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R Iyengar

Publications and source records attributed to R Iyengar.

At least 109 records · Page 6Linked to original sources
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↗
PubMed1983

Human chorionic gonadotropin-induced heterologous desensitization of adenylyl cyclase from highly luteinized rat ovaries: attenuation of regulatory N component activity.

We injected hCG into superovulated rats on the seventh day of pseudopregnancy and confirmed previous findings that this results in both homologous desensitization of luteal adenylyl cyclase (loss of responsiveness to LH) and heterologous desensitization of the same adenylyl cyclase system (partial loss of responsiveness to catecholamines), and that these changes are associated with the loss of available unoccupied LH receptors (down-regulation) but not with any discernible loss of beta-adrenergic receptors. We tested the hypothesis that the heterologous component of the above changes might be due to alterations in the function of the nucleotide-binding N component of adenylyl cyclase that intervenes between receptors and catalytic units of adenylyl cyclase. This was done by assessing N component activity in reconstitution assays that measured the capacity of luteal N to mediate, in cyc- S49 lymphoma membranes, stimulation of adenylyl cyclase independently by the guanine nucleotide guanylyl imidodiphosphate, by NaF, or by the lymphoma membrane beta-adrenergic receptor. By all of these modes of assay, heterologous desensitization of luteal adenylyl cyclase to beta-adrenergic stimulation was found to be associated with a proportionally similar decrease in N component activity. This change in N component activity could be due to either quantitative or qualitative alterations. It is speculated that if the change is of a qualitative nature, the alteration may be a cAMP-dependent phosphorylation reaction of one of the subunits of the N component.

Adenylyl Cyclases↗
PubMed1982

Hormone receptor modulates the regulatory component of adenylyl cyclase by reducing its requirement for Mg2+ and enhancing its extent of activation by guanine nucleotides.

N-Ethylmaleimide treatment of rat liver plasma membranes results in an adenylyl cyclase (EC 4.6.1.1) system that shows no measurable cyclizing activity but retains both an active glucagon receptor and a receptor-sensitive regulatory component N as assessed by reconstitution into cyclase-negative (cyc-) membranes from S49 murine lymphoma. Treatment of such N-ethylmaleimide-treated membranes, termed C- liver membranes, with guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S] ) and Mg2+, followed by the removal of GTP[gamma S] by washing, yields an activated N which upon mixing with cyc- S49 membranes reconstitutes the cyc- S49 membrane adenylyl cyclase in the absence of added GTP[gamma S]. It was found that GTP[gamma S] activation of the N at saturating concentrations of GTP[gamma S] is slow at low Mg2+ concentration and accelerated by increasing Mg2+ concentrations. Addition of glucagon during the activation results in a lowering of the Mg2+ requirement for full activation from 25 mM to around 10 muM and in concomitant increases in both the rate and the extent of N activation. In contrast to its dramatic effect on Mg2+ requirement, glucagon has little (less than 2-fold) effect on the GTP[gamma S] requirement of N activation. These experiments indicate that the glucagon receptor facilitates activation of N by: (i) decreasing the apparent Km of N for Mg2+, and (ii) increasing the extent of activation that can be elicited by saturating concentrations of guanine nucleotide. It is postulated that the mechanism by which Mg2+ and receptors facilitate N activation involves dissociation of n alpha activated ADP-ribosylatable subunits (with guanine nucleotide bound to them) from n beta non-ADP-ribosylatable subunits (with receptor and Mg2+ bound to them).

Adenylyl Cyclases↗
PubMed1981

Hysteretic activation of adenylyl cyclases. II. Mg ion regulation of the activation of the regulatory component as analyzed by reconstitution.

