Properties of the separated catalytic and regulatory units of brain

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 77, No. 11, pp , November 1980 Biochemistry Properties of the separated catalytic and regulatory units of brain adenylate cyclase (guanine nucleotide regulation/detergent solubilization/enzyme reconstitution) STEPHEN STRITTMATTER* AND EVA J. NEERt Department of Medicine, Peter Bent Brigham Hospital and Harvard Medical School, Boston, Massachusetts Communicated by Eugene Braunwald, July 14, 1980 ABSTRACT Adenylate cyclase from bovine brain cortex was solubilized with 14 mm cholate and 1 M (NH4)2SO4. Gel filtration over a column of Sepharose 6B separated the catalytic unit (CU) from a factor (G/F) that confers responsiveness to 5'- guanylyl imidophosphate (pfnhlppc) or fluoride. The separated CU, which elutes with a K., of 0.48 ± 0.01 (n = 5), is not responsive to p[nh ppg or fluoride and is relatively inactive when MgATP is the substrate but activated 8-15-fold by Mn2. The separated G/F elutes with a Kav of (n = 4). It restores the responsiveness of the CU to p[nhlppg and fluoride. Activation of the enzyme by p[nhlppg before solubilization does not decrease the amount of G/F eluting with a K., of 0.7. Therefore, the G/F is probably present in brain cortex in excess over the CU. p[nhbppg stabilizes the G/F but not the CU against thermal inactivation, suggesting that it interacts with C/F and not with CU. Incubation of the G/F with plnh]ppg before addition of CU markedly increases the rate of activation of the reconstituted enzyme by pgnijppg. We propose, therefore, that the rate-limiting step in adenylate cyclase activation is a process in G/F alone and not a slow conformational change in CU or a slow association of G/F with CU. Binding of ptnh^ppg to the isolated G/F appears to be readily reversible; the ability of fully activated G/F to stimulate CU can be blocked if GDP is added before CU. In contrast, after the CU has been activated by interaction with G/F, GDP cannot reverse the activation. This suggests that association with the CU increases the affinity of G/F for p[nhlppg. The hormone-responsive adenylate cyclase system in the plasma membrane of animal cells has at least three components: a hormone receptor, a catalytic subunit (CU) that converts ATP to cyclic AMP, and a guanine nucleotide-binding unit (G/F) that mediates the response of the CU to guanine nucleotides, fluoride ion, and hormones (for review, see ref. 1). The CU has been separated from the G/F by GTP affinity chromatography of enzyme solubilized from pigeon erythrocytes (2), a method that has also been useful for other avian erythrocytes (3) but has not been effective in separating the components of mammalian adenylate cyclase. The two units have been resolved genetically in variant S49 lymphoma cells (4-6). The response of adenylate cyclase from mammalian brain to 5'-guanylyl imidodiphosphate (p[nh]ppg) can be abolished by two different procedures (7, 8) and subsequently restored by addition of a diluted unfractionated detergent extract of brain that contains both the CU and the G/F. Because the G/F is not separated from the CU, the interpretation of the experiments is complicated. In this paper, we report the separation and characterization of the CU and the G/F from bovine cerebral cortex. The method is simple and can be used to produce large amounts of both units. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact METHODS Solubilization of Adenylate Cyclase. The cortices of fresh bovine brains were separated and stored at -70 C. Adenylate cyclase was solubilized by a modification of the method of Hoffmann (9). All steps were performed at 4 C. The thawed cortices were homogenized in 0.05 M Tris, ph 7.6/0.2 M sucrose/i mm dithiothreitol (Sigma)/15 mm MgCl2 (buffer 1) 3 ml/g of wet weight of tissue in a Dounce glass homogenizer. To each ml of the homogenate, we added 0.2 ml of H20, 0.3 ml of saturated (40C) (NH4)2SO4 (adjusted to ph 7.6 with NaOH), and 38,l of 0.5 M cholic acid (Sigma) (adjusted to ph 7.6 with 5 M NaOH). The final concentration of (NH4)2S04 was 1 M (20% of saturation at 40C) and that of cholate was 14 mm. The mixture was stirred for 20 min and then centrifuged at 100,000 X g for 20 min. The supernatant containing the soluble enzyme (preparation A) was collected, and the pellet was resuspended in approximately half the original volume of buffer 