Formation of cholic acid from 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid in human skin fibroblasts.

Whether 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) was converted into cholic acid in human skin fibroblasts was examined. THCA was incubated with subcellular fractions of cultured skin fibroblasts in the presence of NAD+, ATP, CoA, and Mg2+. The reaction products were analyzed by thin-layer chromatography and high-performance liquid chromatography after p-bromophenacyl ester derivatization. The highest specific activity was found in the light mitochondrial fraction (2.71 nmol/mg protein/h). The specific activity was about 9-fold higher than that in heavy mitochondrial fraction. The peroxisomal fraction prepared from the light mitochondrial fraction by sucrose gradient centrifugation was also able to catalyze the conversion of THCA into cholic acid. The specific activity in this fraction was a further 2.2-fold higher than that in the light mitochondrial fraction. These results suggest that cultured human skin fibroblasts are able to convert THCA into cholic acid, and that the activity exists in peroxisomes.     

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.     

Formation of cholic acid from 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid by rat liver peroxisomes

In a previous study, it was shown that the peroxisomal fraction of rat liver, isolated by Percoll gradient centrifugation of a light mitochondrial fraction, was able to catalyze conversion of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) into cholic acid (Pedersen, J. I., and J. Gustafsson, 1980. FEBS Lett. 121: 345-348). In the present work, this peroxisomal THCA-oxidizing system has been studied in more detail. The peroxisomes were prepared by sucrose gradient centrifugation. By use of different marker enzymes, it was confirmed that the major part of the activity in the light mitochondrial fraction was located in the peroxisomes. The reaction was absolutely dependent on the presence of Mg2+, CoA, ATP, and NAD+ in the reaction medium. In addition to cholic acid, small amounts of 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid were detected as product. Provided the peroxisomes were preincubated with ATP and CoA, the reaction was linear with time up to 75 min. It was linear with peroxisomal protein and the pH optimum was 8. The reaction was stimulated by FAD (ca. 50%), by cytosolic protein (about twofold), by microsomal protein (about twofold), bovine serum albumin (about sevenfold), and by KCN (75% at 1 mM). In the absence of bovine serum albumin in the medium the K'm for the overall reaction was 1.4 X 10(-6) M and the maximum rate was 4.3 nmol X mg-1 X hr-1. In the presence of bovine serum albumin, the K'm increased to 6.3 X 10(-6) M and the maximum rate to about 32 nmol X mg-1 X hr-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of chenodeoxycholic acid from 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid by rat liver peroxisomes

The oxidation of the side chain of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA) into chenodeoxycholic acid has been studied in subcellular fractions of rat liver. The product was separated from the substrate by high pressure liquid chromatography and identified by gas-liquid chromatography-mass spectrometry. The highest specific rate of conversion was found in the heavy (M) and the light (L) mitochondrial fractions with the highest enrichment in the L fraction. Washing the M fraction reduced the side chain cleavage activity by 90%. The peroxisomal marker enzyme urate oxidase was reduced to the same extent. The activity found in the M fraction may thus be due to peroxisomal contamination. After centrifugation of the L fraction on a Nycodenz density gradient, the highest specific activity for side chain cleavage of DHCA (31 nmol X mg-1 X h-1) was found in the fraction with the highest peroxisomal marker enzyme activity. This fraction also catalyzed conversion of 3 alpha,7 alpha,12 alpha-5 beta-cholestanoic acid (THCA) into cholic acid at the highest rate (32 nmol X mg-1 X h-1). The peroxisomal oxidation of DHCA into chenodeoxycholic acid required the presence of ATP, CoA, Mg2+, and NAD in the incubation medium. The reaction was not inhibited by KCN. It is concluded that rat liver peroxisomes contain enzymes able to catalyze the cleavage of the side chain of both DHCA and THCA. The enzymes involved are similar to, but not necessarily identical to, those involved in the peroxisomal beta-oxidation of fatty acids.     

