Subcellular localization of 3 alpha, 7 alpha-dihydroxy- and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoyl-coenzyme A ligase(s) in rat liver

Liver peroxisomes from both rat and humans have previously been shown to contain enzymes that catalyze the oxidative cleavage of the C27-steroid side chain in the formation of bile acids. It has not been clear, however, whether the initial step, formation of the CoA-esters of the 5 beta-cholestanoic acids, also occurs in these organelles. In the present work the subcellular localization of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoyl-CoA (THCA-CoA) ligase (THCA-CoA synthetase) and of 3 alpha,7 alpha-dihydroxy-5 beta-cholestanoyl-CoA (DHCA-CoA) ligase in rat liver has been investigated. Main subcellular fractions and peroxisome-rich density gradient fractions from rat liver were incubated with THCA or DHCA, CoA, ATP, and Mg2+. Formation of THCA-CoA and DHCA-CoA was determined after high pressure liquid chromatography of the incubation extracts. The microsomal fraction contained the highest specific (and also relative specific) activity both for the formation of THCA-CoA and DHCA-CoA. The rates of THCA-CoA formation were further increased from 124-159 nmol/mg.hr-1 in crude microsomal fractions to 184-220 nmol/mg.hr-1 when studied in purified rough endoplasmic reticulum fractions. Formation of THCA-CoA in peroxisomal fractions prepared in Nycodenz density gradients could be accounted for by a small contamination (3-7%) by microsomal protein. The distribution of THCA-CoA ligase was different from that of palmitoyl-CoA ligase that was found to be localized also to the peroxisomal fractions.     

Characterization of liver cholic acid coenzyme A ligase activity. Evidence that separate microsomal enzymes are responsible for cholic acid and fatty acid activation.

Investigations on the cholic acid CoA ligase activity of rat liver microsomes were made possible by the development of a rapid, sensitive radiochemical assay based on the conversion of [3H]choloyl-CoA. More than 70% of the rat liver cholic acid CoA ligase activity was associated with the microsomal subcellular fraction. The dependencies of cholic acid CoA ligase activity on pH, ATP, CoA, Triton WR-1339, acetone, ethanol, magnesium, and salts were investigated. The hypothesis that the long chain fatty acid CoA ligase activity and the cholic acid CoA ligase activity are catalyzed by a single microsomal enzyme was investigated. The ATP, CoA, and cholic (palmitic) acid kinetics neither supported nor negated the hypothesis. Cholic acid was not an inhibitor of the fatty acid CoA ligase and palmitic acid was not a competitive inhibitor of the cholic acid CoA ligase. The cholic acid CoA ligase activity utilized dATP as a substrate more effectively than did the fatty acid CoA ligase activity. The cholic acid and fatty acid CoA ligase activities appeared to have different pH dependencies, differed in thermolability at 41 degrees, and were differentially inactivated by phospholipase C. Moreover, fatty acid CoA ligase activity was present in microsomal fractions from all rat organs tested while cholic acid CoA ligase activity was detected only in liver microsomes. The data suggest that separate microsomal enzymes are responsible for the cholic acid and the fatty acid CoA ligase activities in liver.     

Subcellular distribution of cholic acid:coenzyme a ligase and deoxycholic acid:Coenzyme a ligase activities in rat liver

Cholic acid:CoA ligase (EC 6.2.1.7, choloyl-CoA synthetase) and deoxycholic acid:CoA ligase catalyze the synthesis of choloyl-CoA and deoxycholoyl-CoA from their respective bile acids in rat liver. A modification of the phase partition assay was introduced which yields significantly (3-fold) higher specific activities for cholic acid:CoA ligase than previously reported. An independent method of separating choloyl-CoA from the substrates by high-pressure liquid chromatography was also developed and validates the modification. Both enzymic activities were found to be localized predominantly in the endoplasmic reticulum of rat liver. The level of either ligase in other purified, active subcellular fractions is consistent with the level of contamination by endoplasmic reticulum, estimated by using marker enzymes. Hence, the ligase assay can be used as a sensitive enzymic marker for endoplasmic reticulum in rat liver. The kinetic parameters of both enzymic activities were determined by using purified rough endoplasmic reticulum from rat liver. While the apparent maximal velocities for the two substrates are similar, the Michaelis constant for deoxycholate is significantly lower than that for cholate. Taurocholate and deoxycholate are shown to be competitive inhibitors of cholic acid:CoA ligase. The inhibition constant of deoxycholate is similar to its Michaelis constant for the deoxycholoyl-CoA-synthesizing reaction, suggesting that the same enzyme is responsible for both ligase activities.     

