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Inhibition of adenylate cyclase by a membrane protein from rat cerebrum
157
Brain Research, 264 (1983) 157-159 Elsevier Biomedical Press
Inhibition of adenylate cyclase by a membrane protein from rat cerebrum MAMORU SANO, SATOKO KITAJIMA and AKIRA MIZUTANI
Institute for Developmental Research, A ichi Prefectural Colony, Kamiya-cho, Kasugai, A ichi, 480-03(Japan) (Accepted November 16th, 1982)
Key words: adenylate cyclase inhibitor - detergent- guanine nucleotide
The inhibitor protein of adenylate cyclase was partially purified from detergent-extract of rat brain. The inhibition occurred without lag phase. Calmodulin, GTP, ~uanyl 5'-yl imidodiphosphate (Gpp (NH) p) and forskolin did not change the inhibition, but activities stimulated by NaF or Mn 2+ were more resistant for the inhibition. The inhibitor might include an essential sulfhydryl group.
It is known that adenylate cyclase system includes three independent components, a hormone receptor, guanine nucleotide regulatory unit and a catalytic unit ~1,~2,]4. However, several endogenous factors have been found to affect the activity of adenylate cyclase in the brain. Calmodulin is present in high concentrations and has been shown to regulate adenylate cyclase in this tissuea,3. Brain also contains several calmodulin binding proteins, which inhibit adenylate cyclase by specific association with calmodulin L5,]6. Additionally, soluble protein factor which stimulates adenylate cyclase has been shown in the developing rat brain I°. Brain adenylate cyclase is not fully understood. Recently, we have shown that the detergent-solubilized particulate fraction from rabbit cerebral cortex contained an inhibitor protein(s) of adenylate cyclase, which was inactivated by heat-treatment and by incubation with trypsin 13. Inhibitory action was not reversed by Ca 2÷ plus calmodulin and therefore, it does not appear to be a calmodulin binding protein. Inhibition was not due to the destruction of cyclic AMP by phosphodiesterase or of ATP by ATPase. In the present study, we have attempted to purify the inhibitor protein from the rat cerebrum and to examine the nature of this enzyme inhibition. Adenylate cyclase was assayed as described previouslyt3. Since the inhibition occurred both
in the presence or absence of Ca 2+ , experiments were performed in the presence of EGTA to avoid the effect of calmodulin or its binding protein. Particulate preparations from rat tissues were obtained from male Sprague-Dawley rats. Tissues were homogenized in 9 vol 0.1 M sucrose, 0.5 mM dithiothreitol (DTT), 1 mM EDTA, 1 mM EGTA, 20 mM Tris-HC1, pH 7.5 by a polytron homogenizer at 80% velocity for 1 min. The homogenates were centrifuged at 100,000 g for 30 min. The resulting pellets were washed three times by homogenization in 9 vol of the buffer followed by centrifugation. The final pellets were resuspended in 5 vol buffer and stored at - 8 0 °C. The particulate preparation from cerebrums was dispersed with 1% Lubrol PX by homogenization for 30 s, insoluble material was removed by centrifugation at 38,000 g for 20 min. The supernatant fluid was further centrifuged at 100,000 g for 1 h, the clear fluid was collected and was immediately concentrated by an Amicon membrane (PM 10). Ten to twenty ml ofsolubilized fraction (75-150 mg protein) was applied to a 2.8 × 50 cm of Ultrogel AcA 34 previously equilibrated with 0.1% Lubrol PX, 0.1 M sucrose, 0.1 mM EGTA, 0.5 mM DTT, 20 mM Tris-HCl, pH 7.5 (buffer A). Adenylate cyclase activity appeared near the void volume from the column. Aliquots from various tubes from the column were heated at 45 °C for 5 min, then
0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
158 added to the assay of the main peak of adenylate cyclase. Inhibition was found to occur by addition of fractions eluted after the major peak of the cyclase as we showed previously using rabbit cerebral cortex '3. The heat treatment at 45 °C selectively destroyed adenylate cyclase activity without significant loss of activity of the inhibitor. In an attempt to achieve further purification, fractions from Ultrogel AcA 34 column which inhibit adenylate cyclase were subjected to DEAE-Sepharose CL 6B column (1.2 x 10 cm). Inhibitory activity was eluted by a salt gradient (0-300 mM NaC1 in buffer A)just after the main protein peak. The inhibitor fractions from DEAE-Sepharose CL 6B were pooled and concentrated by an Amicon membrane (PM 10) and subsequently passed through a Sephadex G-200 column (1.5 x 64 cm) previously equilibrated with buffer A containing 0.1 M NaC1. The inhibitory activity was obtained as a broad peak slightly after the main protein peak from Sephadex G200 (Molec. wt. 4 0 ~ 6 0 Kdalton). The inhibitor
% 'To~ E
o
j, LIJ
tl3 ...I
p"J~"
Z tl.l Q
TABLE I
Effect of various stimulating agents on particulate adenylate cyclase with or without inhibitor Particulate adenylate cyclase activity from rat cerebrum was assayed in the presence of various stimulating agents with or without inhibitor protein partially purified. A hundred ~g of enzyme and 10/~g of inhibitor were used. When calmodulin was added, 40 ~tM Ca 2+ was present in assay. Data are representative of three separate experiments.
