共查询到20条相似文献,搜索用时 31 毫秒
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A.C. Simmonds J.M. East O.T. Jones E.K. Rooney J. McWhirter A.G. Lee 《生物化学与生物物理学报:生物膜》1982,693(2):398-406
Quenching of the fluorescence of the purified from muscle sarcoplasmic reticulum can be used to measure relative binding constants of hydrophobic compounds to the phospholipid-protein interface. We show that the binding constant for cholesterol is considerably less than that for phosphatidylcholine, so that cholesterol is effectively excluded from the phospholipid annulus around the ATPase. However, dibromocholestan-3β-ol causes quenching of the fluorescence of the ATPase, and so has access to other, non-annular sites. We suggest that these non-annular sites could be at protein/protein interfaces in ATPase oligomers. Oleic acid can bind at the phospholipid/protein interface, although its binding constant is less than that for a phosphatidylcholine, and it can also bind at the postulated non-annular sites. The effects of these compounds on the activity of the ATPase depend on the structure of the phospholipid present in the systems. 相似文献
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An enriched fraction of plasma membranes was prepared from canine ventricle by a process which involved thorough disruption of membranes by vigorous homogenization in dilute suspension, sedimentation of contractile proteins and mitochondria at followed by sedimentation of a microsomal fraction at . The microsomal suspension was then fractionated on a discontinuous sucrose gradient. Particles migrating in the density range 1.0591–1.1083 were characterized by ( activity and [3H]ouabain binding as being enriched in sarcolemma and were comprised of nonaggregated vesicles of diameter approx. 0.1 μm. These fractions contained which appeared endogenous to the sarcolemma. The enzyme was solubilized using Triton X-100 and 1 M KCl and partially purified. Optimal Ca2+ concentration for enzyme activity was 5–10 μM. Both Na+ and K+ stimulated enzyme activity. It is suggested that the enzyme may be involved in the outward pumping of Ca2+ from the cardiac cell. 相似文献
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Showdomycin inhibited pig brain ( with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na+, K+, Mg2+ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1), (2) , , and none, (3) , and , (4) and , , , and ATP. The highest rate was obtained in the presence of Na+, Mg2+ and ATP. The apparent concentrations of Na+, Mg2+ and ATP for half-maximum stimulation of inhibition () were 3 mM, 0.13 mM and 4μM, respectively. The rate was unchanged upon further increase in Na+ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E1, E2, ES, E1-P and E2-P, i.e., E2 has higher reactivity with showdomycin than E1, while E2-P has almost the same reactivity as E1-P. We conclude that the reaction of ( proceeds via at least four kinds of enzyme form (E1, E2, E1 · nucleotide and EP), which all have different conformations. 相似文献
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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
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Renal basal-lateral and brush border membrane preparations were phosphorylated in the presence of [γ-32P]ATP. The 32P-labeled membrane proteins were analysed on SDS-polyacrylamide gels. The phosphorylated intermediates formed in different conditions are compared with the intermediates formed in well defined membrane preparations such as erythrocyte plasma membranes and sarcoplasmic reticulum from skeletal muscle, and with the intermediates of purified renal enzymes such as and alkaline phosphatase. Two Ca2+-induced, hydroxylamine-sensitive phosphoproteins are formed in the basal-lateral membrane preparations. They migrate with a molecular radius of about 130 000 and 100 000. The phosphorylation of the 130 kDa protein was stimulated by La3+-ions (20 μM) in a similar way as the from erythrocytes. The 130 kDa phosphoprotein also comigrated with the erythrocyte . In addition in the same preparation, another hydroxylamine-sensitive 100 kDa phosphoprotein was formed in the presence of Na+. This phosphoprotein comigrates with a preparation of renal . In brush border membrane preparations the Ca2+-induced and the Na+-induced phosphorylation bands are absent. This is consistent with the basal-lateral localization of the renal Ca2+-pump and Na+-pump. The predominant phosphoprotein in brush border membrane preparations is a 85 kDa protein that could be identified as the phosphorylated intermediate of renal alkaline phosphatase. This phosphoprotein is also present in basal-lateral membrane preparations, but it can be accounted for by contamination of those membranes with brush border membranes. 相似文献
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Lionel Lelievre Alain Zachowski Daniele Charlemagne Patrice Laget Alain Paraf 《生物化学与生物物理学报:生物膜》1979,557(2):399-408
Treatment of plasma membrane isolated from murine plasmocytoma MOPC 173 with an EDTA-containing buffer resulted in a 300-fold increase in sensitivity of Mg2+-ATPase to ouabain. This phenomenon was associated with the solubilization by EDTA of phospholipid free proteins (approx. 30 000–34 000 daltons) from the cytoplasmic face of the plasma membrane and with removal of about 90% of the membrane bound Ca2+. The recovery of the original resistance to ouabain required specifically Ca2+ and was associated with a binding of the solubilized proteins to the membrane. 相似文献
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The calmodulin activation of the ( (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied in the range of 1 nM to 40 μM of purified calmodulin. The apparent calmodulin-affinity of the ATPase was strongly dependent on Ca2+ and decreased approx. 1000-times when the Ca2+ concentration was reduced from 112 to 0.5 μM. The data of calmodulin (Z) activation were analyzed by the aid of a kinetic enzyme model which suggests that 1 molecule of calmodulin binds per ATPase unit and that the affinities of the calcium-calmodulin complexes (CaiZ) decreases in the order of . Furthermore, calmodulin dissociates from the calmodulin-saturated Ca2+-ATPase in the range of 10?7–10?6 M Ca2+, even at a calmodulin concentration of 5 μM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 μM, corresponding to 50–80-times the cellular concentration of Ca2+-ATPase, estimated to be approx. 10 nmol/g membrane protein. We therefore conclude that most of the calmodulin id dissociated from the Ca2+-transport ATPase in erythrocytes at the prevailing Ca2+ concentration (probably 10?7 – 10?8 M) in vivo, and that the calmodulin-binding and subsequent activation of the Ca2+-ATPase requires that the Ca2+ concentration rises to 10?6 – 10?5 M. 相似文献
