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1.
Characterization of an ATP-Mg2+-dependent guanine nucleotide-stimulated adenylate cyclase from Neurospora crassa 总被引:2,自引:0,他引:2
A novel adenylate cyclase activity was found in crude homogenates of Neurospora crassa. The adenylate cyclase had substantial activity with ATP-Mg2+ as substrate differing significantly from the strictly ATP-Mn2+-dependent enzyme characterized previously. Additionally, the ATP-Mg2+-dependent activity was stimulated two- to fourfold by GTP or guanyl-5'-yl-imido-diphosphate (Gpp(NH)p). We propose that the ATP-Mg2+-dependent, guanine nucleotide-stimulated activity is due to a labile regulatory component (G component) of the adenylate cyclase which was present in carefully prepared extracts. The adenylate cyclase had a pH optimum of 5.8 and both the catalytic and G component were particulate. The Km for ATP-Mg2+ was 2.2 mM in the presence of 4.5 mM excess Mg2+. Low Mn2+ concentrations had no effect on adenylate cyclase activity whereas high concentrations of Mn2+ or Mg2+ stimulated the enzyme. Maximal Gpp(NH)p stimulation required preincubation of the enzyme in the presence of the guanine nucleotide and the K1/2 for Gpp(NH)p stimulation was 110 nM. Neither fluoride nor any of a variety of glycolytic intermediates or hormones, including glucagon, epinephrine, and dopamine, had an effect on ATP-Mg2+-dependent adenylate cyclase activity. However, the enzymatic activity was stimulated not only by GTP but also by 5'-AMP and was inhibited by NADH. 相似文献
2.
An assessment was made of some of the basic parameters responsible for the modulation of adenylate cyclase activity in a bovine adrenocortical plasma-membrane preparation. When determined at 0.1 mM-ATP, basal adenylate cyclase activity increased with increasing MgCl2 concentrations, whereas in the presence of corticotropin activity was essentially maximal at 10mM-MgCl2; high concentrations (25mM) of MgCl2 inhibited adenylate cyclase activity determined in the presence of both corticotropin and GTP. At all MgCl2 concentrations, corticotropin and GTP activated the enzyme in a synergistic fashion. The magnitude of the stimulation of basal activity produced by corticotropin was a function of Mg2+ concentration, whereas that produced by GTP appeared largely independent of Mg2+ concentration. Adenylate cyclase activity in the bovine adrenal membrane was half-maximally stimulated by corticotropin concentrations in the range 0.3--1.0 nM. The concentration of corticotropin evoking half-maximum response was not significantly affected by raising the free Mg2+ concentration from 0.4 to 4.9 mM, nor by the presence of GTP. In the presence of GTP, high concentrations (over 1 micrometer) of corticotropin inhibited adenylate cyclase activity, although no inhibition was apparent in the absence of guanine nucleotide. 相似文献
3.
1. The activities of the enzymes involved in the metabolism of cyclic nucleotides were studied in sarcolemma prepared front guinea-pig heart ventricle; the enzyme activities reported here were linear under the assay conditions. 2. Adenylate cyclase was maximally activated by 3mM-NaF; NaF increased the Km for ATP (from 0.042 to 0.19 mM) but decreased the Ka for Mg2+ (from 2.33 to 0.9 mM). In the presence of saturating Mg2+ (15 mM), Mn2+ enhanced adenylate cyclase, whereas Co2+ was inhibitory. beta-Adrenergic amines (10-50 muM) stimulated adenylate cyclase (38+/-2%). When added to the assay mixture, guanyl nucleotides (GTP and its analogue, guanylyl imidophosphate) stimulated basal enzyme activity and enhanced the stimulation by isoproterenol. By contrast, preincubation of sarcolemma with guanylyl imidodiphosphate stimulated the formation of an 'activated' form of the enzyme, which did not reveal increased hormonal sensitivity. 3. The guanylate cyclase present in the membranes as well as in the Triton X-100-solubilized extract of membranes exhibited a Ka for Mn 2+ of 0.3 mM; Mn2+ in excess of GTP was required for maximal activity. Solubilized guanylate cyclase was activated by Mg2+ only in the presence of low Mn2+ concentrations; Ca2+ was inhibitory both in the absence and presence of low Mn2+. Acetylcholine as well as carbamolycholine stimulated membrane-bound guanylate cyclase. 4. Cylic nucleotide phosphodiesterase activities of sarcolemma exhibited both high-and low-Km forms with cyclic AMP and with cyclic GMP as substrate. Ca2+ ions increased the Vmax. of the cyclic GMP-dependent enzyme. 相似文献
4.
