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L Birnbaumer

Publications and source records attributed to L Birnbaumer.

At least 217 records · Page 12Linked to original sources
PubMed1984

GTP hydrolysis by pure Ni, the inhibitory regulatory component of adenylyl cyclases.

The stimulatory and inhibitory regulatory components of adenylyl cyclase (Ns and Ni), purified to apparent homogeneity without the use of regulatory ligands such as Mg, NaF, and guanyl-5'-yl imidodiphosphate, were tested for GTPase activity by incubating them with [gamma-32P]GTP and measuring 32Pi liberation using a charcoal adsorption assay to separate hydrolyzed from nonhydrolyzed radioactivity. We found that Ni is capable of hydrolyzing GTP. The activity was shown to be due to Ni itself and not to presence of one of its minor contaminants by correlating activity with abundance of the 40,000 Da alpha i subunit throughout the last stages of purification and by showing co-migration on a sucrose density gradient of the GTP-hydrolyzing activity with the alpha i, beta, and gamma subunits of Ni and not with any one of three minor contaminants present in the preparation tested. Preparations of Ns, free of detectable Ni, exhibited less than 10% the capacity to hydrolyze GTP, as compared to Ni on an equal protein basis. The basic properties of the GTP-hydrolyzing activity of Ni were determined. The activity is dependent on Mg ion (apparent Km = 5 to 15 nM), and is rapidly lost upon incubation with Mg2+ in the absence of GTP. MgGTP and free GTP serve equally well as substrate (apparent Km about 40 nM). Isotopic dilution studies indicate that the GTP binding site has a relative affinity for guanine nucleotides in the order GTP = GTP gamma S greater than GDP = GMP-P(NH)P greater than GDP beta S with the highest difference (GTP versus GDP beta S) being about 10-fold. NaF inhibited GTP hydrolysis by Ni at concentrations at which it activates Ni in intact membranes.

Adenylyl Cyclase Inhibitors↗
PubMed1984

Interaction of the stimulatory and inhibitory regulatory proteins of the adenylyl cyclase system with the catalytic component of cyc-S49 cell membranes.

The mechanism by which Ns and Ni, the stimulatory and inhibitory regulatory components of adenylyl cyclases, regulate the activity of the catalytic component (C) of adenylyl cyclase was investigated using cyc-S49 cell membranes which contain a functional inhibitory regulatory protein (Ni) but not the active subunit of the stimulatory regulatory protein (Ns). To this end, purified Ns protein was preactivated (Ns) in solution with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) and Mg2+, and then added to cyc- membranes under conditions where Ni was either unactivated or activated (Ni) by GTP gamma S and Mg2+. Activation of Ni in cyc- membranes resulted in a lowered expression of Ns activity under all conditions tested. Upon dilution of the reactants (Ns and cyc- membranes) the reconstituted activity declined in proportion to the dilution with an approximate t 1/2 of 30-45 min, being unaffected by activation of Ni. Postactivation of Ni after reconstitution of cyc- membranes with Ns resulted in a time-dependent decline in Ns activity to a level that was the same as that obtained when Ns was added to cyc- membranes with preactivated Ni. These data indicated that the effects of Ns on C are of a reversible type. The following indicated that Ns and Ni affect C activity in a noncompetitive manner: (a) the per cent reduction in Ns activity due to activation of Ni was constant and independent of the concentration of Ns, (b) double reciprocal plots of activities reconstituted in control and Ni-containing cyc- membranes versus Ns concentration were linear with an unaltered apparent Km for Ns, and (c) the onset of inhibition of C prereconstituted with Ns was much faster (approximate t 1/2 = 2-5 min) than expected if it were due to occupancy of a common site on C left vacant by Ns.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylyl Cyclases↗
PubMed1984

The mammalian beta 2-adrenergic receptor: reconstitution of functional interactions between pure receptor and pure stimulatory nucleotide binding protein of the adenylate cyclase system.