Mg(Ca)-ATPase of arterial muscle cells]

We could show an ATPase in mitochondrial and microsomal fractions of sheep arteria carotis communis and arteria coronaria of cattle which can be stimulated by Ca2+ of Mg2+, respectively. The enzyme has a higher affinity for Ca2+ than for Mg2+. The maximum activity of the Mg(Ca)-ATPase was found at 2-4 mM Ca2+ or Mg2+, respectively. Higher concentrations of these ions inhibit the enzyme. Mn2+, Sr2+ and Co2+ can substitute Ca2+ in splitting of ATP by the ATPase of both fractions of ateria coronaria of cattle. The ions K+ and Na+, variation of temperature and pH and a variety of pharmacological active compounds has the same effect on the ATPase stimulated by Ca2+ or Mg2+. These findings prove that Ca2+ and Mg2+ act at the same site of the ATPase of the mitochondrial and microsomal fraction of vascular smooth muscle.     

Bile acids. LXXIII. Synthesis of analogs of 7 alpha-hydroxy-4-cholesten-3-one as substrates for hepatic steroid 12 alpha-hydroxylase

Analogs of 7 alpha-hydroxy-4-cholesten-3-one were prepared to ascertain structural features necessary for maximal activity of hepatic microsomal 12 alpha-steroid hydroxylase. Methyl 3 alpha,7 alpha-dihydroxy-5 beta-cholane-24-carboxylate derived from chenodeoxycholic acid was oxidized at C-3 with silver carbonate/Celite. The product was hydrolyzed and dehydrogenated with SeO2 to provide 3-oxo-7 alpha-hydroxy-4-cholene-24-carboxylic acid. 5 beta-Cholestane-3 alpha,7 alpha,25-triol and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol were similarly oxidized at C-3 and dehydrogenated to provide 7 alpha,25-dihydroxy-4-cholesten-3-one and 7 alpha,12 alpha,25-trihydroxy-4-cholesten-3-one, respectively. The products were characterized by thin-layer and gas chromatography, ultraviolet, infrared, proton resonance and mass spectrometry.     

Synthesis of C-22, C-23-3H-labeled 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestane

This report describes an efficient synthesis of C-22, C-23-(3)H-labeled 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestane. - Somanathan, R., and S. Krisans. Synthesis of C-22, C-23-(3)H-labeled 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestane.     

Adenosine triphosphatases of cestodes Bothriocephalus scorpii

Activities and properties of adenosine triphosphatases (ATPases) have been studied in mitochondrial and microsomal fractions of cestodes Bothriocephalus scorpii parasitizing in pyloric appendages of the Brandt's bullhead Myoxocephalus brandti. The highest activity has been revealed in the mitochondrial fraction. The mitochondrial and microsomal fractions of B. scorpii have the ATPase activity dependent on the presence of cations Mg2+, Mn2+, and Ca2+. Effects of ions and inhibitors on the B. scorpii ATPase activity with various cations have been studied. Both subcellular fractions are able to hydrolyze, apart from ATP, also GTP, CTP, and UTP.     

Biosynthesis of cholic acid in rat liver. 24-Hydroxylation of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid.

Conversion of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-[7beta-3H]cholestanoic acid into 3alpha, 7alpha, 12alpha, 24-tetrahydroxy-5beta-cholestanoic acid in rat liver was catalyzed either by the mitochondrial fraction fortified with the 100,000 times g supernatant fluid or the microsomal fraction fortified with 100,000 times g supernatant fluid and ATP. The microsomal system was more active than the mitochondrial system. With the microsomal system the rate of reaction was considerably faster with free 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid as substrate than with the corresponding coenzyme A ester. Addition of coenzyme A inhibited the activity. Addition of cofactors other than ATP and coenzyme A did not markedly influence the reaction. The 100,000 times g supernatant fluid could be substituted with a protein fraction obtained by ammonium sulfate precipitation and Sephadex chromatography of the 100,000 times g supernatant fluid. The reaction was not catalyzed by a mixed function oxidase since there was no incorporation of 18O into the product when the reaction was performed in an atmosphere containing 18O2. On the other hand, oxygen may be obligatory since there was almost complete inhibition when the reaction was performed in an atmosphere consisting of nitrogen. Carbon monoxide did not inhibit the reaction. One atom of deuterium was incorporated into the product when the reaction was performed in a medium containing deuterated water. It was concluded that microsomal 24-hydroxylation of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid involves the combined action of a desaturase and a hydratase. The reaction catalyzed by the hydratase appears to be stereospecific since the 24alpha epimer of 3alpha, 7alpha,12alpha-trihydroxy-5beta-cholestanoic acid was the predominant product. In contrast to the microsomal system, the mitochondrial system was not stimulated by the addition of ATP and was not inhibited by coenzyme A. The coenzyme A ester of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid was 24-hydroxylated more efficiently than the free acid.     

