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1.
Intact LM cells, a line of cultured mouse fibroblasts, exhibited an adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity in the presence of exogenous [alpha-32P]ATP which was 20--30% of that observed with comparable preparations of lysed cells. The extent of NaF and prostaglandin E1 stimulation was comparable in intact cells and lysed cells. 96% of the added ATP and 92% of the cyclic AMP produced by intact cells could be isolated extracellularly in the incubation medium. Cellular integrity under assay conditions was monitored by trypan blue exclusion. These data suggest that LM cells contain an adenylate cyclase activity which is accessible to extracellular ATP.  相似文献   

2.
The subcellular localization of adenylate cyclase was examined in human skeletal muscle. Three major subcellular membrane fractions, plasmalemma, sarcoplasmic reticulum and mitochondria, were characterized by membrane-marker biochemical studies, by dodecyl sulfate polycrylamide gel electrophoresis and by electron microscopy. About 60% of the adenylate cyclase of the homogenate was found in the plasmalemmal fraction and 10–14% in the sarcoplasmic reticulum and mitochondria. When the plasmalemmal preparation was subjected to discontinuous sucrose gradients, the distribution of adenylate cyclase in different subfractions closely paralleled that of (Na+ + K+)-ATPase. The highest specific activity was found in a fraction which setteled at the 0.6–0.8 M sucrose interface. The electron microscopic study of this fraction revealed the presence of flattened sacs of variable sizes and was devoid of mitochondrial and myofibrillar material. The electron microscopy of each fraction supported the biochemical studies with enzyme markers. The three major membrane fractions also contained a low Km phosphodiesterase activity, the highest specific activity being associated with sarcoplasmic reticulum.The plasmalemmal adenylate cyclase was more sensitive to catecholamine stimulation than that associated with sarcoplasmic reticulum or mitochondria. The catecholamine-sensitive, but not the basal, enzyme was further stimulated by GTP. The plasmalemmal adenylate cyclase had typical Michaelis-Menten kinetics with respect to ATP and the apparent Km for ATP was approx. 0.3. mM. The pH optimum for that enzyme was 7.5. The enzyme required Mg2+, and the concentration to achieve half-maximal stimulation was approx. 3 mM. Higher concentrations of Mg2+ (about 10 mM) were inhibitory. Solubilization of the plasmalemmal membrane fraction with Lubrol-PX resulted in preferential extraction of 106 000- and 40 000-dalton protein components. The solubilized adenylate cyclase lost its sensitivity for catecholamine stimulation, and the extent of fluoride stimulation was reduced to one-sixth of that of the intact membranes. It is concluded that the catalytically active and hormone-sensitive adenylate cyclase is predominantly localized in the surface membranes of the cells within skeletal muscle. (That “plasmalemmal” fraction is considered likely to contain, in addition to plasmalemma of muscle cells, plasmalemma of bloodvessel cells (endothelium, and perhaps smooth muscle) which may be responsible for a certain amount of the adenylate cyclase activity and other propertiesobserved in that fraction.)The method of preparation used in this study provides a convenient material for evaluating the catecholamine-adenylate cyclase interactions in human skeletal muscle.  相似文献   

3.
A latent, as well as an expressed form of adenylate cyclase coupled to β-adrenergic receptors is present in intact crude synaptosomal preparations from bovine cerebellum. The latent adenylate cyclase activity was assayed in Krebs-Ringer buffer by [3H]adenine labeling and was found to be coupled to a β1-like adrenergic receptor. The externally accessible adenylate cyclase assayed in the same with [3H]ATP was stimulated via β2-adrenergic receptors.  相似文献   

