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Biomedical subjects

M Vaughan

Publications and source records attributed to M Vaughan.

At least 181 records · Page 10Linked to original sources
PubMed1983

Regulation of activity of purified guanylate cyclase from liver that is unresponsive to nitric oxide.

Guanylate cyclase was purified from rat liver supernatant. Electrophoresis under denaturing conditions revealed one major peptide of Mr approx. 69 000. On the basis of the Stokes radius (4.7 nm) and S20,w (6.4S), the calculated Mr value of the native enzyme was 133 000, i.e. it is apparently a homodimer. Kinetics of inactivation by diamide (which was reversible with dithiothreitol) suggested that oxidation of a single class of thiol sites was involved. In the absence of other additions, cyclase activity assayed with Mn2+ was over 7 times that assayed with Mg2+; maximal effects were observed with approx. 5 mM of each (with 1 mM-GTP). The purified enzyme was markedly activated by nitrosylhaemoglobin. Relative activation was much greater in assays with Mg2+ than with Mn2+, although maximal activities were similar. When assayed with Mg2+, the enzyme exhibited a single Km (0.35 mM) for GTP; with Mn2+, plots of 1/v versus 1/[S] were non-linear. Activator or nitrosylhaemoglobin increased Vmax, but did not alter Km in the presence of either Mg2+ or Mn2+. The enzyme was inhibited by Na3VO4, Na2WO4 and Na2B4O7. Reduction from VV to VIV abolished the inhibitory effect of vanadate. Na2B4O7 (2 mM) inhibited activity with Mn2+, but not with Mg2+. In assays with Mg2+, but not with Mn2+, FMN, NAD+ and NADH (each 0.5 mM) inhibited activation by protoporphyrin IX and nitrosylhaemoglobin. Rotenone (0.6 mM) inhibited activity with protoporphyrin IX to a greater extent than with nitrosylhaemoglobin. Methylene Blue (1 mM) inhibited activation by nitrosylhaemoglobin, protoporphyrin IX and activator. It appears that this enzyme purified from rat liver lacks haem (and perhaps other components) required for activation by NO, and it should be particularly useful for elucidating the mechanism of action of NO, protoporphyrin IX and other activators.

Animals↗
PubMed1983

Selective regulation by pertussis toxin of insulin-induced activation of particulate cAMP phosphodiesterase activity in 3T3-L1 adipocytes.

Incubation of 3T3-L1 adipocytes with insulin or isoproterenol for 10 min increased particulate "low Km" cAMP phosphodiesterase activity by 42% and 50%, respectively. Pertussis toxin catalyzed the [32P]-ADP ribosylation of a 41,000 dalton protein in adipocyte particulate fractions; prior incubation of adipocytes with toxin markedly reduced incorporation of radiolabel. Exposure of adipocytes to pertussis toxin (0.3 microgram, 18 hr) increased glycerol production and inhibited activation of cAMP phosphodiesterase by insulin, but not by isoproterenol. These results suggest that pertussis toxin can interfere with receptor-mediated processes that stimulate cAMP hydrolysis as well as those that inhibit cAMP formation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

Purification and characterization of cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from calf liver. Effects of divalent cations on activity.

Cyclic GMP-stimulated cyclic nucleotide phosphodiesterase purified greater than 13,000-fold to apparent homogeneity from calf liver exhibited a single protein band (Mr approximately 102,000) on polyacrylamide gel electrophoresis under denaturing conditions. Enzyme activity comigrated with the single protein peak on analytical polyacrylamide gel electrophoresis, sucrose density gradient centrifugation, and gel filtration. From the sedimentation coefficient of 6.9 S and Stokes radius of 67 A, an Mr of 201,000 and frictional ratio (f/fo) of 1.7 were calculated, suggesting that the native enzyme is a nonspherical dimer of similar, if not identical, peptides. The effectiveness of Mg2+, Mn2+, and Co2+ in supporting catalytic activity depended on the concentration of cGMP and cAMP present as substrate or effector. Over a wide range of substrate concentrations, optimal concentrations for Mg2+, Mn2+, and Co2+ were about 10, 1, and 0.2 mM, respectively. At concentrations higher than optimal, Mg2+ inhibited activity somewhat; inhibition by Co2+ (and in some instances by Mn2+) was virtually complete. At low substrate concentrations, activity with optimal Mn2+ was equal to or greater than that with Co2+ and always greater than that with Mg2+. With greater than or equal to 0.5 microM cGMP or 20 to 300 microM cAMP and for cAMP-stimulated cGMP or cGMP-stimulated cAMP hydrolysis, activity with Mg2+ greater than Mn2+ greater than Co2+. In the presence of Mg2+, the purified enzyme hydrolyzed cGMP and cAMP with kinetics suggestive of positive cooperativity. Apparent Km values were 15 and 33 microM, and maximal velocities were 200 and 170 mumol/min/mg of protein, respectively. Substitution of Mn2+ for Mg2+ increased apparent Km and reduced Vmax for cGMP with little effect on Km or Vmax for cAMP. Co2+ increased Km and reduced Vmax for both. cGMP stimulated cAMP hydrolysis approximately 32-fold in the presence of Mg2+, much less with Mn2+ or Co2+. In the presence of Mg2+, Mn2+ and Co2+ at concentrations that increased activity when present singly inhibited cGMP-stimulated cAMP hydrolysis. It appears that divalent cations as well as cyclic nucleotides affect cooperative interactions of this enzyme. Whereas Co2+ effects were observed in the presence of either cyclic nucleotide, Mn2+ effects were especially prominent when cGMP was present (either as substrate or effector).

