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1.
2.
Previous studies in our laboratory had demonstrated that addition of alpha-naphthoflavone (ANF) to lymphocytes from smokers or polychlorinated biphenyls (PCB)s-exposed individuals caused an increase in sister chromatid exchange (SCE) frequency whereas lymphocytes from controls were relatively unaffected. In order to investigate the mechanism responsible, metabolism of ANF by uninduced and 2,3,7,8-tetrachlorodibenzodioxin (TCDD)-induced microsomes was studied as a function of microsomal protein concentration and incubation time. Nonpolar metabolites were analyzed and the amount of conjugated (polar) and protein-bound metabolites determined. The initial ANF-metabolism rate was 10-fold higher in TCDD-induced microsomes (4.9 +/- 0.6 nmol/min per mg TCDD-induced microsomal protein vs. 0.5 +/- 0.2 nmol/min per mg uninduced microsomal protein) than in uninduced microsomes. Moreover, uninduced microsomes no longer metabolize ANF after 30-40 min while TCDD-induced microsomes metabolize ANF for longer than 2 h or until all the ANF is gone. In addition to the metabolites formed by uninduced microsomes [7,8-dihydro-7,8-dihydroxy-ANF (7,8-dihydrodiol); 5,6-dihydro-5,6-dihydroxy-ANF (5,6-dihydrodiol); 5,6-oxide-ANF and 6-hydroxy-ANF], TCDD-induced microsomes from unidentified metabolites. When TCDD-induced microsomes and 40 microM ANF were added to Chinese hamster ovary (CHO) cells, we found a correlation between the concentration of 5,6-oxide-ANF and clastogenicity to CHO cells. However, purified 5,6-oxide-ANF did not induce SCEs in CHO cells in the absence or presence of TCDD-induced microsomes. However, a minor metabolite (identified as the 9,10-dihydro-9,10-dihydroxy-ANF by acid dehydration) formed with TCDD-induced microsomes produces clastogenicity in CHO cells. These data indicate that a minor metabolite of ANF is a potent clastogen which suggests that this metabolite may be responsible for the ANF-mediated increases in SCE frequency in lymphocytes from smokers or PCB-exposed individuals.  相似文献   

3.
Plant constituents such as terpenes are major constituents of the essential oil in Eucalyptus sp. 1,8-Cineole and p-cymene (Terpenes present in high amounts in Eucalyptus leaves) are potential substrates for the CYP family of enzymes. We have investigated tolbutamide hydroxylase as a probe substrate reaction in both koala and terpene pretreated and control brushtail possum liver microsomes and examined inhibition of this reaction by Eucalyptus terpenes. The specific activity determined for tolbutamide hydroxylase in the terpene treated brushtails was significantly higher than that for the control animals (1865+/-334 nmol/mg microsomal protein per min versus 895+/-27 nmol/mg microsomal protein per min). The activity determined in koala microsomes was 8159+/-370 nmol/mg microsomal protein per min. Vmax values and Km values for the terpene treated possum, control, possum and koala were 1932-2225 nmol/mg microsomal protein per min and 0.80 0.81 mM; 1406-1484 nmol/mg microsomal protein per min and 0.87-0.92 mM and 5895-6403 nmol/mg microsomal protein per min and 0.067-0.071 mM, respectively. Terpenes were examined as potential inhibitors of tolbutamide hydroxylase activity. 1,8-Cineole was found to be a competitive inhibitor for the enzyme responsible for tolbutamide hydroxylation (Ki 15 microM) in the possum. In koala liver microsomes stimulation of tolbutamide hydroxylase activity was observed when concentrations of cineole were increased. Therefore, although inhibition was observed, the type of inhibition could not be determined.  相似文献   