1. Samples were frozen in a dry ice-acetone bath and stored at -70 C. They were stable for at least 2 months. Adenylate Cyclase and Protein Assay. Adenylate cyclase activity was measured by a modification of the method of Krishna et al. (10) as described by Neer (11). Activity was measured for 10 min at 300C or for 15 min at 230C. Assays with Mn2+ contained 5 mm MnCl2 in addition to the 15 mm Mg2+ present in all assays. For p[nh]ppg activation, the sample was incubated at 230C with 40-60,uM p[nh]ppg as indicated in the individual experiments before assay. Figs. 2 and 4 show that activation was maximal in about 3 hr, then stable for up to 20 hr. The duration of activation was chosen for convenience. For fluoride activation, the sample was made 10 mm in NaF and incubated for 1 hr at 230C before assay. Protein concentration was determined by the method of Lowry et al. (12) as modified by Bailey (13); bovine serum albumin was used as a standard. To avoid turbidity in the samples, the reaction mixture was made 0.1% in NaDodSO4. All assays were in duplicate; experiments were carried out at least twice and usually four or five times. Sepharose 6B Gel Filtration. Sepharose 6B (Pharmacia) was equilibrated with 0.05 M Tris/phospholipid (7 mg/ml)/14 mm cholic acid/1.2 M (NH4)2S04 (25% saturated, 4 C)/15 mm MgCl2/0.2 M sucrose/i mm dithiothreitol, adjusted to ph 7.7 with 1 M Tris-OH (buffer 2). Soybean phospholipid (Sigma P5638, soybean L-a-phosphatidyl choline) was washed under nitrogen with acetone (10 ml/g of phospholipid), dried under Abbreviations: p[nh]ppg, 5'-guanylyl imidodiphosphate; CU, catalytic unit of adenylate cyclase; G/F, component of the adenylate cyclase system that mediates the response to guanine nucleotides and fluoride. * Present address: The Johns Hopkins University School of Medicine, Baltimore, MD t To whom reprint requests should be addressed.

2 vacuum, stored at 40C, and homogenized in water to a fine emulsion before use. The buffer was always freshly made, and the columns were reequilibrated less than 24 hr before use. Samples from column fractions were mixed with equal volumes of saturated (40C) (NH4)2SO4, ph 7.6 and centrifuged at 45,000 X g for 45 min at 40C. The supernatant was discarded, and the pellet was resuspended in freshly made phospholipid (7 mg/ml)/0.1% Lubrol 12A9 (ICI)/0.2 M sucrose/ 0.05 M Tris, ph 7.7/15 mm MgCl2/1 mm dithiothreitol. The final volume was of the original sample volume. After precipitation, samples were stable at -700C for up to 2 months. Ammonium sulfate precipitation effectively removed the cholate; the pellet contained only 2-5% of the cholate in the sample (determined by inclusion of a trace amount of [3H]- cholate in test precipitations). Attempts to remove salt and detergent by dialysis led to large losses of activity. Blue dextran, lactate dehydrogenase, bovine serum albumin, myoglobin (Sigma) and [3H]ATP were used to calibrate the columns. Based on their diffusion coefficients (14) the Stokes radii were taken to be: lactate dehydrogenase, 43 A; bovine serum albumin, 27 A; myoglobin, 17 A. RESULTS Solubilization of Adenylate Cyclase from Bovine Cerebral Cortex by Using Cholate and Ammonium Sulfate. Cholate and (NH4)2SO4 together activated and effectively solubilized adenylate cyclase from bovine cerebral cortex (Table 1). Solubilization was confirmed by gel filtration of the supernatant enzyme over Sepharose 6B (see below). Cholate alone neither stimulated nor solubilized the enzyme as effectively as cholate plus a high concentration of salt. Activation of the homogenate by incubation with 60,uM p[nh]ppg for 6 hr at 230C before addition of cholate and (NH4)2SO4 did not affect the fraction of the enzyme solubilized but did increase the absolute level of enzymatic activity. Separation of the Catalytic and Guanine Nucleotide Regulatory Units of Adenylate Cyclase by Sepharose 6B Gel Filtration. The pattern of activity obtained after Sepharose 6B gel filtration of adenylate cyclase solubilized by cholate and (NH4)2SO4 is shown in Fig. 1. The solubilized enzyme applied to the column (preparation A) could be activated fold (n = 4) by p[nh]ppg and 4.1 I 0.8-fold (n = 4) by addition of Mn2+.t After gel filtration, the catalytic activity in peak CU could no longer be activated by p[nh]ppg or NaF but was increased 8- to 15-fold by 5 mm Mn2. (The small peak of p[nh]ppg-dependent activity shown in Fig. 1 was not always