Pig Liver Cytosol Research Articles

Phosphatidylinositol movement between isolated microsomal and mitochondrial membranes.

Transfer of membrane-bound phosphatidyl-[2'-3H]inositol from microsomal to unlabelled mitochondrial and from mitochondrial to unlabelled microsomal membranes was studied using partially purified cytosol proteins isolated from guinea pig liver cytosol. In the absence and presence of these proteins the amounts of phosphatidylinositol transfer from microsomal to mitochondrial membranes were approximately 21 and 33%, respectively, and the amounts from mitochondrial to microsomal membranes were approximately 31 and 39%, respectively. The release of phosphatidyl-[2'-3H]inositol from microsomal membranes in the absence of mitochondria was dependent on concentration of cytosol proteins. Two mechanisms for movement between membranes are proposed. In cytosol-protein-independent movement of phosphatidyl-[2'-3H]inositol from microsomal to mitochondrial membranes, a direct contact between membranes is required, since phosphatidyl-[2'-3H]inositol was not detected in the reaction medium. In the cytosol-protein-catalyzed transfer, formation of phosphatidyl-[2'-3H]inositol - cytosol protein complex is postulated, since phosphatidyl-[2'-3H]inositol was released into the reaction medium and its movement proceeded from mitochondrial to microsomal membranes in the presence of partially purified cytosol proteins. Thus, contact between the two membranes is probably not necessary for this transfer. Implications for the movement of phospholipids between biological membranes are discussed.

The subcellular location of isozymes of NADP-isocitrate dehydrogenase in tissues from pig, ox and rat

Antibodies against purified NADP-isocitrate dehydrogenase from pig liver cytosol and pig heart were raised in rabbits. The purified enzymes from these sources are different proteins, as demonstrated by differences in electrophoretic mobility and absence of crossreactivity by immunotitration and immunodiffusion. The NADP-isocitrate dehydrogenase in the soluble supernatant homogenate fraction from pig liver, kidney cortex, brain and erythrocyte hemolyzate was identical with the purified enzyme from pig liver cytosol, as determined by electrophoretic mobility and immunological techniques. The enzyme in extracts of michondria from pig heart, kidney, liver and brain was identical with the purified pig heart enzyme by the same criteria. However, the ‘mitochondrial’ isozyme was the major component also in the soluble supernatant fraction of pig heart homogenate. The ‘cytosolic’ isozyme accounted for only 1–2% of total NADP-isocitrate dehydrogenase in pig heart, as determined by separation of the isozymes with agarose gel electrophoresis and immunotitration. The mitochondrial isozyme was also the predominant NADP-isocitrate dehydrogenase in porcine skeletal muscle. The ratio of cytosolic/mitochondrial isozyme for porcine whole tissue extract, determined by immunotitration, was about 2 for liver and 1 for kidney cortex and brain. The distribution of isozymes in cell homogenate fractions from ox and rat tissues corresponded to that observed in organs of porcine origin. The mitochondrial and cytosolic isozymes from ox and rat tissues exhibited crossreactivity with the antibodies against the pig heart and pig liver cytosol enzyme, respectively, and the electrophoretic migration patterns were similar qualitatively to those found for the isozymes in porcine tissues. Nevertheless, there were species specific differences in the characteristics of each of the corresponding isozymes. NAD-isocitrate dehydrogenase was not inhibited by the antibodies, confirming that the protein is distinct from that of either isozyme of NADP-isocitrate dehydrogenase.

Species Difference in Liver Microsomal Enzymes Involved in Mutagenic Activation <italic>N</italic>-Hydroxy-<italic>N</italic>-2-fluorenylacetamide<xref ref-type="fn" rid="fn2">2</xref><xref ref-type="fn" rid="fn3">3</xref>

