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1.
Yoshiyuki Ichikawa 《生物化学与生物物理学报:生物膜》1975,394(3):406-415
Optical and magnetic studies were made on subfractions of rabbit kidney cortex. Cytochrome and cytochrome mixed function oxidase systems were localized mainly in the brush border membranes and microsomes. Cytochrome mixed function oxidases in the membranes comprised both an NADPH-dependent system and an NADH-dependent system. 相似文献
2.
A smooth microsomal fraction (smooth II microsomes) was earlier isolated and characterized in a number of investigations. Using a three-layer discontinuous sucrose gradient containing Mg2+ this fraction was divided into two subfractions (IIa and IIb) by a single centrifugation. The smooth IIa fraction proved to be a purified smooth microsomal fraction of specific composition. It contains high amounts of cytochromes and P-450, low activities of other electron transport enzymes and glucose-6-phosphatase, and no UDP-glucuronic acid transferase. No membrane or enzyme synthesis is induced in this subfraction by treatment with phenobarbital or methylcholanthrene. It appears that the membranes of smooth IIa microsomes derive from the smooth endoplasmic reticulum and are devoted to specific functions. 相似文献
3.
In previous reports from various laboratories, the levels of the microsomal cytochromes b5 and P-450 in hepatocytes in primary culture have been found to be very low and difficult to measure. The studies reported in this paper demonstrate that cytochromes b5 and P-450 in hepatocytes cultured on floating collagen membranes for periods of at least 10 days are maintained at levels readily measured by conventional techniques and comparable to those of liver . Addition of high levels of hydrocortisone (10?4M) to the culture medium for periods up to 10 days resulted in further increases in the levels of these cytochromes. Cells cultured in the presence of hydrocortisone exhibited the appearance of cytochrome P-448, in contrast to the cells cultured in the absence of hydrocortisone, where cytochrome P-450 was maintained. 相似文献
4.
Exposure of intact brush border membrane vesicles of hog kidney cortex to cholesterol oxidase resulted in 24% oxidation of membrane cholesterol compared with more than 95% oxidation of cholesterol in lipids isolated from membranes, showing that cholesterol is asymmetrically distributed in membranes. Phospholipase C, hydrolyzed 76% of phosphatidylcholine and 10–12% phosphatidylethanolamine while phosphatidylserine was not hydrolyzed, thus indicating that majority of phosphatidylcholine is present on the outer surface of these vesicles while phosphatidylethanolamine and phosphatidylserine are present on the inner surface. Methylation of phospholipids in brush border membrane with resulted in the formation of , and phosphatidylcholine from endogenous phosphatidylethanolamine. The for was 1·10?4 M with an optimum pH 9.0 for the formation of all three methyl derivatives. Mg2+ was without any effect between pH 5 to 10. Addition of exogenous mono- and dimethylphosphatidylethanolamine derivatives enhanced methyl group incorporation by 4–5-fold as compared to the addition of phosphatidylethanolamine. The conversion of endogenous phosphatidylethanolamine to or addition of exogenous phosphatidylmonomethylethanolamine to brush border membrane did not result in a change in bulk membrane fluidity as determined by fluorescence polarization of diphenylhexatriene. Methylation of phosphatidylethanolamine in brush border membrane did not affect the Na+-dependent uptake of either d-glucose or phosphate, although the accessibility of cholesterol in membrane to cholesterol oxidase was diminished by 21%, presumably due to altered flip-flop movement of cholesterol in the membrane. 相似文献
5.
The kinetic behaviours of cytochrome -563 and cytochrome are shown to be consistent with their participation in coupled cyclic electron flow in intact chloroplasts. Electron transfer between cytochromes -563 and cytochrome is antimycin sensitive. Fluorescence induction studies indicate that plastoquinone may function in a coupled step between the cytochromes. 相似文献
6.
