Interference with the determination of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and its properties by a microsomal enzymic activity.

Rat liver microsomes and microsomal extracts contain an enzymic activity which competes with 3-hydroxy-3-methylglutaryl coenzyme A reductase for 3-hydroxy-3-methylglutaryl coenzyme A. The presence of this activity in enzyme preparations causes errors in the determination of reductase activity and its properties. This contaminant can be removed by gel filtration using Bio-Gel A 1.5m, by washing the microsomes, or by incubating the microsomal extract at 37 °C. The K_m's of the reductase (free of this competing enzymic activity) for d-3-hydroxy-3-methylglutaryl coenzyme A and NADPH are 1.3 and 26 μm, respectively.     

Assay of 3-hydroxy-3-methylglutaryl CoA reductase activity using anionic-exchange column chromatography.

A rapid, easy, and sensitive method is described in this paper for the assay of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase, a key enzyme in cholesterol biosynthesis. [14C]HMG CoA was used as the substrate and the product formed, i.e., [14C]mevalonate, was allowed to be converted to its lactone form (mevalonolactone) in the presence of HCl. The reaction mixture was applied to a column containing an anionic exchanger. The column was made up of QAE-Sephadex (A25, formate form) packed to a height of 4 cm in Pasteur pipets. Under these conditions, mevalonolactone was not retained by the column and was eluted with ammonium formate solution while HMG CoA, being negatively charged, was retained by the gel and eluted by HCl above 0.05 M. Determination of the amount of radioactivity in mevalonolactone was then used to quantitate the activity of HMG CoA reductase. This assay has been successfully used for determining the activity of this enzyme in a microsomal fraction prepared from the liver of the rat.     

Properties of purified rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and regulation of enzyme activity

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from rat liver microsomes has been purified to apparent homogeneity with recoveries of approximately 50%. The enzyme obtained from rats fed a diet supplemented with cholestyramine had specific activities of approximately 21,500 nmol of NADPH oxidized/min/mg of protein. After amino acid analysis a specific activity of 31,000 nmol of NADPH oxidized/min/mg of amino acyl mass was obtained. The s20,w for HMG-CoA reductase was 6.14 S and the Stokes radius was .39 nm. The molecular weight of the enzyme was 104,000 and the enzyme subunit after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 52,000. Antibodies prepared against the homogeneous enzyme specifically precipitated HMG-CoA reductase from crude and pure fractions of the enzyme. Incubation of rat hepatocytes for 3 h in the presence of lecithin dispersions, compactin, or rat serum resulted in significant increases in the specific activity of the microsomal bound reductase. Immunotitrations indicated that in all cases these increases were associated with an activated form of the reductase. However activation of the enzyme accounted for only a small percentage of the total increase in enzyme activity; the vast majority of the increase was apparently due to an increase in the number of enzyme molecules. In contrast, when hepatocytes were incubated with mevalonolactone the lower enzyme activity which resulted was primarily due to inactivation of the enzyme with little change in the number of enzyme molecules. Immunotitrations of microsomes obtained from rats killed at the nadir or peak of the diurnal rhythm of 3-hydroxy-3-methylglutaryl-CoA reductase indicated that the rhythm results both from enzyme activation and an increased number of reductase molecules.     

Altered kinetic properties of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase following dietary manipulations