T Braun 《Journal of cyclic nucleotide research》1975,1(6):271-281
The effect of divalent cations on bovine sperm adenylate cyclase activity was studied. Mn2+, Co2+, Cd2+, Zn2+, Mg2+ and Ca2+ were found to satisfy the divalent cation requirement for catalysis of the bovine sperm adenylate cyclase. These divalent cations in excess of the amount necessary for the formation of the metal-ATP substrate complex were found to stimulate the enzyme activity to various degrees. The magnitude of stimulation at saturating concentrations of the divalent cations was strikingly greater with M2+ than with either Ca2+, Mg2+, Zn2+, Cd2+ or Co2+. The apparent Km was lowest for Zm2+ (0.1 - 0.2 mM) than for any of the other divalent cations tested (1.2 - 2.3 mM). The enzyme stimulation by Mn2+ was decreased by the simultaneous addition of Co2+, Cd2+, Ni2+ and particularly Zn2+ and Cu2+. The antagonism between Mn2+ and Cu2+ or Zn2+ appeared to have both competitive and non-competitive features. The inhibitory effect of Cu2+ on Mn2+-stimulated adenylate cyclase activity was prevented by 2,3-dimercaptopropanol, but not by dithiothreitol, L-ergothioneine, EDTA, EGTA or D-penicillamine. Ca2+ at concentrations of 1-5 mM was found to act synergistically with Mg2+, Zn2+, Co2+ and Mn2+ in stimulating sperm adenylate cyclase activity. The Ca2+ augmentation of the stimulatory effect of Zn2+, Co2+, Mg2+ and Mn2+ appeared to be specific. 相似文献
5.
I Morishima 《Journal of biochemistry》1978,84(6):1495-1500
Adenylate cyclase was assayed in a sonicated preparation of silkworm pupal fat body. The adenylate cyclase was found mostly in the particulate fraction. The activity depended upon either Mg2+ or Mn2+, and the degree of stimulation by Mn2+ was 2 times greater than that by Mg2+ compared at the saturating concentrations. In the presence of Mg2+, the enzyme was inhibited by both EGTA and high concentrations of Ca2+, showing biphasical response to Ca2+. The enzyme was stimulated several-fold by NaF. The enzyme exhibited typical Michaelis-Menten kinetics and Km values were 0.13 mM for MgATP and 0.086 mM for MnATP. 相似文献
6.
A C Howlett 《Journal of cyclic nucleotide research》1982,8(2):91-102
Na+ has been implicated as a requirement for the inhibition of adenylate cyclase by hormones and neurotransmitters. This study examines effects of salt concentration on neuroblastoma plasma membranes that occur in the absence of an inhibitory hormone. The adenylate cyclase response to stimulatory agonists (GTP plus PGE1 (3), PGI2 or PGE2) was influenced by NaCl. As the [NaCl] increased to 150 mM, an increase in maximal activity and a decrease in apparent affinity was observed. At concentrations above 150 mM, NaCl decreased prostaglandin affinity and progressively decreased maximal activation. The GTP requirement was not altered by 30 or 150 mM NaCl in the presence of PGE1 or PGI2. The rate of Gpp(NH)p stimulated activity increased as the [NaCl] was increased in the assay. This increased rate was conserved when membranes activated in the presence of Gpp(NH)p and NaCl were reassayed in the absence of guanine nucleotide or salt. The salt evoked rate increase was proportionally greater at submaximal MgCl2 concentrations. The concentration requirement for Mg2+ was reduced by salt for adenylate cyclase in the presence of GTP or Gpp(NH)p. However, the enzyme stimulated by hormone exhibited a Mg2+ requirement that was low in the absence of salt and could not be further reduced by increased [NaCl]. Alternative monovalent cations (150 mM Li+, K+, Cs+, but not choline or tetramethylammonium) and anions (SO4=) substituted for NaCl. The observed effects were reversible upon washing the membranes and neither ouabain nor tetrodotoxin altered the response. These effects may result from a conformational alteration of a protein particularly sensitive to neutral salts in the assay. 相似文献
7.
