone (TSH) while this hormone had very little effect on the enzyme activity in the L-membranes.
Despite the marked difference in TSH stimulation of adenylate cyclase activity in the H-and L-membrane fractions, specific binding of 12"I-TSH was similar in both fractions. The L-membranes had higher specific activities of 5'-nucleotidase and Mg'+ATPase while (Na+ + K+)-ATPase and alkaline phosphatase activities were similar in the two subfractions.
Protein kinase activity of H-membranes was not significantly stimulated by exogenous cyclic adenosine 3':5'-monophosphate (CAMP). Plasma membranes and H-membranes contained a substrate capable of being phosphorylated.
Such phosphorylation was slightly increased by addition of soluble protein kinase. The phosphorylation of exogenous histone-by protein kinase of plasma membranes and H-membranes was augmented by CAMP. In contrast, L-membranes had very little protein kinase activity even when exogenous histone was added. They were not a very good substrate for cytosolic protein kinase.
Thyroid-stimulating hormone regulates thyroid gland function following its initial binding to specific hormone receptors on the plasma membrane and activation of the adenylate cyclase-adenosine 3':5'-monophosphate system (l-11 to provide a final concentration of sucrose of 45%. Sucrose solutions of41% (10 ml) and 10% (4 to 6 ml) were layered over the membrane suspension. The tubes were centrifuged at 25,000 'pm (63,600 x g) for 120 min using a SW 25 rotor in a Beckman ultracentrifuge.
The materials at the upper interface and the lower interface were collected by aspiration and designated plasma membranes and second band, respectively.
One-third of the plasma membranes and the entire second band were diluted 1:l with 1 mM NaHCO, buffer and centrifuged at 18,000 rpm (33,000 x g) for 25 min in a Beckman ultracentrifuge.
The precipitates were suspended in 0.2 to 0.4 ml of 1 mM NaHCO, buffer and used immediately for all enzyme assays except for adenylate cyclase and protein kinase. These enzymes were assayed within 1 week, at which time there was no loss of activity as a result of storage at -20". The remaining plasma membranes in sucrose solution were refolded into 63% sucrose to provide a final concentration of sucrose of 45%. Sucrose solutions of 41% (6.5 ml), 35% (10 ml), and 10% (2 to 3 ml) were layered over it. The tubes were centrifuged at 25,000 'pm for 120 min using a SW 25 rotor in a Beckman ultracentrifuge. After centrifugation, the materials at the interface between the layers of 10% and 35% sucrose solutions and at the interface between the layers of 35% and 41% sucrose solutions were obtained by aspiration and designated light membranes (Lmembranes) and heavy membranes (H-membranes), respectively. These fractions were diluted 1:l with 1 mM NaHCO, buffer and then centrifuged at 18,000 rpm for 25 min. The precipitates were treated as outlined above for the plasma membranes. Enzyme Assays-Enzyme activities were determined in duplicate in fresh preparations except for adenylate cyclase and protein kinase as described above. Adenylate cyclase activity was assayed as previously described

RESULTS
The data in Table I  sheets, and vesicles ( Fig. 1). Occasionally, profiles compatible with the apical plasmalemma of the thyroid cell with its adjacent vesicles were observed (Fig. 2). The Hmembrane fraction was very similar in appearance to the plasma membrane fraction (Fig. 31, whereas the L-membrane fraction appeared as short membrane sheets or as vesicles (Fig. 4). An unusual feature of this fraction, which was observed frequently, was the apparent continuity of the membrane profiles associated with junctional complexes (Fig. 4). An attempt was made to identify the structures which contained 5'-nucleotidase using a cytochemical procedure for visualizing the enzyme in isolated cell fractions (33) (2) 0.144 IT 0.010 (7) 0.078i0.006 (2) (2) (2) (2) (2) (2)   all of the membrane fractions except the L-membranes. Addition of protein kinase prepared from the cytosol of bovine thyroid increased significantly phosphorylation of endogenous substrate in plasma and H-membranes but not in L-membranes. However, such phosphorylation was not significantly augmented by CAMP. The greatest phosphorylation of all the fractions was obtained when both cytosolic protein kinase and histone were added. As would be expected from the experiment in which histone was added, CAMP increased phosphorylation when both protein kinase and histone were added.

