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1.
The specific radioactivity of the γ-phosphorus of ATP has been determined by an indirect method. Galactokinase is employed to transfer the terminal phosphate group of [γ-32P] ATP to [1-3H] galactose. The doubly labeled galactose-1-phosphate is purified by ion exchange chromatography on QAE Sephadex. The specific radioactivity of the phosphorus is calculated from the ratio. The method is extremely sensitive, requiring only 0.005 μmoles of ATP with a specific radioactivity of 1 μCi/μmole, and the chromatographic isolation of galactose-1-phosphate is simple and reproducible. The method is directly applicable to the determination of the specific radioactivity of [γ-32P] ATP in biological samples. 相似文献
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3.
Diketocoriolin B, a sesquiterpene antitumor antibiotic, inhibits particulate (ATP phosphohydrolase, EC 3.6.1.3) of Yoshida sarcoma cells competitively, with respect to ATP, and uncompetitively with respect to Na+ and K+. The inhibition is reduced by the addition of phosphatidylserine.Rat brain , which is solubilized by deoxycholate and requires phosphatidylserine for its activity, is also inhibited by diketocoriolin B competitively with respect to ATP and the inhibition was reversed by increasing the concentration of phosphatidylserine.However, several differences are found between the solubilized and particulate systems: (a) 2 moles of diketocoriolin B interact with the former, while only one mole interacts with the latter, (b) K+-dependent phosphatase activity of the former requires phospholipid and is sensitive to diketocoriolin B while the reverse is true with the latter.Based on these kinetic studies, it is supported that has two binding sites for phospholipid, one being essential for K+-dependent phosphatase activity and when these two sites are filled with the appropriate phospholipids, ATP can bind to the enzyme. 相似文献
4.
The ATP/ADP exchange is shown to be a partial reaction of the by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to , -di(adenosine-5′) pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg2+ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration () required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg2+ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K+ as a function of pH and Mg2+. 相似文献
5.
P T Riquelme R W Fox R G Kemp 《Biochemical and biophysical research communications》1978,81(3):864-870
Phosphofructokinase isolated from mouse skeletal muscle 18 hours after intraperitoneal injection of [ contained 0.12 to 0.15 moles of covalently bound phosphate per protomer on the basis of the specific activity of radiolabel in the γ-position of ATP. Under identical conditions, muscle pyruvate kinase and aldolase had no covalently bound phosphate. 相似文献
6.
F.M.A.H. Schuurmans Stekhoven H.G.P. Swarts Y.-F. Fu G.A.J. Kuijpers J.J.H.H.M. de Pont S.L. Bonting 《生物化学与生物物理学报:生物膜》1984,774(2):277-287
(1) Treatment of from rabbit kidney outer medulla with the labeled thio-analogue of ATP in the presence of and the absence of K+ leads to thiophosphorylation of the enzyme. The value for [γ-S]ATP is 2.2 μM and for Na+ 4.2 mM at 22°C. Thiophosphorylation is a sigmoidal function of the Na+ concentration, yielding a Hill coefficient . (2) The thio-analogue () can also support overall activity, but at 37°C is only h?1 or 0.09% of the specific activity for ATP (). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0°C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5–6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s?1 vs. 180 s?1), spontaneous dethiophosphorylation (0.04 s?1 vs. 0.5 s?1) and K+-stimulated dethiophosphorylation (0.54 s?1 vs. 230 s?1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s?1, ) and is relatively K+-insensitve. The for K+ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75–90% in the ADP-sensitive E1-conformation. 相似文献
7.
A potent inhibitor of activity was purified from Sigma equine muscle ATP by cation- and anion-exchange chromatography. The isolated inhibitor was identified by atomic absorption spectroscopy and proton resonance spectroscopy to be an inorganic vanadate. The isolated vanadate and a solution of V2O5 inhibit sarcolemma with an of 1 μM in the presence of 1 mM acid (EGTA), 145 mM NaCl, 6mM MgCl2, 15 mM KCl and 2 mM synthetic ATP. The potency of the isolated vanadate in increased by free Mg2+. The inhibition is half maximally reversed by 250 μM epinephrine. Equine muscle ATP was also found to contain a second inhibitor which depends on the sulfhydryl-reducing agent dithioerythritol for inhibition. This unknown inhibitor does not depend on free Mg2+ and is half maximally reversed by 2 μM epinephrine. Prolonged storage or freeze-thawing of enzyme preparations decreases the susceptibility of the to this inhibitor. The adrenergic blocking agents, propranolol and phentolamine, do not block the catecholamine reactivation. The inhibitors in equine muscle ATP also inhibit highly purified from shark rectal gland and eel electroplax. The inhibitors in equine muscle ATP have no effect on the other sarcolemmal ATPases, Mg2+-ATPase, Ca2+-ATPase and . 相似文献
8.