4.
Mammalian triokinase, which phosphorylates exogenous dihydroxyacetone and fructose-derived glyceraldehyde, is neither molecularly identified nor firmly associated to an encoding gene. Human FMN cyclase, which splits FAD and other ribonucleoside diphosphate-X compounds to ribonucleoside monophosphate and cyclic X-phosphodiester, is identical to a DAK-encoded dihydroxyacetone kinase. This bifunctional protein was identified as triokinase. It was modeled as a homodimer of two-domain (K and L) subunits. Active centers lie between K1 and L2 or K2 and L1: dihydroxyacetone binds K and ATP binds L in different subunits too distant (≈14 Å) for phosphoryl transfer. FAD docked to the ATP site with ribityl 4′-OH in a possible near-attack conformation for cyclase activity. Reciprocal inhibition between kinase and cyclase reactants confirmed substrate site locations. The differential roles of protein domains were supported by their individual expression: K was inactive, and L displayed cyclase but not kinase activity. The importance of domain mobility for the kinase activity of dimeric triokinase was highlighted by molecular dynamics simulations: ATP approached dihydroxyacetone at distances below 5 Å in near-attack conformation. Based upon structure, docking, and molecular dynamics simulations, relevant residues were mutated to alanine, and kcat and Km were assayed whenever kinase and/or cyclase activity was conserved. The results supported the roles of Thr112 (hydrogen bonding of ATP adenine to K in the closed active center), His221 (covalent anchoring of dihydroxyacetone to K), Asp401 and Asp403 (metal coordination to L), and Asp556 (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His221 point mutant acted specifically as a cyclase without kinase activity.  相似文献   

5.
Adenylyl cyclases catalyze the production of the second messenger cyclic AMP from ATP. Until now, there has been no fluorescent adenylyl cyclase assay known that is applicable to high-throughput screening and kinetic determinations that can directly monitor the turnover of the unmodified substrate ATP. In this study, a fluorescence-based assay is described using the Ca(II)- and calmodulin-dependent adenylyl cyclase edema factor (EF) from Bacillus anthracis and Tb(III)-norfloxacin as probe for the enzyme activity. This assay can be used to study enzyme regulators, allows real-time monitoring of adenylyl cyclase activity, and does not substitute ATP by fluorescent derivatives. These derivatives must be judged critically due to their interference on the activity of enzymes. Furthermore, the new assay makes redundant the application of radioactively labeled substrates such as [α-32P]ATP or fluorescently labeled antibodies such as anti-cyclic AMP. We determined the Michaelis-Menten constant (KM), the v0max value of ATP turnover, and the IC50 values for three inhibitors of EF by this newly developed fluorescent method.  相似文献   

6.
A potent (Ki = 0.01 mM), competitive inhibition of adenylate cyclase activity in particulate fractions of guinea pig lung by 2′O-palmitoyl cyclic AMP has been observed, in striking contrast to the inactivity of cyclic AMP and N6,2′O-dibutyryl cyclic AMP at concentrations of up to 1 mm or more. The possibility that 2′O-palmitoyl cyclic AMP or similar compounds might function as endogenous regulators of the hormonal stimulation of adenylate cyclase activity is discussed. Several 6- and 8- substituted purine 3′,5′-cyclic ribotides also inhibit, probably by direct interaction with enzymatic sulfhydryl groups. A study of the inhibition by purine bases, nucleosides, and 5′ nucleotides suggests that most of the substrate (ATP) binding determinants reside in the nucleoside. The particulate enzyme fractions were found to have lower ATPase activity relative to cyclase activity than cyclase preparations from either guinea pig heart or bronchial smooth muscle. Lung cyclase fractions were maximally stimulated by 5–15 mm Mg2+ in the presence of 1.2 mm ATP as substrate. The percentage of stimulation of cyclase activity by 0.01 mm isoproterenol is dependent on the Mg2+ concentration. Cyclase activity was significantly stimulated not only by the catecholamines (isoproterenol, epinephrine, and norepinephrine) and fluoride ion, but also by prostaglandins E1, E2, and F, histamine, and glucagon.  相似文献   

7.
Arg8-vasopressin inhibited the adenylate cyclase activity of human platelet particulate fraction up to a maximum of 27% (IC50 = 1.2 nM). This inhibition required the presence of 10 μM GTP and was optimal with 100 mM NaCl. Orn8-vasopressin had similar effects. 1-Deamino-Val4, D-Arg8-vasopressin did not by itself affect adenylate cyclase activity but competitively inhibited the action of Arg8-vasopressin (pA2 = 7.74). Arg8-vasopressin did not inhibit adenylate cyclase in intact platelets but instead caused platelet aggregation, an effect that was also competitively inhibited by 1-deamino-Val4, D-Arg8-vasopressin (pA2 = 7.82). Thus, platelets possess vasopressin receptors of the V1 type that, under appropriate conditions, can mediate either inhibition of platelet adenylate cyclase or platelet aggregation.  相似文献   

