Lubrol 12A9 Research Articles

Partial purification and characterization of a pp60v-src-related tyrosine kinase from bovine brain.

We have identified and substantially purified a tyrosine protein kinase from normal bovine brain that is immunologically related to the product of the Rous sarcoma virus oncogene (pp60v-src). The enzyme, a 61-kDa protein (p61), is solubilized with detergent from bovine cerebral cortical membranes and purified by column chromatography. In the purest preparations, this protein is phosphorylated only on tyrosine, but it can also be a substrate for serine- and threonine-specific protein kinases. The p61 protein phosphorylates the heavy chain of immunoglobulins from rabbits bearing Rous sarcoma virus-induced tumors (TBR IgG) but not normal IgG. TBR IgG precipitates the 61-kDa phosphoprotein and protein kinase activity from purified preparations. The activity of the purified brain tyrosine kinase is 10 times higher in the presence of 7-10 mM Mn2+ and 6 mM Mg2+ than it is with 6 mM Mg2+ alone. With Mn2+, the p61 enzyme has a Km for ATP of 2 microM. All preparations of p61 also contain a 64-kDa protein (p64) that is phosphorylated on tyrosine. Measurement of the Stokes radius of p61 and p64 by gel filtration shows that they are not physically associated in buffer containing the nonionic detergent Lubrol 12A9. The p64 protein is not precipitated by TBR IgG. We do not know whether p64 is only a substrate for the p61 tyrosine kinase or is itself a kinase.

Stabilization of glucose-6-phosphatase activity by a 21 000-dalton hepatic microsomal protein.

Hepatic microsomal glucose-6-phosphatase activity was rendered extremely unstable by a variety of techniques: (a) incubation at pH 5.0; (b) extraction of the microsomal fraction in the presence of 1% Lubrol; (c) various purification procedures. These techniques all result in the removal of a 21 kDa polypeptide from the fraction containing glucose-6-phosphatase activity. The 21 kDa protein was purified to apparent homogeneity by solubilization in the detergent Lubrol 12A-9 and chromatography on Fractogel TSK DEAE-650(S) and centrifugation at 105 000 g. The 21 kDa protein stabilizes glucose-6-phosphatase activity, whereas other purified hepatic microsomal proteins do not. The 21 kDa protein appears to be a potential regulator of glucose-6-phosphatase activity.

A simple method for purification of rat hepatic microsomal cytochrome b5.

Rat hepatic microsomal cytochrome b5 was purified to homogeneity by solubilization with the detergent Lubrol 12-A9 and chromatography on Fractogel TSK DEAE-650(S). The protein was obtained in high yield (52-87%) in 8 h, and only one polypeptide band, of Mr 16 600, was visible after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.

Resolution and properties of the catalytic unit of cardiac adenylate cyclase

The catalytic unit (C) of cardiac adenylate cyclase was resolved from the guanine nucleotide-binding regulatory protein (G/F) by fractionation of Lubrol 12A9 extracts with 33% saturated (NH4)2SO4 and by gel filtration in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS). Catalytic activity in both preparations was supported by Mg2+ and Mn2+, Vmax values being 12 fold higher in the presence of the latter cation. NaF-activated preparations were also subjected to gel filtration in the presence of CHAPS. In this case, both the catalytic activity and G/F emerged in the activated state. G/F could be deactivated by dialysis of the preparation in the absence of NaF; the catalyst however remained activated following dialysis. NaF sensitivity was reconstituted in C by nonactivated and NaF-activated preparations of G/F isolated by gel filtration. Reconstitution was dependent upon the amount of both G/F and C in the assay. Nonactivated G/F also reconstituted guanine nucleotide sensitivity in C. Catalytic activity was thermally labile, but was stabilized at 25 degrees C by substrate (Mn2+ ATP). C was stimulated up to 25-fold by forskolin. The NaF-activated catalyst resolved by gel filtration was relatively insensitive to this agent. Forskolin, however, augmented NaF-sensitivity by both non-activated and NaF-activated G/F provided it was added to the assay before G/F. Similarly, forskolin augmented guanine nucleotide sensitivity of nonactivated G/F in the presence of GTP gamma S. The P site agent 2',5'-dideoxyadenosine (DDA) was a weak inhibitor of nonactivated C, but a powerful inhibitor of C stimulated by forskolin or activated by G/F in the presence of NaF. In all respects C precipitated by (NH4)2SO4 appeared to be identical with that resolved by gel filtration. Ammonium sulfate precipitation, because of its simplicity, speed, relatively good yield, and adaptability for large scale operation, may be the preferred method for preparing cardiac C for purification studies

