Soluble consisting predominantly of αβ-units with below 170 000 was prepared by incubating pure membrane-bound (35–48 μmol Pi/min per mg protein) from the outer renal medulla with the non-ionic detergent dodecyloctaethyleneglycol monoether (C12E8). and potassium phosphatase remained fully active in the detergent solution at C12E8/protein ratios of 2.5–3, at which 50–70% of the membrane protein was solubilized. The soluble protomeric was reconstituted to Na+, K+ pumps in phospholipid vesicles by the freeze-thaw sonication procedure. Protein solubilization was complete at C12E8/protein ratios of 5–6, at the expense of partial inactivation, but and potassium phosphatase could be reactivated after binding of C12E8 to Bio-Beads SM2. At C12E8/protein ratios higher than 6 the activities were irreversibly lost. Inactivation could be explained by delipidation. It was not due to subunit dissociation since only small changes in sedimentation velocities were seen when the C12E8/protein ratio was increased from 2.9 to 46. As determined immediately after solubilization, was 7.4 S for the fully active , 7.3 S for the partially active particle, and 6.5 S for the inactive particle at high C12E8/protein ratios. The maximum molecular masses determined by analytical ultracentrifugation were 141 000–170 000 dalton for these protein particles. Secondary aggregation occurred during column chromatography, with formation of enzymatically active or with S and apparent molecular masses in the range 273 000–386 000 daltons. This may reflect non-specific time-dependent aggregation of the detergent micelles. 相似文献
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
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
Homogenates of adult (blood flukes), isolated from the porto-mesenteric veins of infected mice, contain substantial activities of adenylyl cyclase, cyclic AMP phosphodiesterase, and a cyclic AMP stimulated protein kinase. The adenylyl cyclase, which is largely sedimentable at 10,000xg, is stimulated 20-fold by 10mM sodium fluoride and 1.4 to 2-fold by serotonin, glucagon, prostaglandins E1, E2 or B1. The phosphodiesterase, which is largely sedimentable at 10,000xg, is inhibited by both aminophylline and papaverine but is not influenced by 10mM sodium fluoride. The protein kinase, which is present in the 10,000xg supernatant is stimulated 4 to 8-fold by either cyclic AMP or cyclic GMP. There is a preference for cyclic AMP () over cyclic GMP (). If intact worms are incubated in a glucose free medium there is a mobilization of glycogen stores which is preceded by a rise in cyclic AMP concentration. In a medium with 5mM glucose there is neither a rise in cyclic AMP nor mobilization of glycogen. 相似文献
ATPase preparations from rat brain, dog kidney, and human red blood cells also catalyze a K+-dependent phosphatase reaction. K+ activation and Na+ inhibition of this reaction are described quantitatively by a model featuring isomerization between E1 and E2 enzyme conformations with activity proportional to E2K concentration: Differences between the three preparations in for K+ activation can then be accounted for by differences in equilibria between E1K and E2K with dissociation constants identical. Similarly, reductions in produced by dimethyl sulfoxide are attributable to shifts in equilibria toward E2 conformations. Na+ stimulation of K+-dependent phosphatase activity of brain and red blood cell preparations, demonstrable with KCl under 1 mM, can be accounted for by including a supplementary pathway proportional to E1Na but dependent also on K+ activation through high-affinity sites. With inside-out red blood cell vesicles, K+ activation in the absence of Na+ is mediated through sites oriented toward the cytoplasm, while in the presence of Na+ high-affinity K+-sites are oriented extracellularly, as are those of the ATPase reaction. Dimethyl sulfoxide accentuated Na+-stimulated K+-dependent phosphatase activity in all three preparations, attributable to shifts from the E1P to E2P conformation, with the latter bearing the high-affinity, extracellularly oriented K+-sites of the Na+-stimulated pathway. 相似文献
In many instances the effect of superoxide () trapping agents in suppressing the net rate of O2 consumption of activated PMN's is not in accordance with theoretical expectations. We offer here an alternate explanation to those previously presented by Segal and Meshulam (FEBS Letters 100, 27–32) and Babior (Biochem. Biophys. Res. Comm. 91, 222–226). The paradoxical results previously presented can be explained by recognizing that shortly after activation of resting cells an O2 diffusion layer is established at or near the outer surface of these cells. The presence of this diffusion layer can markedly alter the anticipated stochiometric relationship between trapped and apparent O2 consumed by these cells when they are exposed to trapping agents. 相似文献