Stimulating agents
Control No addition 50.5 GTP (10 -5 M) 58.7 Gpp(NH)p (10-5 M) 112.5 Calmodulin (2/~g) 86.0 NaF ( 10 mM) 151.9 Forskolin (10- 4 M) 484.0 Mn 2+ (5 mM) 287.6
,0" .......
. _ ,..0
41- ....
"x 0 I
0
4
I
8 TIME (rnin)
I
I
12
16
Fig. 1. Time course studies. Adenylate cyclase (50 ~g) from Ultrogel AcA 34 was assayed at 30°C for various incubation times indicated in the presence ( 0 ) or absence (©) of the inhibitor (10 ~tg) from Sephadex G-200. Arrows indicate the addition of inhibitor following the incubation without inhibitor for 4 or 8 min.
Inhibition (%)
Inhibitor 25.3 28.5 66.6 43.9 106.9 276.2 241.4
50.2 51.4 40.8 49.0 29.6 42.9 16.1
seems to be a separable entity from other membrane components, however purification of this inhibitor appears to be difficult. The partially purified inhibitor from Sephadex G-200 was about 40 times as potent as the crude detergent extract in terms of amount of protein. When the inhibitor was added to assay of adenylate cyclase, enzyme inhibition occurred without apparent lag phase (Fig. 1). The addition of the inhibitor immediately reduced the rate of cyclic AMP formation suggesting that the inhibition may not be due to the degradation of cyclic AMP, ATP or enzyme protein itself. The inhibition was not affected by detergent concentration, Mg 2÷ concentration or various pH in assay. The substrate concentration dependence for the effect of the inhibitor was also tested. In the form of Lineweave~Burk plots, the maximum velocity was decreased by the addition of the inhibitor without any change of the Km for substrate ATP. Inhibitor was more potent to the enzyme in the solubilized fraction than to that in particulate. It appears that particulate enzyme includes endogenous inhibitor, and that inhibitor may easily come into contact with the detergent-solubilized enzyme, although it is difficult to know the interaction between cyclase and inhibitor in situ. In Table I, the effects of the inhibitor on particulate adenylate cyclase from various rat tissues were
!iiiiiiiii;ii2 0
A denylate cyclase activity (pmol / min / mg)
159 e x a m i n e d . N o t only a d e n y l a t e cyclase f r o m b r a i n tissues b u t the e n z y m e f r o m h e a r t or liver were also inhibited b y the inhibitor. G u a n i n e nucleotide is n o w believed to be involved in the inhibition o f a d e n y l a t e cyclase b y opiate, a d e n o sine a n d a - a d r e n e r g i c or m u s c a r i n i c cholinergic agonists 1,8.6,7. It can be p r e s u m e d that present inhibitor m a y be i n v o l v e d in the regulatory process by those i n h i b i t o r y h o r m o n e s . H o w e v e r , G T P a n d G p p ( N H ) p were not able to facilitate the inhibition (Table I). It was also s h o w n that forskolin which was recently f o u n d to be a m a r k e d a c t i v a t o r o f the cyclase 4,9 a n d Ca 2+ plus c a l m o d u l i n also did not c h a n g e the inhibition, indicating that the inhibitor decreases e n z y m e
activity i n d e p e n d e n t with these stimulating ligands. In contrast, the inhibitor exerts only a small effect o n fluoride- or M n 2÷ s t i m u l a t e d adenylate cyclase. W e c a n n o t explain these facts at present. T h e inhibitor was c o m p l e t e l y inactivated by the i n c u b a t i o n with 200 ~M N - e t h y l m a leimide at 30 °C for 10 min, indicating that the inhibitor m i g h t include an essential sulfhydryl group. Others did not o b s e r v e the inhibitor in the s t u d y for the detergent-solubilized a d e n y l a t e cyclase f r o m r a b b i t skeletal muscle 5. F u r t h e r investigations in o t h e r tissue sources are r e q u i r e d to d e t e r m i n e w h e t h e r the inhibitor specifically exists in n e u r o n a l tissue or not.
