共查询到20条相似文献,搜索用时 531 毫秒
1.
G. Brunner H.-G. Heidrich J.R. Golecki H.C. Bauer D. Suter P. Plückhahn E. Ferber 《生物化学与生物物理学报:生物膜》1977,471(2):195-212
Free-flow electrophoresis was used to subfractionate membrane vesicles from calf thymocyte plasma membranes. The fractionation resulted in a separation of vesicle populations bearing four different enzymes: alkaline nitrophenylphosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum) EC 3.1.3.1), γ-glutamyltransferase (EC 2.3.2.2), (ATP phosphohydrolase, EC 3.6.1.3) and acyl-CoA:lysophosphatidylcholine acyltransferase (, EC 2.3.1.23). The specific content of cholesterol and total phospholipid coincided with the distribution of membrane-bound protein. However, vesicles migrating towards the cathode had a higher molar ratio of cholesterol to phospholipid (0.75) compared to those migrating to the anode (0.55). Sodium dodecyl sulphate-gel electrophoresis of pooled vesicle fractions also demonstrates distinct differences in their protein pattern. Electron-micrographic thin sections show that the vesicle populations have a similar morphology and size distribution.These results are discussed in terms of heterogeneity of the original thymocytes, contamination with intracellular membranes and a heterogeneous structure of the plasma membrane. 相似文献
2.
J A Babitch T B Breithaupt T C Chiu R Garadi D L Helseth 《Biochimica et biophysica acta》1976,433(1):75-89
A method is described for the preparation of synaptosomes and synaptosomal membranes from chicken brain. Procedures for isolating rat synaptosomal membranes could not be used directly; several modifications of existing procedures are reported. Purity of the subcellular and subsynaptosomal fractions was monitored by electron microscopy and measurements of ferrocytochrome c: oxygen oxidoreductase (EC 1.9.3.)), monoamine: oxygen oxidoreductase (deaminating) EC 1.4.3.4), rotenone-insensitive NADH: cytochrome c oxidoreductase (EC 1.6.99.3), NADPH: cytochrome c oxidoreductase (EC 1.6.99.1), orthophosphoric monoester phosphohydrolase (EC 3.1.3.2), ATP phosphohydrolase (EC 3.6.1.4), and levels of RNA. Microsomes are the main contaminant of the synaptosomal membrane fraction. Mitochondrial and lysosomal enzymes occur in lesser amounts. No myelin contamination was observed. Marker enzymes for contaminants suggest that these synaptosomal membranes are as pure as membranes described by others, and the specific activity of a neuronal membrane marker, (Na+ -K+)-activated ATPase, is as high as other preparations. Levels of this enzyme in the membrane fraction are enriched 13-fold over homogenate ATPase levels. 相似文献
3.
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
4.
Maura Floreani Anna Chiara Bonetti Francesca Carpenedo 《Biochemical and biophysical research communications》1981,101(4):1337-1344
Intact synaptosomes prepared from rat brain were incubated with phosphatidylserine vesicles. The synaptosomes incorporated the phospholipid in proportion to its concentration in the preincubation medium. The activity of membrane-bound enzyme ATPase increased proportionally after treatment with phosphatidylserine liposomes.When breaking phosphatidylserine-enriched synaptosomes by osmotic shock or by sonication and when preparing synaptosomal membranes, the expected increase of ATPase activity was not seen. Therefore, cellular integrity was fundamental in order to see the effect of phosphatidylserine on ATPase activity. 相似文献
5.
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
6.
The subcellular distribution of adenyl cyclase was investigated in small intestinal epithelial cells. Enterocytes were isolated, disrupted and the resulting membranes fractionated by differential and sucrose gradient centrifugation. Separation of luminal (brush border) and contra-luminal (basolateral) plasma membrane was achieved on a discontinuous sucrose gradient.The activity of adenyl cyclase was followed during fractionation in relation to other enzymes, notably those considered as markers for luminal and contraluminal plasma membrane. The luminal membrane was identified by the membrane-bound enzymes sucrase and alkaline phosphatase and the basolateral region by ()-ATPase. Enrichment of the former two enzymes in purified luminal plasma membrane was 8-fold over cells and that of ()-ATPase in purified basolateral plasma membranes was 13-fold. F?-activated adenyl cyclase co-purified with ()-ATPase, suggesting a common localization on the plasma membrane. The distribution of K+-stimulated phosphatase and 5′-nucleotidase also followed ()-ATPase during fractionation. 相似文献
7.
