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Biomedical subjects

F Murad

Publications and source records attributed to F Murad.

At least 199 records · Page 11Linked to original sources
PubMed1981

Production and characterization of monoclonal antibodies to soluble rat lung guanylate cyclase.

Four monoclonal antibodies to rat lung soluble guanylate cyclase [GTP pyrophosphate-lyase (cyclizing) EC 4.6.1.2] have been produced by fusing spleen cells from immunized BALB/c mice with SP-2/0 myeloma cells. The antibodies were detected by their ability to bind immobilized guanylate cyclase and by immunoprecipitation of purified enzyme in the presence of second (rabbit anti-mouse) antibody. After subcloning by limiting dilution, hybridomas were injected intraperitoneally into mice to produce ascitic fluid containing 2-5 mg of antibody per ml. The four antibodies obtained had titers of between 1:1580 and 1:3160 but were detectable at dilutions greater than 1:20,000. Soluble guanylate cyclase from several rat tissues were crossreactive with the four monoclonal antibodies, suggesting that the soluble enzyme from different rat tissues is antigenically similar. The antibodies also recognized soluble lung enzyme from rat, beef, and pig, while enzyme from rabbit was not crossreactive and mouse enzyme was recognized by only one of the antibodies. Particulate guanylate cyclase from a number of tissues had only minimal crossreactivity with the antibodies. Immunoprecipitated guanylate cyclase retained catalytic activity, could be activated with sodium nitroprusside, and was inhibited by cystamine. None of the antibodies were inhibitory under the conditions examined. These antibodies will be useful probes for the study of guanylate cyclase regulation and function under a variety of physiological conditions.

Animals↗
PubMed1981

Guanylate cyclase: purification, properties, free radical activation, radiolabeling, and preparation of hybridoma antibodies.

In summary, we have succeeded in purifying soluble guanylate cyclase from several rat tissues. These methods are being used to purify the enzyme from other tissues and other species. We have also been able to purify partially the particulate form of guanylate cyclase 100- to 200-fold. We expect that our experience with the soluble enzyme and with the antibodies that we are developing should enable us to purify the particulate form of the enzyme in the near future. We can activate the purified enzyme with nitric oxide and other agents such as hydroxyl radical and unsaturated fatty acids (5). Both the activation and the reversal to the basal state can occur without the presence of other macromolecules, suggesting that these activators interact directly and reversibly with the protein. Both basal activity and the capacity to activate with nitric oxide require free sulfhydryl groups on the enzyme. The pure enzyme can be radiolabeled with either [35S] or 125I, and labeled enzyme has assisted us in various ways including purification, examination of the enzyme's subunit structure, and screening sera and media from hybridoma cultures for antibodies to the protein. Thus, as we anticipated several years ago, the availability of purified guanylate cyclase has allowed us to develop a number of new techniques and approaches to examine the regulation and function of guanylate cyclase and cyclic GMP. We expect that there will be much new information and exciting progress in the field forthcoming as a result of these and other new methods.

Animals↗
PubMed1980

Activation of intestinal guanylate cyclase by heat-stable enterotoxin of Escherichia coli: studies of tissue specificity, potential receptors, and intermediates.

Heat-stable enterotoxin (ST) of Escherichia coli increased guanylate cyclase activity in homogenates of rat and rabbit intestinal mucosa and stimulated intestinal fluid secretion in suckling mice. The ST effect on guanylate cyclase was dose-dependent, occurred without a time lag, and was confined to the particulate fraction. ST activation of guanylate cyclase was tissue-specific; ST did not alter activity of soluble or particulate rat liver, lung, heart, kidney, or cerebral cortex enzyme. The ST activity on guanylate cyclase and secretion was methanol-soluble and alkali-labile, and its effects were not altered by phentolamine, propranolol, or atropine. Monosialoganglioside did not reduce ST-induced secretion. However, indomethacin and butylated hydroxyanisole decreased the ST effect on both guanylate cyclase and secretion. Fluid secretion with ST sppears to result from specific activation of particulate intestinal guanylate cyclase. While adrenergic and cholinergic events are probably not involved in this process, the effects of ST may be mediated through prostaglandin synthesis or oxidative mechnanisms.

Alkalies↗
PubMed1980

Inhibition of Escherichia coli heat-stable enterotoxin by indomethacin and chlorpromazine.