4.
The total organic base fraction purified from fried ground beef is metabolized by human-liver microsomes to form mutagens detectable by the Ames/Salmonella bacterial assay. The mutagens produced have an absolute requirement for metabolic activation; without it, no increase in the number of revertants over background is seen. Microsomes from human liver activate the mutagens significantly more than microsomes from uninduced mouse or rat liver; the microsomes from one individual were nearly as active as those of Aroclor-induced mice and rats. alpha-Naphthoflavone (ANF) inhibits activation of these mutagenic bases, implying that the metabolism is mediated by the inducible form(s) of cytochrome P-448. Thus, the human liver has the potential to metabolize the cooked beef mutagen(s) to active intermediates, posing a possible mutagenic risk. However, unlike the animal metabolizing system, which needs to be artificially induced, the human system appears to be naturally induced through diet or environmental exposure.  相似文献   

5.
The metabolism of benzo(a)pyrene [BP], a model carcinogenic PAH, by hepatic microsomes of two duck species, mallard (Anas platyrhynchos) and common merganser (Mergus merganser americanus) collected from chemically-contaminated and relatively non-contaminated areas was investigated. The rate of metabolism of BP by liver microsomes of common merganser and mallard collected from polluted areas (2,650 +/- 310 and 2,200 +/- 310 pmol/min per mg microsomal protein, respectively) was significantly higher than that obtained with liver microsomes of the two species collected from non-polluted areas (334 +/- 33 and 231 +/- 30 pmol/min per mg microsomal protein, respectively). The level of cytochrome P-450 1A1 was significantly higher in the liver microsomes of both duck species from the polluted areas as compared to the ducks from the non-polluted areas. The major BP metabolites, including BP-9, 10-diol, BP-4, 5-diol, BP-7, 8-diol, BP-1, 6-dione, BP-3, 6-dione, BP-6, 12-dione, 9-hydroxy-BP and 3-hydroxy-BP, formed by liver microsomes of both duck species from polluted and non-polluted areas, were qualitatively similar. However, the patterns of these metabolites were considerably different from each other. Liver microsomes of ducks from the polluted areas produced a higher proportion of benzo-ring dihydrodiols than the liver microsomes of ducks from the non-polluted areas, which converted a greater proportion of BP to BP-phenols. The predominant enantiomer of BP-7,8-diol formed by hepatic microsomes of the two duck species had an (-)R,R absolute stereochemistry. The data suggest that duck and rat liver microsomal enzymes have different regioselectivity but similar stereoselectivity in the metabolism of BP.  相似文献   

6.
The Ames assay employing Salmonella typhimurium TA100 and TA98 was used to investigate potential interactions between aflatoxin B1 (AFB1) and the phenolic antioxidants butylated hydroxytoluene, butylated hydroxyanisole, and propyl gallate. AFB1 doses were within the linear response range, and the antioxidants were used at levels of 0 to 50 micrograms per plate. All three antioxidants were nonmutagenic in either bacterial tester strain, with or without the hepatic S-9 enzyme preparation; toxic effects were observed at doses higher than 20 micrograms per plate. Butylated hydroxytoluene and butylated hydroxyanisole substantially increased AFB1-induced mutagenesis in the two tester strains with microsomal activation. The addition of 5 to 20 micrograms of butylated hydroxytoluene or hydroxyanisole to 5 to 20 ng of AFB1 per plate caused more than a twofold increase in the number of His+ revertants. Addition of propyl gallate resulted in only a moderate increase in the number of revertants. Whereas several anticarcinogenic and antimutagenic effects by phenolic antioxidants have been reported, particularly in studies with polycyclic aromatic hydrocarbons, the enhancement of mutagenic potency of AFB1 by these compounds suggests a specificity with respect to the chemical nature of AFB1.  相似文献   

