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Biomedical subjects

A G Gilman

Publications and source records attributed to A G Gilman.

At least 163 records · Page 9Linked to original sources
PubMed1982

Requirements for cholera toxin-dependent ADP-ribosylation of the purified regulatory component of adenylate cyclase.

The requirements for cholera toxin-catalyzed ADP ribosylation of the purified regulatory component of adenylate cyclase are described. In addition to the toxin, this reaction is dependent on or is facilitated by NAD, GTP, phospholipid, and a factor found associated with plasma membranes from several sources. Factor activity is heat-labile and protease-sensitive but is unaffected by treatment with N-ethylmaleimide. Gel filtration indicates that the factor behaves as a monodisperse species with a Stokes radius of 3.2 nm. The factor thus appears to be a protein that is distinct from any of the known components of adenylate cyclase. Factor activity was also detected in the cytoplasm of S49 cells. The cytoplasmic factor was smaller (Stokes radius = 2.0 nm) than the membrane-derived factor, and it was inactivated in the presence of sodium cholate. The initial rate of activation of the regulatory component of adenylate cyclase by toxin was found to be linearly related to the amount of factor present in the reaction. This has allowed the quantitation and partial purification (33-fold from detergent extracts) of the factor from turkey erythrocyte membranes.

Adenosine Diphosphate Ribose↗
PubMed1982

Aluminum: a requirement for activation of the regulatory component of adenylate cyclase by fluoride.

Activation of the purified guanine nucleotide-binding regulatory component (G/F) of adenylate cyclase by F- requires the presence of Mg2+ and another factor. This factor, which contaminates commercial preparations of various nucleotides and disposable glass test tubes, has been identified as Al3+. In the presence of 10 mM Mg2+ and 5 mM F-, AlCl3 causes activation of G/F with an apparent activation constant of approximately 1-5 muM. The requirement for Al3+ is highly specific; of 28 other metals tested, only Be2+ promoted activation of G/F by F-.

Adenosine Triphosphate↗
PubMed1982

The guanine nucleotide-binding regulatory component of adenylate cyclase in human erythrocytes.

The guanine nucleotide-binding regulatory component of adenylate cyclase (G/F) has been purified from human erythrocyte membranes. It is composed of two major polypeptides with molecular weights of 35,000 and 45,000. When cyc- S49 lymphoma cell plasma membranes are reconstituted with purified human erythrocyte G/F, stimulation of adenylate cyclase by beta-adrenergic agonists, guanine nucleotides, and fluoride is restored. Binding of GTP gamma S to human erythrocyte G/F and GTP gamma S-mediated activation of the protein are closely correlated. The agreement between the apparent dissociation constants for these two reactions suggests that the measured binding site is identical to the site responsible for activation. A 41,000-dalton protein has been identified as a contaminant of preparations of G/F that have been purified by four successive chromatographic steps. This protein serves as a specific substrate for ADP-ribosylation and labeling by islet activating protein (IAP) and [32P]NAD, and it appears to contribute an additional high-affinity guanine nucleotide binding site to such preparations.

Adenylyl Cyclases↗
PubMed1981

The regulatory component of adenylate cyclase. Purification and properties of the turkey erythrocyte protein.

The regulatory component (G/F) of adenylate cyclase has been purified from turkey erythrocyte plasma membranes by adaptation of procedures developed for purification of the rabbit liver protein. The major modifications entail inclusion of high concentrations of NaCl to facilitate extraction and reconstitution of the protein. A typical preparation yields 200 micrograms of protein with a reconstitutive specific activity of 3-4 mumol . min-1 mg-1. Turkey erythrocyte G/F contains two putative subunits of 35,000 and 45,000 daltons. The 52,000-dalton polypeptide that appears to be a component of rabbit liver G/F is lacking. In solution, G/F behaves as a particle with Mr = 81,000. This value is reduced to 50,000 in the presence of activating ligands, suggesting dissociation of subunits. Activation of G/F by guanine nucleotide analogs is markedly accelerated in the presence of high concentrations of Mg2+. Reconstitutive and physical properties of the protein are also affected by fluoride. Cyc- S49 lymphoma membranes reconstituted with turkey erythrocyte G/F acquire properties that are characteristic of the turkey adenylate cyclase system; at least certain differing characteristics of adenylate cyclase systems are thus dictated by the nature of their G/F.