seen.) The increase in Mn2+-dependence and the refractoriness to p[nh]ppg suggests that the region marked CU in Fig. 1 contains the catalytic unit from which the G/F has been separated. Some enzyme activity always eluted in the void volume. This was taken to be incompletely solubilized enzyme and was not studied further. Responsiveness to p[nh]ppg could be restored to the fractions in peak CU by addition of fractions from the region marked G/F in Fig. 1, after they had been precipitated with (NH4)2SO4 and taken up in buffer 3. The activity shown by the closed circles in Fig. 1 is that measured when samples from the precipitated fractions were added to a constant amount of CU. The G/F had little or no effect on the basal activity or on the t Biochemistry: Strittmatter and Neer These ratios are higher than those shown for the solubilized enzyme in Table 1 because the samples were prepared in a different way. To be a valid control for the activity in the column effluent, the unfractionated soluble enzyme was diluted 1:10 into column buffer, precipitated with (NH4)2SO4 and taken up in buffer 3 before assay. 0) E 0 'i) 0 U '-4 U U0. ci U I-,._I :>4 *Q Proc. Nati. Acad. Sc. USA 77 (1980) ~.075 Ca I 6 LO.050 " 0 A- a, Fraction FIG. 1. Sepharose 6B gel filtration of solubilized adenylate cyclase. Preparation A (11 ml) was applied to a 400-ml Sepharose 6B column equilibrated at 40C with buffer 2. Fraction size was 2.3 ml; 65 ml were collected before the first fraction. Blue dextran, lactate dehydrogenase (LDH), and a trace amount of [3H]ATP were included in the sample. Fractions were precipitated, and 50-ul samples were assayed at 30 C for 10 min.,, Basal activity;,, activity in the presence of 5 mm Mn2+; 0, activity after incubation with 60,M p[nh]ppg for 6.5 hr at 230C. Recoveries of enzyme activity were basal, ; Mn2+, ; p[nh]ppg-stimulated, 5 + 2, as percentage of preparation Aactivity measured after similar precipitation and resuspension in buffer 3. 0, Ability of fractions to restore p[nh]ppg responsiveness to adenylate cyclase. Sample (25,l) to be tested was mixed with 25 Ml of pooled precipitated CU. Activity shown is normalized to 50 Ml of precipitated CU. *, Protein was determined without precipitation; recovery was 83%. activity in the presence of 5 mm Mn2+. p[nh]ppg-stimulated activity increased as the amount of G/F added to a constant amount of CU increased. We were not able to saturate the enzyme with the G/F (not shown). Addition of G/F to CU also restored the ability of fluoride to activate the enzyme. In the presence of the G/F, the CU could be activated 5-fold by 10 mm NaF. The Kav of the CU was 0.48 ± 0.01 (n = 5), and that of the G/F was (n = 4). However, the high concentration of ionic detergent and salt in the column buffer made it difficult to obtain precise values for the Stokes radii of either unit. We could assay lactate dehydrogenase as an internal marker, and we were able to calibrate separate but identical columns with this enzyme, bovine serum albumin, and myoglobin. By extrapolating from the straight line given by a plot of Ka. versus the Stokes radii of these three proteins, we determined the Stokes radius of the CU to be t68 A and that of the G/F to be -40 A. Properties of the Catalytic and Guanine Nucleotide Regulatory Units. The factor that restores the responsiveness of the CU to p[nh]ppg and fluoride appears to be a protein. It can be inactivated by treatment with trypsin at (1 mg/ml) 30"C for 15 min. Inclusion of soybean trypsin inhibitor (1 mg/ml) during the incubation with trypsin completely protects the activity of the G/F. The G/F can also be inactivated by treatment with 17 mm N-ethylmaleimide for 15 min at 23 C, an inactivation that is blocked by previous addition of 17 mm dithiothreitol. The G/F is heat sensitive and is not active after

