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R Iyengar

Publications and source records attributed to R Iyengar.

At least 91 records · Page 5Linked to original sources
PubMed1988

Beta gamma subunits of GTP-binding proteins inhibit muscarinic receptor stimulation of phospholipase C.

This study examines the mechanism of guanine nucleotide-binding protein (G protein) coupling of receptors to phospholipase C. The Xenopus oocyte has a muscarinic receptor-activated Cl- current that is mediated by inositol 1,4,5-trisphosphate. Modulation of the muscarinic receptor-evoked Cl- current was examined under voltage clamp in oocytes injected with resolved G-protein subunits. The presence of an alpha subunit of G proteins in oocytes was shown by pertussis toxin-labeling of a 41-kDa band in oocyte membranes. The presence of the beta subunit of G proteins was demonstrated by immunoblotting experiments with an antiserum (U-49) that is specific for the beta subunit. Pertussis toxin treatment of oocytes resulted in the uncoupling of muscarinic receptors from activation of the Cl- current. Cells microinjected with 1.5 ng of human erythrocyte beta gamma-subunit complex or 1.0 ng of bovine brain beta gamma-subunit complex showed approximately a 95% reduction in the evoked Cl- current. Cells injected with equal volumes of protein storage vehicle showed no change in response. Cells injected with boiled beta gamma subunits, bovine serum albumin, or resolved alpha subunits also showed no reduction in response. Cells injected with various concentrations of beta gamma subunits showed a concentration dependence with half-maximal inhibition of the muscarinic activated Cl- current at about 10 nM. Cells injected with 1.0 ng of bovine brain beta gamma subunits could not respond to bath-applied agonist but could generate the Cl- current on intracellular injection of inositol 1,4,5-trisphosphate. These observations suggest that there is a G protein responsible for muscarinic receptor-mediated signal transduction through phospholipase C and that it is an alpha beta gamma heterotrimer. It appears that the mode of action of the G protein in the phospholipase C system may be similar to that of the hormone-activated adenylyl cyclase.

Animals↗
PubMed1987

Identification of a new GTP-binding protein. A Mr = 43,000 substrate for pertussis toxin.

In purified preparations of human erythrocyte GTP-binding proteins, we have identified a new substrate for pertussis toxin, which has an apparent molecular mass of 43 kDa by silver and Coomassie Blue staining. Pertussis toxin-catalyzed ADP-ribosylation of the 43-kDa protein is inhibited by Mg2+ ion and this inhibition is relieved by the co-addition of micromolar amounts of guanine nucleotides. GTP affects the ADP-ribosylation with a K value of 0.8 microM. Addition of a 10-fold molar excess of purified beta gamma subunits (Mr = 35,000 beta; and Mr = 7,000 gamma) of other GTP-binding proteins results in a significant decrease in the pertussis toxin-mediated ADP-ribosylation of the 43-kDa protein. Treatment of the GTP-binding proteins with guanosine 5'-O-(thiotriphosphate) and 50 mM MgCl2 resulted in shifting of the 43-kDa protein from 4 S to 2 S on sucrose density gradients. Immunoblotting analysis of the 43-kDa protein with the antiserum A-569, raised against a peptide whose sequence is found in the alpha subunits of all of the known GTP-binding, signal-transducing proteins (Mumby, S. M., Kahn, R. A., Manning, D. R., and Gilman, A. G. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 265-259) showed that the 43-kDa protein is specifically recognized by the common peptide antiserum. A pertussis toxin substrate of similar molecular weight was observed in human erythrocyte membranes, bovine brain membranes, membranes made from the pituitary cell line GH4C1, in partially purified GTP-binding protein preparations of rat liver, and in human neutrophil membranes. Treatment of neutrophils with pertussis toxin prior to preparation of the membranes resulted in abolishment of the radiolabeling of this protein. From these data, we conclude that we have found a new pertussis toxin substrate that is a likely GTP-binding protein.

Adenosine Diphosphate Ribose↗
PubMed1987

Macrophage synthesis of nitrite, nitrate, and N-nitrosamines: precursors and role of the respiratory burst.