DISCUSSION
Two distinct subfractions of thyroid plasma membranes were obtained by discontinuous sucrose gradient centrifugation of plasma membranes. These subfractions, designated Hand L-membranes, had significant differences in some of their enzyme activities indicating that they represent different parts of the plasma membrane and not just different size fragments of the same membrane. The electron microscopic 'I p i 0.001 as compared to control. " p < 0.02 as compared to that of H-membrane. ' p < 0.001 as compared to that of 0.4 mM ATP. J p < 0.001 as compared to that of L-membrane. lip < 0.01 as compared to control. 1 p < 0.01 as compared to that of L-membrane. examination showed that the plasma membrane fraction contained junctional complexes, which must have originated from the lateral plasmalemma (41), together with membranes which probably originated from the apical plasmalemma.
At present, it is much harder to identify membranes from the basal plasmalemma morphologically; the conclusion that these membranes are present in the plasma membrane fraction is based solely on the biochemical results. The origin of the vesicles which are enriched in 5'-nucleotidase is also not certain, even though the enzyme is concentrated in the plasma membrane of a variety of cells (23). The identification of the region of the plasma membrane from which the vesicles are derived must await the results of cytochemical studies which are in progress on intact cells. The results do, however, indicate that the fragmentation and subsequent fractionation of the plasma membrane need not be the same in different cells. Wisher and Evans prepared six different subfractions of hepatic plasma membranes and tentatively identified them as being from different areas of the cell (42). The blood-sinusoidal face was identified by its high concentration of adenylate cyclase activity and its responsiveness to glucagon. Such membranes were composed mostly of vesicles and were of lower density than those fractions which were considered representative of the contiguous faces of the hepatocytes. Another light membrane fraction, designated Z-L, and also composed primarily of vesicles, was felt to represent bile-canicular face membranes since they were very rich in 5'-nucleotidase, alkaline phosphatase, and Mg2+ATPase.
The results obtained with the H-and L-membrane fractions from the thyroid do not permit such definitive identification. The H-membranes might represent basal membranes because of the marked TSH stimulation of adenylate cyclase. Although Wisher and Evans identified a light membrane fraction as the blood-sinusoidal membrane based on high adenylate cyclase activity and its response to glucagon, five of their six subfractions contained some adenylate cyclase activity which was  3 (bottom lefi). Representative field of the heavy membrane FIG. 1 (top). Representative field of the plasma membrane fracfraction. The appearance is similar to that of the plasma membrane tion. Junctional complexes (long arrows), large and small vesicular fraction.
x 22,500. profiles (short arrow and arrowhead, respectively) are clearly evi- FIG. 4 (bottom right). Representative field of the light membrane dent. x 22,500.
Plasma membrane fraction field selected to illusincluding those associated with junctional complexes (arrow). trate a profile which resembles the apical plasmalemma of an intact x 22,500.   stimulated by glucagon (42). In contrast, Toda et al. reported that most of the hepatic adenylate cyclase activity of plasma membranes was in a heavy membrane fraction but the percentage of increase induced by glucagon was similar in both the heavy and light subfractions (34). The procedure which they used was similar to the one which we utilized except they employed isotonic solutions. Toda et al. observed a similar distribution of 5'-nucleotidase, Mg2+ATPase, and alkaline phosphatase as did Wisher and Evans (42); that is these activities were considerably higher in the light membrane fractions than in the heavy ones. Although the distribution of these enzyme activities in the light membranes of thyroid plasma membranes is similar to that reported in the liver, it does not help to localize such fractions in the thyroid.