Sex differences in the hydroxylation of cholecalciferol and of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol in rat liver.

The effect of sex hormones on hydroxylation of cholecalciferol ('vitamin D3') and of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol has been investigated in female- and male-rat livers. The mitochondrial cholecalciferol 25-hydroxylase and C27-steroid 27-hydroxylase activities were respectively 4.6- and 2.7-fold higher in female- than in male-rat livers. The microsomal 1 alpha-hydroxycholecalciferol 25-hydroxylase was 2.8-fold higher in male- than in female-rat liver. No significant difference was found in the microsomal 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. Liver microsomes (microsomal fractions) from male, but not from female, rats also catalysed 1-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. Injection of testosterone into female rats decreased the mitochondrial cholecalciferol 25-hydroxylase and C27-steroid 27-hydroxylase activities, but not to a statistically significant extent. Testosterone treatment had no effect on the microsomal hydroxylases in female-rat liver. Injection of oestradiol valerate to male rats resulted in increased activities of both mitochondrial hydroxylases to the same levels as those of control females, while the microsomal enzyme activities decreased. The present results indicate that sex hormones exert a regulatory control on the mitochondrial cholecalciferol 25-hydroxylase and C27-steroid 27-hydroxylase activities.     

Hepatic enzymes, CoASH and long-chain acyl-CoA in subcellular fractions as affected by drugs inducing peroxisomes and smooth endoplasmic reticulum

1. The activities of acyl-CoA hydrolase, catalase, urate oxidase and peroxisomal palmitoyl-CoA oxidation as well as the protein content and the level of CoASH and long-chain acyl-CoA were measured in subcellular fractions of liver from rats fed diets containing phenobarbital (0.1% w/w) or clofibrate (0.3% w/w). 2. Whereas phenobarbital administration resulted in increased microsomal protein, the clofibrate-induced increase was almost entirely attributed to the mitochondrial fraction with minor contribution from the light mitochondrial fraction. 3. The specific activity of palmitoyl-CoA hydrolase in the microsomal fraction was only slightly affected while the mitochondrial enzyme was increased to a marked extent (3-4-fold) by clofibrate. 4. Phenobarbital administration mainly enhanced the microsomal palmitoyl-CoA hydrolase. 5. The increased long-chain acyl-CoA and CoASH level observed after clofibrate treatment was mainly associated with the mitochondrial, light mitochondrial and cytosolic fractions, while the slight increase in the levels of these compounds found after phenobarbital feeding was largely of microsomal origin. 6. The findings suggest that there is an intraperoxisomal CoASH and long-chain acyl-CoA pool. 7. The specific activity of palmitoyl-CoA hydrolase, catalase and peroxisomal palmitoyl-CoA oxidation was increased in the lipid-rich floating layer of the cytosol-fraction. 8. The changes distribution of the peroxisomal marker enzymes and microsomal palmitoyl-CoA hydrolase after treatment with hypolipidemic drugs may be related to the origin of peroxisomes.     