The microsomal enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the rate-limiting step in the cholesterogenic pathway and was proposed to be composed in situ of 2 noncovalently linked subunits (Edwards, P.A., Kempner, E.S., Lan, S.-F., and Erickson, S.K. (1985) J. Biol. Chem. 260, 10278-10282). In the present report, the activities and kinetic properties of HMG-CoA reductase in microsomes isolated from livers of rats fed on diets supplemented with either ground Amberlite XAD-2 ("X"), cholestyramine/mevinolin ("CM"), or unsupplemented, normal rat chow ("N"), were compared. The specific activities of HMG-CoA reductase in X and CM microsomes were, respectively, 5- and 83-fold higher than that of N microsomes. In NADPH-dependent kinetics of HMG-CoA reductase activated with 4.5 mM GSH, the concentration of NADPH required for half-maximal velocity (S0.5) was 209 +/- 23, 76 +/- 23, and 40 +/- 4 microM for the N, X, and CM microsomes, respectively. While reductase from X microsomes displays cooperative kinetics toward NADPH (Hill coefficient (nH) = 1.97 +/- 0.07), the enzyme from CM microsomes does not (nH = 1.04 +/- 0.07). Similarly to HMG-CoA reductase from CM microsomes, the freeze-thaw solubilized enzyme ("SOL") displays no cooperativity toward NADPH and its Km for this substrate is 34 microM. At 4.5 mM GSH, HMG-CoA reductase from X, CM, and SOL preparations has a similar Km value for [DL]-HMG-CoA, ranging between 13-16 microM, while reductase from N microsomes had a higher Km value (42 microM) for this substrate. No cooperativity towards HMG-CoA was observed in any of the tested enzyme preparations. Immunoblotting analyses of the different preparations demonstrated that the observed altered kinetics of HMG-CoA reductase in the microsomes is not due to preferential proteolytic cleavage of the native 97-100 kDa subunit of the enzyme to the noncooperative 50-55 kDa species. Moreover, it was found that the ratio enzymatic activity/immunoreactivity of the reductase increased in the order N less than X less than CM approximately equal to SOL, indicating that the activity per reductase molecule increases with the induction of the enzyme. These results are compatible with a model suggesting that dietary induction of hepatic HMG-CoA reductase may change the state of functional aggregation of its subunits.     

3-Hydroxy-3-methylglutaryl coenzyme A reductase activity in beef adrenal cortex.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) activity (mevalonate:NADP+ oxidoreductase )CoA-acylating) EC 1.1.1.34) was demonstrated in beef adrenal cortex. Most of the HMG-CoA reductase activity is in the microsomal fraction while a small percentage of the activity is associated with the mitochondria, Mitochondria purified on a linear sucrose gradient are enriched in HMG-CoA reductase and cytochrome c oxidase activities. The reductase present in microsomal preparations from the whole adrenal cortex has an apparent Km of 5.6 X 10(-5) M for (R,S)-HMG-CoA. Reductase activities found in the microsomal fractions from the zona glomerulosa, the zona fasciculata, and the zona reticularis were 1.32, 7.37, and 9.74 nmol mevalonate formed per milligram protein in 30 min respectively.     

Regulation of luteal cell 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by estradiol

Recent studies have suggested that estradiol or androgen precursor may stimulate steroidogenesis in the luteal cell by modulating intracellular sterol availability and metabolism. This investigation was performed to examine the effect of estradiol on de novo synthesis of cholesterol. Pregnant rats hypophysectomized and hysterectomized on Day 12 were treated for 72 h with either estradiol or testosterone. De novo cholesterol synthesis was determined by measurement of the specific activity of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the rate limiting enzyme in cholesterol biosynthesis, in microsome-enriched preparations of luteal tissue and incorporation of [14C] acetate into cholesterol by corpora lutea incubated in vitro. Estradiol or testosterone treatment caused a 4- to 5-fold stimulation of luteal cholesterol biosynthesis, as measured by these techniques. NaF, an inhibitor of phosphatase which blocks the conversion of the inactive enzyme to the active form, reduced the HMG CoA reductase activity to 30% in corpora lutea obtained from either steroid or vehicle-treated rats. However, an increase in enzyme activity of comparable magnitude by steroids was observed whether microsomes were isolated with or without NaF. The effect of estradiol appears to be enzyme-specific, since it failed to affect the microsomal marker, NADPH-cytochrome c reductase. Since the cholesteryl ester content of corpora lutea falls in response to steroid treatment, rats were treated with 4-aminopyrazolo-[3,4d]pyrimidine (4-APP) to deplete cellular cholesterol content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Triacylglycerol synthesis in isolated fat cells. An effect of insulin on microsomal fatty acid coenzyme A ligase activity.

Fatty acid CoA ligase (AMP) (EC 6.2.1.3) specific activity was increased approximately 2-fold in microsomes prepared from isolated rat fat cells incubated with 400 microunits of insulin/ml (2.9 nM) for 45 to 60 min compared to paired controls using an assay based on the conversion of [3H]oleic acid to [3H]oleoyl-CoA. Similar insulin-dependent increases in microsomal fatty acid CoA ligase specific activities were observed using an assay based on the conversion of [3H]CoA to fatty acyl-[3H]CoA. Fatty acid CoA ligase activity was predominately (about 80%) associated with the microsomal fraction. The insulin-dependent increase in microsomal fatty acid CoA ligase specific activity was maximal in 2 to 5 min at 400 microunits/ml. At 10 min, 80 to 100 microunits of insulin/ml caused a maximal increase in fatty acid CoA ligase specific activity. Similar apparent Km values for ATP, CoA, and fatty acid were observed for fatty acid CoA ligase activity in microsomal preparations from control and insulin-exposed cells. These data suggest that fatty acid CoA ligase activity is regulated in adipose tissue by insulin. Such regulation may serve to promote the capture of fatty acid and thereby, triacylglycerol synthesis in adipose tissue.     