7.
The capacity of three B-lymphocyte cell lines to generate superoxide (O2.-) was examined. The Burkitt lymphoma lines P.3HR-1 and Jijoye gave no response to phorbol 12-myristate 13-acetate (PMA) at 100 ng/ml but produced up to 0.35 nmol of O2.-/min per mg of protein when stimulated with 5 micrograms of PMA/ml; the cell line RPMI 1788 produced Nitro Blue Tetrazolium-positive responses to low PMA concentrations and approx. 0.4 nmol of O2.-/min per mg of protein at 5 micrograms of PMA/ml. Each cell line contained approx. 10 pmol of low-potential cytochrome b (cytochrome b-245)/mg of protein. Homogenates of PMA-activated cells gave 10-20-fold greater rates of O2.- produced per mg of protein. The Km for NADPH varied between approx. 250 microM for P3.HR-1 and RPMI 1788 cell lines and 30.5 +/- 6.5 microM for the Jijoye cell line; the Km values for NADH were higher. Determination of intracellular NADPH concentration showed that this might limit the rate of O2.- production since in each cell line it was at or below the Km concentration.  相似文献   

8.
The flavin monooxygenases (FMO) catalyse the NADPH and oxygen-dependent oxidation of a wide range of nucleophilic nitrogen-, sulfur-, phosphorus-, and selenium heteroatom-containing chemicals, drugs, and agricultural agents. In the present study, sheep liver microsomal FMO activity was determined by measuring the S-oxidation rate of methimazole and the average specific activity obtained from different microsomal preparations was found to be 3.8 +/- 1.5 nmol methimazole oxidized min(-1) mg(-1) microsomal protein (mean +/- SE, n = 7). The presence of 0.1% Triton X-100 in the reaction mixture caused an increase of specific sheep liver microsomal FMO activity towards methimazole to 6.1 +/- 1.4 nmol methimazole oxidized min(-1) mg(-1) microsomal protein (mean +/- SE, n = 6). Metabolism of imipramine and chlorpromazine was measured by following the oxidation of cofactor NADPH spectrophotometrically at 340 nm. Sheep liver microsomal FMO activity towards imipramine and chlorpromazine was found to be 10.7 and 12.3 nmol NADPH oxidized min(-1) mg(-1) microsomal protein, respectively. Characterization of sheep liver enzyme was carried out using methimazole as substrate and the maximum FMO enzyme activity was detected at 37 degrees C and at pH 8.0. The apparent K(m) value of sheep liver microsomal FMO for methimazole was 0.118 mM. Effects of the detergents Triton X-100, Cholate, and Emulgen 913, on FMO activity were determined and FMO activity was found to increase with the addition of detergents to the reaction medium. Sheep liver microsomal FMO-catalysed methimazole oxidation was inhibited by imipramine and chlorpromazine when these drugs were used at high concentrations. Western blot-immunochemical analysis revealed the presence of FMO3 in sheep liver microsomes.  相似文献   

9.
Progesterone 21-hydroxylation in hepatic microsomes from adult male sheep is a quantitatively important metabolic pathway (0.27 +/- 0.08 nmol deoxycorticosterone formed/min/mg protein; representing 13-25% of total progesterone conversion). This study was undertaken to determine whether the ovine hepatic progesterone 21-hydroxylase may be another member of the P450 2C subfamily, normally associated with progesterone 21-hydroxylation in rodent liver. An IgG preparation raised in rabbits against purified rat liver microsomal cytochrome P450 2C6 was found to recognize a single antigen (MW 52 kDa) in sheep liver microsomes. This protein was present in sheep liver (apparent concentration 16 +/- 4 ng/micrograms microsomal protein) representing approx. 28% of the corresponding content of P450 2C6 in untreated rat liver. Preincubation of the anti-P450 2C6 IgG with hepatic microsomes was found to decrease the rate of progesterone 21-hydroxylation to 50-80% of uninhibited control. Taken together, from these findings it is apparent that a P450 enzyme, most likely from the 2C subfamily, catalyses deoxycorticosterone formation from progesterone in sheep liver and that this is a quantitatively important pathway of progesterone hydroxylation in these fractions.  相似文献   