1 Blume, A. J., Lichtstein, D. and Boone, G., Coupling of opiate receptors to adenylate cyclase: requirement for Na + and GTP, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 5626-5630. 2 Brostrom, C. O., Haung, Y. C., Breckenridge, B. M. and Wolff, D. J., Identification of calcium-binding protein as a calcium-dependent regulator of adenylate cyclase, Proc. nat. Acad Sci. U.S.A., 72 (1975) 64-68. 3 Cheung, W. Y., Bradham, L. S., Lynch, T. J.,Lin, Y. M. and Tallant, E. A., Protein activator of cyclic 3',5'-nucleotide phosphodiesterase of bovine or rat brain also activate adenylate cyclase, Biochem. Biophys. Res. Comm., 66 (1975) 1055-1062. 4 Daly, J. W., Padgett, W.and Seamon, K. B., Activation of cyclic AMP generating system in brain membranes and slices by the diterpene forskolin; augmentation of receptor-mediated responses, J. Neurochem., 38 (1982) 552544. 5 Drummond, G. I.,Sano, M. and Nambi, P., Skeletal muscle adenylate cyclase: reconstitution of fluoride and guanine nucleotide sensitivity, Arch. Biochem. Biophys., 201 (1980) 286-295. 6 Jacobs, K. H., Faktories, K. and Shultz, G., GTP-dependent inhibition of cardiac adenylate cyclase by muscarinic cholinergic agonists, Naunyn-Schmiedeberg's Arch. PharmacoL, 310 (1979) 113-119. 7 Jacobs, K. H. and Schultz, G., Different inhibitory effect of adrenaline on platelet adenylate cyclase in the presence of GTP plus cholera toxin and of stable GTP analogues, Naunyn-Schmiedeberg's Arch. Pharmacol., 310 (1979) 121-127. 8 Londos, C., Cooper, D. M. F., Schlegel, W. and Rodbell, M., Adenosine analogs inhibit adipocyte adenylate cy-
clase by a GTP-dependent process: basis for actions of adenosine and methylxanthines on cyclic AMP production and lipolysis, Proc. nat. Acad. Sci. U.S.A., 75 (1978) 5362 5366. 9 Metzger, H. and Lindner, E., The positive inotropic-acting forskolin a potent adenylate cyclase activator, Drug Res., 31 (1981) 1248-1250. 10 Nijjar, M. S., Nature of cytoplasmic factor(s) modulating adenylate cyclase in the developing rat brain, J. Neurochem., 34 (1980) 1304-1308. 11 Pfeuffer, T., GTP-binding proteins in membranes and the control of adenylate cyclase activity, J. biol. Chem., 252 (1977) 7224-7234. 12 Ross, E. M.,Howlett, A. C., Ferguson, K. M. and Gilman, A. G., Reconstitution of hormone-sensitive adenylate cyclase activity,./, biol. Chem., 253 (1978) 6401 6412. 13 Sano, M. and Drummond, G. I., Properties of detergentdispersed adenylate cyclase from cerebral cortex: presence of an inhibitor protein, J. Neurochem., 37 (1981) 558 566. 14 Sternweis, P. C., Northup, J. K., Smigel, M. D. and Gilman, A. G., The regulatory component of adenylate cyclase: purification and properties, J. biol. Chem., 256 (1981) 11512 11526. 15 Wallance, R. W., Lynch, T. J., Tallant, E. A. and Cheung, W. Y., Purification and characterization of an inhibitor protein of brain adenylate cyclase and cyclic nucleotide phosphodiesterase, J. biol. Chem., 254 (1978) 372382. 16 Wang, J. H. and Desai, R., Modulator binding protein: bovine brain exhibiting the Ca2+ -dependent association with the protein modulator of cyclic nucleotide phosphodiesterase, J. biol. Chem., 252 (1977) 4175- 4184.