Influence of follicular maturation on 3-hydroxy-methylglutaryl coenzyme A reductase activity in hen granulosa cells

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase: EC 1.1.1.34) was measured in a microsomal preparation of the granulosa of rapidly growing ovarian follicles of laying hens in the late preovulatory period (2-3 h before expected ovulation). The specific activity of the enzyme was measured in the five largest (F1-F5) preovulatory follicles, F1 being the follicle destined to ovulate first. Enzyme activity increased concomitantly with follicle size. The apparent Km of the enzyme decreased 60-80% from the smallest to the largest preovulatory follicle. There was no significant change in the Vmax during follicle development. Although our results have demonstrated the presence of HMG/CoA reductase in chicken granulosa cells and the progressive increase of its activity with follicular maturation, the quantitative significance of de-novo synthesized cholesterol as steroid hormone precursor remains to be ascertained.     

Phosphorylation of native 97-kDa 3-hydroxy-3-methylglutaryl-coenzyme A reductase from rat liver. Impact on activity and degradation of the enzyme

Immunoprecipitation of native rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, phosphorylated by [gamma-32P]ATP in the presence of reductase kinase, revealed a major 97-kDa 32P band which disappeared upon competition with pure unlabeled 53-kDa HMG-CoA reductase. A linear correlation between the expressed/total HMG-CoA reductase activity ratio (E/T) and the fraction of 32P released from the 97-kDa enzyme established the validity of the E/T ratio as an index of HMG-CoA reductase phosphorylation state in isolated microsomes. Incubation of rat hepatocytes with mevalonolactone resulted in a rapid increase in phosphorylation of microsomal reductase (decrease in E/T) followed by an enhanced rate of decay of total reductase activity which was proportional to the loss of 97-kDa enzyme mass determined by immunoblots. Inhibitors of lysosome function dampened both basal and mevalonate-induced reductase degradation in hepatocytes. In an in vitro system using the calcium-dependent protease calpain-2, up to 5-fold greater yields of soluble 52-56-kDa fragments of reductase (immunoblot and total activity) were obtained when the substrate 97-kDa reductase was phosphorylated before proteolysis. Immunoblots of unlabeled phosphorylated reductase compared with gels of immunoprecipitated 32P-labeled reductase resolved a 52-56-kDa doublet which contained 32P solely in the upper band. These data suggest that a major phosphorylation site of HMG-CoA reductase lies within the "linker" segment joining the membrane spanning and cytoplasmic domains of the native 97-kDa protein.     

Biochemical properties of short- and long-chain rat liver microsomal trans-2-enoyl coenzyme A reductase