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
8.
To determine the mechanism of the maturation of the brush border membrane in intestinal epithelial cells, purification of the plasma membrane from undifferentiated rat crypt cells and of the basal-lateral membrane from villous cells has been performed. The method is based on density perturbation of the mitochondria to selectively disrupt their association with the membrane. With both cell populations, two membrane subfractions displaying the same respective density on sucrose gradient have been obtained with an overall yield of 15–20% and a 10-fold enrichment of the plasma membrane markers 5′-nucleotidase and , ouabain-sensitive ATPase chosen to follow their purification. The four fractions constituted by sheets and apparently closed vesicles of various sizes. Each fraction was characterized by a distinct protein composition and different levels of enzyme activities. The cells, used for the preparation of the membranes, were isolated as a villus to crypt gradient. This separation and that of the membranes led to the conclusion that the ATPase is localized principally in the plasma membrane of all cells whatever their state of maturation, while 5′-nucleotidase is predominantly located in the basal-lateral membrane of the villous cells and may serve as a specific marker for the purification of this membrane. Finally it has been shown that aminopeptidase, disaccharidases and alkaline phosphatase do not appear simultaneously in the maturation process of the cells, alkaline phosphatase being absent from the crypt cells and aminopeptidase being the first to be synthesized. This enzyme seems to appear in the crypt cells membrane before being integrated into the mature brush border membrane. 相似文献
9.
R.P. Brivio-Haugland S.L. Louis K. Musch N. Waldeck M.A. Williams 《生物化学与生物物理学报:生物膜》1976,433(1):150-163
To determine whether changes in unsaturation of fatty acids in rat liver plasma membranes might alter activities of membrane-associated enzymes, liver plasma membranes were prepared from rats fed purified diets lacking or supplemented with essential fatty acids. Two methods of membrane purification were used. A similar degree of purification was obtained with both methods for both depleted and control membranes, as indicated by marker enzyme purification. The proportion of essential fatty acids of the linoleate series was significantly lower in phospholipids from depleted rats. The specific activity of 5′-nucleotidase was lower, and the activity, and apparent for total were higher in the depleted liver plasma membranes. Arrhenius plots of total ATPase activity showed a discontinuity at the same temperature for both the depleted and control membranes. Activity with the depleted membranes was higher at all temperatures tested. Supplementation of deficient rats with a source of essential fatty acids (corn oil) restored and apparent values to normal. Adenylate cyclase activity in the presence of fluoride, glucagon or glucagon plus GTP was significantly lower in the depleted plasma membranes. 相似文献
10.
The content of cytochrome c-420 in Rhodospirillum rubrum chromatophores prepared by grinding with alumina is 5–10% of that in whole cells, and 20–40% in chromatophores by ‘French’ pressing.Flash-induced phosphorylation of various chromatophores which varied in cytochrome content from 7 to 40% is proportional to the cytochrome content. Extrapolating the cytochrome c-420 content to that observed in whole cells, a ratio near 1 is calculated. At low flash intensity the phosphorylation per flash is proportional to flash energy.Photophosphorylation in flashes given after a time of several minutes is only slightly dependent on the number of flashes. If the flashes are spaced from 0.1 to 10 s, relative phosphorylation in the first flash is about 70% and in the second 90% of that observed in the following flashes. Proton binding is not affected by the cytochrome c-420 content and a ratio of of 2.3 was found.These results can be explained by a working hypothesis in which charge separation occurring at one reaction centre and the resulting electron transport mediated amongst others by c-420, results in the injection of two protons into an ATPase, this in contrast to a chemiosmotic mechanism, where the protons are released in the chromatophore inner space. 相似文献
11.
The phosphohydrolase activity of the membrane-associated adenosine triphosphatase (ATPase) of the human erythrocyte can be inhibited by micromolar or nanomolar concentrations of cyclic AMP. Millimolar concentrations of cyclic AMP are less effective. The inhibitory effect of cyclic AMP is potentiated in the presence of the phosphodiesterase inhibitor, theophylline. 相似文献
12.