The human liver-specific homolog of very long-chain acyl-CoA synthetase is cholate:CoA ligase

Unconjugated bile acids must be activated to their CoA thioesters before conjugation to taurine or glycine can occur. A human homolog of very long-chain acyl-CoA synthetase, hVLCS-H2, has two requisite properties of a bile acid:CoA ligase, liver specificity and an endoplasmic reticulum subcellular localization. We investigated the ability of this enzyme to activate the primary bile acid, cholic acid, to its CoA derivative. When expressed in COS-1 cells, hVLCS-H2 exhibited cholate:CoA ligase (choloyl-CoA synthetase) activity with both non-isotopic and radioactive assays. Other long- and very long-chain acyl-CoA synthetases were incapable of activating cholate. Endogenous choloyl-CoA synthetase activity was also detected in liver-derived HepG2 cells but not in kidney-derived COS-1 cells. Our results are consistent with a role for hVLCS-H2 in the re-activation and re-conjugation of bile acids entering liver from the enterohepatic circulation rather than in de novo bile acid synthesis.     

Subcellular distribution and properties of acyl/alkyl dihydroxyacetone phosphate reductase in rodent livers

On subcellular fractionation, the enzyme acyl/alkyl dihydroxyacetone phosphate (DHAP) reductase (EC 1.1.1.101) in guinea pig and rat liver was found to be present in both the light mitochondrial (L) and microsomal fractions. By using metrizamide density gradient centrifugation, it was shown that the alkyl DHAP reductase activity in the "L" fraction is localized mainly in peroxisomes. From the distribution of the marker enzymes it was calculated that about two-thirds of the liver reductase activity is in the peroxisomes and the rest in the microsomes. The properties of this enzyme in peroxisomes and microsomes are similar with respect to heat inactivation, pH optima, sensitivity to trypsin, and inhibition by NADP+ and acyl CoA. The enzyme activity in the peroxisomes and microsomes from mouse liver is increased to the same extent by chronically feeding the animals clofibrate, a hypolipidemic drug. The kinetic properties of this enzyme in these two different organelles are also similar. From these results it is concluded that the same enzyme is present in two different subcellular compartments of liver.     

Regulatory effects of sterols and bile acids on hepatic 3-hydroxy-3-methylglutaryl CoA reductase and cholesterol 7alpha-hydroxylase in the rat

Specific activities of the hepatic microsomal enzymes 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase and cholesterol 7alpha-hydroxylase were studied in rats fed sterols and bile acids. The administration of bile acids (taurocholate, taurodeoxycholate, taurochenodeoxycholate) at a level of 1% of the diet for 1 wk reduced the activity of HMG CoA reductase. Taurocholate and taurodeoxycholate, but not taurochenodeoxycholate, inhibited cholesterol 7alpha-hydroxylase. Dietary sitosterol produced increases in the specific activity of HMG CoA reductase (3.6-fold) and cholesterol 7alpha-hydroxylase (1.4-fold), and biliary cholesterol concentrations in this group more than doubled. Compared with controls fed the stock diet, the simultaneous administration of sitosterol and taurochenodeoxycholate resulted in a 60% decrease of HMG CoA reductase activity and no change in cholesterol 7alpha-hydroxylase activity or biliary cholesterol concentration. Rats fed sitosterol plus taurocholate had nearly normal HMG CoA reductase activity, but cholesterol 7alpha-hydroxylase was inhibited and biliary cholesterol remained high. Bile acid secretion rates and biliary bile acid composition were similar in controls and sterol-fed animals. In all groups receiving bile acids, biliary secretion of bile acids was nearly doubled and bile acid composition was shifted in the direction of the administered bile acid. It is concluded that the composition of the bile acid pool influences the hepatic concentrations of the rate-controlling enzymes of bile acid synthesis.     