10.
1. ATP-dependent calcium uptake by a rabbit brain vesicular fraction (microsomes) was studied in the presence of phosphate or oxalate. These anions, which are known to form insoluble calcium salts, increased the rate of calcium uptake and the capacity of the vesicles for calcium accumulation. 2. The degree of activation depended on the concentration of phosphate or oxalate. Under optimal conditions, phosphate promoted a 5-fold increase in the amount of calcium stored at steady state. This level was 200-250 nmol Ca-2+/mg protein. 3. Initial rate of calcium uptake followed Michaelis-Menten kinetics with an apparent Km for calcium of 6.7-10-minus 5 M and a V of 44 nmol/min per mg protein. Optimal pH was 7.0. With 2 mM ATP, optimal Mg-2+ concentration was 2 mM. 4. Dintrophenol and NaN3 inhibited calcium uptake in a mitochondria-enriched fraction but not in the microsomal fraction. 5. Calcium uptake activity was compared in the six subfractions prepared from the whole microsomal fraction by means of a sucrose density gradient fractionation. 6. The Mg-2+-dependent ATPase activity of brain microsomes was activated by calcium. Maximal activation was attained with 100 muM CaCl2. Greater calcium concentrations caused a progressive inhibition. 7. The data suggest that the ATP-dependent calcium uptake in brain microsomes, as in muscle microsomes, is brought about by an active transport process, calcium being accumulated as a free ion inside the vesicles.  相似文献   

11.
20-HETE is a potent, vasoconstrictive arachidonic acid metabolite with a limited number of published methods for quantitative assessment of microsomal formation rate. The purpose of this study was to evaluate the utility of HPLC-MS (negative ESI) for quantitation of rat microsomal 20-HETE enzyme kinetics. Calibration curves were linear over 0.75-16 ng on-column (r(2)>0.996). The intra- and inter-assay precision and accuracy were <15%. Microsomal 20-HETE revealed saturable (100 microM) kinetics (brain K(m) and V(max): 39.9+/-6.0 microM and 8.7+/-0.6 pM/min per mg; liver K(m) and V(max): 23.5+/-3.2 microM and 775.5+/-39.8 pmol/min per mg; kidney K(m) and V(max): 47.6+/-8.5 microM and 1933+/-151 pM/min per mg). This paper demonstrates HPLC-MS as an efficient method for quantitating 20-HETE enzyme kinetics in microsomes from rat tissues.  相似文献   

12.
Preparations of rat lung microsomes containing 0.030-0.050 nmole of cytochromes P-450 and b5 per mg microsomal protein have been observed to contain significant levels of fatty acid desaturase activity. Both stearoyl CoA and palmitoyl CoA are desaturated to their monounsaturated analogues, oleic acid and palmitoleic acid, respectively. Activity (per mg microsomal protein) of the lung preparations varied according to the diet of the animals prior to killing in the order: fat free diet greater than normal rat chow greater than starvation. All preparations exhibited approximately 50% inhibition when incubated in the presence of 0.10 mM CN-. Maximal activity was obtained with the 0.50 mM NADH less activity with equal amounts of NADPH, and there was no synergistic interaction of NADH and NADPH together. The rate of desaturation was linear with protein concentrations between 0.15-1.5 mg microsomal protein/incubation at incubation times up to 8 min. A pH optimum range of 7.0-7.4 was observed. For all variables of fatty acid desaturase activity which were examined, the rate of desaturation of stearoyl CoA was approximately twice that for palmitoyl CoA. These results indicate that the same fatty acid desaturation system which is functional in the liver is also present in significant amounts in mammalian lungs.  相似文献   