Pure beta-adrenergic receptors (beta-AR) isolated from guinea pig lung and pure guanine nucleotide binding regulatory protein (NS) of adenylate cyclase isolated from human erythrocytes have been inserted into phospholipid vesicles, resulting in the functional coupling of these two components. The reconstitution of receptor and NS interactions results in the establishment of a guanine nucleotide sensitive state of the receptor that binds agonists with high affinity. Competition curves of isoproterenol for labeled antagonist binding to vesicles containing both beta-AR and NS are biphasic and reveal two affinity states, one of high (approximately 2 nM) and the other of low affinity (approximately 300 nM). In the presence of guanine nucleotides, the competition curves become monophasic and are shifted to a single low-affinity state for the agonist similar to the situation observed in membrane preparations. In addition, the interactions of the receptor and NS lead to the induction of a GTPase activity in NS. The GTPase activity can be stimulated by beta-adrenergic agonists such as isoproterenol (2-5-fold) and is completely blocked by antagonists such as alprenolol in a stereoselective manner. The established hormone responsive activity retains the beta 2-adrenergic specificity conferred by the pure receptor, and similar extents of stimulation (up to 4-fold) are observed with pure receptor from frog erythrocytes, indicating a similar efficiency of coupling between receptors from different species and NS.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1984

Effects of guanine nucleotides and Mg on human erythrocyte Ni and Ns, the regulatory components of adenylyl cyclase.

The effect of GTP analogs and Mg on the structure of Ns and Ni, the stimulatory and inhibitory regulatory components of adenylyl cyclase, were studied in a comparative manner. Both N proteins, which are alpha beta gamma heterotrimers that differ in their alpha subunits, when exposed to GTP analogs underwent a Mg-dependent conformational change that was not dependent on subunit dissociation. This was seen both as change in sedimentation behavior at 4 degrees C from 4 S to about 3 S and by a property of the new conformation to retain guanine nucleotide tightly bound to it. Warming to 32 degrees C promoted subunit dissociation, each protein giving a mixture of 2 S alpha G and 2 S beta gamma complexes. For both Ns and Ni, these reactions were reversible: 2 S complexes of Ns and Ni associated to 3 S forms on cooling to 4 degrees C, provided the Mg concentration was at or below 10 mM and detergent concentration was below 1%, and the 3 S complexes of these proteins reverted to 4 S forms and released the nucleotide in the cold on chelation of free Mg with EDTA. Reconstitution assays with Ns-deficient membranes from cyc- S49 lymphoma cells revealed that the 3 S form of Ns is a "pre-active" form of the protein. The scheme below summarizes these findings, where G represents a guanine nucleotide. (Formula: see text) Ns and Ni differ in that more Mg is necessary to promote the 4 S to 3 S conversion of Ns than of Ni, and in that both the 2 S to 3 S to 4 S conversions proceeded more readily with Ni than with of Ns. Mg could not be shown to promote subunit dissociation. The above scheme is suggested as a plausible description of the reaction sequence leading from an unactivated to an activated N protein.

Adenylyl Cyclases↗
PubMed1984

Oocyte adenylyl cyclase contains Ni, yet the guanine nucleotide-dependent inhibition by progesterone is not sensitive to pertussis toxin.

Membranes were obtained from Xenopus laevis oocytes after removal of follicular cells by collagenase treatment. [32P]ADP-ribosylation with pertussis toxin showed them to contain a single Mr = 40000 substrate for this toxin that co-migrates on sodium dodecylsufate-polyacrylamide gel electrophoresis with pure human erythrocyte Ni, the inhibitory regulatory component of adenylyl cyclase. [32P]ADP-ribosylation of oocyte membranes with cholera toxin also showed presence of a single substrate but of Mr = 42000. These results indicate, that the adenylyl cyclase system of oocytes, like that of somatic cells and unlike that of spermatozoids, contains the catalytic unit C and both of the known regulatory N components. The possible susceptibility to pertussis toxin of the guanine nucleotide-dependent inhibition of oocyte adenylyl cyclase by progesterone was investigated. This action of progesterone is mediated by a membrane bound receptor as opposed to a receptor of cytosolic or nuclear localization. However, the inhibitory effect of progesterone was unaffected by pertussis toxin, even though the oocyte membrane Ni was fully ADP-ribosylated with pertussis toxin, as revealed by lack of further [32P]ADP-ribosylation on subsequent re-incubation with pertussis toxin. These results indicate that the action of progesterone, in spite of being nucleotide-dependent, is either not mediated by Ni, suggesting the existence of an additional nucleotide regulatory component, or if mediated by Ni, involves a mode of regulation of this coupling protein that is different from that by which all other inhibitory hormones act on adenylyl cyclase.

