Supernatant Fraction Of Rat Liver Research Articles

Rat liver alcohol dehydrogenase. Purification and properties.

Alcohol dehydrogenase (EC 1.1.1.1) from the rat liver supernatant fraction has been purified 200-fold and partially characterized. The isolation procedure involved ammonium sulphate fractionation, DEAE-Sephadex chromatography and gel filtration. The purified enzyme behaved as a homogeneous preparation as evaluated by cellulose acetate and polyacrylamide-gel disc electrophoresis. Sulphoethyl-Sephadex chromatography and immunoelectrophoresis with rabbit antiserum indicated the presence of a minor component. Rat liver alcohol dehydrogenase appears to contain 4mol of zinc/mol, has an estimated molecular weight of 65000 and consists of two subunits of similar molecular weight. Heavy-metal ions, thiol-blocking reagents, urea at concentrations below 8m, low pH (5.5) and chelating agents deactivate the enzyme but do not dissociate it into subunits. Deactivated enzyme could not be reactivated. The enzyme is strictly specific for NAD(+) and has a broad specificity for alcohols, which are bound at a hydrophobic site. Inhibition occurred with the enzyme equilibrated with Zn(2+) at concentrations above 0.1mm.

Immunochemical studies on malate dehydrogenase (decarboxylating) (NADP)

1. 1. Malate dehydrogenase (decarboxylating) (NADP) ( l-malate:NADP oxidoreductase (decarboxylating), EC 1.1.1.40) was purified from the supernatant fraction of rat liver. 2. 2. Antibody against this purified enzyme was prepared. This antibody inhibited the activity of the enzyme and precipitated it. 3. 3. Malate dehydrogenase (decarboxylating) (NADP) in supernatant fractions from kidney, heart, brain and adipose tissue of rats reacted with the antibody in the same manner as that of the ra tliver enzyme in the inhibition tests. Supernantant malate dehydrogenases from all these tissues except adipose tissue formed single precipitin bands with the antibody on Ouchterlony double-diffusion analysis which fused with each other. 4. 4. The enzyme from the livers of different species differed immunologically from that of rat liver. 5. 5. Mitochondrial enzyme was immunologically quite distinct from supernatant enzyme. 6. 6. Increase and decrease in enzyme activities in the cytoplasm of liver under various dietary and hormonal conditions were accompanied by proportional changes in the quantity of immunologically reactive protein.

The oxidation of cholesterol in rat liver sub-cellular particles. The cholesterol-7-alpha-Hydroxylase enzyme system.

Various sub‐cellular fractions of rat liver exert catalytic effects on the oxidation of [4‐14C]‐cholesterol to 7α‐hydroxy‐[4‐14C]cholesterol and to other [4‐14C]cholesterol oxidation products. Some of these oxidation products created in these studies in vitro appear to be aberrant oxidation products in that there is no evidence that they are intermediates in the physiological oxidation of cholesterol. These aberrant oxidation products have been classified as autoxidation products despite the fact that the autoxidation reactions in these cell fractions appear to require the participation of NADPH, native proteins and perhaps certain nucleotides for the production of these compounds.The enzyme system catalysing the oxidation of [4‐14C]cholesterol to 7α‐hydroxy‐[4‐14C]cholesterol could be assayed in a 18000 ×g supernatant fraction of rat liver. This enzymic reaction was adversely affected by extensive homogenisation of liver tissue and by prolonged incubation periods, both of which tended to increase the autoxidation reactions. The cholesterol‐7α‐hydroxylase enzyme showed optimal activity in the presence of NADPH and oxygen.In the washed liver microsomal fraction, [4‐14C]cholesterol was extensively oxidised in the presence of NADPH, ADP and ferrous ions to 7β‐hydroxycholesterol, cholestan‐3β, 5α, 6β‐triol and 7‐ketocholesterol. Further investigation showed that another compound was also formed, having the same mobility as 7‐ketocholesterol in the thin layer solvent systems used. Reduction and further metabolism of this compound compared with reference standards suggested that it may be a cholesterol oxide, formed under conditions where peroxidation of microsomal lipids could occur. A comparison of the effects of Tris and phosphate buffers, and various nucleotides suggests that cholesterol autoxidation in the microsomal fraction may be related to the general lipid peroxidation systems.The boiled 100000 ×g liver supernatant preparation or 2‐mercaptoethylamine both effectively quenched cholesterol peroxidation, at the same time stimulating the cholesterol‐7α‐hydroxylase system. A scheme has been suggested to explain the liver microsomal cholesterol autoxidation or peroxidation and to contrast this process with the mixed function oxidase involved in the enzymic cholesterol‐7α‐hydroxylase reaction.

