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1.
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
2.
The interaction between the and the adenylate cyclase enzyme systems was examined. Cyclic AMP, but not 5′-AMP, cyclic GMP or 5′-GMP, could inhibit the enzyme present in crude rat brain plasma membranes. On the other hand, the cyclic AMP inhibition could not be observed with purified preparations of enzyme. Rat brain synaptosomal membranes were prepared and treated with either NaCl or cyclic AMP plus NaCl as described by Corbin, J., Sugden, P., Lincoln, T. and Keely, S. ((1977) J. Biol. Chem. 252, 3854–3861). This resulted in the dissociation and removal of the catalytic subunit of a membrane-bound cyclic AMP-dependent protein kinase. The decrease in cyclic AMP-dependent protein kinase activity was accompanied by an increase in activity. Exposure of synaptosomal membranes containing the cyclic AMP-dependent protein kinase holoenzyme to a specific cyclic AMP-dependent protein kinase inhibitor resulted in an increase in enzyme activity. Synaptosomal membranes lacking the catalytic subunit of the cyclic-AMP-dependent protein kinase did not show this effect. Reconstitution of the solubilized membrane-bound cyclic AMP-dependent protein kinase, in the presence of a neuronal membrane substrate protein for the activated protein kinase, with a purified preparation of , resulted in a decrease in overall activity in the presence of cyclic AMP. Reconstitution of the protein kinase alone or the substrate protein alone, with the has no effect on activity in the absence or presence of cyclic AMP. Preliminary experiments indicate that, when the activated protein kinase and the substrate protein were reconstituted with the enzyme, there appeared to be a decrease in the Na+-dependent phosphorylation of the Na+-ATPase enzyme, while the K+-dependent dephosphorylation of the was unaffected. 相似文献
3.
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
4.
The of rabbit sarcoplasmic reticulum, when labelled at two Ca2+-protected sites with (NCD-4) retains Ca2+ binding capacity at the sites with values of approx. 3 μM and 0.12 mM as assessed by fluorescence titration. The sites correspond to the two high-affinity Ca2+ binding sites present in the native ATPase. The NCD-4 labelled ATPase exhibits slow conformational changes at each site on addition of Ca2+. It retains the ability to form phosphoenzyme, and can most likely translocate Ca2+. 相似文献
5.
from dog kidney lost its activity when heated at 55°C in the presence of 0.3 M 2-mercaptoethanol. Either heat treatment alone or addition of reducing agent at around 25°C caused little inactivation. One disulfide bond per protomer (mol. wt. 146000) was reduced in the inactivated sample but in active samples no reduction occurred. Neither K+-dependent phosphatase activity nor phosphoenzyme formation in the presence of Na+ was detected in the inactivated sample, suggesting that the disulfide bond was essential for the catalytic cycle of . This essential disulfide bond belonged to the β-subunit, the glycoprotein component of the enzyme, indicating that the β-subunit may be an integral component of the system. 相似文献
6.
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
7.
J.J. Schrijen W.A.H.M. Van Groningen-Luyben H. Nauta J.J.H.H.M. De Pont S.L. Bonting 《生物化学与生物物理学报:生物膜》1983,731(2):329-337
(1) A containing membrane fraction, isolated from pig gastric mucosa, has been further purified by means of zonal electrophoresis, leading to a 20% increase in specific activity and an increase in ratio of to basal Mg2+-ATPase activity from 9 to 20. (2) The target size of , determined by radiation inactivation analysis, is 332 kDa, in excellent agreement with the earlier value of 327 kDa obtained from the subunit composition and subunit molecular weights. This shows that the Kepner-Macey factor of 6.4·1011 is valid for membrane-bound ATPases. (3) The target size of is 444 kDa, which, in connection with a subunit molecular weight of 110000, suggests a tetrameric assembly of the native enzyme. The ouabain-insensitive K+-stimulated activity has a target size of 295 kDa. (4) In the presence of added Mg2+ the target sizes of the and its phosphatase activity are decreased by about 15%, while that for the is not significantly changed. This observation is discussed in terms of a Mg2+-induced tightening of the subunits composing the molecule. 相似文献
8.