Insulin degradation V. Unmasking of glutathione-insulin transhydrogenase in rat liver microsomal membrane

Glutathione-insulin transhydrogenase (glutathione:protein disulfide oxidoreductase, EC 1.8.4.2) inactivates insulin by cleaving its disulfide bonds. The distribution of GSH-insulin transhydrogenase in subcellular fractions of rat liver homogenates has been studied. From the distribution of insulin-degrading activity and marker enzymes (glucose-6-phosphatase and succinate-INT reductase) (INT, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) after cell fractionation by differential centrifugation, the immunological analysis of the isolated subcellular fractions with antibody to purified rat liver GSH-insulin transhydrogenase, and chromatographic analysis (on a column of Sephadex G-75 in 50% acetic acid) of the products formed from ¹²⁵I-labelled insulin after incubation with the isolated subcellular fractions, it is concluded that GSH-insulin transhydrogenase is located primarily in the microsomal fraction of rat liver homogenate. An enzyme(s) that further degrades insulin by proteolysis is located mainly in the soluble fraction; a significant amount of the protease(s) activity is also present in the mitochondrial fraction. The possibility has been discussed that the protease(s) acts upon the intermediate product of insulin degradation, A and B chains of insulin, rather than upon the intact insulin molecule itself.The GSH-insulin transhydrogenase in intact microsomes occurs in a latent state; it is readily released from the microsomal membrane and its activity is greatly increased by treatments which affect the lipoprotein membrane structure of microsomal vesicles. There include homogenization with a Polytron homogenizer, sonication, freezing and thawing, alkaline pH, the nonionic detergent Triton X-100, and phospholipases A and C.     

Bile acid: CoASH ligases from guinea pig and porcine liver microsomes. Purification and characterization

A procedure for the purification of the enzyme bile acid:CoA ligase from guinea pig liver microsomes was developed. Activity toward chenodeoxycholate, cholate, deoxycholate, and lithocholate co-purified suggesting that a single enzyme form catalyzes the activation of all four bile acids. Activity toward lithocholate could not be accurately assayed during the earlier stages of purification due to a protein which interfered with the assay. The purified ligase had a specific activity that was 333-fold enriched relative to the microsomal cell fraction. The purification procedure successfully removed several enzymes that could potentially interfere with assay procedures for ligase activity, i.e. ATPase, AMPase, inorganic pyrophosphatase, and bile acid-CoA thiolase. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified ligase gave a single band of approximately 63,000 Mr. A molecular size of 116,000 +/- 4,000 daltons was obtained by radiation inactivation analysis of the ligase in its native microsomal environment, suggesting that the functional unit of the ligase is a dimer. The purified enzyme was extensively delipidated by adsorption to alumina. The delipidated enzyme was extremely unstable but could be partially stabilized by the addition of phospholipid vesicles or detergent. However, such additions did not enhance enzymatic activity. Kinetic analysis revealed that chenodeoxycholate, cholate, deoxycholate, and lithocholate were all relatively good substrates for the purified enzyme. The trihydroxy bile acid cholate was the least efficient substrate due to its relatively low affinity for the enzyme. Bile acid:CoA ligase could also be solubilized from porcine liver microsomes and purified 180-fold by a modification of the above procedure. The final preparation contains three polypeptides as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The three peptides range in size from 50,000 to 59,000, somewhat smaller than the guinea pig enzyme. The functional size of the porcine enzyme in its native microsomal environment was determined by the technique of radiation inactivation analysis to be 108,000 +/- 5,000 daltons. Thus, the functional form of the porcine enzyme also appears to be a dimer.     

Peroxisomal bile acid-CoA:amino-acid N-acyltransferase in rat liver

Bile acid-CoA:amino-acid N-acyltransferase activity was measured in subcellular fractions of rat liver homogenate. The conversion of [14C]choloyl-CoA and [14C]chenodeoxycholoyl-CoA into the corresponding [14C]tauro- and glyco-bile acids was calculated after isolation of the product by high performance liquid chromatography. There was an enrichment of bile acid-CoA:amino-acid N-acyltransferase activity in the light mitochondrial (L) fraction and to a lesser extent in the microsomal fraction. Surprisingly, no enrichment was found in the cytosolic fraction. Subfractionation of the L-fraction by Nycodenz gradient centrifugation, showed that the activity of the N-acyltransferase had a bimodal distribution and co-sedimented with peroxisomes (particulate catalase) and microsomes (esterase). The highest specific amidation activity of both choloyl-CoA and chenodeoxycholoyl-CoA was always found in the most peroxisome-rich fractions. [14C]Taurocholate formation in the peroxisomal fraction was 2.2 mumol/mg of protein/min. Striking differences were observed in the Km values and the saturation concentrations for glycine and taurine. The peroxisomal amidation of [14C]choloyl-CoA had a Km for taurine of 0.9 x 10(-3) M and for glycine of 17 x 10(-3) M. The results are consistent with the possibility that most of de novo synthesized bile acids conjugate to taurine by a peroxisomal bile acid-taurine N-acyltransferase in rat liver. The bile acids deconjugated in the gut and recirculating to the liver may be activated and amidated by the microsomal enzyme system prior to biliary secretion.     