The mutagenic activations of N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) by subcellular fractions of the livers of the Sprague-Dawley rat, C57BL mouse, Hartley guinea pig, Syrian golden hamster, and Macaca fuscata fuscata monkey were examined for sensitivity to paraoxon, which inhibits a deacetylase but not an arylhydroxamic acid acyltransferase. The mutagenic activation by liver microsomes was almost entirely mediated by a paraoxon-sensitive enzyme in all the animals tested. In contrast, the mutagenic activation by liver cytosol was mediated mostly by a paraoxon-sensitive enzyme in mice and guinea pigs, mostly by a paraoxon-resistant enzyme in rats and hamsters, and by both enzyme types in the monkey. In rats and guinea pigs, attempts were made to identify the enzymes causing mutagenic activation in the liver cytosol by a comparison of the elution positions of these enzymes in gel filtration and DEAE-cellulose column chromatography with those of known enzymes. In the rat liver cytosol, the mutagenic activation was mediated not only by acyltransferase but also by an unknown enzyme, which was resistant to paraoxon and differed from acyltransferase in chromatographic behavior. In the guinea pig liver cytosol, the activation was due to deacetylase activities, which could be separated into four fractions by gel filtration. These data indicate that species differ in the kinds of liver cytosolic enzymes involved in mutagenic activation of N-OH-2-FAA but not in the kind of liver microsomal enzyme involved.

Involvement of non-oxidative enzymes in mutagenic activation of urine from rats, given benzidine and some other aromatic amines

Urinary metabolites of rats treated with benzidine and some other genotoxic aromatic amines became mutagenic in the Ames assay after activation with liver cytosol from rat, mouse and guinea pig. Most of the mutagenic metabolites appeared in urine as glucuronides. Strong evidence was found that N,O- acyltransferase is responsible for the mutagenic activation by rat liver cytosol. The inhibitory effect of paraoxon and sodium fluoride indicates that the activation by mouse liver cytosol is due to the action of deacetylase. Mutagenic activation by guinea pig liver cytosol seemed to be mediated in part by deacetylase. The metabolite activated by these enzymes most likely is a glucuronidated, N-acetylated, N-hydroxylated product.

Studies on the oxidation of ω-hydroxyprostaglandins by an NAD-dependent dehydrogenase from mammalian liver cytosol

The oxidation of 12-hydroxylauric acid methyl ester (12-OH-L-Me) and of ω-hydroxy-prostaglandins (ω-OH-PGs) such as 20-OH-PGB 1 and 20-OH-PGE 1, was demonstrated with liver cytosol from rat, rabbit, and guinea pig in the presence of NAD; however, NADP did not support this oxidation. (ω-1)-Hydroxy-compounds (11-OH-laurate and 19-OH-PGB 1) and PGE 1, PGF 1α, and PGB 1, all lacking the terminal (ω)-hydroxyl, did not reduce NAD. However, at pH 10, PGE 1 slightly enhanced NAD reduction, suggesting that at this pH PGE 1, could be a substrate for 15-hydroxy-PG dehydrogenase (PGDH). The oxidation products from incubations of 12-OH-L-Me, 20-OH-PGB 1-Me, and 20-OH-PGE 1 with guinea pig liver cytosol were isolated and identified by gas chromatography/mass fragmentation spectrometry as being the corresponding dicarboxylic acids. In contrast to the liver cytosol, guinea pig kidney cytosol had only a minimal effect on NAD reduction by 12-OH-L-Me but nevertheless did support the stimulation of NAD reduction by PGE 1, and PGF 1α, but not by PGB 1, indicating the participation of kidney cytosolic PGDH in PGE 1 and PGF 1α oxidation and demonstrating that the oxidation of ω-OH to the carboxylic acid is not mediated by PGDH. Though the in vivo rate of oxidation of ω-OH-PGs has not been established, these results suggest that the urinary dicarboxylic-PG metabolites involve a multiple sequentialstep oxidation of PGs involving ω-hydroxylation by an NADPH-cytochrome P-450 system in the endoplasmic reticulum and the subsequent oxidation of the ω-OH by an NAD-dependent dehydrogenase in the cytosol.

Partial purification and characterization of a triacylglycerol lipase from rat liver cytosol

A triacylglycerol lipase (EC 3.1.1.3) was purified about 60-fold from rat liver cytosol by delipidation with acetone and ethyl ether, hydroxyapatite and Sephadex G-100 column chromatographies and isoelectrofocusing electrophoresis. The partially purified enzyme had a molecular weight of approximately 42 000 and an isoelectric point of 7.2. The K m for trioleylglycerol was 0.33 mM and the pH optimum was around 8.0. The activity of the enzyme was not dependent on serum lipoproteins, but was stimulated about 2-fold by several proteins such as serum albumin, lipoproteins, γ-globulin and ovalbumin. The lipase hydrolyzed trioleylglycerol to oleic acid and glycerol. NaCl had no effect on the enzymatic activity. Some physical and kinetic properties of the partially purified lipid-free lipase were different from those of crude non-delipidated lipase and also from those of a neutral triacylglycerol lipase which was recently purified partially from pig liver cytosol (Ledford, J.H. and Alaupovic, P. (1975) Biochim. Biophys. Acta 398, 132–148).

