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1.
The adenylate cyclase activity of sperm membrane fragments isolated from Lytechinus pictus sperm according to Cross [20] has been studied. Two distinct fractions preferentially coming from the flagellar plasma membrane are obtained. Surface I125-labeling experiments performed by Cross [20] indicate that these membranes are representative of the entire sperm plasma membrane. Both fractions are enriched in their adenylate cyclase activity: the specific activity of the top membranes is eightfold higher than in whole sperm, whereas that of the middle membranes is 15-fold higher. The cyclase seems to be associated with the membranes. Lytechinus pictus egg jelly has no effect or slightly inhibits the adenylate cyclase activity of the isolated sperm plasma membrane fragments. Mg++ and Na+ stimulated their cyclase activity about sevenfold at 2.5 mM Mn++ and 3.2 mM ATP. At this ATP to Mn++ ratio, high concentrations of Ca++ have a small stimulatory effect.  相似文献   

2.
Calcium-dependent activation of tryptophan hydroxylase by ATP and magnesium   总被引:10,自引:0,他引:10  
Tryptophan hydroxylase [EC 1.14.16.4; L-tryptophan, tetrahydropteridine: oxygen oxidoreductase (5-hydroxylating)] in rat brainstem extracts is activated 2 to 2.5-fold by ATP and Mg++ in the presence of subsaturating concentrations of the cofactor 6-methyltetrahydropterin (6MPH4). The activation of tryptophan hydroxylase under these conditions results from a reduction in the apparent Km for 6MPH4 from 0.21 mM to 0.09 mM. The activation requires Mg++ and ATP but is not dependent on either cAMP or cGMP. The effect of ATP and Mg++ on enzyme activity was enhanced by μM concentrations of Ca++ and totally blocked by EGTA. These data suggest that tryptophan hydroxylase can be activated by a cyclic nucleotide independent protein kinase which requires low calcium concentrations for the expression of its activity.  相似文献   

3.
Adenylate and guanylate cyclase activities were confirmed in crude homogenates from rat peritoneal mast cells. Both enzyme activities were associated with the 105, 000 X g particulate fractions, but not detected in the supernatant fractions. The optimal pH for both cyclase activities was 8.2. Mn++ was essentially required for guanylate cylcase activity, while adenylate cyclase activity was observed in the presence of either Mg++ or Mn++. The apparent Km values of adenylate cyclase for Mn++-ATP and Mg++-ATP were 160 μM and 340 μM, respectively, whereas the value of guanylate cyclase for Mn++-GTP was 100 μM. Adenylate cyclase was activated by 10 mM NaF. However, both adenylate and guanylate cyclase activities were neither stimulated nor inhibited by the addition of various kinds of agents which stimulate or inhibit the release of histamine from mast cells.  相似文献   

4.
Adenylate cyclase activities were studied in porcine sperm in the presence and absence of Mn++ before and after incubation in vivo and in vitro. Incubation of sperm in vivo for 30 min increased the Mg++-stimulated adenylate cyclase activity from 35.1 pmoles cyclic AMP formed per mg protein per 10 min to 50.4 pmoles. The activity stimulated by Mg++ and Mn++ increased from 392 to 729 pmoles after 30 min of in vivo incubation. Activity after incubation in vivo for 120 min was not different from activity after 30 min. In vitro incubation of porcine sperm in Ca++-free Ringer-fructose resulted in no change, but incubation in oviductal and uterine flushings obtained from gilts soon after ovulation increased Mg++-stimulated activity by 24% and Mg++?+ Mn++-stimulated activity by 49%. In vitro incubations in preovulatory flushings plus follicular fluid or in bovine serum albumin also increased adenylate cyclase activity.  相似文献   