GTPase activity of the stimulatory GTP-binding regulatory protein of adenylate cyclase, Gs. Accumulation and turnover of enzyme-nucleotide intermediates.

The GTPase activity of the stimulatory guanine nucleotide-binding regulatory protein (Gs) of hormone-sensitive adenylate cyclase was investigated using purified rabbit hepatic Gs and either [alpha-32P]- or [gamma-32P] GTP as substrate. The binding of [35S]guanosine 5'-O-(thiotriphosphate) (GTP gamma S) was used to quantitate the total concentration of Gs. 1) GTPase activity was a saturable function of the concentration of GTP, with Km = 0.3 microM. MgCl2 monotonically increased the activity. The maximum observed turnover number was about 1.5 min-1. 2) During steady-state hydrolysis, 20-40% of total Gs could be trapped as a Gs-GDP complex and 1-2% could be trapped as Gs-GTP. The hydrolysis of Gs-GTP to Gs-GDP occurred with t 1/2 less than or equal to 5 s at 30 degrees C and t 1/2 approximately 1 min at 0 degrees C. Hydrolysis of Gs-GTP was inhibited by 1.0 mM EDTA in the absence of added Mg2+. 3) The rate of formation of Gs-GDP and the initial GTPase rate varied in parallel as functions of the concentrations of either GTP or MgCl2 (above 0.1 mM Mg2+). The ratio of the rate of accumulation of Gs-GDP to the GTPase rate was constant at 0.3-0.4. 4) The rate of dissociation of assayable Gs-GDP was biphasic. The initial phase accounted for 60-80% of total assayable Gs-GDP and was characterized by a t 1/2 of about 1 min. 5) Lubrol 12A9 potently inhibited the GTPase reaction and the dissociation of Gs-GDP in parallel, and inhibition of product release may account for the inhibition of steady-state hydrolysis. 6) The beta and gamma subunits of Gs markedly inhibited the dissociation of GDP from Gs in contrast to their ability to stimulate the dissociation of GTP gamma S. 7) GDP, GTP gamma S, and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) competitively inhibited the accumulation of Gs-GDP. GTP gamma S and Gpp(NH)p inhibited the GTPase reaction noncompetitively, GDP displayed mixed inhibition, and Pi did not inhibit. These data are interpretable in terms of the coexistence of two specific mechanistic pathways for the overall GTPase reaction.