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1983

Ca2+-independent cyclic GMP phosphodiesterases from rat liver and HTC hepatoma cells.

We have separated and characterized a Ca2+- and calmodulin-insensitive cyclic nucleotide phosphodiesterase from rat liver supernatant as well as an analogous enzyme from HTC hepatoma cells. Chromatography of rat liver supernatant on DEAE-cellulose in the presence and subsequently in the absence of 0.1 mM-CaCl2 resulted in the separation of two distinct phosphodiesterase activities, both of which preferentially hydrolysed cyclic GMP rather than cyclic AMP. One enzyme, E-Ib, was activated in the presence of Ca2+ and calmodulin, and the other, E-Ia, was not. The E-Ia enzyme, which did not bind to calmodulin-Sepharose, had Mr 325 000 and displayed anomalous kinetic behaviour [Km (cyclic GMP) 1.2 microM; Km (cyclic AMP) 15.4 microM]. The E-Ib enzyme, which bound to calmodulin-Sepharose in the presence of Ca2+, had Mr 150 000 and exhibited Michaelis-Menten kinetics for hydrolysis of cyclic GMP [Km (basal) 6.5 microM; Km (activated) 12.0 microM]. E-Ia activity was diminished by incubation with alpha-chymotrypsin and was unaffected by the action of a rat kidney lysosomal proteinase. Partial hydrolysis of E-Ib enzyme by alpha-chymotrypsin or the kidney proteinase resulted in irreversible activation of the enzyme. The E-I enzyme isolated from HTC hepatoma cells was similar to the rat liver E-Ia enzyme in many respects. Its apparent Mr was 325 000. Its activity was unaffected by calmodulin in the presence of Ca2+ or by incubation with the kidney proteinase, and was decreased by digestion with alpha-chymotrypsin. Unlike the liver E-Ia enzyme, however, the hepatoma enzyme exhibited normal kinetic behaviour, with Km (cyclic GMP) 3.2 microM. Although HTC cells contain two other phosphodiesterases analogous to those in rat liver and a calmodulin-like activator of phosphodiesterase, no calmodulin-sensitive phosphodiesterase was detected.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1983

Hormone-sensitive particulate cAMP phosphodiesterase activity in 3T3-L1 adipocytes. Regulation of responsiveness by dexamethasone.