Mechanism of the ATP-dependent phosphatidylserine synthesis in liver subcellular fractions

It has been shown that the ATP-dependent incorporation of [14C]serine into phosphatidylserine in rat liver mitochondrial and microsomal fractions is prevented by EGTA. On the other hand, at low (microM) Ca2+ concentrations, serine incorporation is strongly stimulated by ATP and Mg2+. This stimulatory effect is reduced by calcium ionophore A23187. It is therefore suggested that the ATP-dependent process is that of serine base-exchange reaction, stimulated by endogenous Ca2+ accumulated inside the microsomal vesicles by Ca2+,Mg2+-ATPase. The mitochondrial activity can be accounted for by contamination by the endoplasmic reticulum.     

Characterization of CDPdiacylglycerol hydrolase in mitochondrial and microsomal fractions from rat lung

CDPdiacylglycerol pyrophosphatase (E.C. 3.6.1.26) activity has been examined in rat lung mitochondrial and microsomal fractions. While the mitochondrial hydrolase exhibited a broad pH optimum from pH 6-8, the microsomal activity decreased rapidly above pH 6.5. Apparent Km values of 36.2 and 23.6 microM and Vmax values of 311 and 197 pmol.min-1.mg protein-1 were observed for the mitochondrial and microsomal preparations, respectively. Addition of parachloromercuriphenylsulphonic acid led to a marked inhibition of the microsomal fraction but slightly stimulated the mitochondrial activity at low concentrations. Mercuric ions were inhibitory with both fractions. Although biosynthetic reactions utilizing CDPdiacylglycerol require divalent cations, addition of Mg2+, Mn2+, Ca2+, Zn2+, Co2+, and Cu2+ all inhibited the catabolic CDPdiacylglycerol hydrolase activity in both fractions. EDTA and EGTA also produced an inhibitory effect, especially with the mitochondrial fraction. Although addition of either adenine or cytidine nucleotides led to a decrease in activity with both fractions, the marked susceptibility to AMP previously reported for this enzyme in Escherichia coli membranes, guinea pig brain lysosomes, and pig liver mitochondria was not observed. These results indicate that rat lung mitochondria and microsomes contain specific CDPdiacylglycerol hydrolase activities, which could influence the rate of formation of phosphatidylinositol and phosphatidylglycerol for pulmonary surfactant.     

Effects of oestradiol, testosterone and medroxyprogesterone on subcellular fraction marker enzyme activities from rat liver and brain

The following enzymes have been studied (subcellular fractions are shown between parentheses): NAG and beta-glucuronidase (lysosomes); SDH (mitochondrial); glucose-6-phosphatase (endoplasmic reticulum); 5'-nucleotidase and (Na+, K+)Mg2+ ATPase (plasma membranes). Alterations on their activities were observed after subcutaneous injection of sex hormones, compared with controls. NAG activity from liver was always significantly decreased in lysosomal and microsomal fractions after the hormonal treatment. In the same conditions, NAG from brain was always increased. beta-Glucuronidase behaves like NAG in brain; in liver it was not modified by testosterone and it was slightly increased in lysosomal fraction after oestradiol treatment. SDH activity was not modified in mitochondrial fractions from liver, but this activity was always significantly increased in brain. Glucose-6-phosphatase activity was always significantly decreased in microsomal fractions from liver. It was increased in brain after oestradiol and testosterone injection, but medroxyprogesterone treatment caused a decreased activity. 5'-Nucleotidase and (Na+, K+)Mg2+ ATPase from brain were significantly increased in microsomal fractions by oestradiol and testosterone. Medroxyprogesterone, however, caused an increase in ATPase, but did not affect 5'-nucleotidase. Both activities in liver were decreased by oestradiol and increased by testosterone, but medroxyprogesterone caused (Na+, K+)Mg2+ ATPase to rise and 5'-nucleotidase to fall.     

