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M R Prasad

Publications and source records attributed to M R Prasad.

At least 73 records · Page 4Linked to original sources
PubMed1987

Distribution of dietary trans-octadecenoate among acyl-CoA and other lipid fractions of rat liver and heart.

Groups of rats were fed diets containing 10% of either corn oil, partially hydrogenated soybean oil, or a mixture of the two. The partially hydrogenated oil contained a high level of trans-octadecenoate and a low level of linoleate, and all diets were adjusted to contain similar levels of cis-octadecenoate. The fatty acid compositions of five tissue lipid fractions from liver and heart (non-esterified fatty acids, acyl-CoA, diacylglycerols, triacylglycerols and phospholipids) were analyzed to measure the effect of the dietary supply on the accumulation of trans-octadecenoates and other fatty acids at different steps of glycerolipid synthesis. Although trans-octadecenoate was increased in all of the lipid fractions when the dietary supply was increased, the accumulation did not exceed 15% of the acyl chains in any of the lipid pools even when the dietary trans acid accounted for 46% of the fatty acids supplied in the diet. The trans-octadecenoate accumulated in a similar manner in the lipids of both liver and heart, and the amounts found in the acyl-CoA esters of both tissues were relatively low compared to the diet. A high dietary supply of trans-octadecenoate appeared to diminish the relative content of stearate in the acyl-CoA and phospholipid fractions. The level of cis-octadecenoate maintained in tissue phospholipids was similar to that in the acyl-CoA fractions, whereas the trans-octadecenoate content in phospholipids more closely resembled that in the diacylglycerols. Normal proportions of arachidonate were maintained in the tissue phospholipids during high intake of trans acids, even though lower levels were observed in the acyl-CoA and diacylglycerols of liver.

Acyl Coenzyme A↗
PubMed1987

Quantitative determination of acyl chain composition of subnanomole amounts of cellular long-chain acyl-coenzyme A esters.

A procedure for the quantitative determination of the acyl chain composition of cellular long-chain acyl-CoA esters in subnanomole amounts is described. The abundant cellular lipids of samples are removed by extraction with organic solvents, and the proteins are precipitated from the aqueous phase by the addition of acetonitrile. The CoA thiolesters are adsorbed on neutral aluminum oxide and reduced with sodium borohydride to the corresponding alcohols that are then converted to t-butyldimethylsilyl ethers and analyzed quantitatively by gas chromatography. Saturated and unsaturated acyl chains behaved similarly throughout the procedure, and the common lipid esters do not interfere with the analysis of the CoA esters in the final assay procedure described. This simple and relatively rapid method is suitable for analyzing a large number of samples at a time.

Acyl Coenzyme A↗
PubMed1986

Do rat hepatic microsomes contain multiple NADPH-supported fatty acid chain elongation pathways or a single pathway?

[2-14C]-trans-2-hexadecenoyl CoA (16:1) and [2-14C]-trans-2-cis-8,11,14-eicosatetraenoyl CoA (20:4) were chemically synthesized and employed as competitive substrates for the liver microsomal trans-2-enoyl CoA reductase component of the fatty acid chain elongation system. Both 7.5 microM and 15 microM 20:4 competitively inhibited the reduction of 16:1 CoA to palmitoyl CoA. In addition, the reduction of both substrates was identically inhibited to the same extent by the acetylenic derivative, dec-2-ynoyl CoA. Furthermore, trypsin, chymotrypsin and subtilisin inhibited trans-2-enoyl CoA reductase activity when three different substrates were employed--16:1, 20:4 and trans-2-cis-11-octadecadienoyl CoA (18:2). These results are consistent with the hypothesis of multiple condensing enzymes connected to a single elongation pathway.

Acyl-CoA Dehydrogenases↗
PubMed1986

Effect of the peroxisomal proliferator di(2-ethylhexyl)phthalate on component reactions of the rat hepatic microsomal fatty acid chain elongation system and on other hepatic lipogenic enzymes.

The feeding of 2% di(2-ethylhexyl)phthalate (DEHP) to rats increased the hepatic microsomal elongation of palmitoyl-CoA by about twofold, while those of palmitoleoyl-CoA and gamma-linolenoyl-CoA decreased to 83 and 63%, respectively, of the control values. When component reactions of the elongation pathway were measured, it was observed that only the activity of condensing enzyme was increased by twofold, while those of beta-ketostearoyl-CoA reductase, beta-hydroxypalmitoyl-CoA dehydrase, and trans-2-hexadecenoyl-CoA reductases were not affected. Furthermore, the time course for induction of both condensation and elongation of palmitoyl-CoA was similar. In vitro addition of DEHP had no effect on either condensation or elongation. Thus, these results indicate that the peroxisomal proliferator induces only the condensing enzyme which is the regulatory and rate-limiting step of elongation sequence. The DEHP treatment also markedly enhanced the cytosolic NADPH-generating activities of glucose-6-PO4 dehydrogenase (2.2-fold) and malic enzyme (7.3-fold). Unexpectedly, the activities of fatty acid synthetase and citrate cleavage enzyme were unaffected. These results are discussed in light of the fact that these lipogenic enzymes are coordinately induced by diet or hormones.

