共查询到20条相似文献,搜索用时 31 毫秒
1.
Jeffrey B. Smith Paul C. Sternweis Leon A. Heppel 《Journal of cellular biochemistry》1975,3(3):248-255
We have partially purified active delta and epsilon subunits of the E. coli membranebound Mg2+ -ATPase (ECF1). Treating purified ECF1 with 50% pyridine precipitates the major subunits (α, β, and γ) of the enzyme, but the two minor subunits (δ and ϵ), which are present in relatively small amounts, remain in solution. The delta and epsilon subunits were then resolved from one another by anion exchange chromatography. The partially purified epsilon strongly inhibits the hydrolytic activity of ECF1. The epsilon fraction inhibits both the highly purified five-subunit ATPase and the enzyme deficient in the δ subunit. The latter result indicates that the delta subunit is not required for inhibition by epsilon. By contrast, two-subunit enzyme, consisting chiefly of the α and β subunits, was insensitive to the ATPase inhibitor, suggesting that the γ subunit may be required for inhibition by epsilon. The partially purified delta subunit restored the capacity of ATPase deficient in delta to recombine with ATPase-depleted membranes and to reconstitute ATP-dependent transhydrogenase. Previously we reported (Biochem. Biophys. Res. Commun. 62:764 [1975]) that a fraction containing both the delta and epsilon subunits of ECF1 restored the capacity of ATPase missing delta to recombine with depleted membranes and to function as a coupling factor in oxidative phosphorylation and for the energized transhydrogenase. These reconstitution experiments using isolated subunits provide rather substantial evidence that the delta subunit is essential for attaching the ATPase to the membrane and that the epsilon subunit has a regulatory function as an inhibitor of the ATPase activity of ECF1. 相似文献
2.
Methods are described for purification of a vesicular membrane fraction of hog gastric mucosa using differential centrifugation, density gradient separation on zonal rotors and free-flow electrophoresis. As a result a fraction is obtained enriched 40-fold in terms of K+-ATPase and free of any other enzyme marker other than K+-activated phosphatase.the 5′-nucleotidase and basal Mg2+-ATPase are clearly separated from the latter enzymes.Osmotic shock, Triton X-100 treatment or K+ ionophores increased the K+-ATPase activity in isotonic conditions, but phosphatase is not affected by these treatments, nor is the ATPase activity in the presence of NH4+. The results suggest that the electrophoretic fraction contains a major population of tight vesicles, whose permeability to K+ is rate limiting for the ATPase activity but not for the phosphatase activity. It is concluded that K+ site for the ATPase is internal whereas the K+ site for the phosphatase is external, hence, the K+ site must be mobile across the membrane. 相似文献
3.
Christian J. Le Peuch Danielle A.M. Le Peuch Sidney Katz Jacques G. Demaille Maxwell T. Hincke R. Bredoux J. Enouf S. Levy-Toledano Jacques Caen 《生物化学与生物物理学报:生物膜》1983,731(3):456-464
Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a activity of about 10 nmol (min·mg)?1 and an ATP-dependent Ca2+ uptake of about 10 nmol (min·mg)?1. When incubated in the presence of Mg[γ-32P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [32P]phosphate-labeled Ca2+ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca2+ or Ca2+ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of 125I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol (). Some minor calmodulin-binding proteins were enriched in the membrane fractions (). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca2+ uptake is essentially unaltered, while the Ca2+ capacity is diminished as a consequence of a doubling in the rate of Ca2+ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca2+ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 μM calmodulin result in increased levels of vesicle phosphorylation. 相似文献
4.
The DCCD-binding polypeptide is close to the F1 ATPase-binding site on the cytoplasmic surface of the cell membrane of Escherichia coli 总被引:2,自引:0,他引:2
Removal of the F1 ATPase from membrane vesicles of resulted in leakage of protons across the membrane through the FO portion of the ATPase complex. The leakage of protons was prevented by antiserum to the N,N′-dicyclohexylcarbodiimide (DCCD)-binding polypeptide in everted but not in “right-side out” membrane vesicles. The antiserum prevented the rebinding of F1 ATPase to F1-stripped everted membrane vesicles. It is concluded that in F1-depleted vesicles the DCCD-binding polypeptide is exposed on the cytoplasmic surface of the cell membrane at or close to the binding site of the F1 ATPase. 相似文献
5.
Cholinergic synaptic vesicles from the electric organ of have been subjected to analytical scale separation techniques not utilized in the isolation procedure, and the ATPase activity of separated fractions determined. Most of the ATPase activity migrated with the vesicles. Sensitivity of the ATPase activity to 16 potential inhibitors also was determined. Most of the ATPase activity was inhibited by low concentrations of 4-chloro-7-nitrobenzo-oxadiazole (NBD-C1) and dicyclohexylcarbodiimide (DCCD), but not by a water soluble carbodiimide. The close association of the ATPase with the vesicles and the pattern of inhibition obtained provide further support for the authentic presence of a membrane bound ATPase in the cholinergic synaptic vesicle. 相似文献
6.