5.
Summary Insulin decreased markedly the adenylyl cyclase activity associated with fat cell membranes purified by centrifugation in sucrose gradients. The hormone effect was not readily evident in crude membrane preparations.The kinetics of this effect indicate that some time was required for the onset of the insulin-induced inactivation. This lag period decreased when the insulin concentration was increased. The hormone dose dependence for adenylyl cyclase inactivation measured at a fixed time (3 min) showed a 10 to 15% decrease in activity at 1 to 30 U per ml insulin; 30 to 40% at 100 to 1000 U per ml; and 75% at 0.1 U per ml.The insulin effect was completely abolished by 0.1mm GMP-P(NH)P, 10mm fluoride, or 50 ng per ml glucagon, or by increasing the Mn++ concentration to 4mm. In addition, it was partially reversed by the addition of a fraction from the sucrose gradient, which contained soluble factors.The kinetics of the adenylyl cyclase-catalyzed reaction were studied using ATP or AMP-P(NH)P as adenylyl donor, and Mn++ or Mg++ as divalent cation, in the absence or presence of insulin. With ATP and Mg++ there was a striking reduction of the transient reaction rates after 1.5 min of incubation. Under these conditions the insulin effect was not evident. On the contrary, with ATP and Mn++ this spontaneous reduction of activity was less evident; however, in the presence of insulin there was a clear and marked reduction of the transient reaction rate measured after 1.5 min of incubation. With AMP-P(NH)P the kinetic data were qualitatively similar to those observed with ATP.It is concluded that under certain assay conditions adenylyl cyclase may be converted to an inactive enzyme form, and that such a conversion is more evident in the presence of Mg++ than with Mn++. In the latter case, insulin appears to enhance the rate of this conversion.  相似文献   

6.
The flagellar motion of impaled bull spermatozoa can be maintained by external ADP or ATP. The post-impalement flagellar frequency depends sharply on the external Mg2+ concentration. 0.3 mM Mg2+ is required for half-optimal activity with ADP, 0.05 mM Mg2+ with ATP as external power source. Mn2+ can substitute partially for Mg2+ as ionic co-factor. Ca2+ cannot substitute for Mg2+, and at concentrations above 0.5 mM it inhibits motility slightly. Zn2+ acts only as inhibitor of post-impalement flagellar activity, reducing it to zero at concentrations above 1 mM.  相似文献   

7.
Intestinal guanylate cyclase C is activated by guanylin, an endogenous peptide. This activity seems to be modulated by adenine nucleotides, the ions Mg2+ and Mn2+, and pH. In this study, we report an ultracytochemical method for the localization of guanylate cyclase C activity at the electron microscope level. We studied the enzymatic activity in the presence or absence of guanylin and/or ATP, in the presence of the ions Mg2+ or Mn2+, and at different pH levels. The greatest distribution of enzymatic activity was detected in samples incubated at pH 8 and 7.4 in the presence of guanylin, Mg2+ and ATP. Guanylate cyclase C activity was detected at the surface epithelium of stomach and intestine, and in liver, exocrine pancreas and parotid gland. In the intestine, enzymatic activity was more widely distributed in the duodenum than in the jejunum–ileum and colon. In the small intestine, activity was more evident in the upper portion than in the basal portion of the villus. In samples incubated at pH 8 and 7.4 in the absence of ATP, enzymatic activity was detected only in small intestine, liver and exocrine pancreas. Enzymatic activity was present in duodenum incubated at pH 8 and 7.4 in the presence of Mn2+ and in the presence or absence of ATP. No samples incubated in all these experimental conditions but at pH 5 or samples incubated in the presence of guanylin only or in the absence of guanylin, displayed guanylate cyclase C activity. Our results suggest that a complete ultracytochemical detection of guanylate cyclase C activity requires guanylin as stimulator, and incubation in the presence of Mg2+ and ATP atbreak pH 8 and 7.4.  相似文献   

8.
These studies have shown that in the crude system of rat liver mitochondria the branched-chain α-keto acid dehydrogenase activities are activated at high (10.0mM) Mg++ concentrations favoring dephosphorylation, and are inactive at low (1.0mM) Mg++ concentrations favoring phosphorylation. In this crude system, α-Ketoglutarate dehydrogenase activity was also regulated in this manner. In general, the optimum Mg++ and ATP levels for activation were 10mM and 1.0mM respectively.  相似文献   