This study describes the biochemical properties of the rat hepatic microsomal NADPH-specific short-chain enoyl CoA reductase and NAD(P)H-dependent long-chain enoyl CoA reductase. Of the substrates tested, crotonyl CoA and trans-2-hexenoyl CoA are reduced by the short-chain reductase only in the presence of NADPH. The trans-2-octenoyl CoA and trans-2-decenoyl CoA appear to undergo reduction to octanoate and decanoate, respectively, catalyzed by both enzymes; 64% conversion of the C8:1 is catalyzed by the short-chain reductase, while 36% conversion is catalyzed by the long-chain enzyme. For the C10:1 substrate, 45% is converted by the short-chain reductase, while 55% is reduced by the long-chain reductase. trans-2-Hexadecenoyl CoA is a substrate for the long-chain enoyl CoA reductase only. Reduction of C4 and C6 enoyl CoA's was unaffected by bovine serum albumin (BSA), whereas BSA markedly stimulated the conversion of C10 and C16 enoyl CoA's to their respective saturated product. Reduction rates as a function of microsomal protein concentration, incubation time, pH, and cofactors are reported including the apparent Km and Vmax for substrates and cofactors. In general, the apparent Km's for the substrates ranged from 19 to 125 microM. The apparent Vmax for the short-chain enoyl CoA reductase was greatest with trans-2-hexenoyl CoA, having a turnover of 65 nmol/min/mg microsomal protein, while the apparent Vmax for the long-chain enzyme was greatest with trans-2-hexadecenoyl CoA, having a turnover of 55 nmol/min/mg microsomal protein. With respect to electron input, NADPH-cytochrome P-450 reductase, either alone, mixed with phospholipid, or incorporated into phospholipid vesicles, possessed no enoyl CoA reductase activity. Cytochrome c did not affect the NADPH-dependent conversion of the trans-2-enoyl CoA. In addition, anti-NADPH-cytochrome P-450 reductase IgG did not inhibit the reduction of trans-2-hexadecenoyl CoA in hepatic microsomes. Finally, the NADPH-specific short-chain and NAD(P)H-dependent long-chain enoyl CoA reductases were solubilized and completely separated from NADPH-cytochrome P-450 reductase by employing DE-52 column chromatography. These studies demonstrate the noninvolvement of NADPH-cytochrome P-450 reductase in either the short-chain (13) or long-chain enoyl CoA reductase system. Thus, the role of NADPH-cytochrome P-450 reductase in the microsomal elongation of fatty acids appears to be at the level of the first reduction step.     

Purification and properties of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase.

3-Hydroxy-3-methylglutaryl coenzyme A reductase has been purified from rat liver microsomes with a recovery of approx. 25%. The enzyme was homogeneous on gel electrophoresis and enzyme activity comigrated with the single protein band. The molecular weight of the reductase determined by gel filtration on Sephadex G-200 was 200,000. SDS-polyacrylamide gel electrophoresis gave a subunit molecular weight of 52,000 +/- 2000, suggesting that the enzyme was a tetramer. The specific activities of the purified enzyme obtained from rats fed diets containing 0% or 5% cholestyramine were 11,303 and 19,584 nmol NADPH oxidized/min per mg protein, respectively. The reductase showed unique binding properties to Cibacron Blue Sepharose; the enzyme was bound to the Cibacron Blue via the binding sites for both substrates, NADPH and (S)-3-hydroxy-3-methylglutaryl coenzyme A. Antibodies prepared against purified reductase inactivated 100% of the soluble and at least 91% of the microsomal enzyme activity. Immunotitrations of solubilized enzyme obtained from normal and cholestyramine-fed rats indicated that cholestyramine feeding both increased the amount of enzyme protein and resulted in enzyme activation. Administration of increasing amounts of mevalonolactone to rats decreased the equivalence point obtained from immunotitration studies with solubilized enzyme. These data indicate that the antibody cross-reacts with the inactive enzyme formed after mevalonolactone treatment.     

Gonadotropin modulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in desensitized luteinized rat ovary

These studies were done to examine the effect of gonadotropin on rat luteal 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity (the rate-limiting step in cholesterol biosynthesis) in ovaries of pregnant mare's serum gonadotropin (PMSG)-human chorionic gonadotropin (hCG) primed rats. Administration of hCG stimulated HMG CoA reductase activity in a time- and dose-dependent manner: significant increases were noted within 4 h, with maximum effects (30-40-fold increases) seen 24 h after hCG (25 IU) administration. This effect was specific in that only LH, of several hormones tested, was as effective as hCG in stimulating HMG CoA reductase activity, and no change in the activity of either liver microsomal HMG CoA reductase or luteal microsomal NADPH-cytochrome c reductase was seen after hCG. The gonadotropin-induced increase in HMG CoA reductase activity seemed to be due to a net increase in enzyme activity, not to a change in the phosphorylated/dephosphorylated state of the enzyme. Pretreatment of animals with aminoglutethimide, an inhibitor of the conversion of cholesterol to steroid (pregnenolone), prevented the hCG-induced rise in HMG CoA reductase activity, whereas treatment with 4-aminopyrazolo[3,4-d]pyrimidine (4-APP), which depletes cellular cholesterol content, led to striking increases in enzyme activity. However, the combined effects of 4-APP and hCG were additive, suggesting that the stimulating effect of hCG on HMG CoA reductase activity is not entirely due to a depletion of cellular sterol content of luteinized ovaries. Similarly, cholesteryl ester and cholesterol syntheses as measured by [14C]acetate conversion were also increased by hCG and 4-APP treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

3-Hydroxy-3-methylglutaryl coenzyme A reductase from avian liver. Catalytic properties.