Acyl Coenzyme A↗
PubMed1986

Evidence for multiple condensing enzymes in rat hepatic microsomes catalyzing the condensation of saturated, monounsaturated, and polyunsaturated acyl coenzyme A.

The condensation of palmitoyl-CoA with malonyl-CoA by rat hepatic microsomes was competitively inhibited by myristoyl-CoA, whereas it was noncompetitively inhibited by palmitoleoyl and gamma-linolenoyl-CoA. Furthermore, the condensation of palmitoleoyl-CoA with malonyl-CoA was also noncompetitively inhibited by gamma-linolenoyl-CoA. Replacement of normal diet by a fat-free high carbohydrate diet resulted in 8-, 2.5-, and 2.3-fold increases in the condensation rates of both palmitoyl- and myristoyl-CoA, palmitoleoyl-CoA, and gamma-linolenoyl-CoA, respectively. On the other hand, administration of di-(2-ethylhexyl)phthalate (DEHP) resulted in a 2-fold stimulation of the condensation activities with myristoyl- and palmitoyl-CoA, while those with palmitoleoyl- and gamma-linolenoyl-CoA decreased to about 83 and 63%, respectively. Similar results following dietary changes or DEHP administration were obtained for total elongation activities. Finally condensation activities of 16:0, 16:1, and gamma-18:3 CoA were differently affected by the proteolytic enzyme, chymotrypsin. The competitive substrate studies, those of dietary and DEHP administration, and the differential action of chymotrypsin strongly suggest the existence of at least three discrete condensing enzymes catalyzing the condensation of saturated, monounsaturated, and polyunsaturated acyl-CoAs. These studies also indicate that the condensation reaction is the regulating and rate-limiting step of the fatty acid chain elongation system.

Acyl Coenzyme A↗
PubMed1986

Hepatic subcellular distribution of short-chain beta-ketoacyl coenzyme A reductase and trans-2-enoyl coenzyme A hydratase: 25- to 50-fold stimulation of microsomal activities by the peroxisome proliferator, di-(2-ethylhexyl)phthalate.

The present study demonstrates unequivocally the existence of short-chain trans-2-enoyl coenzyme A (CoA) hydratase and beta-ketoacyl CoA reductase activities in the endoplasmic reticulum of rat liver. Subcellular fractionation indicated that all four fractions, namely, mitochondrial, peroxisomal, microsomal, and cytosolic contained significant hydratase activity when crotonyl CoA was employed as the substrate. In the untreated rat, based on marker enzymes and heat treatment, the hydratase activity, expressed as mumol/min/g liver, wet weight, in each fraction was: mitochondria, 684; peroxisomes, 108; microsomes, 36; and cytosol, 60. Following di-(2-ethylhexyl)phthalate (DEHP) treatment (2% (v/w) for 8 days), there was only a 20% increase in mitochondrial activity; in contrast, peroxisomal hydratase activity was stimulated 33-fold, while microsomal and cytosolic activities were enhanced 58- and 14-fold respectively. A portion of the cytosolic hydratase activity can be attributed to the component of the fatty acid synthase complex. Although more than 70% of the total hydratase activity was associated with the mitochondrial fraction in the untreated rat, DEHP treatment markedly altered this pattern; only 11% of the total hydratase activity was present in the mitochondrial fraction, while 49 and 29% resided in the peroxisomal and microsomal fractions, respectively. In addition, all four subcellular fractions contained the short-chain NADH-specific beta-ketoacyl CoA (acetoacetyl CoA) reductase activity. Again, in the untreated animal, reductase activity was predominant in the mitochondrial fraction; following DEHP treatment, there was marked stimulation in the peroxisomal, microsomal, and cytosolic fractions, while the activity in the mitochondrial fraction increased by only 39%. Hence, it can be concluded that both reductase and hydratase activities exist in the endoplasmic reticulum in addition to mitochondria, peroxisomes, and soluble cytoplasm.

Alcohol Oxidoreductases↗
PubMed1986

Isolation of rat liver microsomal short-chain beta-ketoacyl-coenzyme A reductase and trans-2-enoyl-coenzyme A hydratase: evidence for more than one hydratase.