The presence of Ca2+-ATPase activities with high-affinity sites for Ca2+ in brush border as well as basolateral plasma membranes of rat duodenal epithelium has been reported previously (Ghijsen, W.E.J.M. and van Os, C.H. (1979) Nature 279, 802–803). Since both plasma membranes contain alkaline phosphatase (EC 3.1.3.1), which also can be stimulated by Ca2+, the substrate specificity of Ca2+-induced ATP-hydrolysis has been studied to determine whether or not alkaline phosphatase and Ca2+-ATPase are two distinct enzymes. In basolateral fragments, the rate of Ca2+-dependent ATP-hydrolysis was greater than that of ADP, AMP and at Ca2+ concentrations below 25 μM. At 0.2 mM Ca2+ the rates of ATP, ADP, AMP and hydrolysis were not significantly different. In brush border fragments the rates of ATP, ADP and AMP hydrolysis were identical at low Ca2+, but at 0.2 mM Ca2+, Ca2+-induced hydrolysis of ADP and AMP was greater than either ATP or . Alkaline phosphatase in brush border and basolateral membranes was inhibited by 75% after addition of 2.5 mM theophylline. Ca2+-stimulated ATP hydrolysis at 1 μM Ca2+ was not sensitive to theophylline in basolateral fragments while the same activity in brush border fragments was totally inhibited. At 0.2 mM Ca2+, Ca2+-induced ATP hydrolysis in both basolateral and brush border membranes was sensitive to theophylline. Oligomycin and azide had no effect on Ca2+-stimulated ATP hydrolysis, either at low or at high Ca2+ concentrations. Chlorpromazine fully inhibited Ca2+-stimulated ATP hydrolysis in basolateral fragments at 5 μM Ca2+, while it had no effect in brush border fragments. From these results we conclude that, (i) Ca2+-ATPase and alkaline phosphatase are two distinct enzymes, (ii) high-affinity Ca2+-ATPase is exclusively located in basolateral plasma membranes, (iii) alkaline phosphatase activity, present on both sides of duodenal epithelium, is stimulated slightly by low Ca2+ concentrations, but this Ca2+-induced activity is inhibited by theophylline and shows no specificity with respect to ATP, ADP or AMP. 相似文献
7.
Mitochondria isolated from rats chronically fed ethanol demonstrated a marked inability to produce energy. The respiratory control ratio, the ADP/O ratio and state 3 respiration rates were all decreased. Coupled with other data, a progression of ethanol-induced changes is proposed with site I being altered prior to site II. Quantitation of mitochondrial cytochromes revealed decreases in cytochromes and and an increase in . Evaluation of respiration activity in relation to temperature showed ethanol-induced changes in the transition temperature () which may have been related to changes in the lipid composition of the inner membrane. Mitochondrial membranes were separated, and analysis of fatty acids and phospholipids was performed. Various fatty acids were altered in both membranes; however, the outer membrane was altered more severely. A decrease in the arachidonate : linoleate ratio was observed only in the outer membrane; however, there was no ethanol-induced change in degree of unsaturation in either membrane. Phospholipid quantitation showed a reduction of total lipid phosphorous/mg protein in both membrane fractions; however, the inner membrane was most affected. Cardiolipin was the only phospholipid in this membrane which remained unaltered. The evidence indicates that the mechanism for ethanol-induced damage to the liver mitochondrion involves lipid compositional changes as well as changes in cytochromes and possibly other proteins. 相似文献
8.
The purpose of this report is to review the current literature on cytochrome 5 in hepatic microsomes and to draw conclusions as to its role in microsomal electron transfer pathways. For details concerning the history of cytochrome 5 the reader is reffered to reviews by C. F. Strittmatter (1) and P. Strittmatter (2). For information on the chemistry of cytochrome 5 the reader is reffered to the papers by Ozols and Strittmatter (3), Kajihara and Hagihara (4), and Ehrenberg and Bois-Poltoratsky (5). For more recent studies on the isolation and properties of detergent solubilized cytochrome 5, which contains a hydrophobic peptide enabling reincorporation into membranes, the reader is referred to references 6-12.For simplicity, this minireview is divided into four parts, reflecting areas of study on the role of cytochrome 5 in the microsomes. One major area is in fatty acid 9 desaturation. Two other areas concern cytochrome 5 involvement in cytochrome P-450 mediated mixed function oxidations. The fourth section deals with other non-cytochrome P-450 pathways in which cytochrome 5 is suggested as being a component. 相似文献
9.
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
10.