3 6346 Biochemistry: Strittmatter and Neer Proc. Natl. Acad. Sci. USA 77 (1980) Preparation Table 1. Cholate and (NH4)2SO4 Homogenate 38 Homogenate plus cholate and (NH4)2SO * Supernatant t Pellet t Cholate but not (NH4)2SO4t Homogenate 38 Homogenate plus cholate * Supernatant 43 26t Pellet t Cholate and (NH4)2SO4 after activation with 60 um p[nh]ppg* Homogenate 291 Homogenate plus cholate and (NH4)2SO * Supernatant t Pellet t Solubilization of adenylate cyclase No addition Mn2+ p[nh]ppg Activity Recovery, % Activity Recovery, % Activity Recovery, % Adenylate cyclase activity was assayed at 30 C for 10 min. The samples were diluted 1:21 in buffer 1 before assay or activation with p[nh]ppg. This experiment is representative of five similar ones. Specific activities are expressed as (pmol cyclic AMP/mg protein)/min; total activities were calculated by multiplying the specific activity by the total amount of protein * Compared with activity in the starting homogenate. t Compared with activity in the homogenate plus cholate from which they were derived. I H20 was substituted for (NH4)2SO4. 371* 48t 36t 201* 30t 113t 130* 72t 39t * 43t 24t 115* gt 69t 106* 39t 37t 10-min incubation at 50 C. These properties are similar to those reported by Ross and Gilman for the G/F from S49 lymphoma cells (4). The G/F unit does not stabilize or activate the CU unless p[nh]ppg is present (Fig. 2A). p[nh]ppg does not stabilize the basal or Mn2+-dependent activity of the CU without the G/F (Fig. 2B), suggesting that the CU does not bind p[nh]ppg. In contrast, when p[nh]ppg and the G/F are present together, adenylate cyclase is stabilized and activated (Fig. 2B). This is true whether the activity is measured in the presence of Mn2+ or of Mg2+. This shows that the activity measured in the presence of either metal is produced by a catalytic unit that can interact with the G/F. To determine whether the G/F can bind p[nh]ppg in the absence of the CU, we measured its thermal stability in the presence and absence of p[nh]ppg (Fig. 3). At each time point, we assayed the ability of the G/F to restore p[nh]ppg-responsiveness to the CU. The reconstituting capacity decreased rapidly when the G/F was incubated at 370C in the absence of p[nh]ppg but was stable for at least 60 min at 37 C in its presence. The p[nh]ppg-concentration dependence of the reconstituted enzyme was similar to that of other adenylate cyclases (15). Half-maxial activation occurred at 2,uM p[nh]ppg (not shown). The standard concentration used in these studies, AM, was saturating. Gel Filtration of Solubilized Adenylate Cyclase After Activation by p[nhlppg. To determine whether activation of preparation A by p[nh]ppg changed the elution pattern of the CU or the amount of separated G/F, we passed p[nh]- ppg-activated soluble adenylate cyclase over a column of Sepharose 6B exactly as in Fig. 1 (not shown). The peak enzyme activity measured in the presence of Mg2+ was 25 (pmol cyclic AMP/10 min)/50-ml sample compared with 5 in Fig. 1. Because p[nh]ppg was included in the buffer and the sample was already activated, the activity measured in this way represents p[nh]ppg-stimulated activity. The ratio of activity in the presence of Mn2+ to activity in the presence of Mg2+ was -3-4 in the preactivated peak rather than 8-15 as in the separated CU shown in Fig. 1. The elution position of activated adenylate cyclase was not noticeably changed in comparison with that of unactivated enzyme. The Kay was (n = 2) for the activity measured in the presence and absence of Mn2+. Fractions from the G/F region of such a column reconstituted p[nh]ppg responsiveness of the separated CU taken from a column effluent like that shown in Fig. 1. We could detect no decrease in the amount of G/F. The elution position of the G/F was not changed by prior activation by p[nh]ppg. Kinetics of p[nhlppg Activation of Reconstituted Adenylate Cyclase. Activation of native soluble brain adenylate cyclase by p[nh]ppg is a slow process that requires several hours of incubation with the guanine nucleotide analogue to reach maximal stimulation (1). We have shown that activation of the reconstituted enzyme by p[nh]ppg is also slow. If the rate-limiting step in this activation were the rate of association of CU and G/F or a slow conformational change in the CU, then treatment of the G/F with p[nh]ppg before recombination with the CU would not increase the rate of activation of the CU. We found that incubation of the G/F with p[nh]ppg I7i7 k Time (of incubation at 23 C), hr FIG. 2. Stability of the catalytic unit at 23 C. Pooled, precipitated fractions from the CU and GJF regions of columns similar to that shown in Fig. 1 were used. CU was mixed with an equal volume of buffer 3 (---) or of G/F (-) and assayed for adenylate cyclase activity after incubation. Stability of the CU in the absence (A) and presence (B) of p[nh]ppg. Presence of 15 mm Mg2+: A-A, presence of G/F; A - --, absence of G/F. Presence of 5 mm Mn2+: A-A, presence of G/F; A --- A, absence of G/F.