The catalytic properties of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase from avian liver have been investigated. Solubilized and highly purified reductase preparations were not cold labile, and enzymic activity remained unchanged following preincubation at 37 degrees C. The pH optimum was 6.8--7.0 and maximal catalytic activity was achieved with 2 mM dithiothreitol and 0.75 M KCl. The heat stability of the enzyme was studied and the addition of 0.75 M KCl, 0.8 mg/ml bovine serum albumin and 5 mM NADPH reduced the inactivation of the purified reductase associated with heat treatment at 65 degrees C. At 37 degrees C, 0.8 mg/ml bovine serum albumin enhanced the purified reductase activity by 100 (+/- 20)%. An improved assay was developed for the avian hydroxymethylglutaryl-CoA reductase and the specific activity of the purified enzyme increased from 1550 to 3300 nmol . min-1 . mg-1. The Km values of solubilized and purified reductase for D-hydroxymethylglutaryl-CoA were 1.05 micrometer and 1.62 micrometer, and for NADPH, 1 mM and 263 micrometer, respectively. The activities of the reductase preparations were non-competitively inhibited by coenzyme A, acyl-CoA esters, and hydroxymethylglutarate. MgATP also reduced avian reductase activity. These modulators may play a role in the cellular regulation of the reductase activity.     

Phosphorylation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and modulation of its enzymic activity by calcium-activated and phospholipid-dependent protein kinase

A calcium-activated and phospholipid-dependent protein kinase (protein kinase C) catalyzes the phosphorylation of both insoluble microsomal (Mr approximately 100,000) and purified soluble (Mr = 53,000) 3-hydroxy-3-methylglutaryl coenzyme A reductase. The phosphorylation and concomitant inactivation of enzymic activity of HMG-CoA reductase was absolutely dependent on Ca2+, phosphatidylserine, and diolein. Dephosphorylation of phosphorylated HMG-CoA reductase was associated with the loss of protein bound radioactivity and reactivation of enzymic activity. Maximal phosphorylation of purified HMG-CoA reductase was associated with the incorporation of 1.05 +/- 0.016 mol of phosphate/mol of native form of HMG-CoA reductase (Mr approximately 100,000). The apparent Km for purified HMG-CoA reductase and histone H1 was 0.08 mg/ml, and 0.12 mg/ml, respectively. The tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate stimulated the protein kinase C-catalyzed phosphorylation of HMG-CoA reductase. Increased phosphorylation of HMG-CoA reductase by phorbol 12-myristate 13-acetate suggests a possible in vivo protein kinase C-mediated mechanism for the short-term regulation of HMG-CoA reductase activity. The identification of the protein kinase C system in addition to the reductase kinase-reductase kinase kinase bicyclic cascade systems for the modulation of the enzymic activity of HMG-CoA reductase may provide new insights into the molecular mechanisms involved in the regulation of cholesterol biosynthesis.     

Improved methods for the study of hepatic HMG CoA reductase: one-step isolation of mevalonolactone and rapid preparation of endoplasmic reticulum.

Two new methods are described for the study of hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. (1) Endoplasmic reticulum was rapidly prepared by diluting a 10,000 g supernatant with buffer containing 8 mM calcium chloride. The yield of protein and the specific activity of HMG CoA reductase in the pellet subsequently obtained by low speed centrifugation were nearly identical to those in the microsomal pellet prepared by ultracentrifugation. This technique may be particularly useful in studies of the rapid, in vitro modulation of the enzyme. (2) Mevalonolactone was extracted into benzene from the HMG CoA reductase assay mixture with an efficiency of 58%. There was less than 1% extraction of HMG CoA, acetoacetate, or beta-hydroxybutyrate. The extracted mevalonolactone was at least 98% pure as judged by thin-layer chromatography with four different solvent systems. These improved methods should significantly aid studies of the physiological importance of HMG CoA reductase.     