A microsomal fraction rich in has been isolated from the outer medulla of pig kidney. ouabain-sensitive phosphatase activity was studied in this preparation treated with arylsulphatase, an enzyme that specifically hydrolyzes ceramide galactose-3-sulphate. The activity of phosphatase was inactivated in proportion to the amount of sulphatide hydrolyzed. A maximum inactivation of ouabain-sensitive activity was obtained with 60% of the sulphatide content hydrolyzed. The inactivation caused by arylsulphatase was partially reversed by the sole addition of sulphatide. The evidence offered in this paper about sulphatide function in the sodium pump mechanism supports the idea that sulphatides are involved in the K+-activated phosphatase, a partial reaction of the . 相似文献
9.
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
10.
The modulating effect of membrane lipids on enzyme function has been described by several investigators. We have used the spin probe methyl stearate (M 12-NSE) to study this interaction in ox brain membranes enriched with . This methyl ester of stearic acid is practically insoluble in aqueous media, and consequently spectra of M 12-NSE-labelled preparations are free of “liquid lines”.At least two types of spectra may be obtained when ox brain microsomes are spin labelled with M 12-NSE, indicating the presence of two distinct binding sites. At one site the spin label is relatively unrestricted and gives rise to an isotropic spectrum. A second spectrum, which is obtained from spin label at another site, is similar to that which is observed after incorporation of M 12-NSE into phospholipid bilayers. This suggests that this latter site is within the core of the microsomal membrane.The two binding sites differ in their affinity for the spin probe. The low affinity site is both more abundant in crude preparations and is more easily removed by detergent treatment; spin labels at this site produce isotropic spectra. The high affinity sites are fewer in number and produce broad spectra. In addition these high affinity sites increase in concentration as the enzyme undergoes purification.The two sites are quite distinct in their sensitivity to ascorbic acid, the low affinity site showing a considerably greater rate of reduction by this agent.This study also demonstrates that the delipidation effects of sodium dodecyl sulfate and sodium deoxycholate on microsomes from ox brain are not identical.It is suggested that the two spin probe binding sites represent two different lipid domains, one of which is very closely associated with the enzyme and may reflect a protein-directed phospholipid specificity for this enzyme. 相似文献
11.
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
12.
1. Extensive treatment of rabbit kidney microsomes with phosphatidylinositol-specific phospholipase C under various conditions never resulted in more than 75% hydrolysis of the substrate. 2. The non-degraded fraction of the phosphatidylinositol (10–12 nmol per mg microsomal protein) could be recovered only by an acidic extraction procedure. 3. The activity found in those membranes was not affected by this treatment. 4. Complete degradation of phosphatidylinositol could be easily achieved when the phospholipase was applied to rat liver microsomes which do not contain any detectable activity. 5. It is concluded that in rabbit kidney microsomes a close association exist between the and that fraction of the phosphatidylinositol that is directly involved in the maintenance of its activity. 相似文献
13.
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
14.
Activation of lymphocytes by antigens and mitogens can effectively be prevented by ouabain, a known inhibitor of . Recently it was shown that lowering of intracellular levels of monovalent cations is not involved in the inhibitory effect of ouabain. was found to be closely associated with acylCoA: lysophosphatidylcholine acyltransferase in the plasma membrane of lymphocytes. Both enzymes are activated as an immediate consequence of mitogen binding. Human peripheral lymphocytes were stimulated with concanavalin A. Ouabain suppressed the induction of RNA and DNA synthesis in a concentration-dependent way. Increase of RNA synthesis was suppressed only if the glycoside were added within the first hours of activation. If ouabain was added later, incorporation of uridine remained at the rate that was reached at the time of glycoside administration, pointing to an early event where ouabain may be operative. Ouabain, in a dose-dependent manner similar to that affecting RNA and DNA synthesis, inhibited the increase in the incorporation of oleate into phospholipids in stimulated lymphocytes, whereas the turnover of phospholipid fatty acids in resting lymphocytes was unaffected. Increasing extracellular K+ concentrations reversed the binding of ouabain to lymphocytes. Simultaneously, the inhibition of stimulated RNA synthesis was decreased and the inhibition of oleate incorporation was reversed. These results suggest that the suppression of lymphocyte activation by ouabain is due to the inhibition of membrane phospholipid metabolism mediated by the . 相似文献
15.
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
16.