The sugar composition of the growth medium influenced the ratio, pyruvate and lactate production, and ATP levels in both normal and transformed fibroblast cell lines growing in tissue culture. Removal of glucose led to a rapid three- to fourfold rise in the ratio, followed by a slower decline in the content of ATP. However, there was no change in the adenylate energy charge [] over a 2-h period. The ratio was restored to the original level within 10 s of glucose readdition. The ratios in cell lines growing on galactose were as high as for those incubated without sugars; growth on mannose or fructose produced intermediate ratios. There was an inverse relationship between the ratio and pyruvate-lactate production for glucose, fructose and galactose. Thus, all cell lines had a high production of pyruvate and lactate but a low ratio when grown on glucose. In contrast, when galactose served as the sugar source, acid production was low, while the ratio was high. All cell lines had comparable hexokinase activity, and glucose was the best substrate, mannose intermediate and fructose poorest. Hexokinase activity did not correlate with the relative degree of utilization of the sugars. These results suggest that the sugar composition of the growth medium affects the metabolic pattern of a cell line, including the ratio, the ATP content and the production of pyruvate and lactate. 相似文献
9.
Quercetin inhibited a dog kidney ( preparation without affecting for ATP or for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E2 enzyme conformations, blocked this inhibition, while the K+-phosphatase activity was at least as sensitive to quercetin as the ( activity, all consistent with quercetin favoring E1 conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the for ATP and the for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K+-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E2 conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E1 conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on , for substrate, and for K+, as well as for stimulation of phosphatase activity by both these reagents at low K+ but high Na+ concentrations. 相似文献
10.
Modulation of human erythrocyte Ca2+/Mg2+ ATPase activity by phospholipase A2 and proteases. A comparison with calmodulin 总被引:6,自引:0,他引:6
The human erythrocyte membrane ATPase responded to the presence of an acidic phospholipase A2 and to low levels of trypsin (and chymotrypsin) in much the same way as it did to calmodulin isolated from human erythrocytes. The increased concentration of ATP hydrolyzed in 1 hour was similar to that observed when calmodulin had been added to a suspension of membranes during the assay. The observations reported here strongly suggest that activation of the ATPase can proceed by introducing apparently distinct perturbations either to the protein or to phospholipid domains of the erythrocyte membrane. 相似文献
11.
A Ziegelhoffer M B Anand-Srivastava R L Khandelwal N S Dhalla 《Biochemical and biophysical research communications》1979,89(4):1073-1081
The sarcolemmal membrane obtained from rat heart by hypotonic shock-LiBr treatment method was found to incorporate 32P from [γ-32P] ATP in the absence and presence of cyclic AMP and protein kinase. The phosphorylated membrane showed an increase in Ca2+ ATPase and Mg2+ ATPase activities without any changes in Na+K+ ATPase activity. The observed increase in ATPase activity was found to be associated with an increase in Vmax value of the reaction whereas Ka value for was not altered. These results provide information concerning biochemical mechanism for increased calcium entry due to hormones which are known to elevate cyclic AMP levels in myocardium and produce a positive inotropic effect. 相似文献
12.
G.E. Breitwieser 《生物化学与生物物理学报:生物膜》1982,689(3):457-463
(1) A membrane fraction enriched in (EC 3.6.1.3) was obtained from optic ganglia of the squid (Loligo pealei) by density gradient fractionation of membranes followed by treatment with either SDS or Brij-58. The resulting membrane had an specific activity of approx. 2 units/mg and was ouabain-sensitive. (2) The had a for ATP of and a pH optimum of 7.0. It was inhibited by ouabain with a of . (3) Optimum monovalent cation concentrations were: 240 mM NaCl, 60 mM KCl, tested with . (4) The Mg2+ dependence of hydrolysis varied with the absolute ATP concentration. At 3 mM ATP, the for Mg2+ was , and at 6 mM ATP, the was . High levels of Mg2+ caused inhibition of hydrolysis. (5) The interactions of Na+ and K+ were examined over a range of conditions. K+ levels caused modulations in the Na+ dependence in the range of 1–150 mM. (6) The prepared from squid optic ganglion displays properties similar to those of the sodium pump in injected nerves. 相似文献
13.