Purified heat-stable enterotoxin (ST) from a procine strain of enterotoxigenic Escherichia coli activates quanylate cyclase in particulate fractions of rat intestinal tissue and induces fluid accumulation in suckling mice. These effects of ST were examined in the presence of either indomethacin or chlorpromazine. We also examined the effects of these two drugs on fluid accumulation in suckling mice induced by the 8-bromo analog of cyclic guanosine monophosphate. Either indomethacin or chlorpromazine reduced ST activation of guanylate cyclase. Both drugs also reduced intestinal fluid accumulation in suckling mice that resulted from submaximal doses of ST (both P < 0.001). However, there was no reduction in fluid secretion by either drug when a maximally effective dose of ST was used, suggesting that inhibition of fluid secretion by both drugs can be overcome by increasing the ST dose and that a threshold level of guanylate cyclase activity results in maximal secretory response. Both drugs also reduced basal guanylate cylase activity in rat intestinal tissue and fluid secreton in suckling mice. Chlorpromazine also reduced intestinal secretion mediated by 8-bromo cyclic guanosine monophosphate (P < 0.001). These findings indicate that chlorpromazine interferes with the effects of ST both before and after its activation of guanylate cyclase, whereas indomethacin interfers with ST only before its activation of guanylate cyclase.

Animals↗
PubMed1980

Purified guanylate cyclase: characterization, iodination and preparation of monoclonal antibodies.

Guanylate cyclase was purified from the soluble fraction of rat lung using a modification of procedures published previously. The purified enzyme exhibited specific activities, at pH 7.6, of 219-438 nmoles/mg protein/min and 34-60 nmoles/mg protein/min with Mn2+ and Mg2+ as cation cofactors, respectively. The specific activity changed as a function of the protein concentration due to a change in Vmax with no alteration of the Km for GTP. The enzyme migrated as a single band coincident wih guanylate cyclase activity on nondenaturing polyacrylamide and isoelectric focusing gels (isoelectric point = 5.9). Purified guanylate cyclase had an apparent molecular weight of 150,000 daltons as determined by gel filtration chromatography and polyacrylamide gel electrophoresis. Electrophoresis in the presence of sodium dodecyl sulfate revealed a single subunit of 72,000 daltons, suggesting that the enzyme is a dimer of an identical subunit. The purified enzyme could be activated by nitric oxide, indicating that this compound interacts directly with the enzyme.

Animals↗
PubMed1980

Automated "Gamma-flo" radioimmunoassay of urinary cyclic AMP.

The "Gammaflow" automated assay concept of Brooker et al. (Science 194:270, 1976) has been adapted to the assay for urinary cyclic AMP in patients' samples on the now commercially available Squibb Gamma-FloTM system, a totally automated continuous-flow immunoassay instrument. The instrument aspirates the unknown sample, combines it with radioligand and specific antiserum, incubates the mixture, separates antibody-bound radioligand from free radioligand, counts the radioactivity of the bound ligand, computes the standard curve, and determines subsequent unknown samples by comparison to the standard curve. The urinary cyclic AMP assay we report here is rapid, accurate, and concentrations of cyclic AMP as low as 15 nmol/L can be detected. The assays, which can be done at 60 determinations per hour, are accurate at all dilutions of urine tested and inter-assay (day-to-day) CVs were 3.9% at the midpoint of the standard curve (B/B0 = 0.49).

Autoanalysis↗
PubMed1979

Effects of thiols, sugars, and proteins on nitric oxide activation of guanylate cyclase.

Purification of soluble guanylate cyclase from rat liver resulted in an apparent loss of enzyme activation by nitric oxide that could be restored by dithiothreitol. methemoglobin, bovine serum albumin, or sucrose. Although hemoglobin also permitted some activation with nitric oxide, the effect of other agents to restore enzyme activation was prevented with hemoglobin. As a result of enzyme purification, there is an alteration of the dose-response relationship for nitric oxide activation. After partial enzyme purification, relatively high concentrations of nitric oxide that were stimulatory in crude enzyme preparations had no effect on enzyme activity. However, partially purified or homogeneous enzyme was activated by lower concentrations of nitric oxide. The bell-shaped dose-response curve for nitric oxide was shifted to the left with guanylate cyclase purification. The addition of dithiothreitol, methemoglobin, bovine serum albumin, or sucrose to enzyme markedly broadens the dose-response curve for nitric oxide. Thus, the apparent loss of responsiveness to nitric oxide with purification is a function of increased sensitivity of guanylate cyclase to nitric oxide. Increased sensitivity to nitric oxide with enzyme purification probably results from the removal of heme, proteins, and small molecules that can serve as scavengers or sinks for nitric oxide and prevent excessive oxidation of the enzyme.

Animals↗
PubMed1979

Synthesis of adenosine 3',5'-monophosphate by guanylate cyclase, a new pathway for its formation.