The macrophage cell line RAW 264.7 when activated with Escherichia coli lipopolysaccharide and interferon-gamma synthesized nitrite (NO3-) and nitrate (NO3-). Medium change after the activation showed that L-arginine was the only amino acid essential for this synthesis. D-Arginine would not substitute for L-arginine. Other analogues that could replace L-arginine were L-homoarginine, L-arginine methyl ester, L-arginamide, and the peptide L-arginyl-L-aspartate. L-Argininic acid, L-agmatine, L-ornithine, urea, L-citrulline, and ammonia were among the nonprecursors, while L-canavanine inhibited this L-arginine-derived NO2-/NO3- synthesis. When morpholine was added to the culture medium of the activated RAW 264.7 macrophages, N-nitrosation took place, generating N-nitrosomorpholine. GC/MS experiments using L-[guanido-15N2]arginine established that the NO2-/NO3- and the nitrosyl group of N-nitrosomorpholine were derived exclusively from one or both of the terminal guanido nitrogens of arginine. Chromatographic analysis showed that the other product of the L-arginine synthesis of NO2-/NO3- was L-citrulline. The role of the respiratory burst in NO2-/NO3- synthesis was examined using the macrophage cell lines J774.16 and J774 C3C. Both cell lines synthesized similar amounts of NO2-/NO3-. However, J774 C3C cells do not produce superoxide and hence do not exhibit the respiratory burst. Additional experiments also ruled out the involvement of the respiratory burst in NO2-/NO3- synthesis.

Arginine↗
PubMed1987

The hepatic glucagon receptor: a comparative study of the regulatory and structural properties.

Guanine nucleotide and Mg2+ ion regulation of [125I-Tyr10]monoiodoglucagon ([125I]MIG) binding to liver plasma membranes from chicken, rat, and rabbit was studied. It was found that [125I]MIG binding to chicken liver membranes was increased by the addition of Mg2+ ion, while binding to rat and rabbit liver membranes was unaffected. In the chicken liver membranes, the Mg2+ ion induced high affinity binding which was sensitive to guanine nucleotides, while the low affinity binding in the absence of Mg2+ ion was not. Maximal effects of Mg2+ ion were observed at 1 mM. Glucagon binding to rat liver membrane receptors was GTP sensitive regardless of whether Mg2+ ion was added. Glucagon binding to rabbit liver membranes was insensitive to both Mg2+ ions and GTP. This lack of GTP effect was not due to degradation of GTP; no effect of the nonhydrolyzable analog guanyl-5'-yl-imidodiphosphate was observable. Glucagon stimulation of rabbit liver adenylyl cyclase, however, was dependent on GTP, as was the case with all of the other liver adenylyl cyclases studied here. The Kact of GTP for the rabbit liver system was very similar to that for rat liver membranes. The glucagon receptor was covalently labeled with [125I]MIG using p-hydroxysuccinimidyl azidobenzoate and analyzed by sodium dodecyl sulfate-gel electrophoresis. In all cases, a major labeled band at 63,000 daltons was observed. The levels of glucagon receptor and stimulatory (Ns) and inhibitory (Ni) regulatory proteins of adenylyl cyclase were measured. The highest levels of glucagon receptor were measured in rat liver membranes, while the levels in chicken and rabbit membranes were 30-40% lower. Rabbit liver membrane had the highest levels of Ns, while rat liver membranes had 2-fold lower and chick liver membrane 4-fold lower levels than rabbit liver membranes. The levels of Ni was similar in the three systems. Thus, the ratio of Ns to glucagon receptor was highest in the rabbit. In the rat, this ratio was 3-fold lower than that in the rabbit. In the chicken membranes, the ratio was about 60% of that in the rat. These data suggest that the observed differences in effects of GTP on hormone binding can be explained by alterations in the ratio of the receptor and Ns proteins among the various species.

Adenylate Cyclase Toxin↗
PubMed1986

Gentamicin nucleotidyltransferase. Stereochemical inversion at phosphorus in enzymatic 2'-deoxyadenylyl transfer to tobramycin.