Properties of the resolved catalytic unit of skeletal muscle adenylate cyclase

Attempts were made to resolve the catalytic unit (C) of adenylate cyclase from the guanine nucleotide-binding regulatory protein (G/F) in detergent extracts of pigeon breast muscle. When preparations solubilized in 1% Lubrol 12A9 were fractionated at 33% saturated (NH 4) 2SO 4, catalytic activity precipitated which was insensitive to NaF and guanine nucleotide. G/F was concentrated in a fraction precipitating between 33 and 47% saturated (NH 4) 2SO 4, but was not obtained free of C. C was also resolved from G/F by gel filtration in the presence of 13 m m 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps), but recoveries of C were lower than those obtained by salt fractionation. NaF-Activated preparations were also subjected to gel filtration in the presence of Chaps, in which case both the catalytic activity and G/F emerged in the activated state. G/F from such columns was deactivated by removal of NaF by dialysis. NaF and guanine nucleotide sensitivity could be reconstituted in nonactivated preparations of C by G/F in the presence of NaF or guanylylimidodiphosphate. Reconstitution was dependent upon both the amount of C and G/F in the assay. C in both preparations was strongly stimulated by the diterpene, forskolin, whereas the NaF-activated enzyme resolved by gel filtration was only marginally stimulated by this agent. C was only weakly inhibited by the P-site agent 2′,5′-dideoxyadenosine. However, when C was stimulated by forskolin, dideoxyadenosine was a potent inhibitor. The NaF-activated catalytic unit was also strongly inhibited by this agent. Preparations of C obtained by (NH 4) 2SO 4 precipitation may be suitable starting material for attempted purification of this component of adenylate cyclase.

Effect of different detergent systems on the molecular size of UDP glucuronosyltransferase and other microsomal drug-metabolizing enzymes.

Mouse liver microsomes were solubilized in various detergent systems, and the resulting aggregate structures associated with cytochrome P-450, cytochrome c reductase, and UDP glucuronosyltransferase were sized by gel filtration chromatography. Cholate or its derivative, CHAPS, in combination with Emulgen 911 or Lubrol 12A9 were necessary to generate a particle of about 140 k daltons, the smallest structure associated with cytochrome P-450. Cholate or CHAPS alone was sufficient to generate a minimally sized aggregate of 200 k daltons associated with NADPH cytochrome c reductase activity. Cholate in combination with Emulgen 911 or Lubrol 12A9 generated particles of about 280 k daltons associated with UDP glucuronosyltransferase activity. CHAPS alone also generated similarly sized particles under conditions in which UDP glucuronosyltransferase activity toward 1-naphthol and morphine was two to about twenty times greater, respectively, than with the combination of detergents. This finding suggests that the zwitterionic CHAPS is superior to other detergent systems for studies concerned with the purification of transferase enzymes, a microsomal system in which investigation of the number of different forms has been hampered by the instability of the enzyme upon solubilization and subsequent manipulation.

Retinol dehydrogenase from bovine retinal rod outer segments. Kinetic mechanism of the solubilized enzyme.

Retinol dehydrogenase solubilized by Lubrol 12A9 from bovine retinal rod outer segments forms mixed micelles of Stokes radius 8.5 nm. The kinetic properties of the solubilized retinol dehydrogenase were examined and retinaldehyde reduction and retinol oxidation were seen to proceed at pH 8.3 by a sequential Ordered Bi Bi mechanism. This conclusion was supported by bisubstrate initial velocity studies, dead-end and product inhibition. The kinetic mechanism of retinol dehydrogenase is not altered by the effect of Lubrol until a concentration of 2 mM is reached, at which the detergent lowers the values of the Michaelis and dissociation constants. The catalytic rate of the retinol dehydrogenase is significantly lowered by detergent in the range of pH 3 to 9.

Phosphatidylcholine-promoted interaction of the catalytic and regulatory proteins of adenylate cyclase.

The catalytic protein of rabbit hepatic adenylate cyclase, after chromatographic separation from the GTP-binding regulatory protein (G/F) (Ross, E. M. (1981) J. Biol. Chem. 256, 1949-1953), is essentially free of endogenous phospholipids. This preparation is active in the presence of Mn2+ and is markedly stimulated by forskolin, but it is stimulated only slightly by the addition of purified G/F plus an activator (GTP gamma S or NaF). The ability of activator-liganded G/F to stimulate the activity of the resolved catalyst is increased up to 8-fold by the addition of either dimyristoylphosphatidylcholine (0.3-1.5 mM optimal concentration) or several other phosphatidylcholines. Phosphatidylcholine stabilizes the catalyst to denaturation but has little effect on its basal activity or on its stimulation by Mn2+ or forskolin. It also had no stimulating effect on the activation of G/F by GTP gamma S. These data are interpreted as showing that phosphatidylcholine promotes or is required for the stimulatory interaction of activator-liganded G/F with the catalytic protein of adenylate cyclase. Lubrol 12A9, Triton X-100, cholate, lysophosphatidylcholine, digitonin, and phosphatidylserine could not substitute for phosphatidylcholine. The detergents inhibited stimulation by liganded G/F even in the presence of phosphatidylcholine. Removal of cholate from a mixture of soluble catalytic protein and phosphatidylcholine by dialysis and sucrose density gradient centrifugation caused the binding of catalytic protein to large unilamellar vesicles. This preparation was further reconstituted with increasing amounts of G/F to yield vesicles with varied G/F:catalyst ratios and similarly varied responses to G/F-mediated activating ligands.