S D Dunn 《Biochemical and biophysical research communications》1978,82(2):596-602
ATPase activity was restored to the inactive coupling factor, F1ATPase, of strain AN120 () by reconstitution of the dissociated complex with an excess of wild-type α subunit. Large excesses of α gave the highest levels of activity. The other subunits which are required for the reconstitution of ATPase activity, β and γ, did not complement the mutant enzyme. These results indicate that the α polypeptide of the AN120 ATPase is defective. 相似文献
7.
J E Ekholm S D Shukla D J Hanahan 《Biochemical and biophysical research communications》1981,103(2):407-413
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
8.
Membrane vesicles from Escherichia coli wild type and an otherwise isogenic mutant were used to immunize rabbits. In addition, a membrane protein fraction, containing the material found deficient in A mutants, was purified by preparative polyacrylamide gel electrophoresis in sodium dodecylsulfate, and used for immunization. The antisera produced were analyzed by immunoelectrophoresis and immunofluorescence microscopy. The antisera obtained by immunization with membrane vesicles from either wild type or mutant membrane preparations were qualitatively similar in the precipitin bands seen after immunoelectrophoresis. The antisera obtained by immunization with the purified protein fraction contained a subset of the antibodies seen when whole vesicles were used for immunization. In a semiquantitative precipitin assay, the antisera prepared against whole membrane vesicles or the isolated protein fraction both caused the precipitation of more protein from sodium dodecylsulfate-solubilized membranes of wild type than of mutants. No difference was seen by immunoelectrophoresis between the protein composition of wild type or membrane preparations. Thus, the mutant appears to differ from the wild type in the quantitative composition of its membrane proteins, whereas no qualitative differences were detected.Fluorescein-conjugated antiserum preparations were employed to assess the reactivity of intact cells, spheroplasts and membrane vesicles with the antisera studied above. Wild type cells of E. coli have a barrier to reaction with the antisera; this barrier is removed when the cells are converted to spheroplasts or to membrane vesicle. Similarly, a highly permeable mutant of E. coli permits reaction of the antisera with unaltered cells. Antisera to both whole membrane vesicles and to the isolated protein fraction react identically with the cellular and subcellular preparations. Thus, antisera prepared from membrane proteins isolated after sodium dodecylsulfate-polyacrylamide gel electrophoresis can still recognize some antigens present in membrane vesicle preparations. 相似文献
9.
Barry J. Bowman Stanley E. Mainzer Kenneth E. Allen Carolyn W. Slayman 《生物化学与生物物理学报:生物膜》1978,512(1):13-28
A comparative study has been made of the effects of a variety of inhibitors on the plasma membrane ATPase and mitochondrial ATPase of Neurospora crassa. The most specific inhibitors proved to be vanadate and diethylstilbestrol for the plasma membrane ATPase and azide, oligomycin, venturicidin, and leucinostatin for mitochondrial ATPase. , octylguanidine, triphenylsulfonium chloride, and quercetin and related bioflavonoids inhibited both enzymes, although with different concentration dependences. Other compounds that were tested (phaseolin, fusicoccin, deoxycorticosterone, alachlor, salicyclic acid, , triiodobenzoic acid, cyclic AMP, cyclic GMP, theobromine, theophylline, and histamine) had no significant effect on either enzyme. Overall, the results indicate that the plasma membrane and mitochondrial ATPases are distinct enzymes, in spite of the fact that they may play related roles in H+ transport across their respective membranes. 相似文献
10.
Hiromichi T. Narahara Vincent G. Vogrin John D. Green Ronald A. Kent Michael K. Gould 《生物化学与生物物理学报:生物膜》1979,552(2):247-261
A new technique for isolating fragmented plasma membranes from skeletal muscle has been developed that is based on gentle mechanical disruption of selected homogenate fractions. , Mg2+-dependent ATPase was used as an enzymatic marker for the plasma membrane, Ca2+-stimulated, Mg2+-dependent ATPase as a marker for sarcoplasmic reticulum, and succinate dehydrogenase for mitochondria. Cell Cell segments in an amber low-speed () pellet of a frog muscle homogenate were disrupted by repeated gentle shearing with a Polytron homogenizer. Sarcoplasmic reticulum was released into the low-speed supernatant, whereas most of the plasma membrane marker remained in a white, fluffy layer of the sediment, which contained sarcolemma and myofibrils. Additional gentle shearing of the white low-speed sediment extracted plasma membranes in a form that required centrifugation at for pelleting. This pellet, the fragmented plasma membrane fraction, had a relatively high specific activity of ATPase compared with the other fractions, but it had essentially no Ca2+-stimulated ATPase activity and only a small percentage of the succinate dehydrogenase activity of the homogenate.Experimental evidence suggests that the fragmented plasma membrane fraction is derived from delicate transverse tubules rather than from the thicker, basement membrane-coated sarcolemmal sheath of muscle cells. Electron microscopy showed small vesicles lined by a single thin membrane. Hydroxyproline, a characteristic constituent of collagen and basement membrane, could not be detected in this fraction. 相似文献
11.