9.
A phosphoprotein phosphatase preparation which showed activity towards glycogen synthase, phosphorylase, phosphorylase kinase, and phosphohistones was reversibly inhibited (70–90%) by preincubation with free ATP (apparent Ki about 0.3 mM). Other nucleotides (ADP2 (apparent Ka 3μM) prior to assay. Other divalent metals (Co++ > Zn++ > Mg++) were partially effective in reversing the inhibition. It is concluded that ATP by virtue of its special structure and metal binding capacity possibly removes a catalytically important metal ion from the enzyme.  相似文献   

10.
Summary Nonenzymatic ATP hydrolysis in medium of Wachstein and Meisel for histochemical demonstration of ATPase activity was investigated. In this medium considerable amounts of phosphorus are released without the participation of the enzyme. ATP hydrolysis in Wachstein-Meisel's medium increase with the concentration of Pb++ and decrease at its small concentrations. The degree of ATP hydrolysis appeared to increase with increase both temperature and pH. At high concentration of ATP (5.76 mM) the degree of ATP hydrolysis in Wachstein-Meisel's medium is lower than at 1.44 mM ATP. 10.0 mM Ca++ or 3.6 mM Fe++ speed up ATP hydrolysis after 30- and 60-minute incubation. In the presence of 3.6 mM Co++ or 2.6 mM Cu++ ATP hydrolysis in Wachstein-Meisel's medium increased throughout the whole period examined. On the contrary, 3.6 mM Fe+++ decreases ATP hydrolysis in this medium.10.0 mM F raises the degree of ATP hydrolysis which is, however, lowered in the presence of 2.5 mM pCMB or 3.6 mM KCN. 2.0 mM cysteine highly inhibits the process of nonenzymatic ATP hydrolysis in Wachstein-Meisel's medium.These data show that the histochemical reaction for ATPase activity in Wachstein-Meisel's medium does not originate exclusively from the hydrolysis of ATP in the presence of Pb++, but take rise, above all, as a result of an enzymatic reaction.  相似文献   

11.
The preparation of cytoplasmic membranes from suspensions of Staphylococcus aureus lysed by an enzyme recently isolated in these laboratories is described. These membranes contained: protein, 34.4%; ribonucleic acid, 6.6%; lipids, 34.5%; and total phosphorus, 1.4%. Such membranes exhibited adenosine 5′-triphosphatase (E.C. 3.6.1.3) activity, liberating orthophosphate at an initial rate of 0.53 μmole per min per mg of protein under optimal conditions. The enzyme was Mg++-dependent and K+- or Na+-stimulated. Maximal activity was observed with a molar adenosine 5′-triphosphate (ATP) to Mg++ ratio of 1. One mole of orthophosphate was liberated per mole of ATP; the other product of digestion was adenosine 5′-diphosphate. Inorganic pyrophosphate and the 5′-triphosphates of guanosine, uridine, and cytidine were also attacked by membrane preparations, but more slowly than ATP. Ouabain, p-chloromercuribenzoate, and 2,4-dinitrophenol did not alter adenosine triphosphatase activity, whereas both Atebrine and chlorpromazine were inhibitory.  相似文献   

12.
Effect of exogenous ATP on the volume of TA3 ascites tumor cells   总被引:1,自引:0,他引:1  
When exogenous ATP is added to suspensions of TA3 ascites tumor cells suspended in Ca++ and Mg++ free media, a significant increase in cell volume can be measured. This increase is reversible upon addition of Ca++ and/or Mg++ back to the media. The enlargement of these cells is temperature sensitive and specific for ATP; no other nucleotides, EDTA or ouabain were effective. The evidence suggest that this phenomena may be due to an alteration in membrane permeability and that the regulation of membrane permeability is an energy dependent process.  相似文献   