Comparison of the rat microsomal Mg-ATPase of various tissues

The microsomal Mg-ATPase from various rat tissues was compared. After fractionating the microsomal vesicles by sucrose gradient centrifugation, the highest specific activity of the Mg-ATPase was found in the low-density vesicles which contained plasma membrane. A large fraction (25-90%) of the microsomal Ca-independent Mg-ATPase found in each tissue had the following properties: (1) the Km for ATP was 0.2 mM; (2) the rate of ATP hydrolysis by the Mg-ATPase was nonlinear due to an ATP-stimulated inactivation of the enzyme; (3) wheat germ agglutinin, concanavalin A, glutaraldehyde, and antiserum prevented inactivation induced by ATP or AdoPP[NH]P; (4) detergents at relatively low detergent:protein ratios increased the rate of inactivation with little change in the initial rate of ATP hydrolysis; (5) the Mg-ATPase was inactivated by irradiation in the presence of 8-azido ATP. (6) in addition to ATP, the Mg-ATPase was able to hydrolyze CTP, GTP, UTP, ITP, and GTP but was unable to hydrolyze any of the 10 nonnucleotide phosphocompounds which were tested; (7) the bivalent cation requirement of the Mg-ATPase could be provided by Mg2+, Ca2+, Mn2+, Zn2+, or Co2+ but the enzyme was inactive in the presence of Cu2+, Sr2+, Ba2+, or Be2+; (8) the Mg-ATPase activity was not altered by ionophores or inhibitors of the Na,K-ATPase, the Ca,Mg-ATPase or the mitochondrial F1ATPase. These data suggest that a major portion of the microsomal, basal Mg-ATPase activity is due to one unique enzyme found in most if not all tissues.     

Mitochondrial and Peroxisomal β-Oxidation of Stearic and Lignoceric Acids by Rat Brain

Crude subcellular fractions were prepared from adult rat brains by differential centrifugation of brain homogenates. Greater than 98% of the cellular mitochondrial marker enzyme activity sedimented in the heavy and light mitochondrial pellets, and less than 1% of the activity sedimented in microsomal pellets. Lysosomal marker enzyme activities mainly (71-78% of cellular activity) sedimented in the heavy and light mitochondrial pellets. Significant amounts of the lysosomal marker enzyme activity also sedimented in the crude microsomal pellets (9-13% of total) and high-speed supernatants (14-16% of total). The specific activities of microsomal and peroxisomal marker enzyme activities were highest in the crude microsomal pellets. Fractionation of the crude microsomal pellets on Nycodenz gradients resulted in the separation of the bulk of the remaining mitochondrial, lysosomal, and microsomal enzyme activities from peroxisomes. Fatty acyl-CoA synthetase activities separated on Nycodenz gradients as two distinct peaks, and the minor peak of the activities was in the peroxisomal enriched fraction. Fatty acid beta-oxidation activities also separated as two distinct peaks, and the activities were highest in the peroxisomal enriched fractions. Mitochondria were purified from the heavy mitochondrial pellets by Percoll density gradients. Fatty acyl-CoA synthetase and fatty acid beta-oxidation activities were present in both the purified mitochondrial and peroxisomal enriched fractions. Stearoyl-CoA synthetase activities were severalfold greater compared to lignoceroyl-CoA synthetase, and stearic acid beta-oxidation was severalfold greater compared to lignoceric acid beta-oxidation in purified mitochondrial and peroxisomal enriched fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bile acids of snakes of the subfamily Viperinae and the biosynthesis of C-23-hydroxylated bile acids in liver homogenate fractions from the adder, Vipera berus (Linn.).

1. Analysis of bile salts of four snakes of the subfamily Viperinae showed that their bile acids consisted mainly of C-23-hydroxylated bile acids. 2. Incubations of 14C-labelled sodium cholate (3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholan-24-oate) and deoxycholate (3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oate) with whole and fractionated adder liver homogenates were carried out in the presence of molecular oxygen and NADPH or an NADPH-generating system. The formation of C-23-hydroxylated bile acids, namely bitocholic acid (3 alpha, 12 alpha, 23xi-trihydroxy-5 beta-cholan-24-oic acid) and 3 alpha, 7 alpha, 12 alpha, 23 xi-tetrahydroxy-cholanic acid (3 alpha, 7 alpha, 12 alpha, 23 xi-tetrahydroxy-5 beta-cholan-24-oic acid), was observed mainly in the microsomal fraction and partly in the mitochondrial fraction. 3. Biosynthetic pathways of C-23-hydroxylated bile acids are discussed.     