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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
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The action of ATP and its analogs as well as the effects of alkali ions were studied in their action on the ouabain receptor. One single ouabain receptor with a dissociation constant () of 13 nM was found in the presence of () and (). pH changes below pH 7.4 did not affect the ouabain receptor. Ouabain binding required Mg2+, where a curved line in the Scatchard plot appeared. The affinity of the receptor for ouabain was decreased by K+ and its congeners, by Na+ in the presence of (), and by ATP analogs (ADP-C-P, ATP-OCH3). Ca2+ antagonized the action of K+ on ouabain binding. It was concluded that the ouabain receptor exists in a low affinity (Rα) and a high affinity conformational state (Rβ). The equilibrium between both states is influenced by ligands of . With 3 mM Mg2+ a mixture between both conformational states is assumed to exist (curved line in the Scatchard plot). 相似文献
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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
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Interaction of lectins with a detergent-solubilized ATPase from eel electric organ was studied. Concanavalin A, which binds to α-mannosides, altered the rate of enzyme migration in agar and inhibited the formation of an antigen-antibody precipitate; other lectins had no such effects. Concanavalin A similar amounts partially inhibited ; this inhibition was reversible by α-methylglucoside. There was no corresponding effect of concanavalin A on the potassium . Concanavalin A also did not interfere with ouabain binding. Thus, concanavalin A binds to an antigenic region also involved in Na+ and/or ATP binding, but does not interact with a K+ site. 相似文献
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(1) A quantitative study has been made of the binding of ouabain to the ( in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain ( will bind ouabain either in the presence of Mg2+ and Pi, (‘Mg2+, Pi’ conditions) or in the presence of Na+, Mg2+, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain (. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the ) of ouabain to the M. sexta brain ( under both binding conditions. However, ouabain binding is more sensitive to K+ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K+ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta ( has a higher for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta ( can bind ouabain. 相似文献
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(1) Eosin bound to the in the presence of K+ has practically the same fluorescence as eosin without enzyme while in the presence of Na+ the fluorescence is higher, the excitation maximum is shifted from 518 to 524 nm, the emission maximum from 538 to 542 nm, and a shoulder appears at about 490 nm on the excitation curve. (2) The amount of eosin bound increases with the K+ concentration but with a low affinity. With equal concentrations of Na+ and K+ more is bound in the presence of Na+, and the difference between 150 mM Na+ and 150 mM K+ shows one high-affinity eosin binding site per 32P-labelling site ( 0.45 μM). With lower concentrations of the cations there are between one and two Na+-dependent high-affinity eosin binding sites per 32P-labelling site. (3) ATP (and ADP) prevents the hig-affinity Na+-dependent eosin binding and there is competition between eosin and ATP for the hydrolysis in the presence of Na+ (+Mg2+). (4) Eosin, like ATP, increases the Na+ relative to K+ affinity ) for Na+ activation of hydrolysis and for Na+ protection against inactivation by . (5) The results suggest that the high affinity eosin binding site is an ATP binding site and that it is located on the enzyme in an environment with a low polarity, i.e., the conformational change induced by Na+ opens a high-affinity site for ATP while K+ closes the site (or decreases the affinity to a low level). The experiments suggest, furthermore, that the ATP which increases the Na+ relative to K+ affinity of the internal sites is not the ATP which is hydrolyzed, i.e., in a turnover cycle in the presence of the system reacts with two different ATP molecules. 相似文献
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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
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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
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J.J. Schrijen A. Omachi W.A.H.M. Van Groningen-Luyben J.J.H.H.M. De Pont S.L. Bonting 《生物化学与生物物理学报:生物膜》1981,649(1):1-12
(1) The total phospholipid content of a gradient purified ()-ATPase preparation from pig gastric mucosa is 105 μmol per 100 mg protein, and consists of 29% sphingomyelin, 29% phosphatidylcholine, 28% phosphatidylethanolamine, 10% phosphatidylserine and 4% phosphatidylinositol. The cholesterol content corresponds to 50 μmol per 100 mg protein. (2) Treatment with phospholipase C (from Clostridium welchii and Bacillus cereus) results in an immediate decrease of the phosphate content. Up to 50% of the phospholipids are hydrolyzed by each phospholipase C preparation alone, without further hydrolysis by increased phospholipase concentration or prolonged incubation time. Combined treatment with the two phospholipase C preparations, sequentially or simultaneously, hydrolyzes up to 65% of the phospholipids. (3) The ()-ATPase and K+ stimulated activities are decreased proportionally with the total phospholipid content, indicating that these enzyme activities are dependent on phospholipids. (4) Phospholipase C treatment does not change optimal pH, value for ATP and temperature dependence of the gastric ()-ATPase, but slightly decreases the value for K+. (5) Phospholipase C treatment lowers the binding and phosphorylation capacities, suggesting that inactivation occurs primarily on the substrate binding level. (6) Most of the results can be understood by assuming that hydrolysis of the phospholipids by phospholipase C leads to aggregation of the membrane protein molecules and complete inactivation of the aggregated ATPase molecules. 相似文献