Purification and properties of N-acetylglucosamine kinase from rat liver.

N-Acetylglucosamine kinase that catalyzes the phosphorylation of N-acetylglucosamine to N-acetylglucosamine-6-phosphate in the presence of ATP and Mg2+ has been purified 110-fold from the 105, 000×g supernatant fraction of rat liver by fractionation involving ammonium sulfate precipitation and DEAE-cellulose column chromatography. The purified enzyme is highly specific for N-acetylglucosamine and differs from the enzymes of bacterial origin in that the former does not catalyze the phosphorylation of glucose as does the latter. The activity is greatest at pH 9.4. The Km for N-acetylglucosamine is 6.0×10-5M and the Km for ATP is 4.8×10-4M. The enzyme is inhibited by ADP, whose effect is partly competitive with respect to ATP and partly noncompetitive. Two rat ascites tumors, namely, Yoshida sarcoma and Yoshida ascites hepatoma (AH 130), possess N-acetylglucosamine kinase at levels comparable to those found in rat liver.

Species-specific Reactions of Cell-free Polypeptide Synthesis

Summary Species-specific steps of polypeptide biosynthesis were studied in cell-free systems with components from E. coli , rat liver, and etiolated pea seedlings. For aminoacyl-tRNA synthesis, enzyme and tRNA fractions from Euglena gracilis and Anacystis nidulans were also included. Both amino acid acceptor and transfer reactions appeared specific in respect to the protokarytic or eukaryotic origins of the macromolecular components. According to the first reaction, the degree of specificity between protokaryotes ( Anacystis ) and eukaryotes ( Euglena , rat liver) was further dependent on the amino acid species. With aminoacyl-tRNA synthetases from the blue-green alga and tRNAs from rat liver and green as well as u. v.-bleached Euglena cells, the rates of arginyl-tRNA formation were at least in the same order of the homologous system from Anacystis. In contrast, a strict class specificity existed for phenylalanine and leucine acceptor activities. Transfer RNA from Anacystis was not charged with arginine by the eukaryotic synthetases, and the Euglena enzymes specific for leucine-tRNA showed a complete homologous behaviour to the tRNAs from any source. Poly U-dependent 14 C-phenylalanine incorporation with the homologous pea seedling system was found inactive, however, the heterologous system consisting of pea ribosomes and rat liver cytoplasm did work. On the other hand, the poly U-directed 14 C-phe-tRNA transfer into polypeptides occurred in all homologous, and in the heterologous systems from eukaryotes. Since the rates of 14 C-phe-tRNA transfer into polypeptides were additive also in combination with rat liver and pea seedling supernatants, it was concluded that the block of synthesis caused by pea supernatant fractions must be localized prior to the transfer step. Transfer activity was absent in heterologous systems with E. coli components. Additional rat liver supernatant fraction to the homologous E. coli system did not disturb the transfer reaction, but E. coli supernatants caused a marked suppression of the activity in the homologous eukaryotic system.

Disulfide reduction in rat liver. II. Chromatographic separation of nucleotide-dependent disulfide reductase and GSH-disulfide transhydrogenase activities of the high-speed supernatant fraction