Structural changes in the purified accompanying detergent inactivation were investigated by monitoring changes in light scattering, intrinsic protein fluorescence, and tryptophan to β-parinaric acid fluorescence resonance energy transfer. Two phases of inactivation were observed using the non-ionic detergents, digitonin, Lubrol WX and Triton X-100. The rapid phase involves detergent monomer insertion but little change in protein structure or little displacement of closely associated lipids as judged by intrinsic protein fluorescence and fluorescence resonance energy transfer. Lubrol WX and Triton X-100 also caused membrane fragmentation during the rapid phase. The slower phase of inactivation results in a completely inactive enzyme in a particle of 400 000 daltons with 20 mol/mol of associated phospholipid. Fluorescence changes during the course of the slow phase indicate some dissociation of protein-associated lipids and an accompanying protein conformational change. It is concluded that non-parallel inhibition of and activity by digitonin (which occurs during the rapid phase of inactivation) is unlikely to require a change in the oligomeric state of the enzyme. It is also concluded that at least 20 mol/mol of tightly associated lipid are necessary for either or activity and that the rate-limiting step in the slow inactivation phase involves dissociation of an essential lipid. 相似文献
17.
The kinetic parameters for the hydrolyses of different l-α-amino acid-β-naphthylamides by Bacillus subtilis aminopeptidase have been measured for the native enzyme and for the enzyme activated in 5 mm Co(NO3)2. In most cases Co2+ activation decreased Km(app) values and increased kcat values, in other cases km(app) and kcat values were increased; for the remainder of the substrates tested km(app) values and kcat values were decreased. In all cases tested the ratios of were increased (2- to 108-fold). For the native enzyme the order of specificity toward the l-amino acid-β-naphthylamides was Arg > Met > Trp > Lys > Leu and for the Co2+ activated enzyme the order of specificity was Lys > Arg > Met > Trp > Leu. The native enzyme hydrolyzed Pro-β-naphthylamide, but not α-Glu-β-naphthylamide; Co2+ activation of the enzyme affected an appreciable rate of hydrolysis of the latter substrate. 相似文献
18.
19.
Treatment by EDTA of purified plasma membranes from MF2S cells (a variant of the murine plasmacytoma MOPC 173) solubilized proteins and increased by a 1000-fold the sensitivity of to ouabain. When added back with Ca2+ to treated plasma membranes, these EDTA-solubilized proteins restored the initial sensitivity of the enzyme to its inhibitor. We report the purification of a protein of 32 000, isolated from the EDTA-treated membrane supernatant. This protein was purified by a one-step procedure involving a preparative polyacrylamide gel electrophoresis without detergent. In the presence of Ca2+ it was able to restore the original sensitivity to ouabain of from EDTA-treated membrane. This protein was shown to be similar to the β-actinin described by Maruyama by the following criteria: (1) molecular weight and amino acid composition; (2) cross-reactivity with their respective antisera; (3) in the presence of Ca2+ the same quantitative biological activity on ouabain sensitivity of the . A possible interaction between β-actinin, calmodulin and membrane-bound is discussed. 相似文献
20.
The effect of the protein structure of ( on its incorporation into liposome membranes was investigated as follows: the catalytic α-subunit of ( was split into low-molecular weight fragments by trypsin treatment and the digested enzyme was reconstituted at the same protein concentration as intact control enzyme. The reconstitution process was quantified by the average number of intramembrane particles appearing on concave and convex fracture faces after freeze-fracture of the ( liposomes. The number of intramembrane particles as well as their distribution on concave and convex fracture faces is not modified by the proteolysis. In contrast, the ATPase activity and the transport capacity of the ( decrease progessively with increasing incubation times in the presence of trypsin and are abolished when the original 100 000 molecular weight α-subunit is no longer visible by sodium dodecylsulfate gel electrophoresis. Apparently, functional ( with intact protein structure and digested, non functional enzyme consisting of fragments of the α-subunit reconstitute in the same manner and to the same extent as judged by freeze-fracture analysis. We conclude that, while trypsin treatment modifies the ( molecule in a functional sense, it appears not to modify its interaction with the bilayer in producing intramembrane particles. On the basis of our results, we propose a lipid-lipid interaction mechanism for reconstitution of (. 相似文献