Very long chain fatty acid beta-oxidation by rat liver mitochondria and peroxisomes

Crude mitochondrial fractions were isolated by differential centrifugation of rat liver homogenates. Subfractionation of these fractions on self-generating continuous Percoll gradients resulted in clearcut separation of peroxisomes from mitochondria. Hexacosanoic acid beta-oxidation was present mainly in peroxisomal fractions whereas hexacosanoyl CoA oxidation was present in the mitochondrial as well as in the peroxisomal fractions. The presence of much greater hexacosanoyl CoA synthetase activity in the purified preparations of microsomes and peroxisomes compared to mitochondria, suggests that the synthesis of coenzyme A derivatives of very long chain fatty acids (VLCFA) is limited in mitochondria. We postulate that a specific VLCFA CoA synthetase may be required to effectively convert VLCFA to VLCFA CoA in the cell. This specific synthetase activity is absent from the mitochondrial membrane, but present in the peroxisomal and the microsomal membranes. We postulate that substrate specificity and the subcellular localization of the specific VLCFA CoA synthetase directs and regulates VLCFA oxidation in the cell.     

Topography of phosphatidylcholine, phosphatidylethanolamine and triacylgycerol biosynthetic enzymes in rat liver microsomes

The topography of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol biosynthetic enzymes within the transverse plane of rat liver microsomes was investigated using two impermeant inhibitors, mercury-dextran and dextran-maleimide. Between 70 and 98% of the activities of fatty acid : CoA ligase (EC 6.2.1.3), sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15), phosphatidic acid phosphatase (EC 3.1.3.4), diacylglycerol acyltransferase (EC 2.3.1.20), diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) were inactivated by mercury-dextran. Dextran-maleimide caused 52% inactivation of the sn-glycerol-3-phosphate acyltransferase. Inactivation of each of these activities except fatty acid : CoA ligase occurred in microsomal vesicles which remained intact as evidenced by the maintenance of highly latent mannose-6-phosphatase activity (EC 3.1.3.9). These glycerolipid biosynthetic activities were not latent, indicating that substrates have free access to the active sites. Moreover, ATP, CDP-choline and CMP appeared unable to penetrate the microsome membrane. These data indicate that the active sites of thease enzymes are located on the external surface of microsomal vesicles. It is concluded that the biosynthesis of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum.     

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.     

Acid:Coenzyme A Ligase in Brain: Fatty Acid Specificity in Cellular and Subcellular Fractions

Abstract: We measured long-chain fatty acid:coenzyme A (CoA) ligase (EC 6.2.1.3) activity with four fatty acids in brain homogenates, and cellular and subcellular fractions to determine whether there are differences in activity that could be correlated with differences in fatty acid composition and metabolism. In rat brain homogenates, ligase activity varied appreciably with the four acids, with 18:2 > 18:1 > 16:0 > 22:1 (nmol acyl-CoA formed/min/mg protein; 1.46, 1.20, 0.96, and 0.57, respectively). This order was similar under all incubation conditions tested, including variable pH and fatty acid concentrations. The relative specific activities (RSA, 16:0 = 1.0) with the four substrates were similar in rat brain homogenate, mitochondria, and microsomes, with the highest specific activities in the latter fraction. The RSA were also similar in ox brain homogenates, in rabbit brain microsomes prepared from gray and white matter, in neurons isolated from rat brain, and in cultured neuroblastoma cells. Rat liver homogenates had a significantly different pattern of RSA. These results indicate that the ligase(s) has a preference for certain fatty acids, but suggest that the major control of fatty acid composition and metabolism is a function of subsequent metabolic steps.     

