Supernatant Fraction Of Liver Homogenate Research Articles

Changes in activity of some enzymes involved in glucose utilization and formation in developing rat liver

1. The activities of some enzymes involved in both the utilization of glucose (pyruvate kinase, ATP citrate lyase, NADP-specific malate dehydrogenase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADP-specific isocitrate dehydrogenase, all present in the supernatant fraction of liver homogenates) and the formation of glucose by gluconeogenesis (glucose 6-phosphatase in the whole homogenate and fructose 1,6-diphosphatase, phosphopyruvate carboxylase, NAD-specific malate dehydrogenase and fumarase in the supernatant fraction) have been determined in rat liver around birth and in the postnatal period until the end of weaning. 2. The activities of those enzymes involved in the conversion of glucose into lipid are low during the neonatal period and increase with weaning. NADP-specific malate dehydrogenase first appears and develops at the beginning of the weaning period. 3. The marked increase in cytoplasmic phosphopyruvate carboxylase activity at birth is probably the major factor initiating gluconeogenesis at that time. 4. The results are discussed against the known changes in dietary supplies and the known metabolic patterns during the period of development.

Metabolism of drugs. LII. Enzymatic study on secobarbital metabolism.

ω-and (ω-1)-Oxidation of secobarbital was studied with cell free systems of rabbit liver. By gas chromatography, secobarbital was found to be metabolized into (ω-1)-hydroxy-secobarbital, ω-carboxysecobarbital, and (ω-1)-oxosecobarbital by incubation with 9000g supernatant fraction of liver homogenate. When secobarbital was incubated with microsomes with addition of NADPH and oxygen, formation of (ω-1)-hydroxysecobarbital and (ω-1)-hydroxymethylhexabital dehydrogenase were not concerned with metabolism of secobarbital in rabbit liver.

DEOXYRIBONUCLEIC ACID POLYMERASE ACTIVITY DURING AZO DYE CARCINOGENESIS.

SummaryDNA polymerase activity was measured in extracts of liver prepared from rats fed a carcinogenic azo dye. Groups of rats were sacrificed during the period of carcinogenic transformation in the liver, i.e., the first 8 weeks of dye-feeding, and the DNA polymerase activities in the soluble ultracen-trifugal supernatant fraction of liver homo-genates were assayed. A doubling of the rather low enzyme activity of normal rat liver was found at 6 days, the earliest time point, after initiation of dye feeding. Activity continued to increase through the third week of dye feeding to almost 3 times the normal value, then it gradually dropped to values slightly above normal at the end of the eighth week.

GLYCINE METHYLTRANSFERASE

The supernatant fraction of liver homogenate brings about the methylation of glycine by S-adenosylmethionine. The secondary amine formed in the reaction appears to be sarcosine. This conclusion is based on (I) the stoichiometry of the reaction, and (II) radioautography of chromatograms of the incubation products. The enzyme, glycine N-methyltransferase, was purified 80-fold, and certain of its characteristics are reported. The possible role of the enzyme in the biological oxidation of the methyl group of methionine is discussed.

Studies on ω-oxidation of fatty acids in vitro I. Overall reaction and intermediate

1. 1. ω-Oxidation of straight-chain fatty acids has been demonstrated with cell-free systems of guinea-pig liver. Cell-fractionation studies have shown that the 6000 × g supernatant fraction is the most active. 2. 2. Using sorbic acid amide, octatrienoic acid amide, or capric acid amide as substrate, formation of the corresponding dicarboxylic acod monoamide has been detected by paper chromatography, following incubation with the 6000 × g supernatant fraction of liver homogenate, together with NAD and the NADPGH 2-generating system. 3. 3. ω-Hydroxy-fatty acid has been found to be the intermediate compound and the hydroxylating reaction occurs in the microsomes. When [2- 14C]sorbic acid amide was incubated with microsomes, the formation of ε-hydroxy [2- 14C]sorbic acid amide was detected. Oxidation of ε-hydroxysorbic acid amide to muconic acid amide has been observed with a supernatant fraction of 105000 × g. 4. 4. The hydroxylating reaction requires NADPH 2 and oxygen. Catalase does not inhibit the reaction. The nature of the hydroxylating reaction is discussed.

GLYCINE METHYLTRANSFERASE

The supernatant fraction of liver homogenate brings about the methylation of glycine by S-adenosylmethionine. The secondary amine formed in the reaction appears to be sarcosine. This conclusion is based on (I) the stoichiometry of the reaction, and (II) radioautography of chromatograms of the incubation products. The enzyme, glycine N-methyltransferase, was purified 80-fold, and certain of its characteristics are reported. The possible role of the enzyme in the biological oxidation of the methyl group of methionine is discussed.

Studies on ω-oxidation of fatty acids in vitro I. Overall reaction and intermediate

1. 1. ω-Oxidation of straight-chain fatty acids has been demonstrated with cell-free systems of guinea-pig liver. Cell-fractionation studies have shown that the 6000 × g supernatant fraction is the most active. 2. 2. Using sorbic acid amide, octatrienoic acid amide, or capric acid amide as substrate, formation of the corresponding dicarboxylic acod monoamide has been detected by paper chromatography, following incubation with the 6000 × g supernatant fraction of liver homogenate, together with NAD and the NADPGH 2-generating system. 3. 3. ω-Hydroxy-fatty acid has been found to be the intermediate compound and the hydroxylating reaction occurs in the microsomes. When [2- 14C]sorbic acid amide was incubated with microsomes, the formation of ε-hydroxy [2- 14C]sorbic acid amide was detected. Oxidation of ε-hydroxysorbic acid amide to muconic acid amide has been observed with a supernatant fraction of 105000 × g. 4. 4. The hydroxylating reaction requires NADPH 2 and oxygen. Catalase does not inhibit the reaction. The nature of the hydroxylating reaction is discussed.