13.
Previous studies have identified a calmodulin-stimulated ATP-dependent Ca2+ pump as the major Ca2+ efflux pathway in enterocytes. Here, we developed methods to quantify the number of Ca2+ pumps in basolateral and intracellular membranes from porcine duodenum. By the use of a pig strain with a genetic defect in renal 1 alpha-hydroxylase, we were able to investigate the influence of 1,25(OH)2D3-deficiency on the number of Ca(2+)-ATPases in porcine duodenum. The amount of Ca(2+)-ATPase in isolated basolateral membranes was 5.5 +/- 0.7 micrograms/mg protein, while the Vmax of ATP-dependent Ca2+ transport into inside-out resealed basolateral membrane vesicles was 2.6 +/- 0.4 nmol/mg protein per min. From these data we estimated roughly about 95 x 10(3) plasma membrane Ca2+ pump sites per enterocyte. In addition, the amount of intracellular Ca(2+)-ATPase in microsomal fractions was 0.41 +/- 0.02 microgram/mg protein. Comparison of these parameters between control and rachitic animals showed that Ca2+ pump capacities in both basolateral membranes and microsomal fractions of porcine duodenum are not influenced by 1,25(OH)2D3-deficiency. In conclusion, stimulatory effects of 1,25(OH)2D3 on intestinal Ca2+ transport most likely result from specific effects on apical influx and facilitation of cytosolic Ca2+ diffusion by Ca(2+)-binding proteins and not from an increase in Ca2+ pumping capacity in basolateral membranes.  相似文献   

14.
The influence of membrane cholesterol content on 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase, EC 1.1.1.34) in rat liver microsomes was investigated. Microsomes were enriched in cholesterol by incubation with egg phosphatidylcholine-cholesterol vesicles and the nonspecific lipid transfer protein from rat liver. By this method, the microsomal cholesterol content was 2.5-fold enhanced up to final concentrations of 140 nmol cholesterol per mg microsomal protein. In another experiment, microsomes isolated from rats fed a cholesterol-rich diet were depleted of cholesterol by incubation with egg phosphatidylcholine vesicles and the transfer protein. Both cholesterol enrichment and depletion had virtually no effect on the microsomal HMG-CoA reductase activity. In another set of experiments, normal rat liver microsomes were incubated with human serum, resulting in a rise of microsomal cholesterol content. This was reflected in an increase of acyl-CoA:cholesterol acyltransferase activity but failed to have an effect on HMG-CoA reductase.  相似文献   

15.
The effects of Ca2+, ionophore A23187, and vasopressin on CTP:phosphocholine cytidylyltransferase were investigated. Cytidylyltransferase is present in the cytosol and in a membrane-bound form on the microsomes. Digitonin treatment caused release of the cytosolic form rapidly. Addition of 7 mM Ca2+ to hepatocyte medium resulted in a 3-fold decrease in cytidylyltransferase released by digitonin treatment (1.7 +/- 0.1 nmol/min per mg compared to 5.1 +/- 0.2 nmol/min per mg in the control). Verapamil, a calcium channel blocker, partially overcame this effect of Ca2+. Ionophore A23187 and vasopressin both mimicked the effect of Ca2+ and resulted in a decrease in cytidylyltransferase release (2.4 +/- 0.1 nmol/min per mg and 2.5 +/- 0.2 nmol/min per mg, respectively) compared to control (3.4 +/- 0.1 nmol/min per mg). In agreement with the digitonin experiments, incubation with 7 mM Ca2+ resulted in a decrease in cytidylyltransferase in the cytosol (from 4.0 to 1.2 mol/min per mg) and a corresponding increase in the microsomes (from 0.6 to 2.4 nmol/min per mg). Verapamil partially blocked this translocation caused by Ca2+. Ionophore A23187 and vasopressin also caused translocation of the cytidylyltransferase from the cytosol to the microsomes. The addition of Ca2+ also resulted in an increase in PC synthesis. With 7 mM Ca2+ in the medium, the label associated with PC increased to 3.8 +/- 0.1.10(6) dpm/dish from 2.7 +/- 0.1.10(6) dpm/dish after 10 min. PC degradation was also affected, since 7 mM Ca2+ in the medium resulted in an increase in LPC formation both in the cell and the medium. We conclude that high concentrations of calcium in the hepatocyte medium can cause a stimulation of CTP:phosphocholine cytidylyltransferase and PC synthesis in cultured hepatocytes.  相似文献   