Confluent 3T3-L1 fibroblasts incubated for 72 h with methylisobutylxanthine, dexamethasone, and insulin differentiate and acquire phenotypic characteristics of mature adipocytes, including hormone-sensitive cAMP phosphodiesterase activity located in a particulate fraction of homogenates. About 10 days after initiating differentiation, a maximally effective concentration of insulin (100 pM) increased particulate cAMP phosphodiesterase activity 40 to 60% in 8 min; activation persisted for at least 30 min in the presence of insulin. Incubation of adipocytes for 6-8 min with agents that increased cAMP, e.g. 1 microM epinephrine, 0.1 microM isoproterenol, corticotropin (2 mu units/ml), or thyroid-stimulating hormone (15 ng/ml), also increased particulate phosphodiesterase activity 40-60%. Changes in phosphodiesterase activity produced by epinephrine tended to lag behind changes in cAMP. Insulin, epinephrine, and corticotropin increased Vmax, not Km (0.5 microM), for cAMP. Particulate phosphodiesterase activity, solubilized with detergent, eluted in a single peak from DEAE-Bio-Gel. Insulin and epinephrine increased the activity eluted in this peak. Neither insulin nor lipolytic hormones increased activity in soluble fractions from differentiated cells or particulate or soluble fractions from undifferentiated cells. Incubation of adipocytes for 48 h with 1 microM dexamethasone prevented insulin-induced activation of the particulate phosphodiesterase and did not alter basal activity. After incubation for 72 h with 0.1 microM dexamethasone, insulin and epinephrine activation were abolished. These effects of dexamethasone on hormonal regulation of particulate phosphodiesterase activity could account for some of the so-called permissive effects of glucocorticoids on cAMP-mediated processes as well as the "anti-insulin" effects of glucocorticoids.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Affinity chromatography of brain cyclic nucleotide phosphodiesterase using 3-(2-pyridyldithio)propionyl-substituted calmodulin linked to thiol-sepharose.

[3-(2-Pyridylthio)propionyl]calmodulin (PDP-CaM), an activated thiol derivative of calmodulin (CaM), was synthesized. Preparations of this derivative containing an average of 2.8 mol of substituent/mol of protein activated purified cyclic nucleotide phosphodiesterase in a manner indistinguishable from that of native CaM. PDP-CaM was covalently coupled to free thiol-Sepharose 4B through formation of a stable mixed disulfide bond for use in affinity chromatography. The binding capacity of the disulfide-linked CaM-Sepharose for phosphodiesterase activity was proportional to substituent level up to 4 mg of CaM/mL of gel; the total capacity of the gel for binding phosphodiesterase was 4 times that of CNBr-coupled CaM-Sepharose. Quantitative recovery was achieved by desorption of both ligand and bound proteins with a reducing agent. The thiolated CaM derivative was then separated from phosphodiesterase by rapid gel filtration; the overall recovery of phosphodiesterase activity was greater than 70%. Preparations of homogeneous enzyme in good yield were obtained after a second chromatography step on CaM-Sepharose. Binding and recovery of phosphodiesterase activity were entirely reproducible, since each preparation of affinity gel was used only once. As it permits separation of interacting species in free solution, this general method may be useful with other ligands for increasing yields from affinity chromatography, particularly when dissociation of molecules in their matrix-bound conformation may be difficult to achieve.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1983

Pertussis toxin inhibits enkephalin stimulation of GTPase of NG108-15 cells.

In neuroblastoma-glioma (NG108-15) hybrid cells, opiates inhibit adenylate cyclase and stimulate a low Km GTPase. It has been postulated that the stimulation of GTPase plays a role in opiate inhibition of adenylate cyclase (Koski, G., and Klee, W. A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4185-4189). Treatment of NG108-15 cells with pertussis toxin attenuates receptor-mediated inhibition of adenylate cyclase. The toxin acts by catalyzing the ADP-ribosylation of a 41,000-dalton substrate believed to be a part of the receptor-adenylate cyclase complex. We have found that toxin treatment of NG108-15 results in inhibition of the opiate-stimulated GTPase. The concentration of toxin required for inhibition of this GTPase was similar to that needed for both attenuation of opiate inhibition of adenylate cyclase and ADP ribosylation of the 41,000-dalton substrate. Inhibition of the opiate-induced GTPase by pertussis toxin in isolated membranes required NAD, consistent with the hypothesis that this effect of the toxin resulted from ADP ribosylation of a protein component of the system. Since the opiate-stimulated GTPase is believed to play a role in the receptor-mediated decrease in adenylate cyclase activity, inhibition of this GTPase may be an important part of the mechanism by which the toxin interferes with opiate action on adenylate cyclase.

Adenosine Diphosphate Ribose↗
PubMed1983

Release of guanyl nucleotides from the regulatory subunit of adenylate cyclase.