EFFECT OF FATTY-ACID DOUBLE-BOND POSITION ON THE SELECTIVITY OF RAT-BRAIN ENZYMES: THE INCORPORATION OF OLEIC AND CIS-VACCENIC ACIDS INTO LYSOLECITHIN IN VITRO

Abstract— —Selectivity in the esterification of fatty acids to lysolecithin by rat-brain enzymes in vitro was investigated using free fatty acids (activation plus esterification) and CoA esters (esterification) of two naturally-occurring monoenoic fatty-acid isomers, oleic acid [18:1 (n - 9)] and cis-vaccenic acid [18:1 (n - 7)]. Esterification of free acids to l-acyl-sn-glycero-3-phosphorylcholine (1-acyl GPC) was dependent on CoA and ATP, and was stimulated by MgCl₂ and NaF. Under comparable conditions, fatty-acid activation (acyl-CoA synthetase [acid: CoA ligase (AMP)] EC 6.2.1.3.) appeared to be rate-limiting to 1-acyl GPC acyltransferase (acyl-CoA:l-acylglycero-3-phosphocholine O-acyltrans-ferase, EC 2.3.1.23.), since rates were always less with free fatty acids than with the CoA esters. A comparison of substrate curves obtained with free fatty acids and CoA esters suggests a preference for oleic acid during activation. Acyltransferase activity with 2-acyl GPC was similar with both acyl-CoA isomers, whereas with 1-acyl GPC, activity with oleoyl-CoA consistently exceeded that with cis-vaccenoyl-CoA. This difference between patterns of selectivity in esterification of positions 1 and 2 of lecithin suggests that separate enzymes catalyze the two reactions. The transfer of the isomers to the 2 position was affected in a similar manner by changes in pH and temperature, as well as in protein, fatty acid (or acyl-CoA), and 1-acyl GPC concentrations. Patterns of incorporation with simultaneous incubation of both isomers suggests one enzyme. Differences in acyltransferase activity with the two isomerie acyl-CoA's were observed in subcellular distribution, activity changes with brain maturation, and loss of activity on preincubation of microsomes at 45C. From these results it is not certain whether oleic and cis-vaccenic acids are esterified to the 2 position by separate enzymes, or by one enzyme with different affinities for the isomers. However, the investigation clearly indicates that acyltransferases, and possibly acyl-CoA synthetases in brain possess selectivity related to subtle differences in double-bond position. These selectivities probably are important in determining the specific fatty-acid composition of the complex lipids of brain.     

Kinetic and biochemical properties of CTP:choline-phosphate cytidylyltransferase from the rat brain

In order to investigate the mechanisms involved in some brain disorders at the membrane level, we studied the kinetics and biochemical properties of brain CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15), the rate-limiting enzyme of the two-step biosynthesis of phosphatidylcholine. This enzyme catalyzes the biosynthesis of CDPcholine from choline phosphate and CTP. We found that its subcellular localization (mainly in microsomal and cytosolic fractions) was different from that of phosphatidylethanolamine N-methyltransferase (EC 2.1.1.17), the enzyme of the alternative pathway for phosphatidylcholine synthesis. CTP:choline-phosphate cytidylyltransferase showed a Km of 10 mM for CTP and 0.3 mM for choline phosphate and exhibited a random mechanism. CDPcholine, the reaction product, was a competitive inhibitor of choline phosphate and CTP utilization and had a Ki of 0.090 mM. Both particulate and soluble enzymes required Mg2+ and exhibited an optimal pH at about 7. Cytosolic activity was enhanced by addition of unsaturated fatty acids or phospholipids extracted from brain membranes. Such an enhancement was increased with the centrifugation time used for preparing the soluble enzyme.     

Enhancement of long-chain acyl-CoA hydrolase activity in peroxisomes and mitochondria of rat liver by peroxisomal proliferators

The present study has confirmed previous findings of long-chain acyl-CoA hydrolase activities in the mitochondrial and microsomal fractions of the normal rat liver. In addition, experimental evidence is presented in support of a peroxisomal localization of long-chain acyl-CoA hydrolase activity. (a) Analytical differential centrifugation of homogenates from normal rat liver revealed that this activity (using palmitoyl-CoA as the substrate) was also present in a population of particles with an average sedimentation coefficient of 6740 S, characteristic of peroxisomal marker enzymes. (b) The subcellular distribution of the hydrolase activity was greatly affected by administration of the peroxisomal proliferators clofibrate and tiadenol. The specific activity was enhanced in the mitochondrial fraction and in a population of particles with an average sedimentation coefficient of 4400 S, characteristic of peroxisomal marker enzymes. Three populations of particles containing lysosomal marker enzymes were found by analytical differential centrifugation, both in normal and clofibrate-treated rats. Our data do not support the proposal that palmitoyl-CoA hydrolase and acid phosphatase belong to the same subcellular particles. In livers from rats treated with peroxisomal proliferators, the specific activity of palmitoyl-CoA hydrolase was also enhanced in the particle-free supernatant. Evidence is presented that this activity at least in part, is related to the peroxisomal proliferation.     