Adenosine Diphosphate Ribose↗
PubMed1984

Reconstitution of a hormone-sensitive adenylate cyclase system. The pure beta-adrenergic receptor and guanine nucleotide regulatory protein confer hormone responsiveness on the resolved catalytic unit.

A hormone responsive adenylate cyclase has been reconstituted in phosphatidylcholine vesicles from its isolated protein components. The proteins used were the affinity chromatography purified (500-2000-fold) or pure Mr = 64,000 beta-adrenergic receptors (beta AR) isolated from hamster and guinea pig lung membranes, the pure heterotrimeric (Mr: alpha = 42,000; beta = 35,000; gamma approximately equal to 5,000) guanine nucleotide regulatory protein (Ns) isolated from human erythrocyte membranes; and the catalytic unit of the adenylate cyclase (C) solubilized from bovine brain caudate nucleus and resolved from beta AR and Ns by gel filtration. Adenylate cyclase activity in vesicles containing C alone was stimulated by forskolin but not by guanine nucleotides or by the beta-adrenergic agonist isoproterenol. Reconstitution of Ns and C interactions in the lipid vesicles resulted in guanine nucleotide but not beta-adrenergic agonist sensitivity. When beta AR was inserted together with Ns and C into lipid vesicles, the catalytic unit became responsive to beta-adrenergic agonists as well and this stimulation was blocked in a stereoselective manner by the beta-adrenergic antagonist alprenolol. Regulation of adenylate cyclase activity in the reconstituted system by beta-adrenergic agonists, guanine nucleotides, and Mg2+ showed properties similar to those observed in native membranes. The interactions of the various protein components in the reconstituted system were also monitored by GTPase activity. Such activity was observed to occur primarily as a result of receptor-Ns interactions. The results described in this report document the feasibility of studying hormone-responsive adenylate cyclase in a totally reconstituted system which retains the major regulatory properties of the enzyme in its native membrane-bound environment.

Adenylyl Cyclases↗
PubMed1984

Ns and Ni, the stimulatory and inhibitory regulatory components of adenylyl cyclases. Purification of the human erythrocyte proteins without the use of activating regulatory ligands.

Methods were developed to adequately extract, separate and, without the use of NaF as stabilizing agent, purify to better than 90% purity human erythrocyte Ns and Ni, the stimulatory and inhibitory guanine nucleotide- and Mg-binding regulatory components of adenylyl cyclases, as well as a protein containing Mr = 35,000 subunits. On the basis of a functional assay for Ns, it was purified about 5,000-fold from starting washed erythrocyte membranes with a yield of about 10%. A typical purification yields from 60 units of outdated human blood, between 500 and 1,000 micrograms of pure Ns, and a similar amount of Ni. Pure Ns and Ni contain each at least one alpha and one beta subunit (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. 74, 6516-6520; Codina, J., Hildebrandt, J.D., Iyengar, R., Birnbaumer, L., Sekura, R.D., and Manclark, C.R. (1983) Proc. Natl. Acad. Sci. U.S.A. 77, 4276-4280). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate at varying acrylamide concentrations yielded Mr values of 42,000 and 40,000 for the alpha subunits of Ns and Ni, and of 35,000 for the beta subunits of Ns and Ni. Two-dimensional thin layer analysis of tryptic peptides obtained from digesting 125I-labeled subunits of Ns and Ni confirmed the finding of Manning, D., and Gilman, A.G. (1983) J. Biol. Chem. 258, 7059-7063) that while their alpha subunits are clearly different, their beta subunits are the same. Hydrodynamic analysis of the molecular weights of the nondenatured proteins showed behavior consistent with Mr = 95,500 for Ns, the same for Ni, and Mr = 40,000 for the protein containing the Mr = 35,000 beta subunit. Sedimentation coefficients and Stokes radii of the purified Ns were indistinguishable from those of Ns activity present in initial cholate extracts from human erythrocyte membranes. Further, the overall kinetics with which Ns activity in cholate extracts and Ns activity in the purified protein reconstituted the Ns-deficient adenylyl cyclase system of cyc- S49 cells was also indistinguishable. We conclude that we have purified the native unactivated form of Ns, and by serendipity the Ni, as well as a protein containing the 35 kDa beta subunit of Ns and Ni.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1984

Identification of a gamma subunit associated with the adenylyl cyclase regulatory proteins Ns and Ni.