4 E6" *o 10,\, Biochemistry: Strittmatter and Neer Proc. Nati. Acad. Sci. USA 77 (1980) G~~~~~~~/F +p[nhippg, 370; CU E '\ /37XC 451 I ~30 25 >,20 - G/F + CU 15 I <DS 10S G/F, 37 + CU Time (of incubation at 3700), min FIG. 3. Stabilization of the guanine nucleotide regulatory unit by p[nhippg. Pooled, precipitated fractions from the CU and G/F regions of columns similar to that shown in Fig. 1 were used. 0-0, G/F was incubated at 370C with 60MgM for the indicated times. An equal volume of CU was added, the p[nh]ppg concentration was readjusted to 60 MM, and the mixture was incubated at 230C for 16 hr before assaying. 0---O, G/F was incubated at 370C without p[nh]ppg for the indicated times. An equal volume of CU was added, and the mixture was made 60 AM in p[nh]ppg and incubated at 230C for 16 hr before assaying , G/F was incubated at 370C for the indicated times without p[nh]ppg. An equal volume of CU was added, and the mixture assayed without further incubation. Adenylate cyclase activity was assayed for 10 min at 300C. before addition to the CU increased the rate of activation of the CU (Fig. 4). This shows that the rate-limiting step is a slow change in the G/F. Activation by p[nh]ppg of soluble adenylate cyclase from brain and other tissues (16, 17) is irreversible or only very slowly reversible. Once the fully activated state has been attained, it is not reversible by incubation with GDP (Table 2). We have shown that incubation of the G/F with p[nh]ppg at 370C for 25 min is sufficient to convert it to a state capable of activating the CU rapidly (data not shown). Thus, the interaction of p[nh]ppg and the G/F appears to be complete in that time. However, incubation of activated G/F with GDP before addition of the CU will prevent the subsequent activation of the CU. DISCUSSION Cholate in combination with a high concentration of (NH4)2S04 solubilizes about half of the adenylate cyclase activity from bovine cerebral cortex. The cholate and (NH4)2S04-solubilized enzyme responds to p[nh]ppg and fluoride, indicating that both the catalytic and the guanine nucleotide regulatory units are in a functionally intact state. After filtration over a column of Sepharose 6B, the properties of adenylate cyclase are quite different from those of the unfractionated supernatant. The enzyme is now virtually inactive with Mg-ATP as the substrate but is activated 15-fold by addition of Mn2+ to the assay. It can no longer be activated by p[nh]ppg or NaF. Responsiveness to p[nh]ppg or fluoride Time (of incubation at 230C), hr FIG. 4. Effect of incubation of the G/F with p[nh]ppg on the rate of activation of adenylate cyclase. Pooled precipitated fractions from the CU and G/F regions of a column similar to that shown in Fig. 1 were used.,, G/F was incubated with 60,M p[nh]ppg for 25 min at 370C. An equal volume of CU was added, and the concentration of p[nh]ppg was readjusted to 60 AM. Samples were incubated at 230C for the times indicated before measurement of adenylate cyclase activity for 15 min at 230C. 0, CU and G/F were mixed in equal volume and made 60MuM in p[nh]ppg. Adenylate cyclase activity was measured as above. A, G/F was incubated for 25 min at 370C without p[nh]ppg. CU was then added together with p[nh]ppg (60,M). Adenylate cyclase activity'was measured as above. Horizontal lines indicated the time during which enzyme activity was assayed. can be restored to the catalytic unit by recombination with fractions eluting later from the Sepharose 6B column. We (18) and others (5, 19, 20) have proposed that when the G/F is physically or functionally absent, the enzymatic activity of all CU is expressed with Mn-ATP and not with Mg.ATP. The properties of adenylate cyclase activity after gel filtration are consistent with this proposal. The separated CU has properties very similar to those of adenylate cyclase from variant S49 lymphoma cells [which lack a functional G/F; (5)] and separated catalytic unit from pigeon erythrocytes (21). The simplest explanation of our data is that the CU has been entirely separated from the G/F. However, because an excess of G/F seems to be required for activation of the CU, it is possible that our separated CU is still associated with some G/F, but at too low a concentration to be activating. The G/F appears to be present in excess over that which can associate with the CU in the presence of p[nh]ppg. Activation of adenylate cyclase by p[nh]ppg seems to stabilize or promote the formation of a CU-G/F complex (22-24). Gel filtration in cholate and (NH4)2SO4 of p[nh]ppg-activated enzyme does not seem to dissociate this complex because the activity measurable with Mg-ATP is greater than that after gel filtration without prior activation and is approximately equal to that otained after reconstitution of the CU with G/F. Also, the ratio of activity in the presence of Mn2+ to that in the presence of Mg2+ is -3-4 rather than 8-15 as in the separated CU. Despite the apparent association of G/F and C, there is approximately the same amount of G/F activity in the later fractions from gel