The proton translocating properties of cytochrome c oxidase have been studied in artificial phospholipid vesicles into the membranes of which the isolated and purified enzyme was incorporated.Initiation of oxidation of ferrocytochrome c by addition of the cytochrome, or by addition of oxygen to an anaerobic vesicle suspension, leads to ejection of H+ from the vesicles provided that charge compensation is permitted by the presence of valinomycin and K+. Proton ejection is not observed if the membranes have been specifically rendered permeable to protons.The proton ejection is the result of true translocation of H+ across the membrane as indicated by its dependence on the intravesicular buffering power relative to the number of particles (electrons and protons) transferred by the system, and since it can be shown not to be due to a net formation of acid in the system.Comparison of the initial rates of proton ejection and oxidation of cytochrome c yields a quotient close to 1.0 both in cytochrome c and oxygen pulse experiments. An approach towards the same stoichiometry is found by comparison of the extents of proton ejection and electron transfer under appropriate experimental conditions.It is concluded that cytochrome c oxidase is a proton pump, which conserves redox energy by converting it into an electrochemical proton gradient through electrogenic translocation of H+. 相似文献
13.
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
14.
The proton translocating properties of cytochrome c oxidase in whole cells of Paracoccus denitrificans have been studied with the oxidant pulse method. quotients have been measured with endogenous substrates, added methanol and added ascorbate (+TMPD) as reductants, and oxygen and ferricyanide as oxidants. It was found that both the observed with ascorbate (+TMPD) as reductant, and the differences in proton ejection between oxygenand ferricyanide pulses, with endogenous substrates or added methanol as a substrte, indicate that the P. denitrificans cytochrome c oxidase translocates protons with a stoichiometry of . The results presented in this and previous papers are in good agreement with recent findings concerning the mitochondrial cytochrome c oxidase, and suggest unequal charge separation by different coupling segments of the respiratory chain of P. denitrificans. 相似文献
15.
16.
Ole Scharff 《生物化学与生物物理学报:生物膜》1978,512(2):309-317
The additional activation by monovalent cations of the ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied.The Ca2+-ATPase occurs in two different states. In the A-state the enzyme is virtually free of protein activator and the kinetics of Ca2+ activation is characterized by low apparent Ca2+ affinity and low maximum activity. In the B-state the enzyme is associated with activator and the kinetics is characterized by high Ca2+ affinity and high maximum activity.At optimum concentrations of Ca2+ the additional activation of the B-state by K+, NH4+, Na+ and Rb+ exceeded the corresponding activations of the A-state, and half-maximum activations by K+, NH4+, and Na+ were achieved at lower concentrations in the B-state than in the A-state. Li+ and Cs+ activated the two states almost equally but maximum activation was obtained at lower cation concentrations in the B-state than in the A-state.The activation of the B-state by the various cations decreased in the order . The A-state was activated almost equally by K+, Na+, NH4+, and Rb+ and to a smaller extent by Li+ and Cs+.At sub-optimum concentrations of Ca2+ high concentrations of monovalent cations (100 mM) activated the Ca2+-ATPase equally in the A-state and the B-state. In the absence of Ca2+ the monovalent cations inhibited the Mg2+-dependent ATPase in both types of membranes. This dependence on Ca2+ indicates that the monovalent cations interact with the Ca2+ sites in the B-state.The results suggest that K+ or Na+, or both, contribute to the regulation of the Ca2+ pump in erythrocytes. 相似文献
17.
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
18.
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
19.
(i) Purified bovine heart mitochondrial cytochrome b-c1 complex (ubiquinone-cytochrome c oxidoreductase) and photosynthetic reaction centres isolated from Rhodopseudomonas sphaeroides strain R-26 have been incorporated into lipid vesicles. In the presence of cytochrome c and ubiquinone-2, light activation caused a cyclic electron transfer involving both components. (2) Since cytochrome c is added outside the vesicles, it is both reduced by the cytochrome b-c1 complex and oxidised by the reaction centre on the outside of the vesicles. Ubiquinone-2, however, is reduced by the reaction centres at a site in contact with the inside of the vesicles, but the reduced form, ubiquinol-2, is oxidised by the cytochrome b-c1 complex at a site in contact with the outer aqueous phase. (3) In the presence of valinomycin plus K+, initiation of cyclic electron flow causes protons to move from inside the vesicles to the outer medium and the ratio was calculated to be close to 4. 相似文献
20.
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