16.
The characteristics and kinetics of calcium uptake activity were studied in isolated hepatic microsomes. The sustained accumulation of calcium was ATP- and oxalate-dependent. Glucagon increased microsomal Ca2+ uptake upon either in vivo injection, or in vitro perfusion of the hormone in the liver. In contrast, the effect of insulin depended on the route of administration. Calcium accumulation by subsequently isolated hepatic microsomes increased when insulin was injected intraperitoneally whereas it decreased when the hormone was perfused directly into the liver. These effects of glucagon and insulin were dose dependent. When insulin was added to the perfusate prior to the addition of glucagon, insulin blocked the glucagon-stimulated increase in microsomal Ca2+ uptake. Cyclic AMP mimicked the effect of glucagon on microsomal Ca2+ accumulation when the cyclic nucleotide was perfused into the liver. The effects of glucagon and insulin on the kinetics of hepatic microsomal Ca2+ uptake were investigated. In microsomes isolated from perfused rat livers treated with glucagon the V of the uptake was significantly increased over the control values (12.2 vs. 8.6 nmol Ca2+ per min per mg protein, P less than 0.02). In contrast, the addition of insulin to the perfusate significantly decreased the V of Ca2+ uptake by subsequently isolated microsomes (6.8 vs. 8.3 nmol Ca2+ per min per mg protein, P less than 0.05). However, neither hormone had an effect on the apparent Km for Ca2+ (4.1 +/- 0.5 microM) of the reaction. The effect of these hormones on the activity of Ca2+-stimulated ATPase was also studied. No significant changes in either V or Km for Ca2+ of the enzymatic reaction were detected.  相似文献   

17.
Rat liver microsomes were immobilized by entrapment in a chemically crosslinked synthetic gel obtained by crosslinking prepolymerized polyacrylamide-hydrazide with glyoxal. Approximately 88% of the microsomal fraction was entrapped in the gel. The specific rate of O-demethylation of p-nitroanisole was used to assay the microsomal cytochrome P-450 activity of the immobilized microsomal preparations. The gel entrapped microsomes showed monooxygenase activity at 37 degrees C of Vmax = 2.3 nmol p-nitrophenol/min per nmol cytochrome P-450, similar to that of microsomes in suspension. The Km value for the p-nitroanisole-immobilized microsomal cytochrome P-450 system (1.2 X 10(-5) M) was rather close to that of microsomes in suspension (0.8 X 10(-5) M). Under the experimental conditions used the pH activity curve of the immobilized preparation was shifted towards more alkaline values by approx. 0.5 pH unit in comparison with microsomes in suspension. The rate of cytochrome c reduction by the immobilized microsomal system (11.7 nmol/min per mg protein) at 25 degrees C was considerably lower than that of the control (microsomes in suspension, 78 nmol/min per mg protein). Enzyme activity in both preparations showed the same temperature dependence at the temperature range of 10 to 37 degrees C. The immobilized microsomal monooxygenase system could be operated continuously for several hours at 37 degrees C provided that adequate amounts of an NADPH-generating system were added periodically. Under similar conditions a control microsomal suspension lost its enzymic activity within 90 min.  相似文献   

18.
Various cell fractions from rat liver were tested for their ability to convert nitrosopyrrolidine (NO-PYR) to products which were mutagenic to E. coli in liquid-incubation assays. Microsomes alone produced only a small number of tyr+ revertants, approximately 40/10(8) survivors), while the S100 supernatant produced none at all. However, the S8 Fraction or combinations of microsomes and the S100 supernatant, yielded 300-400 tyr+ revertants/10(8) survivors. Neither products of the microsomal, nor microsome + supernatant reactions were mutagenic in the absence or presence of cellular fractions. These results suggest that bacterial mutagens are formed during the microsomal metabolism of NO-PYR to 2-hydroxytetrahydrofuran by alpha-hydroxylation, but not during the metabolism of 2-hydroxytetrahydrofuran by the S100 supernatant enzymes. Possible roles of the supernatant enzymes in the formation of mutagenic intermediates during the initial alpha-hydroxylation of NO-PYR are discussed.  相似文献   

