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1.
The distribution of activity was studied in cell fractions prepared from homogenates of rat liver. The level of mitochondrial contamination in the microsomal fraction depended on the fractionation procedure and on the method of homogenization. With proper care, microsomes with undetectable mitochondrial contamination could be prepared. These microsomes had no detectable activity. Approximately 85 % of the total activity of the post 6000 x g · min supernatant was recovered in the mitochondrialfraction. The properties of this mitochondrial were not distinguishable from those of the various microsomal previously described by other investigators. 相似文献
2.
Julián Gómez-Cambronero María L. Nieto JoséM. Mato Mariano Sánchez-Crespo 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1985,845(3):511-515
The enzyme lyso-platelet-activating factor:acetyl-CoA acetyltransferase (EC 2.3.1.67) was assayed in microsomal fractions from rat spleens. The addition of micromolar Ca2+ rapidly enhanced acetyltransferase activity and this activation was reversed by the addition of EGTA in excess of Ca2+. The effect of Ca2+ was on the apparent of the enzyme for the substrate acetyl-CoA without showing any significant effect on the of the acetylation reaction. When microsomes were isolated in the presence of 5 mM EGTA, to remove endogenous calmodulin, the same enhancing effect of Ca2+ on the acetylation reaction was observed. The addition of exogenous calmodulin to this preparation had no effect on the enzyme activity. Preincubation of spleen microsomes with the calmodulin inhibitor trifluoperazine decreased acetyltransferase in both the presence and the absence of Ca2+, indicating an effect of this drug independently of calmodulin. The addition of Mg-ATP to the assay mixture also had no effect on the acetylation reaction. These data suggest that Ca2+ modulates acetyltransferase activity from rat spleen microsomes by a mechanism that seems to be independent of calmodulin or protein phosphorylation. 相似文献
3.
NADPH reduces both liver microsomal cytochrome P-450 and cytochrome . In the presence of CO, ferrous cytochrome P-450 can slowly transfer electrons to amaranth, an azo dye. This reaction is followed by the reoxidation of cytochrome which proceeds at essentially the same rate as does cytochrome P-450 oxidation. It is suggested that cytochrome directly reduces cytochrome P-450 in rat liver microsomes. 相似文献
4.
M J Modak S L Marcus L F Cavalieri 《Biochemical and biophysical research communications》1973,55(1):1-7
Incubation of liver microsomes with cytochrome , purified after solubilization with detergents, caused an effective incorporation of the cytochrome into the microsomal membranes. The incorporated cytochrome was reducible by NADH and could not be removed by repeated washing with 0.3 M KCl or 10 mM EDTA. The incorporation was much more efficient at 37°C than at 0°C. Trypsin-solubilized cytochrome , which lacks the hydrophobic tail of the native protein, could not be inserted into the membranes. These findings confirm the view that the hydrophobic tail of the cytochrome molecule is responsible for its tight binding to the microsomal membranes. 相似文献
5.
Highly purified divalent and monovalent antibodies against cytochrome , anti- immunoglobulin G (IG) and anti- Fab', were used in elucidating the role of this cytochrome in the drug-oxidizing enzyme system of mouse liver microsomes. Anti- IG strongly inhibited not only NADH-supported but also NADPH-supported oxidation of 7-ethoxycoumarin and benzo(a)pyrene, but had no inhibitory action on the oxidation of aniline. Anti- Fab' also inhibited NADH-supported and NADPH-supported benzo(a)pyrene hydroxylation. These observations indicate an essential role of cytochrome in the transfer of electrons not only from NADH but also from NADPH to cytochrome P-450 in the microsomal oxidation of some drugs, but not of aniline. 相似文献
6.
An ATPase is demonstrated in plasma membrane fractions of goldfish gills. This enzyme is stimulated by Cl? and HCO3?, inhibited by SCN?.Biochemical characterization shows that HCO3? stimulation () is specifically inhibited in a competitive fashion by SCN? (). The residual Mg2+-dependent activity is weakly is weakly affected by SCN?.In the microsomal fraction chloride stimulation of the enzyme occurs in the presence of HCO3? (); no stimulation is observed in the absence of HCO3?. Thiocyanate exhibits a mixed type of inhibition () towards the Cl? stimulation of the enzyme.Bicarbonate-dependent ATPase from the mitochondrial fraction is stimulated by Cl?, but this enzyme has a relatively weak affinity for this substrate (). 相似文献
7.
Dorothy A. Schafer Donald E. Hultquist 《Biochemical and biophysical research communications》1980,95(1):381-387
Microsomal NADH-cytochrome reductase has been purified from bovine liver by an improved procedure which employs affinity chromatography on ADP-agarose in combination with anion exchange chromatography. The reductase was extracted from a 105,000 × microsomal pellet with Triton X-100. The overall purification from isolated microsomes was 98-fold and the yield was 10%. The preparation was nearly homogeneous on SDS-PAGE. This procedure requires less time and effort than previously described procedures. Partially purified cytochrome is also obtained. 相似文献
8.