Brain Research, 264 (1983) 157-159 Elsevier Biomedical Press
Inhibition of adenylate cyclase by a membrane protein from rat cerebrum MAMORU SANO, SATOKO KITAJIMA and AKIRA MIZUTANI
Institute for Developmental Research, A ichi Prefectural Colony, Kamiya-cho, Kasugai, A ichi, 480-03(Japan) (Accepted November 16th, 1982)
Key words: adenylate cyclase inhibitor - detergent- guanine nucleotide
The inhibitor protein of adenylate cyclase was partially purified from detergent-extract of rat brain. The inhibition occurred without lag phase. Calmodulin, GTP, ~uanyl 5'-yl imidodiphosphate (Gpp (NH) p) and forskolin did not change the inhibition, but activities stimulated by NaF or Mn 2+ were more resistant for the inhibition. The inhibitor might include an essential sulfhydryl group.
It is known that adenylate cyclase system includes three independent components, a hormone receptor, guanine nucleotide regulatory unit and a catalytic unit ~1,~2,]4. However, several endogenous factors have been found to affect the activity of adenylate cyclase in the brain. Calmodulin is present in high concentrations and has been shown to regulate adenylate cyclase in this tissuea,3. Brain also contains several calmodulin binding proteins, which inhibit adenylate cyclase by specific association with calmodulin L5,]6. Additionally, soluble protein factor which stimulates adenylate cyclase has been shown in the developing rat brain I°. Brain adenylate cyclase is not fully understood. Recently, we have shown that the detergent-solubilized particulate fraction from rabbit cerebral cortex contained an inhibitor protein(s) of adenylate cyclase, which was inactivated by heat-treatment and by incubation with trypsin 13. Inhibitory action was not reversed by Ca 2÷ plus calmodulin and therefore, it does not appear to be a calmodulin binding protein. Inhibition was not due to the destruction of cyclic AMP by phosphodiesterase or of ATP by ATPase. In the present study, we have attempted to purify the inhibitor protein from the rat cerebrum and to examine the nature of this enzyme inhibition. Adenylate cyclase was assayed as described previouslyt3. Since the inhibition occurred both
in the presence or absence of Ca 2+ , experiments were performed in the presence of EGTA to avoid the effect of calmodulin or its binding protein. Particulate preparations from rat tissues were obtained from male Sprague-Dawley rats. Tissues were homogenized in 9 vol 0.1 M sucrose, 0.5 mM dithiothreitol (DTT), 1 mM EDTA, 1 mM EGTA, 20 mM Tris-HC1, pH 7.5 by a polytron homogenizer at 80% velocity for 1 min. The homogenates were centrifuged at 100,000 g for 30 min. The resulting pellets were washed three times by homogenization in 9 vol of the buffer followed by centrifugation. The final pellets were resuspended in 5 vol buffer and stored at - 8 0 °C. The particulate preparation from cerebrums was dispersed with 1% Lubrol PX by homogenization for 30 s, insoluble material was removed by centrifugation at 38,000 g for 20 min. The supernatant fluid was further centrifuged at 100,000 g for 1 h, the clear fluid was collected and was immediately concentrated by an Amicon membrane (PM 10). Ten to twenty ml ofsolubilized fraction (75-150 mg protein) was applied to a 2.8 × 50 cm of Ultrogel AcA 34 previously equilibrated with 0.1% Lubrol PX, 0.1 M sucrose, 0.1 mM EGTA, 0.5 mM DTT, 20 mM Tris-HCl, pH 7.5 (buffer A). Adenylate cyclase activity appeared near the void volume from the column. Aliquots from various tubes from the column were heated at 45 °C for 5 min, then
0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
158 added to the assay of the main peak of adenylate cyclase. Inhibition was found to occur by addition of fractions eluted after the major peak of the cyclase as we showed previously using rabbit cerebral cortex '3. The heat treatment at 45 °C selectively destroyed adenylate cyclase activity without significant loss of activity of the inhibitor. In an attempt to achieve further purification, fractions from Ultrogel AcA 34 column which inhibit adenylate cyclase were subjected to DEAE-Sepharose CL 6B column (1.2 x 10 cm). Inhibitory activity was eluted by a salt gradient (0-300 mM NaC1 in buffer A)just after the main protein peak. The inhibitor fractions from DEAE-Sepharose CL 6B were pooled and concentrated by an Amicon membrane (PM 10) and subsequently passed through a Sephadex G-200 column (1.5 x 64 cm) previously equilibrated with buffer A containing 0.1 M NaC1. The inhibitory activity was obtained as a broad peak slightly after the main protein peak from Sephadex G200 (Molec. wt. 4 0 ~ 6 0 Kdalton). The inhibitor
% 'To~ E
o
j, LIJ
tl3 ...I
p"J~"
Z tl.l Q
TABLE I
Effect of various stimulating agents on particulate adenylate cyclase with or without inhibitor Particulate adenylate cyclase activity from rat cerebrum was assayed in the presence of various stimulating agents with or without inhibitor protein partially purified. A hundred ~g of enzyme and 10/~g of inhibitor were used. When calmodulin was added, 40 ~tM Ca 2+ was present in assay. Data are representative of three separate experiments.