The rate of appearance and extent of reconstituted adenylyl cyclase activity in the presence of guanyl-5'-yl imidodiphosphate (GMP-P(NH)P) upon mixing cholate extract of rat liver 10,000 X g particulate preparation and S49 cyc- cell membranes was dependent on Mg ion concentration. Increasing concentrations of Mg ion lead to progressively shorter lag times in the appearance of reconstituted activity. In contrast, at 20 mM MgCl2, the rate of appearance of reconstituted activity was insensitive to concentrations of GMP-P(NH)P, cyc- membrane protein, and untreated cholate extract protein, but became sensitive to cholate extract protein concentration after treatment with GMP-P(NH)P and Mg ion. Treatment of cholate extract with GMP-P(NH)P and Mg ion resulted in a faster appearance of reconstituted activity upon exposure to S49 cyc- membranes as compared to treatment in the presence of GMP-P(NH)P and EDTA. Activation of the regulatory component by GMP-P(NH)P occurred only in the presence of Mg ion. Mn2+, Ca2+, or Ba2+ did not appear to substitute for Mg. The effect of Mg ion allowing for the rapid appearance of reconstituted activity was reversible in the presence or absence of S49 cyc- membranes. However, while dilution of GMP-P(NH)P in the absence of S49 cyc- membranes resulted in relatively rapid reversal, treatment of cholate extract with 5 microM GMP-P(NH)P and 20 mM Mg ion followed by reconstitution at 5 or 0.1 microM GMP-P(NH)P showed similar reconstituted activities for up to 15 min. The rate of activation of the regulatory component was faster at 20 mM Mg ion as compared to that at 1 mM Mg ion. Similarly, the rate of activation of the regulatory component by NaF was also accelerated by increasing the concentration of Mg ion. These data indicate that the rate-limiting step in the activation of adenylyl cyclase is conversion of the regulatory component from its inactive to active form. In the presence of saturating concentration of guanine nucleotides and NaF, both the rate and extent of this activation is regulated by Mg ion.

Adenylyl Cyclases↗
PubMed1981

Concentration of activated intermediates of the fructose-1,6-bisphosphate aldolase and triosephosphate isomerase reactions.

As discovered by Grazi & Trombetta [Grazi, E., & Trombetta, G. (1978) Biochem. J. 175, 361], fructose-1,6-bisphosphate aldolase of rabbit muscle causes the slow formation of inorganic phosphate (Pi) and methylglyoxal when incubated with dihydroxyacetone phosphate (DHAP). In addition, these authors found an acid-labile intermediate in equilibrium with the aldolase-dihydroxyacetone phosphate complexes representing approximately 60% of the enzyme-bound DHAP species. Experiments are reported here which argue that this acid-labile species is the enzyme-bound enamine phosphate or its equivalent that decomposes by beta elimination in acid. A similar mechanism involving an enediol phosphate is proposed to explain a phosphatase action of triosephosphate isomerase that produces methylglyoxal and Pi at the rate of approximately 0.1 s(-1) at pH 5.5. When DHAP with excess isomerase is quenched in strong acid, the formation of Pi indicates that approximately 5% of bound reactant is in the form of enediol phosphate. The remainder of the substrate is about equally distributed between bound forms of DHAP and D-glyceraldehyde 3-phosphate. This equilibrium differs by 300-fold from the appropriate equilibrium in solution. Yeast aldolase, contrary to expectation, does not catalyze formation of inorganic phosphate and methylglyoxal when incubated with DHAP and gives no evidence fro an enediol phosphate intermediate when quenched in acid.

Animals↗
PubMed1981

Liberation of the triosephosphate isomerase reaction intermediate and its trapping by isomerase, yeast aldolase, and methylglyoxal synthase.