F. Bode K. Baumann R. Kinne C. Hornung R. Maier G. Zimmerschied 《生物化学与生物物理学报:生物膜》1976,433(2):294-310
Pinocytic vesicles, brush border microvilli, lysosomes and basolateral plasma membranes were isolated from rat kidney cortex and their biochemical composition and membrane turnover compared. Pinocytic vesicles are devoid of marker enzymes of brush border microvilli, such as alkaline phosphatase and 5′-nucleotidase, and of lysosomes, such as acid phosphatase and β-glucuronidase. The protein pattern as revealed by polyacrylamide gel electrophoresis differs for all four membranes. Analysis of the phospholipid composition shows that pinocytic vesicles are rich in the negatively charged phospholipid phosphatidylserine and have a low content of sphingomyelin and phosphatidylethanolamine.[14C]guanido-arginine, [3H]fucose and were preferentially incorporated into the pinocytic vesicles. Using a double label technique with leucine also, evidence of a more rapid turnover of the pinocytic vesicle membrane proteins was obtained.The results suggest that pinocytic vesicles are not derived from the brush border microvillous membrane but are independent entities that are newly synthesized during the pinocytic process. 相似文献
11.
Åke Elhammer Gustav Dallner Tsuneo Omura 《Biochemical and biophysical research communications》1978,84(3):572-580
The distribution and site of synthesis of cytochrome was studied by antibody precipitation of the enzyme labeled . The enzyme is present in rough and smooth microsomes, Golgi and outer mitochondrial membranes. The cytochrome is synthesized only on bound ribosomes, where glucosamine and galactose moieties are also added. The enzyme seems to be devoid of mannose and sialic acid residues. 相似文献
12.
Evidence for plastoquinol-cytochrome f/b-563 reductase as a common electron donor to P700 and cytochrome oxidase in cyanobacteria 总被引:6,自引:0,他引:6
G A Peschek G Schmetterer 《Biochemical and biophysical research communications》1982,108(3):1188-1195
Membranes isolated from sp. strain MAC and displayed spectral changes in the cytochrome region when examined by reduced oxidized or dual wavelength spectrophotometry under physiological conditions. The same changes accompanied both light-induced (photosynthetic) and oxygen-induced (respiratory) electron transport. Physiological reduction of the cytochrome moiety was abolished after extraction of plastoquinone but reappeared on reconstitution of the depleted membranes with authentic plastoquinone. Moreover, a mutual inhibition of photosynthetic and respiratory activities could be directly demonstrated with the isolated membranes. From the results it is concluded that the membrane-bound plastoquinol-cytochrome reductase functions as a common electron donor to both P700 and the cytochrome oxidase in cyanobacteria. 相似文献
13.
Membrane proteins of the intestinal brush border were labelled in vivo by intraluminal injection of diazotised [125I]iodosulfanilic acid, a highly polar molecule. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of brush border membranes labelled in this manner showed 20 protein bands, 11 of which contained significant radioactivity. The most heavily labelled proteins had molecular weights greater than 150 000, indicating that they were the most exposed to the intestinal lumen. Little radioactivity was detected in proteins with molecular weights of less than 94 000. The majority of these smaller proteins were likely to have been brush border core proteins. The evidence that diazotised [125I]iodosulfanilic acid bound primarily to brush border membrane proteins when administered in this way, was: (a) the specific activity of brush border proteins was up to 3-fold greater than that of total cell particulate proteins (pelleted at 27 000 × from mucosal homogenates); (b) principal peaks in the gel radioactivity profile of total cell particulate proteins corresponded to the most heavily labelled proteins of the isolated brush border membrane; and (c) brush border core proteins showed minimal radioactivity in vivo, but considerably higher radioactivity when brush border membranes were labelled in vitro. A small amount of label was absorbed across the intestinal mucosa. However, secondary labelling of brush border proteins by this absorbed label was minimal, since the specific activity of brush border proteins in jejunum adjacent to the labelled loop was only 0.22% of the level for those proteins in the labelled segment. Since this technique did not affect the cellular morphology, enzyme activity or biochemical integrity of the membrane, it should prove useful as a means of accurately studying in vivo turnover rates of brush border membrane proteins. 相似文献
14.
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
15.
Addition of -nitroanisole to a reaction mixture containing phenobarbital-pretreated rabbit liver microsomes brings about an increase the reoxidation rate of NADH-reduced cytochrome b5. Addition of partially purified cytochrome b5 to a solution containing microsomes results in a marked increase in both NADH- and NADPH-dependent O-demethylation of -nitroanisole. -Nitroanisole also increases the rate of NADH mediated cytochrome P-450 reduction. From these and other results described in the Discussion section, we confirm that electrons required for NADH-dependent O-demethylation of -nitroanisole is transfered from NADH to cytochrome P-450 via cytochrome b5 and that cytochrome P-450 is the enzyme which catalyzes -nitroanisole O-demethylation. 相似文献
16.