Stimulating agents
Control No addition 50.5 GTP (10 -5 M) 58.7 Gpp(NH)p (10-5 M) 112.5 Calmodulin (2/~g) 86.0 NaF ( 10 mM) 151.9 Forskolin (10- 4 M) 484.0 Mn 2+ (5 mM) 287.6
,0" .......
. _ ,..0
41- ....
"x 0 I
0
4
I
8 TIME (rnin)
I
I
12
16
Fig. 1. Time course studies. Adenylate cyclase (50 ~g) from Ultrogel AcA 34 was assayed at 30°C for various incubation times indicated in the presence ( 0 ) or absence (©) of the inhibitor (10 ~tg) from Sephadex G-200. Arrows indicate the addition of inhibitor following the incubation without inhibitor for 4 or 8 min.
Inhibition (%)
Inhibitor 25.3 28.5 66.6 43.9 106.9 276.2 241.4
50.2 51.4 40.8 49.0 29.6 42.9 16.1
seems to be a separable entity from other membrane components, however purification of this inhibitor appears to be difficult. The partially purified inhibitor from Sephadex G-200 was about 40 times as potent as the crude detergent extract in terms of amount of protein. When the inhibitor was added to assay of adenylate cyclase, enzyme inhibition occurred without apparent lag phase (Fig. 1). The addition of the inhibitor immediately reduced the rate of cyclic AMP formation suggesting that the inhibition may not be due to the degradation of cyclic AMP, ATP or enzyme protein itself. The inhibition was not affected by detergent concentration, Mg 2÷ concentration or various pH in assay. The substrate concentration dependence for the effect of the inhibitor was also tested. In the form of Lineweave~Burk plots, the maximum velocity was decreased by the addition of the inhibitor without any change of the Km for substrate ATP. Inhibitor was more potent to the enzyme in the solubilized fraction than to that in particulate. It appears that particulate enzyme includes endogenous inhibitor, and that inhibitor may easily come into contact with the detergent-solubilized enzyme, although it is difficult to know the interaction between cyclase and inhibitor in situ. In Table I, the effects of the inhibitor on particulate adenylate cyclase from various rat tissues were
!iiiiiiiii;ii2 0
A denylate cyclase activity (pmol / min / mg)
159 e x a m i n e d . N o t only a d e n y l a t e cyclase f r o m b r a i n tissues b u t the e n z y m e f r o m h e a r t or liver were also inhibited b y the inhibitor. G u a n i n e nucleotide is n o w believed to be involved in the inhibition o f a d e n y l a t e cyclase b y opiate, a d e n o sine a n d a - a d r e n e r g i c or m u s c a r i n i c cholinergic agonists 1,8.6,7. It can be p r e s u m e d that present inhibitor m a y be i n v o l v e d in the regulatory process by those i n h i b i t o r y h o r m o n e s . H o w e v e r , G T P a n d G p p ( N H ) p were not able to facilitate the inhibition (Table I). It was also s h o w n that forskolin which was recently f o u n d to be a m a r k e d a c t i v a t o r o f the cyclase 4,9 a n d Ca 2+ plus c a l m o d u l i n also did not c h a n g e the inhibition, indicating that the inhibitor decreases e n z y m e
activity i n d e p e n d e n t with these stimulating ligands. In contrast, the inhibitor exerts only a small effect o n fluoride- or M n 2÷ s t i m u l a t e d adenylate cyclase. W e c a n n o t explain these facts at present. T h e inhibitor was c o m p l e t e l y inactivated by the i n c u b a t i o n with 200 ~M N - e t h y l m a leimide at 30 °C for 10 min, indicating that the inhibitor m i g h t include an essential sulfhydryl group. Others did not o b s e r v e the inhibitor in the s t u d y for the detergent-solubilized a d e n y l a t e cyclase f r o m r a b b i t skeletal muscle 5. F u r t h e r investigations in o t h e r tissue sources are r e q u i r e d to d e t e r m i n e w h e t h e r the inhibitor specifically exists in n e u r o n a l tissue or not.