The 105 000 X g gupernatant fractions from homogenates of various rat tissues catalyzed the formation of both cyclic GMP and cyclic AMP from GTP and ATP, respectively. Generally cyclic AMP formation with crude or purified preparations of soluble guanylate cyclase was only observed when enzyme activity was increased with sodium azide, sodium nitroprusside, N-methyl-N'-nitro-N-nitrosoguanidine, sodium nitrite, nitric oxide gas, hydroxyl radical and sodium arachidonate. Sodium fluoride did not alter the formation of either cyclic nucleotide. After chromatography of supernatant preparations on Sephadex G-200 columns or polyacrylamide gel electrophoresis, the formation of cyclic AMP and cyclic GMP was catalyzed by similar fractions. These studies indicate that the properties of guanylate cyclase are altered with activation. Since the synthesis of cyclic AMP and cyclic GMP reported in this study appears to be catalyzed by the same protein, one of the properties of activated guanylate cyclase is its ability to catalyze the formation of cyclic AMP from ATP. The properties of this newly described pathway for cyclic AMP formation are quite different from those previously described for adenylate cyclase preparations. The physiological significance of this pathway for cyclic AMP formation is not known. However, these studies suggest that the effects of some agents and processes to increase cyclic AMP accumulation in tissue could result from the activation of either adenylate cyclase or guanylate cyclase.

Animals↗
PubMed1979

Separation of soluble adenylate and guanylate cyclases from the mature rat testis.

The mature rat testis contains both a soluble guanylate cyclase and a soluble adenylate cyclase. Both these soluble enzymes prefer manganous ion for activity. It is known that guanylate cyclase can, when activated by a variety of agents, catalyze the formation of cyclic AMP. The following experiments were performed to determine whether the testicular soluble adenylate and guanylate cyclase activities were carried on the same molecule. Analysis of supernatants from homogenized rat testis by gel filtration and sucrose density gradient centrifugation showed that the two activities were clearly separable. The molecular weight of guanylate cyclase is 143 000, while that of adenylate cyclase is 58 000. Treatment of the column fractions with 0.1 mM sodium nitroprusside allowed guanylate cyclase activity to be expressed with Mg(2+) as well as with Mn(2+). Sodium nitroprusside did not affect the metal ion or substrate specificity of adenylate cyclase. These experiments show that adenylate and guanylate cyclase activities are physically separable.

Adenylyl Cyclases↗
PubMed1979

Hormonal control of glycogen metabolism.

In summary, we have presented evidence which relates to the action pathway of hormonal control of glycogen metabolism. In the case of insulin, there are changes demonstrable in the cyclic AMP-dependent protein kinase and also in the phosphoprotein phosphatase, under conditions where no direct relationship to either cyclic AMP or cyclic GMP levels are measurable. Therefore, a new unknown intermediate or second messenger system is again proposed. An insulin-generated labile compound(s) which inhibits the protein kinase has been discovered. This may function as an intermediate. Finally, the fact that the glycogen synthase system clearly differs from phosphorylase in its regulation by covalent phosphorylation is discussed. Synthase is now accepted as a multiply phosphorylated subunit, in contrast to phosphorylase which is singly phosphorylated. The inherent theoretical advantages of multiple phosphorylation over single phosphorylation are considered. The advantages of a multistate over a two-state model of enzyme interconversion are mentioned. The importance of the multiple phosphorylations interacting in a nonlinear manner with the control by cellular metabolites is in the explanation of how a small change in covalent phosphorylation signalled by a hormone can be translated in the cell milieu into a much larger change in rate.

Animals↗
PubMed1979

Purification of soluble guanylate cyclase from rat liver.

Soluble guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] has been purified from rat liver and exhibited a single protein band on polyacrylamide gels coincident with activity and indicative of a molecular weight of 150,000. The apparent specific activity of the purified enzyme was 276 nmol of cyclic GMP formed per mg per min with Mn(2+) as the cation cofactor and 23.8 nmol of cyclic GMP formed per mg per min with Mg(2+). This represented 9200-fold and 7400-fold purifications of Mn(2+) and Mg(2+) activities, respectively. The specific activity of soluble guanylate cyclase was not constant with protein concentration. At all stages of purification, increasing the enzyme concentration in the guanylate cyclase assay increased the apparent specific activity of the preparation. The purified enzyme could be activated by nitroprusside, nitric oxide, arachidonate, linoleate, oleate, and superoxide dismutase. However, the degree of activation was dependent upon the concentration of enzyme protein assayed.

Journal Article↗