A new assay for ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) has been devised, the key feature of which is the quantitative precipitation of the unreacted substrate as Na2SO4, in ethanol:water (5:1), leaving the product adenosine 5′-phosphosulfate (APS) in solution with an 80% yield. This separation procedure, coupled with the use of , makes it possible to assay the synthesis of picomole amounts of [35S]APS and thereby determine accurately the initial velocity of the forward reaction catalyzed by ATP sulfurylase. Extracts from tobacco cells synthesized [35S]APS and a negligible amount of one unknown sulfur compond, but they did not synthesize 3′-phosphoadenosine 5′-[35S]phosphosulfate (PAPS) under the assay conditions adopted, so that the 35S remaining in solution after ethanol precipitation of is a valid assay of the ATP sulfurylase reaction in these extracts. This assay is simple, rapid, and suitable for assaying multiple samples. Extracts of four additional species of plants were incubated under the same conditions, and each catalyzed the synthesis of [35S]APS according to this assay. The assay is suitable for use with the enzyme from any organism, provided that the product APS is not acted upon by another enzyme. 相似文献
14.
The partial purification of from pig lens has been achieved by treatment with deoxycholate followed by density gradient centrifugation. The specific activity of the final preparation, ranging from 300 to 500 nmol/h per mg protein, is increased approx. 100-fold compared to the homogenate. A parallel increase in activity is also observed. Sodium dodecyl sulfate (SDS) gel electrophoresis reveals six major protein bands, one of which is the 93 kDa α subunit of which can be phosphorylated by reaction with [γ-32P]ATP. A second band contains a glycoprotein which displays an apparent molecular weight of 51 000 and thus appears to be the β subunit of the enzyme. The enzyme is sensitive to ouabain with the for and inhibition being 1.2 and 1.3 μM, respectively. Several agents which inhibit from other tissues such as oligomycin, Ca2+, vanadate, , (PCMBS) and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) also inhibit the lens enzyme. Monovalent cations other than K+ are partially effective in activating the ()-ATPase and activities. The K+ congeners were relatively more effective in supporting compared to activity. Other kinetic properties of the lens enzyme are also comparable to those of the enzyme from other tissues. Utilizing the partially purified membrane bound enzyme, discontinuities in Arrhenius plots of activity, activity and fluoresence polarization of the fluidity probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), are observed near the physiological temperature of lens. The possible significance of these observations for the mechanism of cataract formation are discussed. 相似文献
15.
Chloroplasts which were rapidly isolated from illuminated leaves showed activity of ATP hydrolysis at a level much higher than that of the dark control. Under the high-intensity illumination or under repetitive flash excitation, the activated chloroplasts synthesized more ATP than those with a low ATP hydrolysis activity. formed under repetitive flashes was smaller in the activated chloroplasts than in the inactive chloroplasts. The inhibition of ATP yield per flash by valinomycin or nigericin in the presence of K+ was stronger in the inactive chloroplasts than in the activated chloroplast. ATP synthesis in the activated chloroplasts seems to have a lower threshold. 相似文献
16.
The influence of the mode of preparation upon some of the characteristics of white adipose tissue plasma membranes and microsomes has been reported. Plasma membrane fractions prepared from mitochondrial pellet were shown to have higher specific activities of than plasma membranes originating in crude microsomes. Isolation of fat cells by collagenase treatment was found to result in a decrease in specific activity of the plasma membrane enzymes; in plasma membranes prepared from isolated fat cells, the specific activity values obtained for and 5′-nucleotidase were only 42% and 6.3% respectively of those obtained in plasma membranes prepared from whole adipose tissue. Purification of whole adipose tissue crude microsomes by hypotonic treatment caused extensive solubilization of the endoplasmic reticulum marker enzymes, NADH oxidase and NADPH cytochrome reductase. The lability of endoplasmic reticulum marker enzymes, however, was found to be greatly diminished in the preparations from isolated fat cells. The possibility that NADH oxidase and NADHPH cytochrome reductase activities found in the plasma membranes are microsomal enzymes adsorbed by the plasma membranes is discussed. The peptide patterns as well as the NADH oxidase and NADPH cytochrome reductase activity patterns of plasma membranes and purified microsomes were compared by means of sodium dodecyl sulfate or Triton X-100 polyacrylamide gel electrophoresis. 相似文献
17.