Rat hepatic microsomal acetoacetyl-CoA reductase. A beta-ketoacyl-CoA reductase distinct from the long chain beta-ketoacyl-CoA reductase component of the microsomal fatty acid chain elongation system

The present study provides evidence for a new rat liver microsomal enzyme, a short chain beta-ketoacyl (acetoacetyl)-CoA reductase, which is separate from the long chain beta-ketoacyl-CoA reductase component of the microsomal fatty acid chain elongation system. This microsomal reductase converts acetoacetyl-CoA to beta-hydroxybutyryl-CoA at a rate of 70 nmol/min/mg of protein; the enzyme has a specific requirement for NADH and appears to obtain electrons directly from the reduced pyridine nucleotide without the intervention of cytochrome b5 and its flavoprotein reductase. The apparent Km of the enzyme of the acetoacetyl-CoA was 21 microM and for the cofactor, 18 microM. The pH optimum was broad, ranging from 6.5 to 8.0. The product formed is the D-isomer of beta-hydroxybutyryl-CoA. High carbohydrate fat-free diet resulted in a small but significant (35%) increase in microsomal acetoacetyl-CoA reductase activity. The cytosol also contains this enzyme activity, measuring approximately 57% of that found in the microsomes. The mitochondrial activity which is 20-25% higher than the microsomal activity appears to be due to L-beta-hydroxyacyl-CoA dehydrogenase which converts acetoacetyl-CoA to L-beta-hydroxybutyryl-CoA. The microsomal acetoacetyl-CoA reductase activity was extracted from the microsomal membrane by 0.4 M KCl, resulting in an 8- to 10-fold purification; in addition, the long chain fatty acid elongation system was unaffected by this extraction procedure. Employing beta- hydroxyhexanoyl -CoA as a substrate, evidence is also provided for a separate dehydratase which acts on short chain substrates. Lastly, the liver microsomes had no detectable acetoacetyl-CoA synthetase or acetyl-CoA acetyltransferase activities. Hence, the possible involvement of the rat hepatic microsomal short chain beta-ketoacyl-CoA reductase, short chain beta-hydroxyacyl-CoA dehydratase, and the previously reported short chain trans-2-enoyl-CoA reductase in the hepatic utilization of acetoacetyl-CoA and in the synthesis of butyryl-CoA for hepatic lipogenesis is discussed.     

Microsomal NADH-cytochrome b5 reductase of bovine brain: purification and properties

Bovine brain microsomal NADH-cytochrome b5 (cyt. b5) reductase [EC 1.6.2.2] was solubilized by digestion with lysosomes, and purified 8,500-fold with a 20% recovery by procedures including affinity chromatography on 5'-AMP-Sepharose 4B. The purified enzyme showed one band of a molecular weight of 31,000 on polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). Polyacrylamide gel electrophoresis of the purified enzyme without SDS revealed a major band with a faint minor band, both of which exhibited NADH-cyt. b5 reductase activity. The isoelectric points of these components were 6.0 (major) and 6.3 (minor). The apparent Km values of the purified enzyme for NADH and ferricyanide were 1.1 and 4.2 microM, respectively. The apparent Km value for cyt. b5 was 14.3 microM in 10 mM potassium phosphate buffer (pH 7.5). The apparent Vmax value was 1,190 mumol cyt. b5 reduced/min/mg of protein. The NADH-cyt. b5 reductase activity of the purified enzyme was inhibited by sulfhydryl inhibitors and flavin analogues. Inhibition by phosphate buffer or other inorganic salts of the enzyme activity of the purified enzyme was proved to be of the competitive type. These properties were similar to those of NADH-cyt. b5 reductase from bovine liver microsomes or rabbit erythrocytes, although the estimated enzyme content in brain was about one-twentieth of that in liver (per g wet tissue). An immunochemical study using an antibody to purified NADH-cyt. b5 reductase bovine liver microsomes indicated that NADH-cyt. b5 reductase from brain microsomes is immunologically identical to the liver microsomal enzyme.     