Effects of guanine nucleotides on the adenylate cyclase activity of thyroid plasma membranes were investigated by monitoring metabolism of the radiolabeled nucleotides by thin-layer chromatography (TLC). When ATP was used as substrate with a nucleotide-regenerating system, TSH stimulated the adenylate cyclase activity in the absence of exogenous guanine nucleotide. Addition of GTP or GDP equally enhanced the TSH stimulation. Effects of GTP and GDP were indistinguishable in regard to their inhibitory effects on NaF-stimulated activities. The results from TLC suggested that GDP could be converted to GTP by a nucleotide-regenerating system. Even in the absence of a nucleotide-regeneration system, addition of GDP to the adenylate cyclase assay mixture resulted in the parallel decrease in ATP levels and formation of GTP indicating that thyroid plasma membrane preparations possessed a transphosphorylating activity. When an ATP analog, App[NH]p, was used as substrate without a nucleotide-regenerating system, no conversion of GDP to GTP was observed. Under such conditions, TSH did not stimulate the adenylate cyclase activity unless exogenous GTP or Gpp[NH]p was added. GDP no longer supported TSH stimulation and caused a slight decrease in the activity. GDP was less inhibitory than Gpp(NH)p to the NaF-stimulated adenylate cyclase activity. These results suggest: (1) TSH stimulation of thyroid adenylate cyclase is absolutely dependent on the regulatory nucleotides. (2) In contrast to GTP, GDP cannot support the coupling of the receptor-TSH complex to the catalytic component of adenylate cyclase. (3) The nucleotide regulatory site is more inhibitory to the stimulation of the enzyme by NaF when occupied by Gpp[NH]p than GDP. 相似文献
8.
Yasuo Totsuka Thor B. Nielsen James B. Field 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,718(2):135-143
Effects of guanine nucleotides on the adenylate cyclase activity of thyroid plasma membranes were investigated by monitoring metabolism of the radiolabeled nucleotides by thin-layer chromatography (TLC). When ATP was used as substrate with a nucleotide-regeneratign system, TSH stimulated the adenylate cyclase activity in the absence of exogenous guanine nucleotide. Addition of GTP and GDP equally enhanced the TSH stimulation. Effects of GTP and GDP were indistinguishable in regard to their inhibitory effects on NaF-stimulated activities. The results from TLC suggested that GDP could be converted to GTP by a nucleotide-regenerating system. Even in the absence of nucleotide-regenerating system, addition of GDP to the adenylate cyclase assay mixture int he parallel decrease in ATP levels and formation of GTP indicating that thyroid plasma membrane preparatiosn possessed a transphosphorylating activity. When an ATP analog, App[NH]p, was used as substrate without a nucleotide-regenerating system, no conversion of GDP to GTP was observed. Under such conditions, TSH did not stimulate the adenylate cyclase activity unless exogenous GTP or Gpp[NH]p was added. GDP no longer supported TSH stimulation and caused a slight decrease in the activity. GDP was less inhibitory than Gpp(NH)p to the NaF-stimulated adenylate cyclase activity. These results suggest: (1) TSH stimulation of thyroid adenylate cyclase is absolutely dependent on the regulatory nucleotides. (2) In contrst to GTP, GDP cannot support the coupling of the receptor-TSH complex to the catalytic componenet of adenylate cyclase. (3) The nucleotide regulatory site is more inhibitory to the stimulation of the enzyme by NaF when occupied by Gpp[NH]p than GDP. 相似文献
9.
A M Tolkovsky 《The Journal of biological chemistry》1986,261(10):4579-4586
A new method was developed to follow the rate of activation of adenylate cyclase in rat brain membranes by rapid freezing and N-ethylmaleimide treatment at 0 degrees C. This method was used to investigate the relationship between the rate of activation of adenylate cyclase by p(NH)ppG and GTP gamma S and their apparent affinities. These studies established the following. 1) The kinetics of activation by p(NH)ppG and GTP gamma S were indistinguishable although the apparent affinity of p(NH)ppG was 20-fold lower than the affinity of GTP gamma S. Activation was first order, kobs varying approximately 1.5-fold (average t 1/2 = 3.5 min, 30 degrees C) between 20-90% occupancy by either guanine nucleotide. 2) Final levels of activity were strictly dependent on the concentration of the nucleotides in a saturable manner. 3) Mg2+ increased the apparent affinity of either guanine nucleotide by 10-20-fold between 0.1 microM and 3 mM free Mg2+ in the presence of 2 mM EDTA but did not enhance the rate or maximal extent of activation. 4) The effects of Mg2+ were expressed through two independent classes of sites with affinities in the nanomolar and micromolar range. 5) A Mg2+ X guanine nucleotide complex was not the substrate for activation. The affinity of Mg2+ for nucleotides was determined as 6.25 mM GTP gamma S, 0.930 mM GTP, 0.156 mM p(NH)ppG. 6) Full activation by p(NH)ppG was completely reversible but activation by GTP gamma S was only partially reversible. These results suggest that: activation of adenylate cyclase in native membranes does not require Mg2+ or irreversible binding of the guanine nucleotide and there are two independent pathways for formation of active adenylate cyclase. A minimal mechanism for activation is discussed in light of current models. 相似文献
10.