8.
ATP, ADP and AMP but not adenosine increased cyclic AMP in dispersed enterocytes prepared from guinea pig small intestine. This action of ATP was augmented by IBMX and was reproduced by App(NH)p or App(CH2)p. ATP also increased the formation of cyclic [14C]AMP in enterocytes that had been preincubated with [14C]adenine. Gpp(NH)p and NaF each caused persistent activation of adenylate cyclase in plasma membranes from enterocytes and ATP caused significant augmentation of this persistent activation. In addition to increasing cellular cyclic AMP and agumenting Gpp(NH)p and NaF-stimulated persistent activation of adenylate cyclase, ATP increased the Isc across mounted strips of small intestine and inhibited net absorption of fluid and electrolytes in segments of everted small intestine. These results indicate that intestinal epithelial cells possess a receptor that interacts with ATP and other adenine nucleotides and that receptor occupation by ATP causes activation of adenylate cyclase, increased cyclic AMP and changes in active ion transport across intestinal mucosa.  相似文献   

9.
Bovine or rat brain adenylate cyclase (EC 4.6.1.1) solubilized by Lubrol-PX, a nonionic detergent, requires a Ca2+-binding protein activator for full activity (Cheung et al., 1975, Biochem. Biophys. Res. Commun.66, 1055–1062). We now show that particulate rat brain adenylate cyclase also required the activator for maximum activity. A brain particulate fraction was extracted with a hypertonic NaCl solution containing [ethyl-enebis(oxyethylenenitrilo)] tetraacetic acid. This procedure removed preferentially the activator, making adenylate Cyclase activator deficient and, consequently, dependent on an exogenous activator for maximum activity. The activator increased the V of adenylate cyclase without affecting its apparent Km for ATP. In the presence of the activator, the enzyme was more stable against thermal inactivation, suggesting that the activator probably induced a conformational change to the enzyme. F? and 5′-guanylylimidodi-phosphate [GMP-p(NH)p] greatly stimulated brain adenylate cyclase. Adenylate cyclase activity obtained in the presence of the activator and F? was comparable to the summed activities of the two agents assayed separately, indicating that their effects were additive. Similarly, the effects of the activator and GMP-p(NH)p were additive. These results suggest that the action of the activator is independent of the other two ligands. Since the activator is present in excess over adenylate cyclase, the cellular flux of Ca2+ is believed to be important in modulating the enzyme activity. The role of the Ca2+/ activator is discussed with respect to cyclic AMP metabolism in brain.  相似文献   