Properties of detergent-dispersed adenylate cyclase from cerebral cortex. Presence of an inhibitor protein.

Adenylate cyclase was solubilized from washed particulate fraction of rabbit cerebral cortex with the nonionic detergent Lubrol 12A9 and subjected to either gel filtration on Ultrogel AcA 34 or chromatography on DEAE Bio-Gel A. By both procedures the enzyme was resolved into two components, one insensitive to guanyl 5'-yl imidodiphosphate [Gpp(NH)p] and NaF but stimulated by Ca2+ and calmodulin, and another that was sensitive to Gpp(NH)p and NaF but relatively insensitive to Ca2+ and calmodulin. The data support the possibility that two independent forms of adenylate cyclase exist in cerebral cortex, one regulated by guanine nucleotide regulatory protein and another by Ca2+-calmodulin. Fractions containing the guanylnucleotide-sensitive activity were found to contain a factor that inhibited basal and Ca2+-stimulated adenylate cyclase in the Ca2+-sensitive fraction. The inhibitor was inactivated by heating at 60 degrees C and by incubation with trypsin. Inhibition was not time-dependent, and it was not due to destruction of cAMP by phosphodiesterase or of ATP by ATPase. Inhibitory action was not reversed by calmodulin and therefore it does not appear to be a calmodulin binding protein. Sucrose density gradient sedimentation indicated a sedimentation coefficient of 4S for the inhibitor; by this technique it co-sedimented with the adenylate cyclase sensitive to Gpp(NH)p and NaF.

Effects of detergents and organic solvents on rat liver microsomal UDP-glucuronosyltransferase activity towards phenolic substrates.

1. The effects of several detergents (Brij 58, deoxycholate and Lubrol 12A9) and ether on the initial rate of UDP-glucuronosyltransferase activity towards fixed concentrations of five phenolic acceptor substrates of widely different octanol-buffer (pH 7.4) partition coefficient have been compared with those observed in non-activated and Triton X-100-and n-pentane-activated rat liver microsomes. 2. Enzymic activity was dependent on the lipid-solubility of acceptor substrate. Each activator, except Triton X-100, enhanced enzymic activity towards all substrates by a similar factor, which was independent of the octanol-buffer partition coefficient. For Triton X-100 microsomes, the activation was also partition-dependent. 3. The highest activation factor was seen with ether. Pre-incubation of ether-activated microsomes for 30 min at 37 degrees C before assay resulted in inactivation of the enzyme towards more water-soluble substrates. Tryptic digestion (30 min at 37 degrees C) of the ether-activated microsomes resulted in marked reduction of enzyme activity towards all substrates. 4. Ether, and the two detergents, Brij 58 and Lubrol 12A9, released small amounts of protein (5-12% total present); both detergents also released some (8-12%) phospholipid. 5. The Kappm towards acceptor substrate also depended on the octanol-buffer partition coefficient, and was largely unchanged on activation by n-pentane. Vmax was not dependent on partition coefficient and was significantly increased on activation.

Increase in the size of soluble brain adenylate cyclase with activation by guanosine 5'-(beta, gamma-imino)triphosphate.