The activity of calcium-stimulated and magnesium-dependent adenosinetriphosphatase which possesses a high affinity for free calcium (high-affinity , EC 3.6.1.3) has been detected in rat ascites hepatoma AH109A cell plasma membranes. The high-affinity had an apparent half saturation constant of for free calcium, a maximum reaction velocity of ATP hydrolyzed/mg protein per min, and a Hill number of 0.8. Maximum activity was obtained at 0.2 μM free calcium. The high-affinity was absolutely dependent on 3–10 mM magnesium and the pH optimum was within physiological range (pH 7.2–7.5). Among the nucleoside trisphosphates tested, ATP was the best substrate, with an apparent of 30 μM. The distribution pattern of this enzyme in the subcellular fractions of the ascites hepatoma cell homogenate (as shown by the linear sucrose density gradient ultracentrifugation method) was similar to that of the known plasma membrane marker enzyme alkaline phosphatase (EC 3.1.3.1), indicating that the ATPase was located in the plasma membrane. Various agents, such as K+, Na+, ouabain, KCN, dicyclohexylcarbodiimide and NaN3, had no significant effect on the activity of high-affinity . Orthovanadate inhibited this enzyme activity with an apparent half-maximal inhibition constant of 40 μM. The high-affinity was neither inhibited by trifluoperazine, a calmodulin-antagonist, nor stimulated by bovine brain calmodulin, whether the plasma membranes were prepared with or without ethylene glycol . Since the kinetic properties of the high-affinity showed a close resemblance to those of erythrocyte plasma membrane , the high-affinity of rat ascites hepatoma cell plasma membrane is proposed to be a calcium-pumping ATPase of these cells. 相似文献
18.
Plasma membrane vesicles of Ehrlich ascites carcinoma cells have been isolated to a high degree of purity. In the presence of Mg2+, the plasma membrane preparation exhibits a Ca2+-dependent ATPase activity of 2 μmol Pi per h per mg protein. It is suggested that this activity is related to the measured Ca2+ transport which was characterized by values for ATP and Ca2+ of and , respectively. Phosphorylation of plasma membranes with [γ-32P]ATP and analysis of the radioactive species by polyacrylamide gel electrophoresis revealed a Ca2+-dependent hydroxylamine-sensitive phosphoprotein with a molecular mass of 135 kDa. Molecular mass and other data differentiate this phosphoprotein from the catalytic subunit of and from the catalytic subunit of of endoplasmic reticulum. It is suggested that the 135 kDa phosphoprotein represents the phosphorylated catalytic subunit of the of the plasma membrane of Ehrlich ascites carcinoma cells. This finding is discussed in relation to previous attempts to identify a Ca2+-pump in plasma membranes isolated from nucleated cells. 相似文献
19.
Kathleen J. Sweadner 《生物化学与生物物理学报:生物膜》1978,508(3):486-499
A microsomal fraction from canine brain gray matter has been extracted with the detergent sodium dodecyl sulfate to partially purify the membrane bound adenosine triphosphatase. Phospholipid, glycolipid, and a family of other glycoproteins are also enriched by the procedure; it is proposed that the product is an intrinsic membrane protein fraction. 6–8-fold purification of is obtained without solubilizing the enzyme and without irreversibly altering its turnover number. Final specific activities are 350–400 μmol of ATP hydrolyzed/h per mg protein. The stimulation and reversible inactivation of the by dodecyl sulfate were examined for information relevant to the mechanism of action of the detergent. 相似文献
20.
The ionic influence and ouabain sensitivity of lymphocyte Mg2+-ATPase and ATPase were studied in intact cells, microsomal fraction and isolated plasma membranes. The active site of 5′-nucleotidase and Mg2+-ATPase seemed to be localized on the external side of the plasma membrane whereas the ATP binding site of was located inside the membrane.Concanavalin A induced an early stimulation of Mg2+-ATPase and both on intact cells and purified plasma membranes. In contrast, 5′-nucleotidase activity was not affected by the mitogen. Although the thymocyte Mg2+-ATPase activity was 3–5 times lower than in spleen lymphocytes, it was much more stimulated in the former cells (about 40 versus 20 %). activity was undetectable in thymocytes. However, in spleen lymphocytes activity can be detected and was 30 % increased by concanavalin A. Several aspects of this enzymic stimulation had also characteristic features of blast transformation induced by concanavalin A, suggesting a possible role of these enzymes, especially Mg2+-ATPase, in lymphocyte stimulation. 相似文献