Gentamicin nucleotidyltransferase-catalyzed reaction of (Sp)-[alpha-17O]dATP with tobramycin produced 2"-(2'-deoxyadenosine 5'-[17O]phosphoryl)tobramycin. The configuration at phosphorus in this product was shown to be Rp by chemical degradation to chiral [17O, 18O]dAMP using a stereochemically defined procedure, and determination of the configuration at phosphorus in this product. Periodate-base treatment of 2"-(2'-deoxyadenosine 5'-[17O]phosphoryl)tobramycin followed by NaBH4 reduction produced (2-glyceryl)-[17O]dAMP, which upon snake venom phosphodiesterase-catalyzed hydrolysis in H(2)18O produced [17O,18O] dAMP. The configuration at phosphorus in this product was shown to be S by enzymatic phosphorylation to [17O,18O]dATP, adenylylcyclase (Bordetella pertussis)-catalyzed cyclization to 3',5'-cyclic [17O,18O]dAMP, and 31P NMR analysis of the ethyl esters. Since snake venom phosphodiesterase-catalyzed hydrolyses proceed with retention of configuration at phosphorus, (Sp)-[17O,18O]dAMP must have been produced from (Rp)-(2-glyceryl)-[17O]dAMP; and since the chemical degradation to the latter compound did not involve cleavage of any bonds to phosphorus, the initial enzymatic product must have been (Rp)-2"-(2'-deoxyadenosine 5'-[17O]phosphoryl)tobramycin. Therefore, nucleotidyl transfer catalyzed by gentamicin nucleotidyl-transferase proceeds with inversion of configuration at phosphorus, and the reaction mechanism involves an uneven number of phosphotransfer steps. Inasmuch as this is an uncomplicated two-substrate group transfer reaction, the mechanism probably involves direct nucleotidyl transfer from the nucleoside triphosphate to the aminoglycoside. The B. pertussis adenylylcyclase reaction was shown to proceed with inversion at phosphorus, as has been established for other adenylylcyclases.

Adenylyl Cyclases↗
PubMed1986

Mevalonate-5-diphosphate decarboxylase: stereochemical course of ATP-dependent phosphorylation of mevalonate 5-diphosphate.

Chicken liver mevalonate-5-diphosphate decarboxylase catalyzes the reaction of mevalonate 5-diphosphate (MVADP) with ATP to produce isopentenyl diphosphate, ADP, CO2, and inorganic phosphate. The overall reaction involves an anti elimination of the tertiary hydroxyl and carboxyl groups. To investigate the mechanism for transfer of the terminal phosphoryl group of ATP to the C-3 oxygen of MVADP, we have carried out the reaction using stereospecifically labeled (Sp)-adenosine 5'-O-(3-thio[3-17O2,18O]triphosphate) [( gamma-17O2,18O]ATP gamma S) in place of ATP. The configuration of the [17O,18O]thiophosphate produced was found to be Rp, corresponding to overall inversion of configuration at phosphorus in the thiophosphoryl group transfer step. This result is consistent with the direct transfer of the thiophosphoryl group from (Sp)-[gamma-17O2,18O]ATP gamma S to MVADP at the active site. Our result does not indicate the involvement of a covalent thiophosphoryl-enzyme on the reaction pathway.

Adenosine Triphosphate↗
PubMed1984

The hepatic glucagon receptor. Solubilization, characterization, and development of an affinity adsorption assay for the soluble receptor.

The hepatic glucagon receptor was covalently labeled with [125I-Try10]monoiodoglucagon [( 125I]MIG) by use of the heterobifunctional cross-linker hydroxysuccinimidyl p-azidobenzoate. Labeling of the Mr = 63,000 peptide was sensitive to glucagon and GTP at concentrations at which they affect [125I]MIG binding to the receptor. The labeled receptor was solubilized with Lubrol-PX, and the hydrodynamic characteristics of the receptor were determined. The molecular parameters of the solubilized receptor are: S20,w = 4.3 +/- 0.1, Stokes radius = 6.3 +/- 0.1 nm, frictional coefficient f/f0 = 1.8, and a calculated Mr = 119,000. Incubation of liver membranes at 32 degrees C for 15 min prior to the addition of [125I]MIG permitted us to identify the high molecular weight form (Mr = approximately 113,000) of the receptor by direct sodium dodecyl sulfate-gel electrophoretic analysis. The Mr = 63,000 peptide can be adsorbed to wheat germ lectin-Sepharose. The glycoprotein nature of the receptor has been utilized to develop an assay for the detergent-solubilized receptor that uses wheat germ lectin-Sepharose as a solid matrix to adsorb the [125I] MIG-receptor complex. The free hormone remains in the liquid phase and is removed in the supernatant after low speed centrifugation. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) solubilizes receptors with retention of [125I]MIG binding activity. [125I]MIG binding to the CHAPS-solubilized receptor is specifically affected by unlabeled glucagon. Interaction of [125I]MIG with the soluble receptor is insensitive to the presence of GTP. IC50 for glucagon using the soluble receptor was 33-70 nM, irrespective of the presence or absence of GTP, while when the membrane-bound receptor was used, the IC50 in the absence of GTP was 2-4 nM and in the presence of GTP was 35-80 nM. These data allow us to conclude that the hepatic glucagon receptor in the membrane and in the nondenaturing detergent solution is a dimer of the Mr = 63,000 hormone-binding subunit and a glycoprotein. The soluble receptor does not display any functional interaction with the stimulatory regulator.