Adenylate cyclase solubilized from bovine brain with Lubrol 12A9 or Triton X-100 can be resolved into two forms by gel filtration or sucrose density gradient centrifugation. The activity of one of these forms is not stimulatable by guanosine 5'-(beta, gamma-imino)triphosphate (Gpp(NH)p) and represents the "basal" adenylate cyclase activity. In Lubrol 12A9, this form has Mr = 330,000 (total, protein and detergent) and Mr (protein only) = 265,000. The other form of adenylate cyclase can be activated by Gpp(NH)p and has a smaller molecular weight: Mr (protein and detergent) = 293,000, Mr (protein only) = 199,000. Upon activation by Gpp(NH)p, the size of the Gpp(NH)p-responsive form of adenylate cyclase increases: Mr (protein and detergent) = 330,000, Mr (protein only) = 251,000. Similar values were obtained in Triton X-100. The kinetics of heat inactivation are different in the two forms of the enzyme. Both forms are activated about 2-fold by 5 mM MnCl2. Neither of the forms is associated with measurable low Km GTPase activity. On the basis of these studies, we propose that the catalytic unit (C) of adenylate cyclase and the guanine nucleotide regulatory unit (G/F) may exist in solution in the following rapid equilibrium: (formula: see text) We propose that activation by Gpp(NH)p stabilizes the C . G/F complex and this accounts for the greater mass of the enzyme measured after activation with Gpp(NH)p. The size of the enzyme which represents the basal activity is very similar to that of the C . G/F complex. We suggest, therefore, that the basal activity is the result of a stable association of the catalytic unit with the guanine nucleotide regulatory site.

Proteolysis of skeletal muscle adenylate cyclase. Destruction and reconstitution of fluoride and guanylnucleotide sensitivity.

Adenylate cyclase was measured in skeletal muscle plasma membranes incubated with subtilisin. Under specific conditions the protease preferentially inactivated fluoride and guanylnucleotide sensitivity. Following protease treatment, membranes were solubilized with Lubrol 12A9 and subjected to ion-exchange chromatography. Adenylate cyclase was eluted with 200 mM NaCl; the enzyme recovered was completely unresponsive to either NaF or guanylyl imidodiphosphate. Responsiveness to the two ligands was restored by adding a heart fraction in which basal activity had been destroyed by heating at 40 degrees C or by adding a soluble skeletal muscle fraction in which basal activity had been largely destroyed by N-ethylmaleimide. The solubilized subtilisin-treated skeletal muscle preparation may serve as a source of catalytic activity for the study and purification of regulatory factors for adenylate cyclase.

The effect of dimethyl 3,3'-dithiobispropionimidate on the adenylate cyclase activity of bovine corpus luteum; stimulation of particulate enzyme activity and the stabilization of activity to dispersion in detergent [proceedings