The subunit composition of the Ns and Ni, the human erythrocyte stimulatory and inhibitory regulatory proteins of adenylyl cyclase, respectively, were analyzed by a sodium dodecyl sulfate-containing discontinuous urea and polyacrylamide gradient gel electrophoresis system designed for the study of low molecular weight polypeptides. This system disclosed that these proteins, in addition to their known alpha and beta subunits, contain an additional small peptide of apparent molecular weight of 5,000 (5K). This "5K peptide" is also present in preparations of another protein which we termed "40K protein" on the basis of its hydrodynamic behavior and whose primary protein constituent is the Mr 35,000 beta subunit of the above regulatory proteins. Analyzing Ni, the 5K peptide was functionally related to the protein by showing that its apparent Stokes radius changes from 5.9 to 5.1 nm after treatment with guanyl-5'-yl imidodiphosphate and magnesium in parallel with the alpha and beta subunits. These data are interpreted as evidence for the existence of a third subunit associated with the regulatory proteins of adenylyl cyclase. We call this subunit gamma and propose a minimum subunit structure for these proteins of the alpha beta gamma type.

Adenylyl Cyclases↗
PubMed1984

Glucagon-stimulable adenylyl cyclase in rat liver. Effects of chronic uremia and intermittent glucagon administration.

The effects of chronic uremia and glucagon administration on glucagon-stimulable adenylyl cyclase in rat liver were assessed by determinations of adenylyl cyclase activities, specific iodoglucagon binding, and the activity of the stimulatory regulatory component of adenylyl cyclase. Glucagon-stimulated adenylyl cyclase was reduced in uremia to 75-80% of control levels (P less than 0.05), in the presence or absence of saturating levels of guanosine triphosphate (GTP) and 5'-guanylylimidodiphosphate [GMP-P(NH)P]. Although these changes were accompanied by a concomitant 20% reduction in sodium fluoride-stimulated activity, basal, GTP-, GMP-P(NH)P-, and manganese-dependent adenylyl cyclase activities were unchanged. Using [125I-Tyr10]monoiodoglucagon as a receptor probe, the number of high affinity glucagon-binding sites was reduced 28% (P less than 0.01) in uremic as compared with control liver membranes. However, the affinity of these binding sites was unaltered. The S49 cyc- -reconstituting activity with respect to both GMP-P(NH)P- and isoproterenol plus GTP-stimulable adenylyl cyclase was unaltered in membranes from uremic as compared with control rats. Intermittent glucagon (80-100 micrograms) injections administered at 8-h intervals to normal rats reproduced all of the above described effects of chronic experimental uremia on the adenylyl cyclase system. It is concluded that changes in the hormone-stimulable adenylyl cyclase complex in uremia and with glucagon treatment result primarily from a decrease in the number of hormone-specific receptor sites in hepatic plasma membranes. Since the changes in liver adenylyl cyclase are qualitatively and quantitatively the same in glucagon-treated and uremic rats, it is suggested that these may be the result of the hyperglucagonemia of uremia. Further, the data reveal an unexpected dissociation between guanine nucleotide and sodium fluoride stimulation of adenylyl cyclase. Possible causes for this dissociation based on the known subunit composition of cyclase coupling proteins are discussed.

Adenylyl Cyclases↗
PubMed1984

Glucagon-stimulable adenylyl cyclase in rat liver. The impact of streptozotocin-induced diabetes mellitus.