Purification and characterization of the enzymes of bile acid conjugation from fish liver

1. The two steps in bile acid conjugation have been studied in subcellular fractions of liver from three species of fish; vermillion rockfish, canary rockfish and ling codfish. 2. The bile acid: coenzyme A (CoA) ligase activity in homogenates and isolated microsomes is undetectable due to indeterminate factors. 3. A purification scheme is presented which eliminates the interfering factors. The purified ligase was found to have a lower affinity for bile acids as compared to the mammalian form and to be present in much lower titer. 4. Since it appears to be the rate controlling enzyme in all species, it is expected that the rate of bile acid conjugation is much slower in non-mammalian liver as compared to mammalian liver. 5. The bile acid-CoA:taurine N-acyltransferase was found to exist as a dimer of molecular weight 100,000, in contrast to the monomeric mammalian forms. 6. The only major kinetic difference is that the fish liver forms have rates of glycine conjugation which are only 1-2% of the rate with taurine, in part due to a very high Km for glycine.     

A study of the subcellular structure of the rabbit liver during the development of a bacterial infection

Lysosomal, microsomal and peroxisomal fractions have been isolated from the liver cells of rabbits infected with Staphylococcus aureus pathogenic strain 274 by differential centrifugation in the density gradient of sucrose. The study of the fractions of cell organelles by electron-cytochemical and biochemical methods in the dynamics of the development of infection has revealed changes in the activity of marker enzymes, which is indicative of structural and functional shifts occurring in subcellular structures.     

Localization and solubilization of a rat liver microsomal carnitine acetyltransferase.

Carnitine acetyltransferase activity had been previously shown to occur in peroxisomes, mitochondria, and a membranous fraction of rat and pig hepatocytes. When components of this third subcellular fraction (plasma membranes, components of the Golgi apparatus, and microsomes) were further separated, carnitine acetyltransferase fractionated with the microsomes. Microsomes isolated by three different methods (isopycnic sucrose density zonal centrifugation, high-speed differential centrifugation, and aggregation with Ca²⁺ followed by low-speed differential centrifugation) all contained carnitine acetyltransferase activity. The lability of carnitine acetyltransferase in microsomes isolated by different methods and in different isolation media is reported.When total microsomes were subfractionated into rough and smooth components, carnitine acetyltransferase activity was found to the same extent in both and was tightly associated with the microsomal membrane. The microsomal enzyme was rapidly inactivated in 0.25 m sucrose or 0.1 m phosphate, but was stable for at least 2 weeks in 0.4 m KCl. Extensive treatment with high ionic strength salt solutions, 1% Triton X-100, or a combination of the two was used to solubilize microsomal carnitine acetyltransferase activity.Carnitine octanoyltransferase activity was also found in the microsomal fractions isolated by three different methods, but no carnitine palmitoyltransferase was detected in the microsomal fractions. It is proposed that microsomal carnitine acetyl- and octanoyltransferases could be involved in the transfer of acyl groups across the microsomal membrane, thereby providing a source of acetyl and other acyl CoA's at sites of acetylation reactions and synthesis.     

Subcellular Compartmentation of the 4-Aminobutyrate Shunt in Protoplasts from Developing Soybean Cotyledons

The subcellular localization of enzymes involved in the 4-ami-nobutyrate shunt was investigated in protoplasts prepared from developing soybean [Glycine max (L.) Merrill cv Maple Arrow] cotyledons. Protoplast lysate was fractionated by differential and continuous Percoll-gradient centrifugation to separate organelle fractions. Glutamate decarboxylase (EC 4.1.1.15) was found exclusively in the cytosol, whereas 4-aminobutyrate:pyruvate transami-nase (EC 2.6.1.19) and succinic semialdehyde dehydrogenase (EC 1.2.1.16) were associated exclusively with the mitochondrial fractions. Mitochondrial fractions also catabolized [U-14C]4-aminobu-tyrate to labeled succinate.