Jose C. Gan 《International journal of biological macromolecules》1980,2(2):97-104
An material has been purified to homogeneity from the soluble fraction of normal human liver by procedures adapted from those employed for plasma . The liver material, in contrast to a previous report1 has the same molecular weight as the corresponding normal plasma . The subunit structure, immunoelectrophoretic and immunological properties of the liver glycoprotein are identical to those of normal plasma . Amino acid and carbohydrate compositions of the liver material are similar to those of obtained from the plasma. The material has also been isolated and purified from the microsomal fraction of liver It has the same molecular weight and immunological properties as glycoprotein obtained from the cytosol. Although inhibitors of lysosmal proteases were added during the homogenization of the liver, the purified glycoprotein is devoid of trypsin-inhibitory capacity. The loss of inhibitory activity could be due to extensive cellular autolysis before autopsy. 相似文献
9.
Shakunthala Narasimhulu 《生物化学与生物物理学报:生物膜》1979,556(3):457-468
The addition of cholate to the microsomes at 37.5°C resulted in a striking decrease in the apparent substrate dissociation constant () and its temperature dependency. The microsomal membranes depleted of 80% of the lipids preserved the temperature dependency of the and exhibited breaks in the Van't Hoff plot at the characteristic temperature of the lipids phase transition. The results indicate that the cytochrome is considerably restrained from expressing its maximum substrate binding potential at physiological temperature. In addition, the results indicate that the majority of the lipids apparently do not play a significant role in imposing constraint on the substratecytochrome binding reaction and in the temperature dependency of the . 相似文献
10.
Walter C. Schneider 《Biochemical and biophysical research communications》1977,74(4):1607-1612
Rat liver nuclear and cytoplasmic DNA samples were denatured and the kinetics of their reassociation was measured. About 85% of the soluble cytoplasmic (mitochondrial) DNA reannealed rapidly with a while 65% of the particulate (microsomal) DNA reassociated with a Both nucleic acids were clearly differentiated from nuclear DNA in their reassociation kinetics. The results indicate that both mitochondrial and microsomal DNA consist mainly of single components or closely related families with repetitive sequences. 相似文献
11.
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
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.
Dorothy A. Schafer Donald E. Hultquist 《Biochemical and biophysical research communications》1981,100(4):1555-1561
A protease which generates a soluble hemepeptide from bovine liver microsomal cytochrome has been isolated from the membrane fraction of rabbit reticulocytes. Inhibition by pepstatin and an acidic pH optimum indicate that the protease belongs to the acid protease class. Little cytochrome -processing activity is observed in rabbit erythrocytes. We suggest that the protease may be involved in the processing which generates the proteins of the methemoglobin reduction system from their membrane-bound precursors during the maturation of the erythroid cell. 相似文献
14.
A. Edelman C.L. Thil M. Garabédian T. Anagnostopoulos S. Balsan 《生物化学与生物物理学报:生物膜》1983,732(1):300-303
Cell membrane potential, , was monitored in rabbit hypertrophic cartilage metatarsals, amphibian proximal tubule and muscle cells during application of 1,25-dihydroxy vitamin D-3, 25-hydroxy vitamin D-3 or cholesterol (10?10M). 1,25-Dihydroxy vitamin D-3 elicited quick variations of (in less than 1 min) in proximal tubular cells (whether injected in the lumen or in peritubular capillaries) and in cartilage. The precursor 25-hydroxy vitamin D-3 and cholesterol produced a small shift of in proximal tubule only when applied from the luminal side, but this change was significantly smaller than that observed with 1,25-dihydroxy vitamin D-3. Muscle cells were unresponsive to both metabolites and cholesterol. It is concluded that rapid effects of 1,25-dihydroxy vitamin D-3 on , in target cells, are specific, most likely due to permeability changes and not related to nuclear protein synthesis; they may contribute to early modulation of cell function. 相似文献
15.
The effect of extra bound cytochrome b-5 on cytochrome P-450-dependent enzyme activities in liver microsomes 总被引:1,自引:0,他引:1
Binding of increasing amounts of detergent-purified cytochrome to rabbit liver microsomes produces a progressive inhibition of NADPH-cytochrome P-450 reductase activity which is accompanied by a similar inhibition of NADPH-supported benzphetamine demethylation. In contrast, NADH-cytochrome P-450 reductase activity in the enriched microsomes is markedly enhanced and this stimulation is accompanied by a similar increase in NADH-peroxidase activity, suggesting that cytochrome in these two reactions functions as an intermediate electron carrier to cytochrome P-450. 相似文献
16.