Adenylyl Cyclases↗
PubMed1981

The regulatory component of adenylate cyclase. Purification and properties.

The regulatory component (G/F) of adenylate cyclase, which has been purified previously, contains three putative subunits with molecular weights of 52,000, 45,000, and 35,000 (Northup, J. K., Sternweis, P. C., Smigel, M. D., Schleifer, L. S., Ross, E. M., and Gilman, A. G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6516-6520). The published procedure has been modified to reduce the time required for preparation and to increase the yield. Application of the improved procedure allows purification of .5 to 1.0 mg of purified G/F from 1.5 kg of frozen rabbit liver. Greater than 95% of the protein observed on sodium dodecyl sulfate polyacrylamide gels is found in the three bands mentioned above. Purified G/F has the following properties: 1. Hydrodynamic measurements in cholate indicate that purified hepatic G/F has a molecular weight of about 70,000. If G/F is activated with either fluoride or GTP analogs, its apparent molecular weight is reduced to 50,000. 2. The measurement of G/F by reconstitution with the catalytic moiety of adenylate cyclase is dependent on the concentrations of both G/F and catalytic moiety. This interaction is consistent with a model derived from a simple bimolecular binding equilibrium. 3. Purified G/F can be activated by fluoride and guanine nucleotide analogs in a Mg2+-dependent reaction. The rate of activation by guanine nucleotides is markedly stimulated by high concentrations of Mg2+, indicating a site of action of divalent metallic cations on G/F. 4. The 52,000- and 45,000-dalton polypeptides can be partially resolved by heptylamine-Sepharose chromatography. G/F fractions that are enriched in the 52,000-dalton protein reconstitute hormone-stimulated adenylate cyclase activity more efficiently and are activated by GTP analogs more rapidly than are fractions that are essentially free of this polypeptide. The 35,000-dalton protein is present in all cases.

Adenylyl Cyclases↗
PubMed1980

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.

Adenylyl Cyclases↗
PubMed1980

The regulatory component of adenylate cyclase from uncoupled S49 lymphoma cells differs in charge from the wild type protein.

The uncoupled (UNC) variant of the S49 lymphoma possesses the components known to be required for a hormone-sensitive adenylate cyclase system, including receptors for hormones, a guanine nucleotide-binding regulatory protein (G/F), and a catalyst; however, hormones are ineffective in stimulating enzymatic activity in these cells. Two proteins (Mr = 45,000 and 55,000) are labeled with [32P]NAD when wild type or UNC membranes are incubated with the labeled nucleotide and cholera toxin (Johnson, G.L., Kaslow, H.R., and Bourne, H.R. (1978) J. Biol. Chem. 253, 7120-7123). The 45,000-dalton component appears to be a constituent of G/F. Two dimensional electrophoresis of the labeled proteins reveals that both are more acidic when derived from UNC cells.

Adenylyl Cyclases↗
PubMed1980

Purification of the regulatory component of adenylate cyclase.

The regulatory component (G/F) of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from rabbit liver plasma membranes has been purified essentially to homogeneity. The purification was accomplished by three chromatographic procedures in sodium cholate-containing solutions, followed by three steps in Lubrol-containing solutions. The specific activity of G/F was enriched 2000-fold from extracts of membranes to 3-4 mumol x min-1 x mg-1 (reconstituted adenylate cyclase activity). Purified G/F reconstitutes guanine nucleotide-, fluoride-, and hormone-stimulated adenylate cyclase activity in the adenylate cyclase-deficient variant of S49 murine lymphoma cells. G/F also recouples hormonal stimulation of the enzyme in the uncoupled variant of S49. Preparations of pure G/F contain three polypeptides with approximate molecular weights of 52,000, 45,000, and 35,000. The active G/F protein behaves as a multisubunit complex of these polypeptides. Treatment of G/F with [32P]NAD+ and cholera toxin covalently labels the molecular weight 52,000 and 45,000 polypeptides with 32P.

Adenylyl Cyclases↗
PubMed1979

Identification and partial characterization of some components of hormone-stimulated adenylate cyclase.