Implication of alanine and aspartate aminotransferases, glutamic dehydrogenase and phosphoenolpyruvate carboxykinase in glucose production in rats, chicken and pigs

1. 1. Blood glucose concentration in rat, pig and chicken was 88, 96 and 216 mg per 100 ml and hepatic glycogen content was 4.6, 2.6 and 2.4 mg/100 mg tissue, respectively. Except in pig, serum ASPTf activity was several times higher than ALAT activity. 2. 2. Total ASPT activity was 3 times higher than the total ALAT activity in rat liver, 14 times higher in chicken liver and 1.5 times higher in pig liver. In all species, ASPT activity in the muscle was roughly 50% of that in the liver. ASPT activity in chicken muscle was 70 times higher than the ALAT activity and 7 and 9 times higher in rat and pig muscle, respectively. 3. 3. ASPT activity in the liver was distributed between the mitochondria and the cytosol by 10% and 87% in rat, 14% and 78% in pig, and 14% and 82% in chicken, respectively, and ALAT activity in 13% and 83% in rat, 55% and 40% in pig and 90% and 6% in chicken, respectively. 4. 4. K m values for ASPT occurring in pig liver mitochondria and cytosol were 0.56 mM and 0.22 mM for α-ketoglutarate. 5. 5. Glutamic dehydrogenase was exclusively located in the mitochondria. Activity of this enzyme in pig liver mitochondria was approx 50 times higher than in the muscle mitochondria. PEPCK activity was distributed between the liver mitochondria and cytosol by 43% and 57%, respectively. 6. 6. Phosphorylase a, phosphoglucomutase, glucose-6-phosphatase, and fructose-1,6-diphosphatase activity in pig liver was not significantly different from the corresponding reported values for rat and chicken livers.

Studies on partially purified pig liver steroid delta 4-5 beta-reductase activity.

Some properties of partially purified steroid Δ4-5β-reductase activity of pig liver cytosol have been studied using testosterone as substrate. The enzymatic activity was stable for 72 h at 4° when stored in 0.05 M Tris–maleate buffer, pH 7.4 or 8.4; storage at pH 8 at 4° resulted in a 25% decrease in activity in 30 days. The pH optimum in Tris–maleate buffers was 6.4. Enzyme activity was completely inhibited by 0.2 mM p-chloromercuribenzenesulfonate and 0.2 mM p-chloromercuribenzoate. Enzyme activity was reduced by 20% and 45% with 1.0 mM iodoacetamide and 1.0 mM N-ethylmaleimide, respectively. The end products of the enzymatic reaction, NADP+ and 5β-dihydrotestosterone, inhibited the rate of reduction of testosterone. Testosterone Δ4-5β-reductase activity was present in protein of molecular weight 25 000–30 000, as determined by gel filtration.The enzyme preparation reduced a variety of C19 and C21 steroids. The highest activity (twice that for testosterone) was found with aldosterone as substrate.

Nicotinamide Adenine Dinucleotide Phosphate-linked Xylitol Dehydrogenase in Guinea Pig Liver Cytosol

Abstract Guinea pig liver has previously been reported to contain a highly specific NADP-linked xylitol dehydrogenase (EC 1.1.1.10) and NAD-linked xylitol dehydrogenases (EC 1.1.1.9) of broad substrate specificity. The NADP-linked enzyme, which interconverts xylitol and L-xylulose, has been obtained from mitochondria, while NAD-linked xylitol dehydrogenases, which interconvert xylitol and D-xylulose, have been obtained from both mitochondria and cytosol. This report describes a fourth xylitol dehydrogenase activity, which occurs in the cytosol of guinea pig liver. This enzyme is similar to the mitochondrial NADP-linked xylitol dehydrogenase in its coenzyme requirement, substrate specificity, and Km for xylitol, and its activity is much higher than that of the mitochondrial enzyme. The two soluble xylitol dehydrogenases were separated from each other on the basis of differential adsorption on alumina Cγ gel. The occurrence of high activities of these enzymes in the cytosol allows the required conversion of L-xylulose to D-xylulose via xylitol in the glucoronate-xylulose pathway without involvement of the mitochondria. Questions regarding the origin and role of the mitochondrial xylitol dehydrogenases are discussed.