Affinity Labels↗
PubMed1984

Glucagon-induced heterologous desensitization of the MDCK cell adenylyl cyclase. Increases in the apparent levels of the inhibitory regulator (Ni).

Treatment of MDCK cells with glucagon results in decreases in glucagon, NaF and prostaglandin E1-stimulated adenylyl cyclase activities, indicating the occurrence of a heterologous desensitization process. The extent of desensitization was time and glucagon concentration dependent. Maximal desensitization (30-50% decrease in stimulation by various effectors) was obtained by 4 h at 100 nM glucagon. Glucagon also induced homologous desensitization since after treatment, the Kact of glucagon was specifically increased. Treatment of cells with 10 microM 8-bromoadenosine 3':5'-monophosphate or 10 microM forskolin resulted in decreased hormonal (glucagon and prostaglandin E1) stimulation without any decrease in the stimulation by nonhormonal effectors (NaF, forskolin, and guanyl-5'-yl imidodiphosphate). The stimulatory regulator (Ns) of the adenylyl cyclase system was analyzed after desensitization with glucagon and no measurable changes in the apparent levels of the alpha s subunits of Ns or the activity of Ns as assessed by reconstitution of the cyc- S49 cell membrane adenylyl cyclase were detected. Levels of the alpha i subunit of the inhibitory regulator (Ni) were monitored by labeling with [32P]NAD and pertussis toxin. Membranes of glucagon-treated cells showed a 2-fold increase in the amount of alpha i labeled. Addition of pure Ns to glucagon-treated MDCK cell membranes restored full stimulation by NaF but did not restore stimulation by prostaglandin E1 or glucagon. It is concluded that glucagon induces heterologous and homologous desensitization of the MDCK cell adenylyl cyclase. The locus of the heterologous desensitization is at the level of the regulatory components. Decreased stimulation is thought to occur due to either an increase in the levels of Ni or due to altered interactions between the subunits of Ni.

Adenylate Cyclase Toxin↗
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

Structural analysis of the hepatic glucagon receptor. Identification of a guanine nucleotide-sensitive hormone-binding region.

[125I-Tyr10]Monoiodoglucagon [( 125I]MIG) was cross-linked to liver membrane glucagon receptors with hydroxysuccinimidyl-p-azidobenzoate, and the products were analyzed by sodium dodecyl sulfate-gel electrophoresis. Autoradiograms of the gel obtained after a 24-h exposure showed one major band at Mr = 63,000 that was sensitive to GTP and excess unlabeled glucagon. Exposure for 7 days showed labeling of an additional Mr = 33,000 species that was also sensitive to excess unlabeled glucagon. The Mr = 33,000 peptide can be obtained by subtilisin, trypsin, elastase, or Staphylococcus aureus V8 protease treatment of the [125I]MIG-occupied receptor in the membrane or in Lubrol-PX solution. In contrast, limited proteolysis of membranes containing vacant receptors results in labeling of a Mr = 24,000 peptide. The Mr = 24,000 peptide specifically binds [125I]MIG in a GTP-sensitive manner. The Mr = 33,000 peptide also retains GTP sensitivity since it releases bound [125I]MIG upon addition of GTP. Elastase treatment of the electroeluted Mr = 33,000 peptide yields the Mr = 24,000 and 15,000 fragments. The Mr = 15,000 peptide is the smallest fragment of the receptor as yet identified. Treatment of the Mr = 63,000 receptor with [125I]MIG cross-linked to it with endo-beta-N-acetylglucosaminidase F results in four distinct fragments with Mr values of 61,000, 56,000, 51,000, and 45,000; prolonged treatment resulted in the accumulation of the last two. Neither the Mr = 33,000 nor the Mr = 24,000 fragment appeared to be substrates for endo-beta-N-acetylglucosaminidase F. These data indicate that glucagon receptor is a glycoprotein of approximately 60,000 daltons which contains at least four N-linked glycans accounting for 18,000 daltons of its mass. Both its glucagon binding function and its capacity to interact with the stimulatory regulator of adenylyl cyclase are contained within a fragment of only approximately 21,000 daltons that does not contain any N-linked glycans. Hormone occupancy of the receptor results in a conformational change so as to expose a region that is susceptible to proteolysis by proteases of varying specificities to yield a peptide of approximately 30,000 daltons that also does not contain N-linked glycans.