In a previous report (Young, 1979) it was shown that dimethyl 3,3'-dithiobispropionimidate inhibited p[NHlppG-stimulated adenylate cyclase of the washed 600g sediment of bovine corpus-luteum homogenate in a dose-dependent manner at concentrations above 0.02m~. This initial series of experiments did not show any statistically signscant effect of dimethyl 3,3'-dithiobispropionimidate concentrations below 0.02 m~ on the activity of the particulate enzyme, although some individual experiments indicated a slight enhancement of activity by 0.0 1 mwdimethyl 3,3'-dithiobispropionimidate. The accumulation of more data has revealed a small, but statistically significant, enhancement of the particulate enzyme activity by dimethyl 3,3'-dithiobispropionimidate at concentrations around 0.0 1 mM, and we now report the biphasic effect of dimethyl 3,3'-dithiobispropionimidate on p[NHlppG-stimulated adenylate cyclase activity. The washed 600g sediment of bovine corpus-luteum homogenate was prepared as described previously (Young & Stansfield, 1977). The adenylate cyclase of the 600g sediment was activated by incubation with 0.1 m~-p{NH]ppG in 4 0 m ~ Tris/HCl (pH7.5)/6rnM-MgSO4 at 37OC and for the times indicated in the Figures, followed by washing twice with p[NHJppG-free buffer at 4OC. Portions of the washed p[NH]ppG-activated 600g sediment and of the native nontreated 600g sediment were resuspended in 40 m~-Tficine/ NaOH buffer, pH 7.5, containing various concentrations of dimethyl 3,3'-dithiobispropionirnidate (see Figure legends). Cross-linking was carried out at 4OC for 18h, followed by washing the sediment twice with buffer (40 mM-Tricine/NaOH, pH 7.5, followed by 40m~-Tris/HCI, pH 7.5). The sediments were resuspended in 40 mM-Tris/HCl buffer, pH 7.5, containing 6mwMgS0, and, where indicated, Lubrol 12A9 (lOg/litre). All tissueextract concentrations were eqiuvalent to 75 mg wet wt. of corpus luteum/ml. Adenylate cyclase was assayed as described previously (Young & Stansfield, 1977, 1978a,b,c). The assay incubation (1 ml) contained 40m~-Tris/HCI, pH 7.5, ~ ~ M M ~ S O , , 1 mMATP, 6.7mlul-catTeine and 5 0 0 ~ 1 of enzyme preparation. Incubation was at 37OC for l0min. The cyclic AMP produced was measured by competitive protein binding (Young & Stansfield, 1977). Assays were performed in duplicate and the mean and range of duplicate values are shown. Fig. 1 demonstrates that there is a biphasic response of p[NHlppG-activated cyclase with respect to dimethyl 3,3'dithiobispropionimidate concentration. Concentrations around 5 p ~ enhanced the activity of the pH1ppG-treated enzyme, whereas higher concentrations (>0.05 mM) inhibited the enzyme. A paired t-test on the normalized data obtained in 12 separate experiments showed that 0.01 mM-dimethyl 3,3'-dithiobispropionimidate causes a significant increase in the activity of p[NH]ppG-treated cyclase (P < 0.01). The mean percentage increase in activity due to treatment with 0.01 mwdimethyl 3,3'-dithiobispropionirnidate was 23.5 (range -9 to 78; S.D. 2 1.7). Dimethyl 3,3'dithiobispropionimidate was without

Hydrodynamic properties of the regulatory component of adenylate cyclase.

Hydrodynamic parameters of the regulatory component of adenylate cyclase, G/F, have been estimated by gel filtration and sucrose density gradient centrifugation. In solutions containing Lubrol 12A9, the protein has an apparent molecular weight of 130,000. G/F from various sources and resolved from the catalytic moiety of the enzyme by different techniques behaves similarly. Consistent with our previous proposal that this protein is the site of action of both guanine nucleotides and fluoride, treatment with these activating ligands causes a reduction in both the sedimentation coefficient and the Stokes radius of G/F. These changes suggest a loss of mass of approximately 40,000 daltons. Nevertheless, this alteration is fully reversible when ligands are removed, even if the liganded protein is first fractionated by gel filtration or sucrose density gradient centrifugation.

Solubilization of membrane-bound adenylate cyclase of isolated rat adrenal cortex cells

The membrane-bound adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) of isolated rat adrenal cortex cells can be rendered soluble using 0.02 M Lubrol 12A9. The solubilized enzyme can be filtered through Millipore filters with pores 0.22 μm in diameter. Using gel filtration, on Sephadex G-200, adenylate cyclase activity was eluted with a distribution coefficient of 0.139, whereas on Sephadex G-100 the activity was eluted in the excluded volume. Half-maximum activation of the postulated guanyl nucleotide regulator site of adenylate was achieved with 5′-guanylyl-imidodiphosphate at a concentration of 1 · 10 −6 M. In contrast, however, using intact isolated rat adrenal cortex cells the guanyl nucleotide regulator site could not be stimulated by 5′-guanylyl-imidodiphosphate.