Choleragen and beta-adrenergic agonists, both of which activate turkey erythrocyte adenylate cyclase, have been reported to accelerate release of bound [3H]guanyl nucleotides from turkey erythrocyte membranes. We have now obtained evidence that choleragen- or isoproterenol-stimulated release reflects a change in the affinity of the regulatory subunit (G/F) of adenylate cyclase for guanyl nucleotides. Solubilized preparations of turkey erythrocytes that had bound radiolabeled GTP were chromatographed on Ultrogel AcA 34. The protein from which guanyl nucleotide was released upon incubation with choleragen or isoproterenol was co-eluted with G/F activity. Furthermore, this protein appears to be the same size as the complex containing the 42,000-dalton peptide, ADP*-ribosylated by choleragen, which is presumably a subunit of G/F. ADP ribosylation of the 42,000-dalton subunit of G/F by choleragen occurred with a half-time of about 5 min, whereas choleragen-stimulated release of guanyl nucleotides was much slower (t1/2 greater than or equal to 60 min). When membranes were treated with choleragen and NAD, the delay in activation of adenylate cyclase by guanylyl imidodiphosphate was decreased but not abolished, a finding consistent with the idea that release of endogenously bound nucleotide (and subsequent binding of the nonhydrolyzable GTP analog) occurs only slowly following ADP ribosylation. In contrast, activation of the adenylate cyclase of either toxin-treated or untreated membranes in the presence of isoproterenol and guanylyl imidodiphosphate was very rapid. These data support the hypothesis that isoproterenol and choleragen may activate adenylate cyclase, at least in part, by increasing the rate of release of guanyl nucleotides from G/F.

Adenylyl Cyclases↗
PubMed1983

Membrane and cytoplasmic changes in 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU)-sensitive and resistant human malignant glioma-derived cell lines.

Human glioma-derived cell lines previously determined by a microtiter chemotherapy assay to be either 'sensitive' or 'resistant' to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) were treated with BCNU (1-80 micrograms/ml) and observed using microcinematography, scanning electron microscopy, and transmission electron microscopy. Striking bleb formation and cell retraction were observed to occur in a dose-dependent relationship within minutes in the cells known to be BCNU-sensitive. At 15 micrograms/ml, 69% of cells showed blebs by 30 min, 87% by 90 min, and 100% by 4 hr. This activity was not seen in BCNU-resistant cells. These morphological changes occur at a time too early to be accounted for by the known BCNU mechanism of DNA alkylation and cross-link formation and suggest that cytoplasmic and/or membrane events may be significant initial events in the cytotoxic actions of BCNU.

Adult↗
PubMed1983

Bradykinin stimulates phospholipid methylation, calcium influx, prostaglandin formation, and cAMP accumulation in human fibroblasts.

The biochemical events that lead to bradykinin stimulation of cAMP accumulation in human fibroblasts were examined. Treatment of human fibroblasts with bradykinin increases phospholipid methylation, Ca2+ influx, arachidonic acid release, prostaglandin formation, and cAMP content. The dose-response curves of bradykinin for the increase in the above changes were similar. In human fibroblasts, exogenous arachidonic acid was mainly incorporated into phosphatidylcholine, followed by phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol. Bradykinin caused a release of arachidonic acid from methylated phospholipids (phosphatidylcholine) and phosphatidylinositol. 3-Deazaadenosine, a methyltransferase inhibitor, almost completely inhibited bradykinin-stimulated phospholipid methylation and Ca2+ influx and partially reduced arachidonic acid release and prostaglandin formation but had no effect on cAMP formation. Mepacrine, a phospholipase inhibitor, blocked bradykinin-induced arachidonic acid release, prostaglandin release, and cAMP accumulation. Indomethacin, a cyclooxygenase inhibitor, blocked the effect of bradykinin on cAMP accumulation. Prostaglandins E1 and E2, but not F2 alpha, increased accumulation of cAMP. These observations indicate that bradykinin generates cAMP via arachidonic acid release and subsequent formation of prostaglandins. Our findings suggest that arachidonic acid can arise from either phosphatidylcholine synthesized by the methylation pathway or phosphatidylinositol.

Arachidonic Acid↗
PubMed1983

Alterations in cyclic AMP phosphodiesterase activities during differentiation of 3T3-L1 cells.