The interactions between calmodulin, ATP and Ca2+ on the red cell Ca2+ pump have been studied in membranes stripped of native calmodulin or rebound with purified red cell calmodulin. Calmodulin stimulates the maximal rate of by 5–10-fold and the rate of Ca2+-dependent phosphorylation by at least 10-fold. In calmodulin-bound membranes ATP activates along a biphasic concentration curve (), but in stripped membranes the curve is essentially hyperbolic (). In calmodulin-bound membranes Ca2+ activates at low concentrations () in stripped membranes the apparent Ca2+ affinities are at least 10-fold lower.The results suggest that calmodulin (and perhaps ATP) affect a conformational equilibrium between E2 and E1 forms of the Ca2+ pump protein. 相似文献
The membrane ATPase of density (age) separated human erythrocytes was examined for its stimulation by the cytosols of these cell groups. On the assumption that the stimulatory activity in the cytosol is only calmodulin, it was consistently observed that the young cytosol had a significantly higher activity towards the membrane ATPase activity (from any age group) than did the old cell cytosol. The data clearly demonstrates decided differences in the expression of calmodulin activity in cytosols from young and old erythrocytes and would support the conclusion that calmodulin activity is altered during aging of these cells. Possible mechanisms for these alterations are discussed. 相似文献
M1 cells, which are cell line cells established from myeloid leukemia cells of the SL strain mouse, can differentiate from blast cells () to mature macrophages () within 48 hr, when they are cultured with conditioned medium (CM) obtained from murine embryonic fibroblasts. While cells have no phagocytic activity nor Fc receptor (FcR), cells possess both characteristics. The appearance of FcR is temperature-dependent and inhibited by a metabolic inhibitor, cycloheximide. FcR on cells is resistant to trypsin and pronase. cells improve the viability of macrophage-depleted SL splenic lymphocytes and restore the in vitro secondary plaque forming cell response of macrophage-depleted spleen cells to particulate and soluble antigens. cells lack this macrophage-substituting capacity. Mm1 cells, mutant cells from M1 cells, having FcR and higher phagocytic activity than cells, are also devoid of this capacity. 相似文献
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
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
The kinetics of pyruvate transport across the isolated red blood cell membrane were studied by a simple and precise spectrophotometric method: following the oxidation of NADH via lactate dehydrogenase trapped within resealed ghosts. The initial rate of pyruvate entry was linear. Influx was limited by saturation at high pyruvate concentration. Pyruvate influx was greatly stimulated by increasing ionic strength in the outer but not the inner aqueous compartment. The ranged from 15.0 mM at to 3.7 mM at , while the went from to . Ionic strength was shown to affect the translocation step and not pyruvate binding. The energy of activation of pyruvate flux into resealed ghosts was 25 kcal/mol, similar to that found in intact red blood cells. Inhibitors of pyruvate influx included such anions as thiocyanate, chloride, bicarbonate, α-cyanocinnamate, salicylate and ketomalonate (but not acetate); noncompetitive inhibitors were phloretin, 1-fluoro-2,4-dinitrobenzene, 4-acetamido-4′-isothiocyanate-stilbene-2,2′-disulfonic acid and o-phenanthroline/CuSO4 mixtures. The last reagent, known to induce disulfide links in certain membrane proteins, blocked the ionic strength stimulation of pyruvate influx in this study. 相似文献
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
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
The contractility of hearts from normal fed rats is decreased by 70% during perfusion with 50 μM chloroquine, which is a potent inhibitor of endogenous lipolysis. In triacylglycerol-rich hearts, obtained by feeding rats rapeseed-oil, chloroquine depresses lipolysis much less, while contractility was found to be inhibited only 30%. In both groups of hearts the effect of chloroquine was decreased by adding fatty acids, prostaglandin E1, the ionophore X-537A or more Ca2+ to the perfusion fluid. Norepinephrine and glucagon also stimulate chloroquine-depressed hearts. The conclusion is therefore reached that fatty acids act as Ca2+-vehicles in heart cells and that chloroquine, by inhibiting lipolysis, decreases Ca2+-transport by lowering unesterified fatty acid levels. 相似文献