When a mixture of triosephosphate isomerase (rabbit muscle) and dihydroxyacetone phosphate (DHAP) is quenched with acid, a compound is liberated, presumed to be the cis-enediol 3-phosphate, that decomposes to inorganic phosphate (Pi) and methylglyoxal [Iyengar, R., & Rose, I.A. (1981) Biochemistry (preceding paper is this issue)]. The decomposition can be prevented by rapid neutralization if a catalytic amount of fresh isomerase is present. Varying the time between acidification and rescue gave a half-life of the liberate compound of approximately 12-17 ms. Varying the concentration of enzyme used for rescue gave a minimum second-order rate constant for trapping of 10(9)M(-1)s(-1). These results add further evidence favoring a stepwise mechanism for the aldose-ketose isomerase reactions in which a chemically defined enzyme-bound intermediate is found. The high rate of trapping over a wide pH range indicates that the enediol phosphate, not the enediolate phosphate, is the intermediate. One property of the enzyme is to stabilize the intermediate with respect to its fragmentation in solution by greater than 1000-fold. Yeast aldolase is also able to rescue all of the isomerase intermediate, though higher concentrations of enzyme are required. Although different enantiotopic protons of DHAP are abstracted by isomerase and aldolase, both enzymes use the same enediol phosphate intermediate. Methylglyoxal synthase at a 50-fold greater concentration was unable to compete with triosephosphate isomerase for cis-enediol phosphate. Either the synthetase has a low V/K for the cis isomer or it uses the trans-enediol phosphate form specifically. A new strategy for the chemical and enzymological characterization of enzyme reaction intermediates is proved here based on the liberation of the intermediate from the reaction equilibrium and its recovery by fresh enzyme or another enzyme species.

Animals↗
PubMed1980

Regulation of hormone-receptor coupling to adenylyl cyclase. Effects of GTP and GDP.

GDP and GTP regulation of receptor-mediated stimulation of adenylyl cyclases in membranes of S49 murine lymphoma cells (S49), NS-20 murine neuroblastoma cells (NS-20), rabbit corpora lutea (CL), and turkey erythrocytes were studied under assay conditions which minimized conversion of added GTP to GDP and of added GDP to GTP. Hormonal stimulation in all systems required guanine nucleotide addition. In the presence of GTP, adenylyl cyclase activity in S49, NS-20, and CL was stimulated respectively by isoproterenol and prostaglandin E1 (PGE1), by PGE1 and the adenosine analog, phenylisopropyladenosine, and by PGE1 and isoproterenol, with the first of the listed stimulants eliciting higher activities than the second. Activity in turkey erythrocyte membranes was stimulated by isoproterenol. GDP was partially effective in promoting hormonal stimulation, being able to sustain stimulation by isoproterenol and PGE1 in S49 cell membranes and by PGE1 in CL membranes. In NS-20 membranes, both GDP and guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) were inhibitory on basal activity, yet promoted limited but significant stimulation by PGE1. In turkey erythrocytes, stimulation by isoproterenol could not be elicited with GDP or GDP beta S. Thus, although less effective than GTP in promoting hormonal stimulation of several adenylyl cyclase systems, GDP was clearly not inactive. Concentration effect curves for active hormone in the presence of GDP had higher apparent Ka values than in the presence of GTP. In spite of differences between the effects of GTP and GDP on hormonal stimulation of adenylyl cyclase activities, GTP and GDP affected equally well isoproterenol binding, regardless of whether or not its receptor could be shown to stimulate adenylyl cyclase in the presence of GDP. Determination of transphosphorylation of GDP to GTP showed that at saturating concentrations, the proportion of GDP converted to GTP is negligible and unaffected by hormonal stimulation. Concentrations giving 50% inhibition were determined for GTP- and GDP-mediated inhibition of guanyl-5'-yl imidodiphosphate stimulation in the absence and presence of stimulatory hormones. In all four systems studied, GTP and GDP interacted with about equal potency and hormonal stimulation was not accompanied by a selective decrease in affinity for GDP. One way to explain all of the results obtained is to view hormonally sensitive adenylyl cyclase systems as two-state enzymes whose activities are regulated by GTP and GDP through an allosteric site related to the catalytic moiety, and receptors as entities that are inactive and hence unable to couple unless occupied by hormones and activated by any guanine nucleotide through a distinct receptor-related process.

Adenylyl Cyclases↗