1 Blume, A. J., Lichtstein, D. and Boone, G., Coupling of opiate receptors to adenylate cyclase: requirement for Na + and GTP, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 5626-5630. 2 Brostrom, C. O., Haung, Y. C., Breckenridge, B. M. and Wolff, D. J., Identification of calcium-binding protein as a calcium-dependent regulator of adenylate cyclase, Proc. nat. Acad Sci. U.S.A., 72 (1975) 64-68. 3 Cheung, W. Y., Bradham, L. S., Lynch, T. J.,Lin, Y. M. and Tallant, E. A., Protein activator of cyclic 3',5'-nucleotide phosphodiesterase of bovine or rat brain also activate adenylate cyclase, Biochem. Biophys. Res. Comm., 66 (1975) 1055-1062. 4 Daly, J. W., Padgett, W.and Seamon, K. B., Activation of cyclic AMP generating system in brain membranes and slices by the diterpene forskolin; augmentation of receptor-mediated responses, J. Neurochem., 38 (1982) 552544. 5 Drummond, G. I.,Sano, M. and Nambi, P., Skeletal muscle adenylate cyclase: reconstitution of fluoride and guanine nucleotide sensitivity, Arch. Biochem. Biophys., 201 (1980) 286-295. 6 Jacobs, K. H., Faktories, K. and Shultz, G., GTP-dependent inhibition of cardiac adenylate cyclase by muscarinic cholinergic agonists, Naunyn-Schmiedeberg's Arch. PharmacoL, 310 (1979) 113-119. 7 Jacobs, K. H. and Schultz, G., Different inhibitory effect of adrenaline on platelet adenylate cyclase in the presence of GTP plus cholera toxin and of stable GTP analogues, Naunyn-Schmiedeberg's Arch. Pharmacol., 310 (1979) 121-127. 8 Londos, C., Cooper, D. M. F., Schlegel, W. and Rodbell, M., Adenosine analogs inhibit adipocyte adenylate cy-
clase by a GTP-dependent process: basis for actions of adenosine and methylxanthines on cyclic AMP production and lipolysis, Proc. nat. Acad. Sci. U.S.A., 75 (1978) 5362 5366. 9 Metzger, H. and Lindner, E., The positive inotropic-acting forskolin a potent adenylate cyclase activator, Drug Res., 31 (1981) 1248-1250. 10 Nijjar, M. S., Nature of cytoplasmic factor(s) modulating adenylate cyclase in the developing rat brain, J. Neurochem., 34 (1980) 1304-1308. 11 Pfeuffer, T., GTP-binding proteins in membranes and the control of adenylate cyclase activity, J. biol. Chem., 252 (1977) 7224-7234. 12 Ross, E. M.,Howlett, A. C., Ferguson, K. M. and Gilman, A. G., Reconstitution of hormone-sensitive adenylate cyclase activity,./, biol. Chem., 253 (1978) 6401 6412. 13 Sano, M. and Drummond, G. I., Properties of detergentdispersed adenylate cyclase from cerebral cortex: presence of an inhibitor protein, J. Neurochem., 37 (1981) 558 566. 14 Sternweis, P. C., Northup, J. K., Smigel, M. D. and Gilman, A. G., The regulatory component of adenylate cyclase: purification and properties, J. biol. Chem., 256 (1981) 11512 11526. 15 Wallance, R. W., Lynch, T. J., Tallant, E. A. and Cheung, W. Y., Purification and characterization of an inhibitor protein of brain adenylate cyclase and cyclic nucleotide phosphodiesterase, J. biol. Chem., 254 (1978) 372382. 16 Wang, J. H. and Desai, R., Modulator binding protein: bovine brain exhibiting the Ca2+ -dependent association with the protein modulator of cyclic nucleotide phosphodiesterase, J. biol. Chem., 252 (1977) 4175- 4184.