An enzyme preparation (IIIB) isolated from liver microsomes of untreated male rats was found to contain two activities--short-chain trans-2-enoyl-CoA hydratase and beta-ketoacyl-CoA reductase. The hydratase was purified more than 1000-fold, while the reductase activity was purified over 600-fold. Employing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, a single band with a molecular weight of 76,000 was observed. Although attempts to separate these two activities have failed, it remains to be established whether the final preparation contains a single enzyme with two activities or two separate enzymes. The hydratase was most active toward crotonyl-CoA, followed by trans-2-hexenoyl-CoA (6:1) and -octenoyl-CoA (8:1); the enzyme was essentially inactive toward substrates containing more than eight carbon atoms. The Vmax for crotonyl-CoA was 2117 mumol/min/mg protein, while the Km was 59 microM. Using acetoacetyl-CoA as substrate, the Vmax for the beta-ketoacyl-CoA reductase was over 60 mumol/min/mg protein and the Km was 37 microM; the Vmax for beta-ketopalmitoyl-CoA was only 15% of that observed with acetoacetyl-CoA, although the Km was 6 microM. During the course of purification, a second short-chain hydratase was discovered (fraction IVA); unlike IIIB, this fraction catalyzed the hydration of 4:1, 6:1, and 8:1 at similar rates. The partially purified preparation yielded maximal activity with 8:1 CoA (apparent Vmax 35 mumol/min/mg), followed by 6:1 CoA, 4:1 CoA, and 10:1 CoA; longer chain CoA's were relatively poor substrates, with trans-2-hexadecenoyl CoA about 0.1 as active as 8:1 CoA. On SDS-gels, fraction IVA contained four bands, all of which were below 60,000 Mr. Proteases, such as trypsin, chymotrypsin, and subtilisin, were found to completely inactivate both enzyme fractions.

Alcohol Oxidoreductases↗
PubMed1985

Solubilization and purification of hepatic microsomal trans-2-enoyl-CoA reductase: evidence for the existence of a second long-chain enoyl-CoA reductase.

The present study describes the solubilization and purification of a NADPH-specific trans-2-enoyl-CoA reductase from rat liver microsomes. The final preparation was purified to near homogeneity and had a minimal molecular weight of 51,000 +/- 2,000, as judged by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. This enzyme specifically used NADPH, as cofactor, and was chromatographically (2',5'-ADP-agarose) separated from another trans-2-enoyl-CoA reductase which utilized either NADH or NADPH as cofactor. The NADPH-specific trans-2-enoyl-CoA reductase catalyzed the reduction of trans-2-enoyl-CoAs from 4 to 16 carbon units. The Km values for crotonyl-CoA, trans-2-hexenoyl-CoA, and trans-2-hexadecenoyl-CoA were 20, 0.5, and 1.0 microM, while the Km value for NADPH was 10 microM. Although N-ethylmaleimide, heat treatment, and limited proteolysis with trypsin affected the reduction of short-chain (C4) and long-chain (C16) substrates equally, and in spite of the fact that a single protein band was observed on SDS-gels, at the present time one cannot state unequivocally that the purified preparation contained only one reductase. trans-2-Hexenoyl-CoA, for example, did not inhibit the reduction of trans-2-hexadecenoyl-CoA to palmitoyl-CoA and trans-2-decenoyl-CoA to decanoyl-CoA whereas it strongly inhibited the conversion of crotonyl-CoA to butyryl-CoA. The potential implications of this finding are discussed. Finally, the reductase preparation was shown not to contain either heme, nonheme iron, or a flavin prosthetic group.

Acyl Coenzyme A↗
PubMed1985

Hepatic microsomal short-chain beta-hydroxyacyl-CoA dehydrase distinct from the fatty acid elongation component: substrate specificity of the membrane-extracted enzyme.

The ability of 0.4 M KCl to extract over 80% of a short-chain beta-hydroxyacyl-CoA dehydrase from rat hepatic endoplasmic reticulum, while more than 80% of the long-chain beta-hydroxyacyl-CoA dehydrase component of the fatty acid chain elongation system remains intact, confirms the existence of more than one hepatic microsomal dehydrase. Following extraction from the microsomal membrane, the short-chain dehydrase undergoes, at least, a two-fold activation. Employing even-numbered trans-2-enoyl-CoA substrates ranging in carbon chain length from 4 to 16, the highest dehydrase specific activity of 16 mumol min-1 mg protein-1 was obtained with trans-2-hexenoyl-CoA; crotonyl-CoA was the second most active substrate, followed by 8 greater than 10 greater than 12 greater than 14 greater than 16. The specific activity of the short-chain dehydrase with trans-2-hexadecenoyl-CoA (C-16) was only 3% of that observed with the trans-2-hexenoyl-CoA. With crotonyl-CoA or beta-hydroxybutyryl-CoA as substrates, HPLC was employed to identify the products, beta-hydroxybutyryl-CoA, of the hydration reaction, or crotonyl-CoA, of the reverse dehydration reaction. It was also observed that the short-chain dehydrase catalyzed the formation of both D(-) and L(+) stereoisomers of beta-hydroxybutyryl-CoA. The equilibrium constant for the dehydrase-catalyzed reaction determined at pH 7.4 and 35 degrees C, was calculated to be 6.38 X 10(-2) M-1, while the standard free energy change was -775 cal/mol, results similar to those obtained with crystalline crotonase. Finally, based on membrane fraction marker enzymes, substrate specificity, and heat lability of the dehydrase, it was concluded that the microsomal membrane contains a short-chain beta-hydroxyacyl-CoA dehydrase which is separate from the mitochondrial crotonase.