10.
The subcellular localization of adenylate cyclase was examined in human skeletal muscle. Three major subcellular membrane fractions, plasmalemma, sarcoplasmic reticulum and mitochondria, were characterized by membrane-marker biochemical studies, by dodecyl sulfate polycrylamide gel electrophoresis and by electron microscopy. About 60% of the adenylate cyclase of the homogenate was found in the plasmalemmal fraction and 10–14% in the sarcoplasmic reticulum and mitochondria. When the plasmalemmal preparation was subjected to discontinuous sucrose gradients, the distribution of adenylate cyclase in different subfractions closely paralleled that of (Na+ + K+)-ATPase. The highest specific activity was found in a fraction which setteled at the 0.6–0.8 M sucrose interface. The electron microscopic study of this fraction revealed the presence of flattened sacs of variable sizes and was devoid of mitochondrial and myofibrillar material. The electron microscopy of each fraction supported the biochemical studies with enzyme markers. The three major membrane fractions also contained a low Km phosphodiesterase activity, the highest specific activity being associated with sarcoplasmic reticulum.The plasmalemmal adenylate cyclase was more sensitive to catecholamine stimulation than that associated with sarcoplasmic reticulum or mitochondria. The catecholamine-sensitive, but not the basal, enzyme was further stimulated by GTP. The plasmalemmal adenylate cyclase had typical Michaelis-Menten kinetics with respect to ATP and the apparent Km for ATP was approx. 0.3. mM. The pH optimum for that enzyme was 7.5. The enzyme required Mg2+, and the concentration to achieve half-maximal stimulation was approx. 3 mM. Higher concentrations of Mg2+ (about 10 mM) were inhibitory. Solubilization of the plasmalemmal membrane fraction with Lubrol-PX resulted in preferential extraction of 106 000- and 40 000-dalton protein components. The solubilized adenylate cyclase lost its sensitivity for catecholamine stimulation, and the extent of fluoride stimulation was reduced to one-sixth of that of the intact membranes. It is concluded that the catalytically active and hormone-sensitive adenylate cyclase is predominantly localized in the surface membranes of the cells within skeletal muscle. (That “plasmalemmal” fraction is considered likely to contain, in addition to plasmalemma of muscle cells, plasmalemma of bloodvessel cells (endothelium, and perhaps smooth muscle) which may be responsible for a certain amount of the adenylate cyclase activity and other propertiesobserved in that fraction.)The method of preparation used in this study provides a convenient material for evaluating the catecholamine-adenylate cyclase interactions in human skeletal muscle.  相似文献   

11.
Direct phosphorylation of purified rat brain guanylate cyclase by cyclic AMP-dependent protein kinase is demonstrated. In the presence of [γ-32P]ATP, 32P was incorporated into the protein to the extent of 0.8 to 0.9 mol/mol of guanylate cyclase. The presence of 32P in the guanylate cyclase molecule was demonstrated by gel-filtration and by autoradiography after gel electrophoresis. The phosphorylation was accompanied by an increase in enzyme activity, characterized by an increase of VM. These results suggest that the activity of guanylate cyclase may be regulated in vivo by phosphorylation.  相似文献   

12.
Certain biochemical characteristics of an adenylate cyclase that is activated by low concentrations of histamine (Ka, 8 μm) and that is present in cell-free preparations from the dorsal hippocampus of guinea pig brain have been studied. Histamine increased the maximal reaction velocity of adenylate cyclase without altering the Km (0.18 mm) for its substrate, MgATP. Increasing concentrations of free Mg2+ stimulated enzymatic activity; the kinetic properties of this activation by Mg2+ suggest the existence of a Mg2+ allosteric site on the enzyme. Histamine increased the affinity of this apparent site for free Mg2+. Free ATP was a competitive inhibitor with respect to the MgATP substrate. The apparent potency of free ATP as an inhibitor increased in the presence of histamine. In the presence of Mg2+, low concentrations of Ca2+ markedly inhibited adenylate cyclase activity; half-maximal inhibition of both basal and histamine-stimulated enzyme activity occurred at 40 μm Ca2+. Other divalent cations, including Zn2+, Cu2+, and Cd2+, were also inhibitory. Of the divalent cations tested, only Co2+ and Mn2+ could replace Mg2+ in supporting histamine-stimulated adenylate cyclase activity. The nucleoside triphosphates GTP and ITP increased basal adenylate cyclase activity and markedly potentiated the stimulation by histamine. Preincubation of adenylate cyclase with 5′-guanylylimidodiphosphate dramatically increased enzyme activity; in this activated state, the adenylate cyclase was relatively refractory to further stimulation by histamine or F?. The subcellular distribution of histamine-sensitive adenylate cyclase activity was studied in subfractions from guinea pig cerebral cortex. The highest total and specific activities were observed in those fractions enriched in nerve endings, while adenylate cyclase activity was not detectable in the brain cytosol fraction. A possible physiological role for this histamine-sensitive adenylate cyclase in neuronal function is discussed.  相似文献   