Insulin exerts actions through a distinct species of guanine nucleotide regulatory protein: inhibition of adenylate cyclase. 总被引:10,自引:9,他引:1
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Insulin failed to exert an effect on the basal and glucagon- and guanosine 5'-[beta, gamma-imido]-triphosphate-stimulated adenylate cyclase activities of hepatocyte membranes. In the presence of high GTP (0.1 mM) concentrations, however, insulin was shown to inhibit adenylate cyclase activity. This effect was dose-dependent, exhibiting an EC50 (median effective concentration) of 3 microM for GTP. Elevated glucagon concentrations blocked the inhibitory effect of insulin in a dose-dependent fashion, with an EC50 of 1 nM. The insulin inhibition was dose-dependent (EC50 = 90 pM). The inhibitory effects of insulin were abolished using membranes from either glucagon-desensitized hepatocytes or cholera-toxin-treated hepatocytes. If either Mn2+ replaced Mg2+ in adenylate cyclase assays or Na+ was removed from the assay mixtures then insulin failed to exert any inhibitory effect. It is suggested that insulin exerts its action on adenylate cyclase through an inhibitory guanine nucleotide protein. This is integrated with the proposal [Heyworth, Rawal & Houslay (1983) FEBS Lett. 154, 87-91; Heyworth, Wallace & Houslay (1983) Biochem. J. in the press] that insulin mediates a variety of cellular effects through a specific guanine nucleotide regulatory protein and associated protein kinase(s). 相似文献
11.
D J Franks 《Canadian journal of biochemistry》1976,54(11):981-987
Adenylate cyclase (EC 4.6.1.1) activity in mouse liver plasma membranes is increased fivefold when animals are pretreated with cholera toxin. The increase in activity is detectable within 20 min of an intravenous injection of the toxin. The response of the control and cholera-toxin-activated adenylate cyclase to hormones, GTP, and NaF is complex. GTP causes the same fold stimulation of control and toxin-activated cyclase, but glucagon and NaF remain the most potent activators of liver adenylate cyclase irrespective of whether the enzyme is activated by cholera toxin. Determination of kinetic parameters of adenylate cyclase indicates that cholera toxin, hormones, and NaF do not change the affinity of the enzyme for ATP-Mg nor do they alter the Ka for free Mg2+. High concentrations of Mg2+ inhibit adenylate cyclase that is stimulated by either cholera toxin, glucagon, or NaF. These same Mg2+ concentrations have no effect on the basal activity of the enzyme or its activity in the presence of GTP. 相似文献
12.
Iu Iu Chirkov I A Tyshchuk N N Belushkina I S Severina 《Biokhimii?a (Moscow, Russia)》1987,52(6):956-963
Human blood platelets were disrupted by ultrasonication, and the guanylate cyclase activity was determined in the 105,000 g supernatant. The guanylate cyclase preparation obtained in the absence of dithiothreitol (DTT) was characterized by a nonlinear dynamics of cGMP synthesis during incubation at 37 degrees C. The use of 0.2 mM DTT during platelet ultrasonication stabilized the guanylate cyclase reaction and did not influence the enzyme activity. With a rise in DTT concentration up to 2 mM the guanylate cyclase activity diminished. Sodium nitroprusside stimulated the enzyme; this effect was enhanced in the presence of DTT. The maximum guanylate cyclase activity was revealed at 4 mM Mn2+ or Mg2+ and with 1 mM GTP. In the presence of Mn2+ the enzyme activity was higher than with Mg2+. The apparent Km values for GTP in the presence of 4 mM Mn2+ and Mg2+ was 30 and 200 microM, respectively. At GTP/cation ratio of 1:4 the Km values for Mn2+ and Mg2+ were nearly the same (249 and 208 microM, respectively). It was assumed that besides being involved in the formation of the GTP-substrate complex, Mn2+ exerts a strong influence on guanylate cyclase by oxidizing the SH-groups of the enzyme. 相似文献
13.