Adenylate cyclase can be resolved into at least two protein components, neither of which by itself catalyzes the formation of cyclic AMP with Mg-ATP as substrate. Mixture of the two reconstitutes Mg-ATP-dependent, fluoride- and Gpp(NH)p-stimulable activity. One, a heat-labile, N-ethylmaleimide-sensitive protein of molecular weight 190,000 can catalyze cyclic AMP formation with Mn-ATP as substrate, and is therefore proposed to be the catalytic moiety of the adenylate cyclase complex. The other protein (or proteins) is more resistant to heating or N-ethylmaleimide, and is proposed to confer upon the catalyst the ability to ultilize Mg-ATP as substrate. It is also required for the regulation of that activity by guanine nucleotides, hormones, and probably fluoride ion. The catalytic protein is found in a phenotypically adenylate cyclase-deficient (AC-) variant of S49 lymphoma cells. The thermostable regulatory protein can be resolved from the catalyst by heat treatment or N-ethylmaleimide treatment of plasma membranes of wild-type S49 cells, rat or rabbit liver, or avian erythrocytes, and is also found in a phenotypically adenylate cyclase-deficient hepatoma cell line. Mixture of AC- S49 membranes, which contain the beta-adrenergic receptor, with a crude detergent-solubilized preparation of the regulatory protein reconstitutes hormone-stimulable adenylate cyclase activity. Binding of the regulatory protein to the membranes is a time- and temperature-dependent process that requires an activating ligand of the adenylate cyclase system [fluoride, Gpp(NH)p].

Adenylyl Cyclases↗
PubMed1978

Reconstitution of hormone-sensitive adenylate cyclase activity with resolved components of the enzyme.

Adenylate cyclase can be resolved into at least two proteins, a thermolabile, N-ethylmaleimide-sensitive component and a second protein (or proteins) that is more stable to either of these treatments. Neither component by itself catalyzes the formation of cyclic AMP using MgATP as substrate. However, mixture of the two reconstitutes MgATP-dependent fluoride- and guanyl-5'-yl imidodiphosphate (Gpp(NH)p)-stimulatable adenylate cyclase activity. The more stable component can be resolved from the first in various tissues or cultured cells by treatment of membrnes or detergent extracts with heat or N-ethylmaleimide. The two proteins have also been resolved genetically in two clonal cell lines that are deficient in adenylate cyclase activity. An adenylate cyclase-deficient variant of the S49 lymphoma cell (AC-) contains only the thermolabile activity, while the activity of the more stable protein is found in a complementary hepatoma cell line (HC-1). In addition, AC-S49 cell plasma membranes contain MnATP-dependent adenylate cyclase activity. The protein that catalyzes this reaction appears to be the same as that which can combine with the thermostable component to reconstitute Mg2+-dependent enzyme activity because both activities co-fractionate by gel exclusion chromatography and sucrose density gradient centrifugation, both activities have identical denaturation kinetics at 30 degrees C, and both activities are stabilized at 30 degrees C and labilized at 0 degree C by various nucleotides and divalent cations with similar specificity. It is thus hypothesized that the thermolabile factor is the catalytic subunit of the physiological adenylate cyclase and that the Mn2+-dependent activity is a nonphysiological expression of the catalytic protein. The thermostable moiety of the enzyme, which is proposed to serve a regulatory function, appears to consist of two functional components, based upon differential thermal lability of its ability to reconstitute hormone-, NaF-, or Gpp(NH)p-stimulated adenylate cyclase activity. These components have not, however, been physically separated. The thermolabile and thermostable components can interact in detergent solution or in a suitable membrane. Mixing of the detergent-solubilized regulatory component with AC-membranes that contain only the catalytic protein and beta-adrenergic receptors reconstitutes catecholamine-stimulatable adenylate cyclase activity; however, addition of the catalytic protein to membranes that contain receptor and the regulatory component yields MgATP-dependent enzymatic activity that is unresponsive to hormone.

Adenylyl Cyclases↗
PubMed1978

S49 lymphoma wild type and variant clones contain normal calcium dependent regulator.

Calcium dependent regulator is present in wild-type S49 lymphoma cells, in the variant that is deficient in adenylate cyclase activity (AC-), and in the uncoupled variant (UNC). The electrophoretic mobility and the ability to stimulate cyclic nucleotide phosphodiesterase of the calcium dependent regulator from each of these three clones are indistinguishable from those of the modulator protein isolated from bovine brain. Calcium dependent regulator does not appear to be involved in the defect responsible for the UNC or AC- variants.

3',5'-Cyclic-AMP Phosphodiesterases↗