A green mutant of Rhodopseudomonas spheroides was isolated in which spectroscopic measurements of the α-band region of cytochromes could be made. It was grown either aerobically or photosynthetically, and the membrane fractions prepared from cells of each type. Anaerobic potentiometric titration at 560 nm minus 542 nm showed the same three redox components, tentatively identified as b-type cytochromes, in membrane fractions from either type of cell. The mid-point potentials were approximately +185, +41 and ?104 mV. In membranes from photosynthetically grown cells the major cytochrome form absorbing at 560 nm had a mid-point potential of +42 mV; in aerobically grown cells the major form had a potential of +185 mV. In both types of cell only one c-type cytochrome was found, with a mid-point potential of +295 mV. An a-type cytochrome was present only in aerobically-grown cells.Substrate-reduced particles from these cells were mixed with air-saturated buffer in a stopped-flow spectrophotometer and the kinetics of oxidation of b- and c-type cytochromes were measured. The same two b-type components, reacting with pseudo first order kinetics, were detected in particles from both aerobically and photosynthetically grown cells ( for oxidation 1.3 s and 0.13 s). The c-type cytochrome of particles from aerobically grown cells was oxidised with of 0.97 s; the c-type cytochrome of photosynthetic cells was oxidised faster, with of 0.27 s.These observations have implications on the adaptive formation of electron transport systems that are discussed. 相似文献
17.
18.
NADPH-dependent reductase solubilized from microsomes by peroxidation and its activity 总被引:1,自引:0,他引:1
Johan Högberg Robert E. Larson Annika Kristoferson Sten Orrenius 《Biochemical and biophysical research communications》1974,56(3):836-842
Isolated rat liver microsomes were subjected to enzymatic or non-enzymatic lipid peroxidation . NADPH-dependent cytochrome reductase activity was released from the microsomes into the media during peroxidation. This activity could be recovered from the media by DEAE-cellulose chromatography. The recovered enzyme retained high activity for the reduction of cytochrome and a lower level of activity for the reduction of cytochrome P-450. The active fractions were capable of enzymatically supporting the peroxidation of isolated mitochondria in the presence of organically complexed Fe+3 and NADPH, and in this respect the specific activity was found to be about ten times higher than in microsomes. 相似文献
19.
Renal basal-lateral and brush border membrane preparations were phosphorylated in the presence of [γ-32P]ATP. The 32P-labeled membrane proteins were analysed on SDS-polyacrylamide gels. The phosphorylated intermediates formed in different conditions are compared with the intermediates formed in well defined membrane preparations such as erythrocyte plasma membranes and sarcoplasmic reticulum from skeletal muscle, and with the intermediates of purified renal enzymes such as and alkaline phosphatase. Two Ca2+-induced, hydroxylamine-sensitive phosphoproteins are formed in the basal-lateral membrane preparations. They migrate with a molecular radius of about 130 000 and 100 000. The phosphorylation of the 130 kDa protein was stimulated by La3+-ions (20 μM) in a similar way as the from erythrocytes. The 130 kDa phosphoprotein also comigrated with the erythrocyte . In addition in the same preparation, another hydroxylamine-sensitive 100 kDa phosphoprotein was formed in the presence of Na+. This phosphoprotein comigrates with a preparation of renal . In brush border membrane preparations the Ca2+-induced and the Na+-induced phosphorylation bands are absent. This is consistent with the basal-lateral localization of the renal Ca2+-pump and Na+-pump. The predominant phosphoprotein in brush border membrane preparations is a 85 kDa protein that could be identified as the phosphorylated intermediate of renal alkaline phosphatase. This phosphoprotein is also present in basal-lateral membrane preparations, but it can be accounted for by contamination of those membranes with brush border membranes. 相似文献
20.
The role of cytochrome 5 in the -nitroanisole O-demethylation was studied with a reconstituted system containing a unique cytochrome P-450, isolated from rabbit liver microsomes as a species with a high affinity for cytochrome 5. The maximal activity was obtained in the complete system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, NADH-cytochrome 5 reductase, and Triton X-100 in addition to cytochrome 5. The omission of cytochrome 5 from the complete system entirely abolished the activity. These results clearly show that cytochrome 5 is obligatory in the reconstitute p-nitroanisole O-demethylation system, and this cytochrome P-450 probably interacts with cytochrome 5 in such a way that the second electron is transferred from cytochrome 5 and thus exhibits the demethylase activity. 相似文献