T J Resink M P Panchenko V A Tkachuk F R Bühler 《European journal of biochemistry》1988,174(3):531-535
In the presence of 1 microM atrial natriuretic factor (ANF) and low (0.1 mM) Mg2+ concentrations, the initial rate of binding of [3H]guanosine 5'-[beta, gamma-imido)triphosphate [( 3H]p[NH]ppG) to rat lung plasma membranes was increased twofold to threefold. ANF-dependent stimulation of the initial rate of [3H]p[NH]ppG binding was reduced at high (5 mM) Mg2+ concentrations. Preincubation of membranes with p[NH]ppG (5 min at 37 degrees C) eliminated the ANF-dependent effect on [3H]p[NH]ppG binding whereas ANF-dependent [3H]p[NH]ppG binding was unaffected by similar pretreatment with guanosine 5'-[beta-thio]diphosphate (GDP[beta S]). An increase in ANF concentration from 10 pM to 1 microM caused a 40% decrease in forskolin-stimulated or isoproterenol-stimulated adenylate cyclase activities (IC50 5 nM) in rat lung plasma membranes. GTP (100 microM) was obligatory for the ANF-dependent inhibition of adenylate cyclase, which could be completely overcome by the presence of 100 microM GDP[beta S] or the addition of 10 mM Mn2+. Reduction of Na2+ concentration from 120 mM to 20 mM had the same effect. Pertussis toxin eliminated ANF-dependent inhibition of adenylate cyclase by catalyzing ADP-ribosylation of membrane-bound Ni protein (41-kDa alpha subunit of the inhibitory guanyl-nucleotide-binding protein of adenylate cyclase). The data support the notion that one of the ANF receptors in rat lung plasma membranes is negatively coupled to a hormone-sensitive adenylate cyclase complex via the GTP-binding Ni protein. 相似文献
14.
We have examined the inhibitory regulation by Ca2+ of the adenylate cyclase activity associated with microsomes isolated from bovine aorta smooth muscle. In the presence of 2 mM MgCl2, Ca2+ (0.8-100 microM) inhibited in a noncompetitive manner activation of the enzyme by GTP, Gpp[NH]p, or forskolin. In all instances the value for half-maximal inhibition was between 2 and 3 microM. In contrast, Ca2+ inhibited the activation by MgCl2 (2-50 mM), alone or in the presence of GTP, in a competitive manner. The inhibition of adenylate cyclase by 10 microM Ca2+ was reversed in the presence of either 5 or 25 microM calmodulin or troponin C. These data show that (i) Ca2+, at concentrations similar to those which activate smooth muscle contraction, inhibits the stimulation of adenylate cyclase by several activators; (ii) Ca2+ and Mg2+ compete for a common site on the smooth muscle adenylate cyclase complex; and (iii) the reversal of Ca2+-dependent inhibition by Ca2+-binding proteins may be produced by chelation of the metal by these proteins. 相似文献
15.
V Macchia S Varrone H Weissbach D L Miller 《The Journal of biological chemistry》1975,250(16):6214-6217
Guanylate cyclase has been purified from extracts of Escherichia coli. After a 1000-fold purification, the enzyme contains only minor contaminants as judged by disc gel electrophoresis. The Km for GTP is approximately 7 times 10(-5) M and the optimal pH is 8.0. More activity is observed with Mn2+ than with Mg2+, and maximal activity is observed at 0.14 mM Mn2+ and 1.4 mM Mg2+. Based on its behavior on Sephadex G-100, the molecular weight of E. coli guanylate cyclase is about 30,000. Disc gel electrophoretic analysis indicates that the enzyme consists of a single polypeptide chain. Guanylate cyclase does not form 3':5'-AMP from ATP, and therefore, is distinct from adenylate cyclase. 相似文献
16.