The equivalent binding of '%I-TSH to the L-and H-membranes does not exclude the possibility that the H-membranes represent basal membranes. Kinne et al. reported that parathyroid hormone bound to both the contraluminal and luminal ' p < 0.01 as compared to histone (-CAMP). Op < 0.05 as compared to histone (-CAMP). h p < 0.001 as compared to histone (-CAMP). ' p < 0.01 as compared to -CAMP.
fractions of rat renal cortical epithelial cells (43). In their experiments adenylate cyclase activity stimulated by parathyroid hormone was localized to the contraluminal plasma membrane.
The increased vesicles in the L-membrane fraction might be indicative of microvilli in the intact membranes as suggested by Wisher and Evans (42). The apical plasma membrane of the thyroid contains numerous microvilli which are of great importance in colloid droplet ingestion (20). The identification of specific regions of the hepatic plasma membrane (42) is facilitated by cytochemical localization of individual enzymes (44); similar studies have yet to be carried out on the thyroid. Since iodination of thyroglobulin occurs at or near the apical end of the thyroid cell (36,37), it was hoped that assay of peroxidase in the H-and L-membranes might assist in their identification. However, peroxidase activity was very low in both the Hand L-membranes. Most of the peroxidase activity was in the material referred to as the second band. This material also Thyroid Plasma Membrane Subfractions contained the highest specific activity of DPNH-cytochrome c reductase, an enzyme characteristic of microsomes. The intracellular function of CAMP involves activation of protein kinase and phosphorylation of appropriate substrates (X-17). It has been speculated that colloid droplet injestion may involve phosphorylation of the apical membranes by protein kinase (45) similar to the mechanism proposed by Schwartz et al. for vasopressin action on the renal medulla (46). They found that vasopressin activates adenylate cyclase in the contraluminal membrane and the CAMP which is generated then activates a protein kinase in the luminal plasma membrane. The subsequent phosphorylation alters the permeability to water. Although the present results do not exclude this formulation, they do not provide unequivocal support for such a mechanism. Although protein kinase activity was demonstrable in plasma membranes and L-and H-membrane subfractions, it was relatively independent of CAMP. These results are different from those reported by Roques et al. (35). These investigators demonstrated increased phosphorylation of thyroid plasma membranes when the incubation was done in phosphate buffer but not when acetate buffer was used. However, when we used phosphate buffer with membrane fractions, CAMP was still apparently without effect." It is possible that a small amount of CAMP-dependent protein kinase was present in the plasma membranes, but it was obscured by the much larger amount of the CAMP-independent activity (47).
The low protein kinase activity in the L-membranes does not reflect the absence of an appropriate substrate since addition of histone did not significantly increase phosphorylation. Phosphorylation of added histone by H-membranes was further augmented by CAMP. Thus with exogenous substrate, in contrast to endogenous substrate, the protein kinase activity of the H-membranes demonstrated some CAMP dependency. The apparent CAMP independence of phosphorylation of endogenous substrate in the H-membranes is further supported by results obtained when cytosolic protein kinase was added. Addition of cytosolic protein kinase to H-membranes caused a small, but significant, increase in phosphorylation of endogenous substrate, which was relatively CAMP-independent.
Cytosolic protein kinase from thyroid is activated by CAMP when histone is the substrate (30). Cytosolic protein kinase did not significantly augment phosphorylation of endogenous substrate of L-membranes.
Addition of both cytosolic protein kinase and histone was associated with increased phosphorylation in both L-and H-membranes, and as would be expected, this was augmented by CAMP (30). Although the basal phosphorylation varied when both histone and protein kinase from the c.ytosol were added. the additional increment in the presence of CAMP was the same in all the subfractions suggesting that it was independent of the membrane subfraction.
Since the protein kinase of the H-membranes was CAMPdependent when histone was added to the incubation, it is possible that this enzyme phosphorylates a substrate in the cytosol which then induces the membrane changes seen in response to TSH. Another possibility is that the endogenous substrate of the protein kinase of the H-membranes was lost during preparation of this fraction. Either of these explanations would preserve the concept of a TSH-induced membrane change as a result of phosphorylation.