13.
In vitro formation of the 35S-labeled Fe-S cluster of ferredoxin (Fd) has been achieved by incubating apo-Fd and [35S]cysteine with osmotically lysed chloroplasts of spinach (Spinacia oleracea). Correct integration of the 35S-labeled Fe-S cluster into Fd was verified on the basis of the following: (a) Under nondenaturing conditions, 35S-labeled holo-Fd showed the same electrophoretic mobility as authentic holo-Fd; (b) 35S-labeled holo-Fd showed an ability to bind Fd-NADP+ reductase; (c) the 35S-labeled moiety was removed from the Fd polypeptide by TCA treatment but not by 2-mercaptoethanol treatment; (d) externally added pea II apo-Fd was converted to 35S-labeled holo-Fd. This reconstitution was dependent on both ATP and light, and formation of the 35S-labeled Fe-S cluster was observed upon addition of ATP or when an ATP generation-system was constructed in the light. In contrast, ATP-consuming systems abolished the Fe-S cluster formation. A non-hydrolyzable ATP analog was unable to serve as an ATP substitute, indicating the requirement of ATP hydrolysis for cluster formation. GTP was able to substitute for ATP, but CTP and UTP were less effective. Fe-S cluster formation in lysed chloroplasts was stimulated by light even in the presence of added ATP. Light stimulation was inhibited by DCMU or methyl viologen but not by NH4+. NADPH was able to substitute for light, indicating that light energy is required for the production of reducing compounds such as NADPH in addition to the generation of ATP. These results confirm the requirement of light for the Fe-S cluster formation observed previously in intact chloroplasts.  相似文献   

14.
Uptake and Degradation of Cyclic AMP by Chloronema Cells   总被引:1,自引:0,他引:1       下载免费PDF全文
Sharma S  Johri MM 《Plant physiology》1982,69(6):1401-1403
Suspension cultures of intact chloronema cells of the moss Funaria hygrometrica take up [3H]cAMP and degrade it rapidly. The increase in total radioactivity accumulated by the cells was linear up to 30 minutes. Initially, the major degradation products were 5′-AMP and adenosine, but later predominantly ADP and ATP. In spite of rapid degradation, the amount of extracellularly applied cAMP retained by the cells is about 4-fold higher than the maximum endogenous level of cAMP reported previously (Handa, Johri 1977 Plant Physiol 59: 490-496). The uptake showed a distinct dependence on the density of the culture. Cells at a lower cell density (1-2 milligrams per milliliter) accumulated 4 to 6 times more radioactivity than the cells at high density (>10 milligrams per milliliter). The cyclic nucleotide phosphodiesterase (cNPDE) activity of whole cells (18 milliunits per milligram protein) was comparable to that of protoplasts (23 milliunits per milligram protein), but about 4-fold lower than that of lysed protoplasts (80 milliunits per milligram protein), indicating an intracellular degradation of cAMP by chloronema cells.  相似文献   

15.
Abstract: The effects of preincubation under phosphorylating conditions on adenylyl cyclase activity were studied in preparations containing synaptic membranes from rat cerebral cortex. Preincubation of the membranes with 2 mM ATP and 10 mM MgCl2 resulted in a 50% increase of adenylyl cyclase activity which withstood sedimentation and washing. This activation was maximal after 5 min of preincubation, was reversed after longer preincubations, and paralleled the time course of endogenous phosphorylation-dephosphorylation of proteins observed under these conditions. The activation showed a critical requirement for Mg2+ ions and was dependent on ATP concentration. Similar activation was observed after preincubation of cerebral-cortical membranes with adenosine-5′-0-(3-thiophosphate) (ATPγS), but this activation was not reversed by prolonged preincubation times. The activation by ATPγS was potentiated severalfold by including synaptoplasm in the preincubation. Further experiments indicated that the activity of nucleoside diphosphokinase, which converts ATPγS to guanosine-5′-0-(3-thiophosphate) (GTPγS), could account for this potentiation. Preincubation of washed membranes for 5 min with 10 μ.M GTP and 10 mM MgCl2 also produced a 50% activation of adenylyl cyclase which withstood sedimentation and washing and was reversed by longer preincubations. Endogenous phosphorylation of specific protein components in the membranes during the preincubation was examined by including radioactively labeled nucleoside thiophosphates in the preincubation medium. Incorporation of 35S from [35S]ATPγS into a protein component with apparent Mr of 54,000 daltons (54K) correlated significantly with the activation of adenylyl cyclase by ATPγS. Thiophosphorylation of the 54K protein was potentiated by addition of GDP to reactions carried out with [35S]ATPγS. Endogenous activity utilizing [γ-32P]GTP as a phosphate donor also preferentially phosphorylated the 54K protein band. These results support previous suggestions that protein phosphorylation plays a role in the regulation of adenylyl cyclase activity. Among the numerous membrane-bound phosphoproteins in rat brain, we have identified a specific protein component with an apparent Mr of 54,000 daltons as the most likely candidate for involvement in this mode of regulation. This 54K protein, which is a principal substrate for a GTP-preferring protein kinase activity in brain membranes, can now be at the focus of investigations attempting to demonstrate a direct role for protein phosphorylation in adenylyl cyclase regulation.  相似文献   