Reconstitution of cholera toxin-activated adenylate cyclase.

Reconstitution of adenylate cyclase activity responsive to stimulation by guanylyl-5'imidodiphosphate or NaF may be achieved by mixing dilute Lubrol 12A9-solubilized extracts of wild-type S49 membranes with membranes of an adenylate cyclase-deficient variant. Experiments using N-ethylmaleimide to inactivate components of the adenylate cyclase system indicate that distinct components from both wild-type detergent extracts and adenylate cyclase-deficient membranes are essential for reconstitution. These results and conclusions confirm those of E. M. Ross and A. G. Gilman [J. Biol. Chem. (1977) 252, 6966-6969]. Detergent extracts of cholera toxin-treated wild-type membranes yield a reconstituted adenylate cyclase as responsive to GTP as to guanylyl-5'-imidodiphosphate whereas, in the absence of cholera toxin treatment, GTP has little or no effect. Cholera toxin-treated adenylate cyclase-deficient membranes and Lubrol 12A9 extracts from them, however, fail to yield a reconstituted adenylate cyclase that responds to GTP with an increase in cyclase activity. Because treatment of the adenylate cyclase-deficient variants with cholera toxin is without effect on the reconstituted cyclase, we propose that the cholera toxin substrate is absent or altered in the adenylate cyclase-deficient phenotype.

Phospholipid content and activity of pure uridine diphosphate-glucuronyltransferase from rat liver.

Rat liver phospholipids were radioactively labeled in vivo before purification of UDP-glucuronyltransferase to homogeneity. The pure enzyme contained very little phospholipid (approx. 0.7 mol of phospholipid/mol of protein). The solubilization detergent Lubrol 12A9 appeared to act as a phospholipid substitute, capable of supporting UDP-glucuronyltransferase activity. Phospholipase C did not inhibit the pure enzyme activity and pure UDP-glucuronyltransferase was stimulated by 40--100% by the addition of phospholipid dispersions.

Resolution of some components of adenylate cyclase necessary for catalytic activity.

Adenylate cyclase activity in a Lubrol 12A9 extract of wild type S49 lymphoma plasma membranes is completely inactivated by incubation at 37 degrees for 20 min. Activity is restored by mixing this heated extract of wild type membranes with an unheated detergent extract of membranes from a variant clone that lacks measureable adenylate cyclase activity (AC-). The factor(s) donated by the AC- extract is labile to heating at 30 degrees (t1/2 = 3 min) or to treatment with N-ethylmaleimide or trypsin. The factor(s) donated by the heated wild type extract is also sensitive to proteases or N-ethylmaleimide. This extract displays more complex inactivation kinetics at 50 degrees, consistent with the existence of separate factors necessary for the stimulatory effects of NaF and guanyl-5'-yl-imidodiphosphate. We suggest that at least two proteins are necessary for adenylate cyclase activity and that one of these is retained in the phenotypically adenylate cyclase-deficient variant.

Reconstitution of catecholamine-sensitive adenylate cyclase activity: interactions of solubilized components with receptor-replete membranes.

Membranes of mouse L cells that contain adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] but lack beta-adrenergic receptors have been solubilized with Lubrol 12A9. Addition of such adenylate cyclase-containing extracts to beta-adrenergic receptor-replete membranes from adenylate cyclase-deficient S49 lymphoma cells results in the production of a catecholamine-sensitive adenylate cyclase system. The effects of beta-adrenergic agonists and antagonists on the reconstituted system reproduce those that are characteristic of the wild-type S49 lymphoma cell. The uncoupled variant of the S49lymphoma contains adenylate cyclase, but donor extracts from this clone fail to reconstitute the hormone-sensitive enzyme activity when added to adenylate cyclase-deficient membranes. Thus, the uncoupled and adenylate cyclase-deficient variants of the S49 cell are not complementary.