Mn2+ and to some degree Fe2+, but not Mg+, Ca2+, ba2+, Sr2+, Co2+, Ni2+, La3+, or Fe3+ were able to serve as effective metal cofactors for sea urchin sperm guanylate cyclase. The apparent Michaelis constant for Mn2+ in the presence of 0.25 mM MnGTP was 0.23 mM. In the presence of a fixed free mn2+ concentration, variation in mngTP resulted in sigmoid velocity-substrate plots and in reciprocal plots that were concave upward. These positive cooperative patterns were observed at both pH 7.0 and 7.8 and in the presence or absence of Triton X-100. When Mn2+ and GTP were equimolar, Ca2+, Ba2+, Sr2+, and Mg2+ increased apparent guanylate cyclase activity. This increase in enzyme activity at least could be accounted for partially by an increase in free Mn2+ concentration caused by the complex formation of GTP with the added metals. However, even at relatively low GTP concentrations and with Mn2+ concentrations in excess of GTP, Ca2+, Sr2+, and Ba2+ significantly increased guanosine 3':5'-monophosphate production. As the total GTP concentration was increased, the degree of stimulation in the presence of Ca2+ decreased, despite maintenance of a fixed total concentration of Ca2+ and a fixed free concentration of Mn2+, suggesting that the concentration of CaGTP and MnGTP were determining factors in the observed response. The concave upward reciprocal plots of velocity against MnGTP concentration were changed to linear plots in the presence of CaGTP or SrGTP. These results suggest that sea urchin sperm guanylate cyclase contains multiple nucleotide binding sites and that stimulation of guanosine 3':5'-monophosphate synthesis by Ca2+, Sr2+, and perhaps other metals may reflect interaction of a metal-GTP complex with enzyme as either an effector or a substrate. 相似文献
17.
For a variety of ligand states, adenylate cyclase activity in the presence of Mn2+ was greater than with Mg2+. Trypsin treatment of intact hepatocytes, under conditions which destroy cell surface glucagon receptors, led to a first order loss of glucagon-stimulated adenylate cyclase activity in isolated membranes assayed in the presence of Mn2+ whether or not GTP (100 microM) was present in the assays. Arrhenius plots of basal activity exhibited a break at around 22 degrees C, those with NaF were linear and those with glucagon +/- GTP (100 microM) were biphasic with a break at around 28 degrees C. It is suggested that Mn2+ perturbs the coupling interaction between the glucagon receptor and catalytic unit of adenylate cyclase at the level of the guanine nucleotide regulatory protein. This appears to take the form of Mn2+ preventing GTP from initiating glucagon's activation of adenylate cyclase through a collision coupling mechanism. 相似文献
18.
Stimulation by thyrotropin of horse thyroid plasma membranes adenylate cyclase: evidence of cooperativity 总被引:1,自引:0,他引:1
J M Boeynaems R Pochet J E Dumont 《Biochemical and biophysical research communications》1974,58(2):446-453
Horse thyroid plasma membranes were prepared by partition in an aqueous two phases system. The membrane fraction was enriched in adenylate cyclase and only slightly contaminated with mitochondria and lysosomes. Adenylate cyclase activity was stimulated by TSH and PGE1. The TSH stimulatory effect was nearly immediate and occured in the same range of concentrations that activates intact cell metabolism. It was potentiated by GTP and ITP. A quantitative analysis of the data suggests that activation of thyroid adenylate cyclase by TSH is a cooperative process. 相似文献
19.
We report that the adenylate cyclase system in human platelets is subject to multiple regulation by guanine nucleotides. Previously it has been reported that GTP is either required for or has little effect on the response of the enzyme to prostaglandin E1. We have found that when platelet lysates were prepared in the presence of 5 mM EDTA, GTP lowered the basal and prostaglandin E1-stimulated adenylate cyclase activity, but at a higher concentration of Mn2+, it caused an increase in enzyme activity exceeding that occurring in the presence of prostaglandin E1. In the presence of Mn2+, dGTP mimics the effect of GTP and is 50% as effective as GTP. Our data suggest that the inhibitory effect of GTP on prostaglandin E1-stimulated adenylate cyclase is mainly due to its direct effect on the enzyme itself, whereas the stimulatory effect of GTP on prostaglandin E1-stimulated adenylate cyclase is due to enhancement of the coupling between the prostaglandin E1 receptor and adenylate cyclase. These studies also indicate that the method of preparation of platelet lysates can profoundly alter the nature of guanine nucleotide regulation of adenylate cyclase. 相似文献
20.
Soluble guanylate cyclase activity from guinea pig heart is inhibited by increasing concentrations of sodium citrate. The Ki value was found to be 2.83 +/- 0.05 mM in the presence of 3 mM Mn2+ and 0.6 mM GTP. Citrate acts by lowering Vmax and increasing the apparent values of Km for GTP and K0.5 for Mn2+ and Mg2+. The soluble guanylate cyclase, activated by sodium nitroprusside, was also inhibited by citrate. This inhibitory action of citrate was not restricted to soluble guanylate cyclase activity of the heart and has been demonstrated also in the supernatant of lung, liver, diencephalon and in the homogenate of blood platelets. Since citrate is known to be an important intermediate of metabolism, its intracellular concentration may be also of relevance for guanylate cyclase activity. 相似文献