16.
Osmotically lysed rat liver mitochondria have been utilized for a study of the biochemical and ultrastructural properties in relation to divalent ion accumulation. Osmotic lysis of mitochondria by suspension and washing in cold, distilled water results in the extraction of about 50% of the mitochondrial protein, the loss of the outer mitochondrial membrane, an increase in respiration, and a marked decrease in the ability to catalyze oxidative phosphorylation. Nevertheless, except for a decrease in the ability to accumulate Sr2+ by an ATP-supported process, these lysed mitochondria retain full capacity to accumulate massive amounts of divalent cations by respiration-dependent and ATP-supported mechanisms. The decreased ability of osmotically lysed mitochondria to accumulate Sr2+ by an ATP-energized process does not appear to be due to a loss or inactivation of a specific Sr2+-activated ATPase. The energy-dependent accumulation processes in lysed mitochondria show an increased sensitivity to inhibition by monovalent cations. Extraction of cytochrome c from osmotically lysed mitochondria results in a complete loss of phosphorylation and the respiration-dependent accumulation of Ca2+; a lesser, but significant, decrease in the ATP-supported accumulation of Ca2+ also was observed. The addition of cytochrome c fully restores the respiration-dependent accumulation of Ca2+ to the level present in unextracted, osmotically lysed mitochondria. The ATP-supported process is not affected by the addition of cytochrome c to extracted mitochondria, indicating that cytochrome c is not involved in ion transport energized by ATP. The osmotically lysed mitochondria are devoid of outer membranes and contain relatively little matrix substance. The accumulation of Ca2+ and Pi by lysed mitochondria under massive loading conditions is accompanied by the formation of electron-opaque deposits within the lysed mitochondria associated with the inner membranes. This finding suggests that the inner membrane plays a role in the deposition of divalent ions within intact rat liver mitochondria. The relevance of these observations to those of other investigators is discussed.  相似文献   

17.
Preillumination of intact cells of the eukaryotic, halotolerant, cell-wall-less green alga Dunaliella salina induces a dark ATPase activity the magnitude of which is about 3–5-fold higher than the ATPase activity observed in dark-adapted cells. The light-induced activity arises from the activation and stabilization in vivo of chloroplast coupling factor 1 (CF1). This activity, 150–300 μmol ATP hydrolyzed/mg Chl per h, rapidly decays (with a half-time of about 6 min at room temperature) in intact cells but only slowly decays (with a half-time of about 45 min at room temperature) if the cells are lysed by osmotic shock immediately after illumination. The activated form of the ATPase in lysed cells is inhibited if the membranes are treated with ferri- but not ferrocyanide, suggesting that the stabilization of the activated form of CF1 is due to the reduction of the enzyme in vivo in the light.  相似文献   