Acetyltransferases↗
PubMed1984

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.

Acetoacetates↗
PubMed1983

Site of participation of cytochrome b5 in hepatic microsomal fatty acid chain elongation. Electron input in the first reduction step.

The present study provides strong evidence for the involvement of rat liver microsomal cytochrome b5 in the first reduction step of fatty acid chain elongation. The rate of reoxidation of NADH-reduced microsomal cytochrome b5 was markedly stimulated (up to 3-fold) by the addition of increasing concentrations of beta-ketohexadecanoyl-CoA (1-8 microM). A quantitative analysis of product formation, the effect of cyanide, and anaerobiosis completely exclude the possibility that desaturase activity accounted for the beta-ketohexadecanoyl-CoA-induced stimulation of the cytochrome b5 reoxidation rate. Using liver microsomes from untreated rats, the beta-keto substrate was found to stimulate the rate of reoxidation of cytochrome b5 by 30%. However, when liver microsomes from fat-free diet rats were employed the stimulation was more than 3-fold, suggesting that the beta-ketoacyl-CoA reductase is inducible by a high carbohydrate, fat-free diet. This study also provides evidence for the noninvolvement of cytochrome b5 in the terminal reaction step (second reduction step of chain elongation), which is catalyzed by the trans-2-enoyl-CoA reductase. Although trans-2-hexadecenoyl-CoA significantly stimulated the NADH-reduced cytochrome b5 reoxidation rate under aerobic conditions, it did not have any stimulatory effect under anaerobic conditions. One interpretation of these results is that the trans-2-hexadecenoyl-CoA is substrate for the microsomal delta 9 desaturase system. Consistent with this conclusion was the fact that the trans-2-hexadecenoyl-CoA inhibited the liver microsomal delta 9 desaturation of stearoyl-CoA to oleoyl-CoA.

Animals↗
PubMed1983

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.

Animals↗
PubMed1983

Kinetic evidence for two separate trans-2-enoyl CoA reductases in rat hepatic microsomes: NADPH-specific short chain- and NAD(P)H-dependent long chain-reductase.

The rat hepatic microsomal conversion of crotonyl- and hexenoyl CoA to butyrate and hexanoate was supported only by NADPH, while both NADH and NADPH were effective cofactors in the conversion of trans-2-hexadecenoyl CoA to palmitate. Experiments using mixtures of long- and short-chain enoyl-CoA substrates and competition experiments support the conclusion that microsomes contain 2 distinct enoyl CoA reductases, (1) a long chain enoyl CoA reductase capable of accepting reducing equivalents from either NADH or NADPH, and (2) a NADPH-specific short chain enoyl CoA reductase.

Animals↗
PubMed1982

Stearoyl-coenzyme A desaturase activity in the mammary gland and liver of lactating rats.

Stearoyl-CoA desaturase activity in microsomes from lactating rat mammary gland is very low (0.05-0.15 nmol/min/mg of protein) regardless of lactating time. In such microsomes, reductase activities and content of cytochrome b5 are several-fold lower than in normal rat liver microsomes. Preincubation of the mammary microsomes with purified terminal desaturase gives a 55-fold stimulation of stearoyl-CoA desaturase activity, whereas preincubation with cytochrome b5 has no effect. However, preincubation of mammary microsomes with both cytochrome b5 and terminal desaturase results in a 200-fold stimulation of overall desaturation. These observations suggest that negligible stearoyl-CoA desaturase activity in lactating rat mammary microsomes is due to a cytochrome b5 content and the absence of terminal enzyme. The hepatic stearoyl-CoA desaturase activity increases 9-fold during lactation. There is little or no change in the NADH-cytochrome c reductase activity or in the concentrations of cytochrome b5 during this period, but the activity of the terminal desaturase increases with the increase of overall desaturation. These results suggest that liver is one of the more important sources of oleic acid for milk triglycerides.

Animals↗