13.
Glycerol kinase is induced in cells ofPseudomonas aeruginosa strain PAO when grown in the presence of glycerol or glycerol-3-phosphate. The enzyme was isolated from the soluble cytoplasmic fraction of cell extracts and purified 500-fold by ammonium sulfate precipitation and chromatography on columns of Sephadex G-25, DEAE-Sephadex, hydroxyapatite, and Sephadex G-200. A molecular weight of 120,000 was estimated by gel filtration of the catalytically active enzyme. In polyacrylamide gel electrophoresis the purified product contained one major band of Coomassie Blue staining material. The enzyme exhibited an apparent Km of 40 M for glycerol and 23 M for ATP. Of the nucleotide triphosphates tested, only ATP served as a phosphoryl group donor. Mg++ or Mn++ was required for activity, although a threefold greater concentration of Mn++ was required when Mn++ substituted for Mg++. In contrast to most other catabolic glycerol kinases in bacteria, the enzyme was not inhibited by fructose-1,6-diphosphate nor by other tested metabolites.  相似文献   

14.
The effect of morphine on ATPase of synaptic plasma membranes (SPM) and synaptic vesicles isolated from the mouse brain was studied. The activity of synaptic vesicle Mg++-dependent ATPase from mice rendered morphine tolerant and dependent by pellet implantation was 40% higher than that from placebo implanted mice. However, the activities of Mg++-dependent ATPase and Na+, K+ activated ATPase of SPM of tolerant and nontolerant mice were not significantly different. The activity of synaptic vesicular Mg++-dependet ATPase was dependent on the concentration of Mg++ but not of Ca++; maximum activity was obtained with 2 mM MgCl2. On the other hand, Mg++-dependent ATPase activity of SPM was dependent on both Mg++ and Ca++, activity being maximum using 2 mM MgCl2 and 10?5 M CaCl2. It is suggested that this stimulation of ATPase activity may alter synaptic transmission and may thus be involved in some aspects of morphine tolerance and dependence.  相似文献   

15.
Guanylate cyclase from crude homogenates of vegetative Dictyosteliumdiscoideum has been characterized. It has a pH optimum of 8.0, temperature optimum of 25°C and requires 1 mM dithiothreitol for optimal activity. It strongly prefers Mn++ to Mg++ as divalent cation, requires Mn++ in excess of GTP for detectable activity, and is inhibited by high Mn++ concentrations. It has an apparent Km for GTP of approximately 517 μM at 1 mM excess Mn++.The specific activity of guanylate cyclase in vegetative homogenates is 50–80 pmoles cGMP formed/min/mg protein. Most of the vegetative activity is found in the supernatant of a 100,000 x g spin (S100). The enzyme is relatively unstable. It loses 40% of its activity after 3 hours storage on ice. Enzyme activity was measured from cells that had been shaken in phosphate buffer for various times. It was found that the specific activity changed little for at least 8 hours. Cyclic AMP at 10?4 M did not affect the guanylate cyclase activity from crude homogenates of vegetative or 6 hour phosphate-shaken cells.  相似文献   

16.
17.
The effect of certain biochemical parameters on transendothelial fluid transport has been studied. Cellular ATP level and (Na+ + K+)-activated as well as Mg2+-activated ATPase activities were measured by ultramicrotechniques using individual rabbit corneal endothelium after they had been subjected to in vitro perfusion with solutions fully supplemented or deficient singly or severally in glucose, adenosine and glutathione (GSH). With the complete medium, the transport system operates in vitro for approx. 6 h. Deletion of glucose alone, glucose and adenosine or glucose, adenosine and GSH brings about a cessation of fluid transport after 3.5 h, 2 to 2.5 h and 0.5 to 1 h, respectively. A marked decrease (62%) of the endothelial ATP level, however, occurs only when all metabolites are omitted. The favorable effect of GSH on transport activity is attributable to its capacity to sustain cellular ATP rather than to protect the functionality of (Na+ + K+)-activated ATPase. Adenosine, in the presence of GSH, maintains normal ATP levels and, additionally, exerts a protective effect on Mg2+-activated ATPase and possibly also on (Na+ + K+)-activated ATPase.  相似文献   