Glucagon receptor levels, glucagon-stimulated and other forms of adenylyl cyclase activity, and regulatory component activity of adenylyl cyclase were determined in hepatic plasma membranes of rats administered streptozotocin without and with insulin to produce varying degrees of hyperglycemia. Receptor levels were assayed by direct binding of the specific probe [125I-Tyr10]-iodoglucagon; regulatory component activity was assayed by the capacity to reconstitute stimulatory regulation in deficient membranes from cyc- S49 murine lymphoma cells. In rats given 150 mg streptozotocin, glucagon stimulation of adenylyl cyclase as well as basal, sodium fluoride, 5' guanylylimidodiphosphate [GMP-P(NH)P] and Mn-dependent activities were reduced 50%, glucagon receptor levels but not affinity were reduced 67%, and regulatory component activity was decreased 50%. In addition, alpha 1-adrenergic receptors and 5'-nucleotidase were similarly reduced in diabetes. However, specific ouabain-inhibitable Na+, K+, ATPase activity was not altered by streptozotocin treatment. The streptozotocin-induced changes were noted within 24 h and became maximal by 120 h after its administration. All of these decreases were partially reversed by in vivo insulin treatment. DNA, cytochrome c oxidase, glucose-6-phosphatase, and N-acetyl-beta-glucosaminidase content in hepatic plasma membrane preparations were not substantially different in diabetic as compared with control animals. The data demonstrate that glucagon-mediated regulation of cyclic AMP formation is deranged in insulin deficiency owing to a combined decrease in receptors, derangement of the coupling mechanism intervening between receptor and adenylyl cyclase, and possibly, an altered basal effector system. Some of these changes appear to reflect a "desensitization-like" phenomenon which may or may not be attributable to the hyperglucagonemia of diabetes mellitus. There also appears to be a concurrent generalized decrease in several but not all plasma membrane receptor and enzymatic proteins. This may be the result of a number of processes among which is the accelerated proteolysis of uncontrolled diabetes.

Adenylyl Cyclases↗
PubMed1984

Updated protocols and comments on the purification without use of activating ligands of the coupling proteins Ns and Ni of the hormone sensitive adenylyl cyclase.

Ns and Ni have been purified without using NaF and Mg as stabilizing agents (Codina, J., Hildebrandt, J.D., Sekura, R.D., Birnbaumer, M., Bryan, J., Manclark, C.R. and Birnbaumer, L. [1984] J. Biol. Chem. 259, in press). Since the submission of that report, several modifications have been introduced to the purification procedure and additional fractions have been processed from which N proteins are obtained. This article describes the updated protocols and presents methodological details not included in the previous publication. The final products are Ns, the stimulatory N, Ni the inhibitory N, both of subunit structure alpha beta gamma, and a Mr = 40,000 protein of beta gamma composition. They are obtained from human erythrocytes.

Adenylyl Cyclases↗
PubMed1983

Inhibitory regulation of adenylyl cyclase in the absence of stimulatory regulation. Requirements and kinetics of guanine nucleotide-induced inhibition of the cyc- S49 adenylyl cyclase.

cyc- S49 cell membranes contain an adenylyl cyclase activity which is stimulated by forskolin and inhibited by guanine nucleotides and NaF. These inhibitory effects are mediated by an inhibitory guanine nucleotide-binding regulatory component (Ni) affecting the adenylyl cyclase catalytic unit (Hildebrandt, J. D., Sekura, R. D., Codina, J., Iyengar, R., Manclark, C. R., and Birnbaumer, L. (1983) Nature (Lond.) 302, 706-709). Since cyc- S49 cells do not contain a stimulatory guanine nucleotide-binding regulatory component (Ns), these membranes were used to study the requirements and kinetics of activation of Ni in the absence of Ns. Activation of Ni by guanyl-5'-yl imidodiphosphate was time-dependent (i.e. hysteretic) and pseudo-irreversible. Although GTP and guanosine 5'-(beta-thio)diphosphate could prevent the inhibition caused by guanyl-5'-yl imidodiphosphate if added simultaneously with it, they could not reverse the inhibited state induced by previous exposure to guanyl-5'-yl imidodiphosphate. Activation of Ni had an absolute requirement for Mg2+. Unlike the activation of Ns, however, which requires millimolar concentrations of Mg2+ in the absence of hormonal stimulation, activation of Ni requires only micromolar concentrations of the divalent cation. These results support the contention that hormones which activate Ni or Ns do so by altering different parameters of a similar activation mechanism.