Glyphosine (N,N-bis(phosphonomethyl)glycine) is known to increase sucrose levels in sugarcane and to cause chlorosis in maize and other plants. It has been suggested (Crofts, S.M., Arntzen, C.J., Vanderhoef, L.N. and Zettinger, C.S. (1974) Biochim. Biophys. Acta 335, 211–217) that its primary mode of action is to inhibit the synthesis of plastid rRNA. Growth of Lemna gibba L. G-3 on 5 · 10?4M glyphosine causes the plants to produce fronds lacking chlorophyll. The plastids in these white fronds contain only a few internal membrane structures, some of which are stacked. Sodium dodecyl sulfate polyacrylamide gel electrophoresis shows an accumulation of substantial amounts of both the large and small subunits of ribulosebisphosphate carboxylase by the white fronds. The membrane fraction from these fronds contains only traces of the light-harvesting chlorophyll apoprotein in comparison to control plants. In vivo labeling and immunoprecipitation show that the large subunit of ribulose-bisphosphate carboxylase is actively synthesized by the white fronds. However, labeling of the chlorophyll apoprotein and a 32000 dalton protein in the membrane fraction is extremely low compared to control plants. We conclude that in Lemna, glyphosine differentially affects the synthesis and/or processing of soluble proteins and some membrane chloroplast proteins, and could be useful in understanding the biogenesis of chloroplast membranes. 相似文献
12.
Hans-Jürgen Misselwitz Horst Will Wolfgang Schulze Liane Will-Shahab Albert Wollenberger 《生物化学与生物物理学报:生物膜》1979,553(2):197-212
Cell surface membrane fragments were isolated and purified by successive rate zonal and isopycnic centrifugation of calcium oxalate-loaded pigeon heart microsomes in sucrose density gradients. The most highly purified cell membrane fraction sediments at a buoyant density of 1.105 g/ml. Some of the membrane pieces are present as open fragments and leaky vesicles, while others form tightly sealed vesicles of both inside-in and inside-out membrane orientation. The pigeon heart cell membrane preparation exhibits high ( and adenylate cyclase activities. Additional activity of these enzymes is uncovered by sodium dodecyl sulfate and alamethicin, respectively. Electron microscopic inspection of the cell surface membrane preparation revealed (a) a predominance of thick-walled vesicles with smooth surfaces on negative staining and (b) binding of concanavalin A to the bulk of isolated membrane pieces following their incubation with the lectin. 相似文献
13.
Ilkka Kouvonen 《生物化学与生物物理学报:生物膜》1981,646(2):268-274
The pig intestinal intrinsic factor receptor has been isolated and dissociated into its α and β subunits. The β subunit was found to be more hydrophobic than the α subunit. In a detergent solution only the α subunit was accessible to digestion with papain. The whole isolated receptor was introduced into artificial single bilayer liposomes where is apparently was randomly oriented. Liposomes containing the receptor were digested with papain and the polypeptide segments that stayed in the lipid fraction were extracted and analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Four species were found with values of 23 000, 45 000, 70 000 and 86 000. 相似文献
14.
Canine trachealis was homogenized and the various membrane fractions isolated by differential centrifugation and discontinuous sucrose gradient centrifugation. A membrane fraction enriched in the plasma membrane marker enzymes 5′-nucleotidase (5-fold) and K+-activated ouabain sensitive p-nitrophenylphosphatase (3-fold) was obtained. The fraction contained very low levels of the inner mitochondrial marker succinate: cytochrome c oxidoreductase. These plasma membrane vesicles showed higher ATP-dependent Ca-uptake (20 μmoles/g protein) than any other submicrosomal fraction. The active Ca-uptake was enhanced by oxalate. The Ca taken up by the plasma membrane vesicles was released instantaneously by dilution in 5m EGTA and 10μ A23187 and more slowly by dilution only in 5m EGTA. 相似文献
15.