18.
Summary To understand the earliest phases of epidermal cell spreading we have sought a defined in vitro system. We studied the divalent cation dependence of guinea pig epidermal cell spreading in media containing varying concentrations of cations. No spreading occurred in calcium-magnesium-free Dulbecco's modified Eagle's medium (CMF-DME) in the presence of cation-free fetal bovine serum; however, significant spreading occurred if the medium was supplemented with Mg++ plus Ca++ or Mg++ alone. Supplementing with Ca++ alone led to much less spreading. These cations in CMF-DME did not support spreading in the absence of serum or the presence of serum albumin. Assaying cell spreading in a simple salt solution consisting of NaCl, KCl, Tris buffer, pH 7.4 plus dialyzed serum and a series of divalent cation supplements (Ca++, Mg++, Mn++, Co++, Zn++, Ni++), showed that only Mg++ and Mn++, and to a lesser extent, Ca++, supported cell spreading. In contrast to Mg++, however, Mn++ could support spreading in the absence of whole serum if serum albumin were present. Although Mn++ plus serum albumin supported more rapid spreading at lower cation concentrations than Mg++ plus serum, equal concentrations of Ca++ completely blocked the Mn++ effect. In contrast to the increasing cell spreading, which occurred in Mg++-containing medium with time, cell death occurred in Mn++-containing medium by 24 h. Consonant with studies from other laboratories, human foreskin fibroblasts spread in Mn++-containing salt solution in the absence of protein supplements. These experiments indicate for epidermal cell spreading that Mg++ is the important cation in tissue culture media, that under proper cation conditions epidermal cells do not need a specific spreading protein (i.e. a protein that has been demonstrated to support cell spreading), that Mn++ and Mg++-induced spreading seem to represent different mechanisms, that fibroblastic and epidermal cells have different cation requirements for in vitro spreading, and that the crucial role cations play in cell spreading remains to be elucidated. This work was supported in part by Public Health Service grant CA34470-01 (KSS) awarded by the National Cancer Institute, Bethesda, Md.  相似文献   

19.
Adenylate and guanylate cyclase activities do not vary in concert during the multiplication of KB cells. Adenylate cyclase activity is low and slightly increases at cell confluency, guanylate cyclase activity, great in sparce cells, decreases during cell multiplication period. These variations are not caused by a modification of catalytic sites because the apparent Km for ATP or GTP is not changed, but by a modification of the dependance on Mg++ or Mn++ ions. Fresh serum increases guanylate cyclase activity but does not affect adenylate cyclase.  相似文献   

20.
The catalytic activity of guanylate cyclase (GCase) coupled to atrial natriuretic peptide (ANP) receptor depends on the metal co-factor, Mn2+ or Mg2+. ATP synergistically stimulates the ANP-stimulated GCase in the presence of Mg2+. We have now shown the ATP regulation of the ANP-stimulated GCase in the presence of Mn2+ in rat lung membranes. ANP stimulated the GCase 2.1-fold compared to the control. ATP enhanced both the basal (basal-GCase) and the ANP-stimulated GCase maximally 1.7- and 2.3- fold compared to the control, respectively, at a concentration of 0.1 mM. The stimulation by ATP was smaller in the presence of Mn2+ than in the presence of Mg2+. The addition of inorganic phosphate to the reaction mixture altered the GCase activities in the presence of Mn2+ with or without ANP and/or ATP. In the presence of 10 mM phosphate, ATP dose-dependently stimulated the basal GCase 5-fold compared to the control at a concentration of 1 mM and augmented the ANP-stimulated GCase, which was 4.2-fold compared to the basal-GCase, 5.5-fold compared to the control at a concentration of 0.5 mM. Protein phosphatase inhibitors, okadaic acid (100 nM), H8 (1 M) and staurosporin (1 M), did not alter the activity. Orthovanadate (1 mM), an inorganic phosphate analogue, significantly stimulated both the basal-GCase and the ANP-stimulated GCase, which were inhibited by ATP. It was assumed that phosphate and orthovanadate might interact with the GCase to regulate the activity in the opposite manner. This was the first report that inorganic phosphate and orthovanadate affected the ATP-regulation of the ANP-stimulated GCase in the presence of Mn2+.  相似文献   

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