Adenylyl Cyclases↗
PubMed1983

Serotonergic and adrenergic regulation of skeletal muscle metabolism in the rat. II. The use of [125I]iodolysergic acid diethylamide and [125I]iodopindolol as probes of sarcolemmal receptor function and specificity.

To evaluate serotonin receptor kinetics in skeletal muscle, we synthesized and developed 2-[125I]iodolysergic acid diethylamide [( 125I]iodoLSD) as a high affinity, high specific activity probe of serotonergic receptor function. The kinetics of binding of this probe and the profile of agonist and antagonist displacement have been compared to results obtained using [125I] iodopindolol as a probe for beta-adrenergic receptor binding. [125I]IodoLSD was prepared by chloramine-T iodination and purified by high pressure liquid chromatography. Fluorescence, ultraviolet, and nuclear magnetic resonance spectra as well as mass spectroscopy demonstrate that the iodinated compound is 2-iodoLSD. This probe bound in a concentration-dependent fashion to sarcolemma in a manner consistent with existence of a single population of specific receptors having a Kd of 1.46 nM and an abundancy of 47 fmol/mg of protein. Half-maximal binding of the probe occurred within 1.0 min and equilibrium binding was observed at 8.0 min. The apparent t 1/2 for the probe-receptor complex was 40 s; the Kd calculated from kinetic data was 1.69 nM. The IC50 for displacement of iodoLSD was 330 +/- 185 nM for methysergide, 757 +/- 309 nM for cyproheptadine, and 2,570 +/- 1,390 nM for serotonin. Adrenergic antagonists such as l-propranolol and oxprenolol also displaced [125I]iodoLSD, but did so with IC50 values of 17,800 +/- 5,100 and 23,300 +/- 5,500 nM, respectively. Using [125I]iodopindolol as a receptor probe, we found the order of potency for adrenergic antagonists to be l-propranolol greater than oxprenolol much much greater than practolol. Serotonergic antagonists, although effective in displacing [125I]iodopindolol, did so only at very high concentrations. These results are consistent with the existence of separate and discrete D-serotonergic and beta 2-adrenergic receptors in skeletal muscle and the finding that adrenergic agonists and antagonists may interact with low affinities with the serotonergic receptor, but that serotonergic agonists and antagonists interact poorly, if at all, with the beta 2-adrenergic receptor.

Animals↗
PubMed1983

Forskolin regulation of liver membrane adenylyl cyclase.

The effects of forskolin on rat liver plasma membrane adenylyl cyclase were studied. The diterpene stimulated the Vmax of the enzyme system with apparent Km values of 3-5 microM. Stimulations were marked both in the absence (20-fold over control) as well as in the presence of various stimulators such as GTP, GuoPP[NH]P, NaF alone or in combination with glucagon. Except with GTP, where stimulations of activities by forskolin and the nucleotide were synergistic (more than additive), stimulations of combinations of GuoPP[NH]P, NaF or glucagon with forskolin were additive. Forskolin did not alter significantly the apparent Km values of the enzyme for MgATP or MnATP or the apparent Ka values (concentrations giving stimulations that are 50% of maximum) for Mg or Mn ions, GTP, GuoPP[NH]P or NaF. Forskolin caused a decrease in the concentration of glucagon required for half-maximal stimulation from 5 microM to 1.5 microM. Except for this effect on the Ka for the glucagon, the only kinetic parameter altered was the Vmax under all conditions tested. Although proteolysis stimulated liver membrane adenylyl cyclase under control conditions, it did not enhance forskolin-stimulated activities. More extensive proteolysis, which resulted in decreased activities in the absence of forskolin, also resulted in reduced forskolin-stimulated activities. 'Uncoupling' of the guanine-nucleotide-binding regulatory component, that mediates guanine nucleotide stimulation by addition of 30 mM MnCl2, did not result in 'uncoupling' of forskolin stimulation. The data indicate that the diterpene forskolin stimulates adenylyl cyclase activity by a novel mechanism that differs from that by which NaF or guanylyl nucleotides affect this membrane-bound system and that the diterpene should be a useful tool with which to explore as yet unrecognized modes of regulation of cyclic AMP production.