5 6348 Biochemistry: Strittmatter and Neer Table 2. Effect of GDP on activation of adenylate cyclase by p[nh]ppg Incubation Second Third Aden- First (370C) (230C) (230C) ylate Time, Time, Time, cylase Component min Add. hr Add. hr activity With p[nh]ppg (Set A) G/F 25 CU 5.3 GDP 0 54 G/F 25 CU 5.3 GDP G/F 25 CU 5.3 GDP G/F 25 CU 5.3 GDP G/F 25 CU With p[nh]ppg (Set B) G/F 25 GDP 0 CU G/F 25 GDP 1.1 CU G/F 25 GDP 2.1 CU G/F 25 GDP 3.7 CU Without p[nh]ppg (Set C) G/F 25 CU* G/F 0 CU* Pooled fractions from regions CU and G/F of columns similar to that shown in Fig. 1 were precipitated with (NH4)2504 and resuspended in 0.5 times their original volume of buffer 3. In the first incubation, G/F was treated as indicated. In the second incubation, either CU or GDP was added and the incubation was continued. In the third incubation, further additions were made, and the reactions were continued. Concentration of p[nh]ppg in the incubations and in the adenylate cyclase assay was 60,M and that of GDP was 0.7 mm. Specific activity is expressed as (pmol cyclic AMP/10 min)/50-,d sample at 300C. * p[nh]ppg (60,M) was added together with the CU. filtration of either p[nh]ppg-activated or unactivated enzyme. If all the available CU is, indeed, in a C-G/F complex, this observation suggests that G/F is present in excess. The ability to separate and reconstitute the catalytic and regulatory components of adenylate cyclase allowed us to design experiments to define the rate-limiting step in activation by p[nh]ppg. Our experiments suggest that the rate-limiting step occurs in the G/F but do not allow us to tell whether the slow step is release of GDP or a slow conformational change in the G/F. However, once the G/F has been activated, it can quickly increase the activity of the CU. Therefore, neither the time required for the CU and G/F to associate nor the time required for the CU to change to an active conformation is rate limiting. p[nh]ppg irreversibly activates soluble adenylate cyclase (20). As shown in Table 2, once the enzyme has been fully stimulated, the activation cannot be reversed by long incubation with excess GDP. This suggests that GDP is not effective in displacing p[nh]ppg from a CU.G/F complex. An alternative, but less likely, explanation is that, once stimulated, the CU no longer requires interaction with G/F to maintain the active state. The situation is different, however, when the isolated G/F, fully charged with p[nh]ppg, is incubated with GDP. GDP can reverse the ability of p[nh]ppg-activated G/F to increase adenylate cyclase activity, presumably by displacing p[nh]ppg. We suggest, therefore, that the association of G/F with CU increases the affinity of G/F for p[nh]ppg or changes the kinetics of nucleotide binding. These results are consistent Proc. Natl. Acad. Sci. USA 77 (1980) with the conclusions of Sayhoun et al. (7) and Nielsen et al. (25) in different kinds of preparations. Our studies with the resolved catalytic and guanine nucleotide regulatory units of brain adenylate cyclase begin to define the functional properties of the individual components and the changes that take place when they interact. Understanding these events in detail will require that the components of the enzyme be structurally and chemically characterized. While this manuscript was in preparation, Ross (26) reported separation of CU and G/F from liver and wild-type S49 lymphoma cells by a similar method. No kinetic studies were described but incorporation of the enzyme into lipid vesicles was reported. This work was supported by a grant from the National Institutes of Health (AM 19277) to E.J.N. 1. Ross, E. M. & Gilman, A. G. (1980) Annu. Rev. Biochem. 49, Pfeuffer, T. & Helmreich, E. J. M. (1975) J. Biol. 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