19.
To define the mechanism responsible for the slow rate of calcium transport by cardiac sarcoplasmic reticulum, the kinetic properties of the Ca2+-dependent ATPase of canine cardiac microsomes were characterized and compared with those of a comparable preparation from rabbit fast skeletal muscle. A phosphoprotein intermediate (E approximately P), which has the stability characteristics of an acyl phosphate, is formed during ATP hydrolysis by cardiac microsomes. Ca2+ is required for the E approximately P formation, and Mg2+ accelerates its decomposition. The Ca2+ concentration required for half-maximal activation of the ATPase is 4.7 +/- 0.2 muM for cardiac microsomes and 1.3 +/- 0.1 muM for skeletal microsomes at pH 6.8 and 0 degrees. The ATPase activities at saturating concentrations of ionized Ca2+ and pH 6.8, expressed as ATP hydrolysis per mg of protein, are 3 to 6 times lower for cardiac microsomes than for skeletal microsomes under a variety of conditions tested. The apparent Km value for MgATP at high concentrations in the presence of saturating concentrations of ionized Ca2+ is 0.18 +/- 0.03 ms at pH 6.8 and 25 degrees. The maximum velocity of ATPase activity under these conditions is 0.45 +/- 0.05 mumol per mg per min for cardiac microsomes and 1.60 +/- 0.05 mumol per mg per min for skeletal microsomes. The maximum steady state level of E approximately P for cardiac microsomes, 1.3 +/- 0.1 nmol per mg, is significantly less than the value of 4.9 +/- 0.2 nmol per mg for skeletal microsomes, so that the turnover number of the Ca2+-dependent ATPase of cardiac microsomes, calculated as the ratio of ATPase activity to the E approximately P level is similar to that of the skeletal ATPase. These findings indicate that the relatively slow rate of calcium transport by cardiac microsomes, whem compared to that of skeletal microsomes, reflects a lower density of calcium pumping sites and lower Ca2+ affinity for these sites, rather than a lower turnover rate.  相似文献   

20.
The effect of phospholipase A2 treatment of rat hepatocytes on CTP: phosphocholine cytidylyltransferase and phosphatidylcholine synthesis was investigated. Cytidylyltransferase is recovered from the cytosol and in a membrane-bound form with the microsomes. Digitonin treatment of cells causes rapid release into the medium of the cytosolic, but not the microsomal form of the cytidylyltransferase. Incubation of hepatocytes for 10 min with phospholipase A2 (0.9 units/dish) in the medium, resulted in a 33% decrease in the cytidylyltransferase activity released by digitonin treatment (2.5 +/- 0.15 nmol/min per mg compared to 3.9 +/- 0.10 nmol/min per mg in the control). In agreement with the digitonin experiments, incubation with 0.9 units/dish of phospholipase A2 resulted in a decrease in the cytidylyltransferase activity in the cytosol (from 4.3 +/- 0.10 nmol/min per mg to 2.6 +/- 0.14 nmol/min per mg) and a corresponding increase in the microsomal fraction (from 0.9 +/- 0.16 nmol/min per mg to 1.8 +/- 0.20 nmol/min per mg). The effect of phospholipase A2 on cytidylyltransferase translocation was concentration- and time-dependent. Incubation of hepatocytes in the presence of phospholipase A2 (0.9 units/dish) for 10 min prior to pulse-chase experiments resulted in an increase in radiolabel incorporation into phosphatidylcholine (from 2.4 +/- 0.02.10(-5) dpm/dish to 3.1 +/- 0.1.10(-5) dpm/dish) and a corresponding decrease in radiolabel associated with the choline (from 2.5 +/- 0.05.10(-5) to 1.4 +/- 0.03.10(-5) dpm) and phosphocholine fractions (from 8.5 +/- 0.07.10(-5) to 6.9 +/- 0.05.10(-5) dpm). We conclude that phospholipase A2 can cause a stimulation of CTP: phosphocholine cytidylyltransferase activity and phosphatidylcholine synthesis in cultured rat hepatocytes.  相似文献   

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