Adenosine Triphosphate↗
PubMed1983

Stimulation and inhibition of adenylyl cyclases mediated by distinct regulatory proteins.

Adenylyl cyclases are under positive and negative control by guanine nucleotides and hormones. Stimulatory responses are mediated by a guanine nucleotide- and Mg-binding regulatory component (Ns), a protein that has been purified to homogeneity. Inhibitory responses have been hypothesized to be mediated by an analogous regulatory component (Ni) distinct from Ns, but definitive proof for this is lacking and these effects may result from modulation of Ns activity. Recently, Bordetella pertussis toxin has been shown to ADP-ribosylate a peptide that is not part of Ns, and this coincides with attenuation of hormonal inhibition of adenylyl cyclase. We show here that cyc- S49 cells contain a substrate for ADP-ribosylation by pertussis toxin and that the toxin alters GTP dependent inhibition of cyc- adenyl cyclase activity. As cyc- S49 cells do not contain Ns by several criteria, we conclude that Ni is a distinct and separate regulatory component of adenylyl cyclase.

Adenosine Diphosphate Ribose↗
PubMed1983

Pertussis toxin substrate, the putative Ni component of adenylyl cyclases, is an alpha beta heterodimer regulated by guanine nucleotide and magnesium.

The final step in a scheme for the purification of the guanine nucleotide- and Mg2+-binding stimulatory regulatory component (Ns) of adenylyl cyclase [adenylate cyclase; ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from human erythrocyte membranes involves chromatography over hydroxylapatite (HAP) which yields two fractions. The first fraction (HAP I) contains predominantly two peptides that, upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis, migrate with Mr values of 39,000 and 35,000. The second fraction (HAP II) contains predominantly Ns formed of two peptides of Mr 42,000 and 35,000. The HAP I, Mr 39,000 peptide is shown to be a substrate for the ADP-ribosylating toxin of Bordetella pertussis (pertussis toxin). Upon sucrose density gradient centrifugation, both the Mr 39,000 and the Mr 35,000 peptides of HAP I migrate at about 4 S. Treatment of HAP I with guanine nucleotide and Mg2+ prior to centrifugation results in a coordinated change in the migration of both peptides to 2 S. It is postulated that HAP I contains an alpha beta heterodimeric protein composed of an alpha subunit of Mr 39,000 and a beta subunit of Mr 35,000. Further, this protein dissociates under the influence of guanine nucleotides and Mg2+ into its individual alpha and beta subunits. Because previous studies have shown that treatment of cells and cell membranes with pertussis toxin results in attenuation of the effects of hormones that inhibit adenylyl cyclase activity, and because this effect correlates with the ADP-ribosylation of a Mr approximately equal to 40,000 peptide, we believe that we have purified a guanine nucleotide- and Mg2+-binding inhibitory regulatory component of adenylyl cyclases--i.e., the Ni.

Adenylate Cyclase Toxin↗
PubMed1983

Monoiodoglucagon: synthesis, purification by high pressure liquid chromatography, and characteristics as a receptor probe.

The synthesis of [125I-Tyr10]monoiodoglucagon from glucagon and carrier-free 125I using 1,3,4,6-tetrachloro-3-6-diphenylglycouril (Iodogen) and its separation in pure form by reverse phase high pressure liquid chromatography (HPLC) over C18-muBondapak columns using two consecutive linear gradients between solvent A [40:60 mixture of methanol and 10 mM H3PO4 in H2O (pH adjusted to 3.0 with triethylamine)] and solvent B (50:50 mixture of acetonitrile and 0.1 M Tris-HCl, pH 9.0) is reported. The newly synthesized [125I]monoiodoglucagon is shown to activate adenylyl cyclase in liver membranes with an EC50 between 5- and 8-fold lower than that of native glucagon. Further, it binds specifically to sites on liver plasma membranes that have the characteristics of glucagon receptors in terms of guanine nucleotide sensitivity and rates of reaction. It is suggested that [125I-Tyr10]monoiodoglucagon is a suitable probe for studying structural and functional properties of glucagon receptors.

Animals↗