3T3-L1 cells contain multiple forms of cyclic nucleotide phosphodiesterase in both supernatant (100,000 X g, 40 min) and particulate fractions. Supernatant fractions from both undifferentiated and differentiated cells contained calmodulin-sensitive activity. In undifferentiated 3T3-L1 cells, only a small fraction of the total cAMP phosphodiesterase activity was found in the particulate fraction and the specific activity of the particulate was lower than the supernatant. With differentiation the specific activity of the particulate doubled, and there was a dramatic increase in total activity in this fraction, while in the supernatant total cAMP phosphodiesterase activity increased less and specific activity decreased. The particulate fraction accounted for approximately 70% of the total cAMP phosphodiesterase activity in differentiated cells in contrast to about one-third in undifferentiated cells. In addition, there was a qualitative change in particulate phosphodiesterase activity. In fractions from 3T3-L1 adipocytes, with either cAMP or cGMP as substrate, Lineweaver-Burk plots were nonlinear, with low Km components of less than 1 microM, and cGMP inhibited cAMP hydrolysis. In particulate fractions from undifferentiated cells, cGMP did not inhibit and often enhanced hydrolysis of cAMP. With differentiation, there was also a marked increase in particulate cGMP phosphodiesterase activity. cAMP and cGMP phosphodiesterase activities solubilized from particulate fraction of differentiated cells coeluted from DEAE-Biogel and exhibited kinetic properties similar to the crude particulate fractions. During differentiation, there seems to be an alteration in the distribution of phosphodiesterase activity as well as the appearance of a particulate phosphodiesterase with kinetic properties similar to a particulate phosphodiesterase found in mature rat adipocytes.

1-Methyl-3-isobutylxanthine↗
PubMed1982

Resolution and activity of adenylate cyclase components in a zwitterionic cholate derivative [3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate].

Bovine brain adenylate cyclase was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), sodium cholate, sodium deoxycholate, or these detergents plus (NH4)2SO4. The specific activity of the extract obtained with 13 mM CHAPS alone was several times those of the other detergent extracts with or without (NH4)2SO4. After solubilization with 13 mM CHAPS, gel filtration completely separated the catalytic unit (C) from the guanine nucleotide binding protein (G/F). C activity when assayed with 5 mM Mn2+ was 5 times that assayed with 10 mM Mg2+ and was unresponsive to GPP(NH)P. C activity was increased approximately 150% by GPP(NH)P in the presence of G/F extracted from human erythrocyte ghosts and approximately 100% by Ca2+ plus calmodulin in assays with Mg2+. On gel filtration and/or density gradient centrifugation, the physical properties of C from brain or AC- cells and G/F from bovine or pig erythrocytes in CHAPs were similar to those observed in other detergents. It appears that the use of CHAPS for solubilization of adenylate cyclase and separation of C and G/F may well prove advantageous in studies of the molecular interactions between the protein subunits and activators of the enzyme as well as for the initial purification of C.

Adenylyl Cyclases↗
PubMed1982

Choleragen-stimulated release of guanyl nucleotides from turkey erythrocyte membranes.

Choleragen stimulates adenylate cyclase by ADP ribosylating a guanyl nucleotide-binding regulatory protein (G/F). beta-Adrenergic hormones also activate the adenylate cyclase of turkey erythrocytes, and it is currently believed that they do so in part by decreasing the affinity of G/F factor for GDP, an effect which is manifested by a hormone-stimulated release of guanyl nucleotides from the membranes. Since choleragen might also activate adenylate cyclase by a similar mechanism, the effect of toxin treatment on the release of guanyl nucleotides from turkey erythrocyte membranes was examined. In the presence of NAD, choleragen was found to stimulate release of guanyl nucleotides from membranes which had been preloaded with radiolabeled GTP. No stimulation of release was observed with cAMP or when NAD was replaced by NADP, which does not serve as a substrate for choleragen-catalyzed ADP ribosylation. While either isoproterenol or choleragen can stimulate release of guanyl nucleotides from the membranes, the amount of guanyl nucleotide released in the presence of both isoproterenol and choleragen was no greater than that released by isoproterenol alone. Furthermore, when membranes were first treated with choleragen and NAD, the subsequent release of guanyl nucleotides induced by isoproterenol was reduced to approximately 15% of that observed with membranes not treated with the toxin. Therefore, choleragen may enhance release of guanine nucleotides from sites on the membranes that are also affected by beta-adrenergic agonists, sites which are thought to correspond to G/F. These data are consistent with the hypothesis that choleragen may stimulate adenylate cyclase, in part, by enhancing release of guanyl nucleotides, a mechanism similar to that of beta-adrenergic agonists.

Animals↗
PubMed1982

Cholera and cell regulation.

The cholera vibrio, unlike most pathogens, neither invades the body's cells nor causes them direct physical damage and thus does not engender inflammation or fever. Rather, the organism attaches itself to the intestinal epithelium, where it secretes a toxin that deranges cellular function. Study of the toxin's structure and mechanism of action is helping to elucidate normal enzymatic processes.

Adenylyl Cyclases↗