18.
The characteristics of the hydrolysis of 5′-adenylylimidodiphosphate [AMP-P(NH)P] by partially purified plasma membranes from rat liver are described. Hydrolysis was less with membranes from fat cells and was poor with a detergent-dispersed preparation from rat cerebellum. The Chromatographic behavior of the principal degradation products suggests that AMP-P(NH)P is first hydrolyzed to 5'?AMP, which is then hydrolyzed further to adenosine. The adenosine is shown to inhibit adenylate cyclase noncompetitively with respect to substrate and in a cation-dependent manner. Sensitivity to inhibition by adenosine was markedly enhanced by agents that stimulated adenylate cyclase. The characteristics of the initial hydrolysis of AMP-P(NH)P fit best those of nucleotide pyrophosphatase and support the conclusion that several of the various phosphatase activities present in membranes may be due to the same enzyme. Under conditions shown to be linear with respect to time and membrane protein concentration, hydrolysis of AMP-P(NH)P exhibited a pH optimum between 9.5 and 10. At pH 9.5, hydrolysis occurred with a Km of about 20 μm and a V of about 220 nmol (min) 1 (mg of protein)?1. The initial hydrolysis of AMP-P(NH)P was inhibited in a linear-competitive manner by ATP, ADP, 5′-AMP, GTP, 5′-guanylylimidodiphosphate, NAD+, and p-nitrophenyl-dTMP and in a noncompetitive manner by UDP-glucose. Adenosine 3′:5?cyclic phosphate and guanosine 3′:5′-cyclic phosphate were not inhibitory at concentrations up to 1 mm. ATP, GTP, and 5′-guanylylimidodiphosphate were also hydrolyzed in a manner comparable to that for AMP-P(NH)P. Hydrolysis of AMP-P(NH)P did not require the presence of added metal, and some metals were inhibitory. Activity was inhibited by dithiothreitol (50% at <1 mm) and by EDTA (50% at about 10 mm). Following pretreatment with EDTA or dithiothreitol, the readdition of certain metals, especially Zn or Co, caused some restoration of hydrolytic activity. The evidence suggests that hydrolytic activity involves the participation of bound metal and that the enzyme is a metallo-protein.  相似文献   

19.
Isolated, intact dermal fibroblasts can transfer the terminal phosphate of adenosine triphosphate, [γ-32P]ATP, to an exogenously added macromolecule (histone). The incorporation of labeled phosphate to histone is attributed to an extracellularly directed protein kinase activity (ecto-kinase) which cannot be accounted for by soluble cytoplasmic protein kinase that might have been released and become bound to cell membranes during the cell preparation. The addition of soluble cytoplasmic enzyme preparations to the cell suspension was fully recoverable in the supernatant and the first wash. The activity of ectokinase was abolished by incubation of intact cells with trypsin for 5 min, whereas the activity of cytoplasmic enzyme was unaffected by the trypsin treatment. These data suggest that dermal fibroblasts contain protein kinase on the outer surface of plasma membrane which can phosphorylate exogenously added macromolecules. The ecto-protein kinase activity is dependent on cell number, time of incubation, and the concentration of Mg2+ in the reaction mixture. Lineweaver-Burk plot analyses yielded Km values for ATP and histone of 7 × 10?5 and 3 × 10?6m, respectively. The ecto-protein kinase activity of normal fibroblasts and fibrosarcoma cells were also compared. The enzyme activity of normal cells was higher than that of the malignant cells and was not significantly affected by cyclic nucleotides, whereas the activity of the malignant cells were stimulated by the addition of micromolar concentrations of the cyclic nucleotides.  相似文献   

20.
Isolated intact chloroplasts of Chlamydomonas reinhardii were found to catalyze photoreduction of CO2 in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea when adapted under an atmosphere of H2 demonstrating the association of a hydrogenase and anaerobic adaptation system with these plastids. The specific activity of photoreduction was approximately one third that detected in cells and protoplasts. Photoreduction was found to have a lower osmoticum optimum relative to aerobically maintained chloroplasts (50 millimolar versus 120 millimolar mannitol). 3-Phosphoglycerate (3-PGA) stimulated photoreduction up to a peak at 0.25 millimolar beyond which inhibition was observed. In the absence of 3-PGA, inorganic phosphate had no effect on photoreduction but in the presence of 3-PGA, inorganic phosphate also stimulated the reaction. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone inhibited photoreduction but inhibition by the former could be partially overcome by exogenously added ATP. The intact plastid can also catalyze photoevolution of H2 while lysed chloroplast extracts catalyzed the reduction of methyl viologen by H2. Both reactions occurred at rates approximately one-third of those found in cells. The oxyhydrogen reaction in the presence or absence of CO2 was not detected.  相似文献   

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