Short, mild treatments of sarcoplasmic reticulum vesicles with aqueous from methanol to caused an inhibition of calcium uptake and an enhancement of ATPase activity. The treatments increased both calcium-dependent (extra) ATPase activity and calcium-independent (basic) ATPase activity of vesicles. The apparent initial reaction rate of ATPase of vesicles was about twice that of control vesicles. With increasing number () of carbon atoms of the , the maximum increment of ATPase activity increased, and both the alcohol concentration () required to inhibit calcium uptake by 50% and the alcohol concentration () required to enhance ATPase activity by 50% of the maximum increment of ATPase activity decreased as follows. The ratio, to , was constant for all values. The apparent free energy of binding of the methylene groups of to sarcoplasmic reticulum vesicles was evaluated (?796 cal/mole) and compared with data from the partition of in octanol and water (?670 cal/mole). The effects of on membrane vesicles are discussed on the basis of these data. 相似文献
16.
A Abrams C Jensen D H Morris 《Biochemical and biophysical research communications》1976,69(3):804-811
ATPase extracted from membranes was purified by preparative slab gel electrophoresis in the presence of Mg++ (plus Mg2+ ATPase) and without Mg2+ (minus Mg2+ ATPase). The subunit composition and membrane binding capacity of both preparations was then examined. The plus Mg2+ ATPase had 5 types of subunits (αβγδ?) and reattached normally to depleted membranes. The minus Mg2+ ATPase had the αβγ and ? chains, but no δ chain, and failed to reattach to membranes. These data indicate that Mg2+ or a similar cationic ligand anchors the δ chain to the core enzyme complex and that the δ chain in turn is needed for membrane attachment. For the plus Mg2+ ATPase the data are consistent with the subunit stoichiometry and arrangement, (α3β3 γ ?)-Mg2+)n?(δ). 相似文献
17.
Adenocarcinoma of the human exocrine pancreas: presence of secretin and caerulein receptors 总被引:2,自引:0,他引:2
A Estival F Clemente A Ribet 《Biochemical and biophysical research communications》1981,102(4):1336-1341
We have measured the cytochrome compositions of subfractions derived from appressed and non-appressed thylakoids by centrifugation and aqueous two-phase partition. Cytochrome -559 (HP) was not detectable in the fraction derived from non-appressed thylakoids. Cytochromes -563 and -559 (LP) were all evenly distributed throughout the thylakoid membrane. This distribution points to plastocyanin as a possible lateral shuttle of reducing equivalents between spatially separated photosystems.Cytochrome was accessible to externally added plastocyanin in the inside-out vesicles but not in vesicles of normal sidedness. This strongly supports a location at the inner side of the thylakoid membrane. Cytochrome -563 was slowly reduced by dithionite in vesicles with both normal and inside-out orientation suggesting a location within the membrane interior. 相似文献
18.
ATPase of Escherichia coli: purification, dissociation, and reconstitution of the active complex from the isolated subunits. 总被引:5,自引:0,他引:5
A simple procedure for the purification of Mg2+-stimulated ATPase of Escherichia coli by fractionation with poly(ethylene glycols) and gel filtration is described. The enzyme restores ATPase-linked reactions to membrane preparations lacking these activities. Five different polypeptides (alpha, beta, gamma, delta, epsilon) are observed in sodium dodecyl sulfate electrophoresis. Freezing in salt solutions splits the enzyme complex into subunits which do not possess any catalytic activity. The presence of different subunits is confirmed by electrophoretic and immunological methods. The active enzyme complex can be reconstituted by decreasing the ionic strength in the dissociated sample. Temperature, pH, protein concentration, and the presence of substrate are each important determinants of the rate and extent of reconstitution. The dissociated enzyme has been separated by ion-exchange chromatography into two major fragments. Fragment IA has a molecular weight of about 100000 and contains the alpha, gamma, and epsilon polypeptides. The minor fragment, IB, has about the same molecular weight but contains, besides alpha, gamma, and epsilon, the delta polypeptide. Fragment II, with a molecular weight of about 52000, appears to be identical with the beta polypeptide. ATPase activity can be reconstituted from fragments IA and II, whereas the capacity of the ATPase to drive energy-dependent processes in depleted membrane vesicles is only restored after incubation of these two fractions with fraction IB, which contains the delta subunit. 相似文献
19.
E Padan S Schuldiner H R Kaback 《Biochemical and biophysical research communications》1979,91(3):854-861
Extraction of ML 308-225 membrane vesicles with cholate yields a particulate fraction containing 10 to 15% of the phospholipid and about 70% of the protein of intact vesicles. Addition of phospholipid to the particulate fraction in the presence of cholate, followed by sonication and removal of detergent by gel filtration yields a vesicular preparation that exhibits carrier function as judged by transient increases in 6′-(N-dansyl)aminohexyl-1-thio-β-D-galactopyranoside fluorescence in the presence of either a lactose diffusion gradient or an artificially-generated membrane potential (interior negative). Activity is not observed in the absence of phospholipid, in the presence of N-ethylmaleimide or in analogous preparations from ML 30 vesicles that do not contain the β-galactoside transport system. 相似文献
20.
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