Acetylglucosaminidase↗
PubMed1984

Characterization of the hepatic glucagon receptor.

The hepatic glucagon receptor was covalently labeled with [125I-Tyr10]-monoiodoglucagon by use of the heterobifunctional crosslinker hydroxysuccinimidyl-p-azidobenzoate and analyzed by SDS-gel electrophoresis. The autoradiogram of the gel showed one band at Mr = 63,000 that was sensitive to excess unlabeled glucagon and GTP. The labeled receptor was solubilized with Lubrol-PX and the hydrodynamic characteristics of the receptor were determined. The molecular parameters of the solubilized receptor are S20,w = 4.3 +/- 0.1, Stokes radius = 6.3 +/- 0.1 nm, frictional coefficient f/f degrees = 1.8 and a calculated Mr = 119,000. Incubation of liver membranes at 32 degrees for 15 min prior to the addition of [125I-Tyr10] permitted us to identify the high molecular weight form (Mr approximately equal to 113,000) by direct SDS-gel electrophoretic analysis. Limited elastase treatment of the hormone-occupied receptor results in the appearance of a Mr = 33,000 fragment, that retains guanine nucleotide sensitivity. Elastase treatment of vacant receptors results in a Mr = 24,000 fragment that binds hormone in a GTP-sensitive manner. The Mr = 24,000 fragment is contained within the Mr = 33,000 fragment. The Mr = 63,000 receptor upon treatment with endo-beta-N-acetylglucosamine F for 4 h yields four fragments of apparent Mr = 61,000, 56,000, 51,000, and 45,000; 24 h treatment results in the accumulation of the last two fragments. Neither Mr = 33,000 and 24,000 fragment appear to be substrates for endo-beta-N-acetylglucosaminidase F. These data allow us to conclude that the hepatic glucagon receptor in the membrane is a dimer of approximately 60,000 dalton hormone binding subunit which is a glycoprotein containing at least four N-linked glycans accounting for 18,000 daltons of its mass. Both the hormone binding function and the capacity for the interaction with the stimulatory regulator of adenylyl cyclase are contained within a fragment of only approximately 21,000 daltons that does not contain any N-linked glycans.

Animals↗
PubMed1983

Functional modification of the guanine nucleotide regulatory protein after desensitization of turkey erythrocytes by catecholamines.

Densensitization of turkey erythrocytes by exposure to the beta-adrenergic agonist (-)isoproterenol leads to decreased activation of adenylate cyclase by agonist, NaF, and guanyl-5'-yl imido diphosphate, with no reduction in the number of beta-adrenergic receptors. Interactions between the receptor and the guanine nucleotide regulatory protein (N protein) also seem to be impaired. These observations suggest that a component distal to the beta-adrenergic receptor may be a locus of modification. Accordingly we examined the N protein to determine whether it was altered by desensitization. The rate at which (-)isoproterenol stimulated the release of [3H]GDP from the N protein was substantially lower in membranes prepared from desensitized cells, providing further evidence for uncoupling of the receptor and the N protein. The amount of N protein in membranes from control and desensitized cells was compared by labeling the 42,000 Mr component of the N protein with [32P]NAD+ and cholera toxin; no significant difference was found. However, significantly more N protein (p less than .001) was solubilized by cholate extraction of desensitized membranes, suggesting an altered association of the N protein with the membrane after desensitization. The functional activity of the N protein was measured by reconstitution of cholate extracts of turkey erythrocyte membranes into S49 lymphoma cyc- membranes. Reconstitution of (-)isoproterenol stimulation of adenylate cyclase activity was reduced significantly (p less than .05) after desensitization. These observations suggest that desensitization of the turkey erythrocyte by (-)isoproterenol results in functional modifications of the guanine nucleotide regulatory protein, leading to impaired interactions with the beta-adrenergic receptor and reduced activation of adenylate cyclase.

Adenylyl Cyclases↗