The apparent Arrhenius energy of activation () of the water osmotic permeability () of the basolateral plasma cell membrane of isolated rabbit proximal straight tubules has been measured under control conditions and after addition of 2.5 mM of the sulfhydryl reagent, para-chloromercuribenzenesulfonic acid (pCMBS), of mersalyl and of dithiothreitol. (kcal/mol) was (controls) and (pCMBS), while decreased with pCMBS to of its control value. Mersalyl also decreased both in vitro and in vivo (using therapeutical doses). These actions of pCMBS and mersalyl were quickly reverted with 5 mM dithiothreitol and prevented by 0.1 M thiourea. for free viscous flow is 4.2 and greater than 10 for non-pore-containing lipid membranes. By analogy with these membranes and with red blood cells, where similar effects of pCMBS on are observed, it is concluded that cell membranes of the proximal tubule are pierced by aqueous pores which are reversibly shut by pCMBS. Part of the action of mercurial diuretics can be explained by their action on . 相似文献
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
Leukemic guinea pig lymphocytes (L2 C) synthesise cholesterol in vitro at a forty-fold greater rate than normal cells. Equilibration (18 h) with lecithin or lecithin-cholesterol liposomes, respectively, enhances or suppresses sterol manufacture by normal lymphocytes but does not influence sterol production by L2 C cells. In contrast, molecules/cell of a nitroxide-derivative of androstane, (17 β-hydroxy-4′,4′-dimethylspiro [5 α-androstan-3,2′-oxazolidin]-3′-yloxyl), commonly used as a membrane spin-probe, drastically inhibit sterol production by both normal and leukemic cells (maximum within 2 h). At molecules/cell, this sterol stimulates cholesterol synthesis. 25-Hydroxycholesterol at low concentrations also stimulates sterol manufacture, whereas high concentrations are also inhibitory in both cell types. 相似文献
(1) +/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O2, NO?2 or N2O. Under optimal H+-translocation conditions, the ratios , , for reduction to N2 and for reduction to N2O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO?2 or N2O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. , for reduction to N2 and for reduction to N2O were ?0.84, ?2.33 and ?1.90, respectively. (3) The ratios, mentioned in item 2, were not altered in the presence of and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O2, NO?2 and N2O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO?2, N2O or O2 pass two H+-translocating sites. The acceptor ratios predicted by this scheme are in good agreement with the experimental values. 相似文献
L1210/R81 lymphoma cells are resistant to methotrexate (MTX) by virtue of a 35-fold elevation in dihydrofolate reductase and an inability to transport the folate antagonist drug effectively. In a phosphate-containing buffer there was little or no influx into the resistant cells at either 1 or 50 μm MTX. Replacement of this buffer with a 4-(2-hydroxyethyl)-1-piperazine-N′-2-ethanesulfonic acid-Mg2+ system resulted in an apparent influx of MTX into the resistant cells. Under these conditions, L1210/R81 cells achieved an apparent steady state at an extracellular MTX concentration of 50 μm. The apparent steady-state level of 5 nmol cells was well below the intracellular level of dihydrofolate reductase (45 nmol/109 cells). Efflux experiments at the apparent steady state indicated that 60% of the MTX was very rapidly removed from the cells by washing. Over the range of the experiment a further 20% of the MTX effluxed more slowly (). The apparent influx into the resistant cells at 5 μm MTX was inhibited 13% by sodium azide (100 μm) and initially stimulated, then inhibited, by p-chloromercuriphenylsulfonic acid (100 μm). 5-Methyltetrahydrofolate (100 μm) had little effect on the process while aminopterin (100 μm) was inhibitory (68%). Kt and V values of 2 × 10?5m and 0.31 nmol cells/min, respectively, were determined for the apparent influx in cells. Comparison of apparent MTX influx in the resistant cells with MTX transport in the sensitive cells indicates profound differences in the two processes. The evidence suggests that the apparent influx in the former cell line may consist of MTX binding to the cell membrane together with a small degree of MTX influx into the intracellular compartment. 相似文献