17.
W.McD. Armstrong W.R. Bixenman K.F. Frey J.F. Garcia-Diaz M.G. ORegan Jeanie L. Owens 《生物化学与生物物理学报:生物膜》1979,551(1):207-219
Na+, K+ and Cl? concentrations () and activities (), and mucosal membrane potentials () were measured in epithelial cells of isolated bullfrog (Rana catesbeiana) small intestine. Segments of intestine were stripped of their external muscle layers, and bathed (at 25°C and pH 7.2) in oxygenated Ringer solutions containing 105 mM Na+ and Cl? and 5.4 mM K+. Na+ and K+ concentrations were determined by atomic absorption spectrometry and Cl? concentrations by conductometric titration following extraction of the dried tissue with 0.1 M HNO3. 14C-labelled inulin was used to determine extracellular volume. was measured with conventional open tip microelectrodes, with solid-state Cl?-selective silver microelectrodes and and with Na+- and K+-selective liquid ion-exchanger microelectrodes. The average recorded was ?34 mV. , and were 51, 105 and 52 mM. The corresponding values for , and were 18, 80 and 33 mM. These results suggest that a large fraction of the cytoplasmic Na+ is ‘bound’ or sequestered in an osmotically inactive form, that all, or virtually all the cytoplasmic K+ behaves as if in free solution, and that there is probably some binding of cytoplasmic Cl?. significantly exceeds the level corresponding to electrochemical equilibrium across the mucosal and baso-lateral cell membranes. Earlier studies showed that coupled mucosal entry of Na+ and Cl? is implicated in intracellular Cl? accumulation in this tissue. This study permitted estimation of the steady-state transapical Na+ and Cl? electrochemical potential differences (Δμ̄Na and Δμ̄Cl). Δμ̄Na (?7000 J · mol?1; cell minus mucosal medium) was energetically more than sufficient to account for Δμ̄Cl (1000–2000 J · mol?1). 相似文献
18.
A gel-electrophoretically homogeneous preparation of cytochrome P-450 from liver microsomes of phenobarbital-pretreated rabbits 总被引:9,自引:0,他引:9
Cytochrome P-450 was purified from liver microsomes of phenobarbital-pretreated rabbits to a specific content of 16 to 17 nmoles per mg of protein with a yield of about 10 %. The purified cytochrome yielded only a single protein band on sodium dodecylsulfate-urea-polyacrylamide gel electrophoresis, and an apparent molecular weight of about 45,000 was estimated for the protein. The preparation was free of cytochrome , NADH-cytochrome reductase, and NADPH-cytochrome reductase activities. Aniline hydroxylase and ethylmorphine N-demethylase activities could be reconstituted upon mixing the purified cytochrome with an NADPH-cytochrome reductase preparation (purified by a detergent method) and phosphatidyl choline. 相似文献
19.
John L. McGregor Kenneth J. Clemetson Elizabeth James Phillippe Clezardin Marc Dechavanne Ernst F. Lüscher 《生物化学与生物物理学报:生物膜》1982,689(3):513-522
Washed platelets were surface-labelled by lactoperoxidase catalyzed iodination and either the platelets or membranes were solubilized in detergent and applied to a wheat germ agglutinin-Sepharose column and a Lens culinaris lectin Sepharose column coupled sequentially. The glycoproteins eluted from the lectin columns were separated by two-dimensional gel electrophoresis. Alternatively, labelled whole platelets or membranes were solubilized and then directly separated by two-dimensional polyacrylamide gel electrophoresis. Spots corresponding to specific glycoproteins identified by apparent isoelectric point (), apparent molecular weight (), staining and labelling characteristics were cut from the gels and analyzed by tryptic peptide mapping. The maps of the individual glycoproteins (GP) Ia, Ib, IIa, IIb, GP132–1354–4.5 IIIa, IIIb and IIIc were all different. Glycoproteins with the same but different were distinct with the exception of regions of GP Ib. There were minor differences in the maps of glycoproteins separated in the reduced or non-reduced state. Tryptic peptide maps provide a valuable additional parameter for the identification and characterization of platelet glycoproteins. 相似文献
20.
José Remacle 《生物化学与生物物理学报:生物膜》1980,597(3):564-576
The in vitro incorporation of cytochrome into purified plasma membranes was investigated by biochemical and immunological methods. Plasma membrane preparations incorporated three times less cytochrome than did microsomal preparations; 60% of this cytochrome could not be reduced by the NADH-cytochrome reductase and was considered as being bound to the plasma membrane. The morphological observations made after the immunochemical labeling of cytochrome clearly showed a good but asymmetrical distribution of the ferritin labeling: only the inner face of the plasma membrane incorporated cytochrome . These results are discussed with respect to theories which concern the subcellular membrane relationships in the cell. 相似文献