Electron-transport cytochrome P-450 system is involved in the microsomal metabolism of the carcinogen chromate

The kinetics of chromate reduction by liver microsomes isolated from rats pretreated with phenobarbital or 3-methylcholanthrene with NADPH or NADH cofactor have been followed. Induction of cytochrome P-450 and NADPH-cytochrome P-450 reductase activity in microsomes by phenobarbital pretreatment caused a decrease in the apparent chromate-enzyme dissociation constant, Km, and an increase in the apparent second-order rate constant, kcat/Km, but did not affect the kcat of NADPH-mediated microsomal metabolism of chromate. Induction of cytochrome P-448 in microsomes by 3-methylcholanthrene pretreatment did not affect the kinetics of NADPH-mediated reduction of chromate by microsomes. The kinetics of NADH-mediated microsomal chromate reduction were unaffected by the drug treatments. The effects of specific enzyme inhibitors on the kinetics of microsomal chromate reduction have been determined. 2'-AMP and 3-pyridinealdehyde-NAD, inhibitors of NADPH-cytochrome P-450 reductase and NADH-cytochrome b5 reductase, inhibited the rate of microsomal reduction of chromate with NADPH and NADH. Metyrapone and carbon monoxide, specific inhibitors of cytochrome P-450, inhibited the rate of NADPH-mediated microsomal reduction of chromate, whereas high concentrations of dimethyl-sulfoxide (0.5 M) enhanced the rate. These results suggest that the electron-transport cytochrome P-450 system is involved in the reduction of chromate by microsomal systems. The NADPH and NADH cofactors supply reducing equivalents ultimately to cytochrome P-450 which functions as a reductase in chromate metabolism. The lower oxidation state(s) produced upon chromate reduction may represent the ultimate carcinogenic form(s) of chromium. These studies provide evidence for the role of cytochrome P-450 in the activation of inorganic carcinogens.     

Salinity adaptation of HCO3--dependent ATPase activity in the gills of blue crab (Callinectes sapidus)

A bicarbonate-dependent ATPase (EC 3.6.1.3) was found in microsomal preparations from blue crab gills. When the crabs were transferred to low salinity (200 mosmolal) from seawater (1000 mosmolal), the HCO3- dependent ATPase increased in all gill pairs, reaching its new steady state in 2 weeks. The greatest increase occurred in the sixth and seventh gill pairs (approx. 2.5-fold). Maximal enzyme activity was observed at an Mg2+ concentration of 2 mM and an optimal pH of 7.8. The apparent Ka for HCO3- was found to be 8.9 mM. Kinetic analysis showed that low-salinity adaptation increased the Vmax without altering the Km for ATP. When the microsomes from high-salinity crab gills were treated with detergent or assayed at different temperatures, the total enzyme activity did not reach the activity levels after adaptation to low salinity. These results suggest that the alteration of HCO3- -ATPase activity may be due to synthesis, rather than modulation of membranes or of the existing enzyme activity.     

Effect of dietary fat saturation on acylcoenzyme A:cholesterol acyltransferase activity of rat liver microsomes

The saturation of the fat contained in the diet has been observed to affect the acylcoenzyme A:cholesterol acyltransferase (ACAT) activity of rat liver microsomes. ACAT activity in microsomes (Mp) prepared from livers of rats fed a polyunsaturated fat-enriched diet containing 14% sunflower seed oil was 70-90% higher than in microsomes (Ms) prepared from livers of rats fed a saturated fat-enriched diet containing 14% coconut oil. This difference was observed within 20 days after the diets were begun, the earliest time tested, and persisted throughout the 70-day experimental period. The difference was noted at all [1-14C]palmitoyl CoA concentrations tested, 2.5-33 micronM, and at temperatures between 18 and 40 degrees C. Arrhenius plots revealed a single transition in enzyme activity, occurring at 29 degrees C in both microsomal preparations. Likewise, the activation energy above this transition was the same in Mp and Ms, 12.5 KCal/mol. Addition of albumin to the incubation medium increased the ACAT activity of both microsome preparations, but the difference between Mp and Ms persisted. Mp was enriched in polyenoic fatty acids, primarily 18:2 and 20:4, while Ms was enriched in monoenoic acids. Although the 20:4 increase in Mp occurred in all phosphoglycerides, it was especially pronounced in the serine and inositol phosphoglyceride fraction. There were no differences in the phospholipid or cholesterol content, phospholipid head group composition, or protein composition of the two microsomal preparations. The possibility is discussed that the changes in ACAT activity result from the differences in fatty acid composition of the microsomes. Other microsomal enzymes exhibited varying responses to these dietary fatty acid modifications. Palmitoyl CoA hydrolase and NADPH cytochrome c reductase activities were unchanged. UDP glucuronyl transferase activity was 50% higher in Mp, but glucose-6-phosphatase and NADH cytochrome b5 reductase activities were 25% higher in Ms. Therefore, dietary fat modifications do not produce a uniform effect on the activity of microsomal enzymes.