Comparative studies of bile salts. 16-Deoxymyxinol, a second bile alcohol from hagfish

1. Material containing the less polar sulphate previously noticed in hagfish bile salts gave, after dioxan-trichloroacetic acid cleavage, 16-deoxymyxinol [3beta,7alpha,-26(27)-trihydroxy-5alpha-cholestane]. 2. Anodic coupling of 3beta-hydroxy-5beta-cholanoic acid and the mixed half esters of dl-methylsuccinic acid, followed by lithium aluminium hydride reduction, yielded 3beta,26(27)-dihydroxy-5beta-cholestane. 3. 16-Deoxymyxinol, the third known bile alcohol having the 3beta-hydroxy-5alpha-hydrogen configuration, poses again the question of how the 3beta-hydroxyl group of cholesterol can be ;retained' in biosynthesis of primitive bile salts.     

Some properties of a microsomal oleate desaturase from leaves.

1. When [1-14C]oleoyl-CoA was incubated with a pea-leaf homogenate oleate was both incorporated into microsomal 3-sn-phosphatidylcholine and released as the unesterified fatty acid. The proportion of oleate incorporated into this phospholipid was dependent on the relative amounts of thiol ester and microsomal preparation present in reactions. 2. At the concentrations of microsomal preparation and [14C]oleoyl-CoA used to study oleate desaturation the metabolism of the thiol ester was essentially complete after 5 min incubation, but the loss of label from 3-sn-phosphatidylcholine oleate and the concomitant increase in radioactivity in the linoleate of this phospholipid proceeded at approximately linear rates over a 60 min period. The kinetics of labelling of unesterified linoleate was consistent with the view that this labelled fatty acid was derived from 3-sn-phosphatidylcholine. 3. Oleate desaturation required oxygen and with unwashed microsomal fractions was stimulated either by NADPH or by the 105 000g supernatant. Washed microsomal preparations did not catalyse desaturation, but actively was restored by the addition of NADPH, 105 000G supernatant or Sephadex-treated supernatant. NADPH could be replaced by NADH or NADP+, but not by NAD+. 4. Microsomal fractions from mature and immature maize lamina and expanding spinach leaves also rapidly incorporated oleate from ([14C]oleoyl-CoA into 3-sn-phosphatidylcholine, but desaturation of 3-sn-phosphatidylcholine oleate was detected only with microsomal preparations from immature maize lamina. 5. It is proposed that leaf microsomal preparations posses an oleate desaturase for which 3-sn-phosphatidylcholine oleate is either the substrate or an immediate precursor of the substrate.     

Comparison of the potencies of (+)- and (−)-2-ethylhexanoic acid in causing peroxisome proliferation and related biological effects in mouse liver

Male C57BL/6 mice were exposed to 1% (w/w) (+)- or (?)-2-ethylhexanoic acid or an equimolar mixture of these enantiomers in their diet for 4 or 10 days. A significant increase in liver weight and a 2- to 3-fold increase in the protein content of the mitochondrial fraction were seen in all cases. Peroxisomal palmitoyl-CoA oxidation was increased 2- to 3.5-fold after 4 days of treatment and 4- to 5-fold after 10 days, while the corresponding increases in peroxisomal lauroyl-CoA oxidase activity were 2- to 3-fold and 9- to 12-fold, respectively. Peroxisomal catalase activity was unchanged, whereas the microsomal and cytosolic activities were increased 2- to 3-fold and 6- to 16-fold, respectively. These treatments also induced microsomal ω-hydroxylation of lauric acid 7-fold and soluble epoxide hydrolase activity in the mitochondrial and cytosolic fractions, as well as microsomal epoxide hydrolase activity about 50–100%. The only significant differences observed between the effects of (+)-2-ethylhexanoic acid and its (?)-enantiomer were on peroxisomal palmitoyl-CoA oxidation and lauroyl-CoA oxidase activity after 4 days of treatment. In both these cases the (+)-enantiomer resulted in increases which were 50–75% greater than those seen with the (?)-form. © 1994 Wiley-Liss, Inc.