1. 1. Three disulfide- reducing activities found in the high-speed supernatant fraction of rat liver, viz. glutathione reductase, GSH-disulfide transhydrogenase, and a NADPH-dependent reductase of broad specificity have been separated from each other by chromatography on DEAE-cellulose. 2. 2. Like the activity present in crude extracts, the chromatographically isolated transhydrogenase appears able to catalyze the reduction of both protein and nonprotein disulfide substrates by GSH in the presence of NADPH and an exogenous source of glutathione reductase. The purified enzyme appears to be quite unstable. 3. 3. The non-specific NADPH-dependent disulfide-reducing activity obtained by DEAE-cellulose chromatography was further purified by gel filtration on Sephadex G-150, using 5,5′-dithiobis-(2-nitrobenzoic acid)(DTNB) as indicator disulfide. The relatively low activity exhibited by this fraction in the reduction of insulin and other disulfides could be greatly increased when reaction mixtures were further supplemented with small amounts of supernatant solution obtained by heat-treatment of crude enzyme preparations at 70° for 20 min. The heat-stable component active in disulfide reduction was further purified on Sephadex G-100 and completely separated from glutathione reductase, whose activity was not affected by the heat treatment. The data presented herein is considered to support earlier speculations on the similarity of the nucleotide-dependent disulfide-reducing activity of normal rat liver to the thioredoxin systems found in the microorganisms and rat hepatoma.

A soluble amino acid-incorporating system from the fat-rich supernatant fractions of rat liver

A soluble amino acid-incorporating system from the fat-rich supernatant fractions of rat liver

Effect of Methylmalonyl Coenzyme A, a Metabolite Which Accumulates in Vitamin B12 Deficiency, on Fatty Acid Synthesis

Abstract We have proposed that the accumulation of methylmalonyl-CoA, which occurs when a deficiency of B12 coenzyme exists, may lead to its incorporation into fatty acids in place of malonyl-CoA. This lead to the production of fatty acids, which are not usually produced and which may be physiologically undesirable. As a first step in the test of this hypothesis, we have examined the effect of methylmalonyl-CoA on fatty acid synthesis in vitro, in rat liver supernatant fractions. It was shown that methylmalonyl-CoA inhibits fatty acid synthesis and is incorporated into fatty acids.

Disulfide reduction in rat liver: I. Evidence for the presence of nonspecific nucleotide-dependent disulfide reductase and GSH-disulfide transhydrogenase activities in the high-speed supernatant fraction

Dialyzed preparations of the 100,000 g supernatant fraction of rat liver were found to promote the reduction of the disulfide bonds of protein and nonprotein substrates when supplemented with NADPH but not with NADH. Extracts of the microsomal and mitochondrial fractions were inactive in this regard when supplemented with either nucleotide. Results of simultaneous spectrophotometric measurements of NADPH utilization and sulfhydryl formation were consistent with the appearance of two sulfhydryl groups per NADPH oxidized. The d- and l-forms of cystine were reduced by the supernatant enzyme system at equal rates. The nucleotide-dependent disulfide-reducing activity of the supernatant fraction was abolished by exposure to 10 −4, m arsenite or by heating at 70 ° for 30 min, treatments which were without effect on the NADPH-specific glutathione reductase activity of these same preparations. Similarities of this disulfide-reducing enzyme activity to the thioredoxin system have been noted. In addition to the foregoing nucleotide-dependent disulfide-reducing system, the supernatant fraction also exhibits an arsenite-insensitive GSH-disulfide transhydrogenase (oxidoreductase) activity capable of catalyzing the reduction of both protein and nonprotein disulfide substrates by GSH when coupled with NADPH and an endogenous or exogenous source of glutathione reductase.

Auxiliary Pathways of Galactose Metabolism: IDENTIFICATION OF REACTION PRODUCTS OF HEXOSE 6-PHOSPHATE DEHYDROGENASE AND OF "GALACTOSE DEHYDROGENASE"

Abstract Hexose 6-phosphate dehydrogenase, a microsomal enzyme form rat liver, has been shown to reduce NAD+ or NADP+ in the presence of galactose-6-P or glucose-6-P. The reaction product of hexose 6-phosphate and galactose-6-P, uniformly labeled with 14C was isolated by Dowex 1-chloride column chromatography. It readily formed lactone on boiling with 1 n HCl and was identified as 6-phosphogalactonic acid by paper chromatography. Incubation of liver fractions with 6-phosphogalactonic acid-1-14C resulted in formation of only minimal amounts of 14CO2, quantities that could readily have come from trace contaminants of 6-phosphogalactonic acid-1-14C. The product of glucose-6-P oxidation by hexose 6-phosphate and NAD+ provided substrate for the reduction of NADP+ in the presence of 6-phosphogluconic acid dehydrogenase, and was hence identified as 6-phosphogluconic acid. Incubation of uniformly labeled galactose-14C with rat liver supernatant fractions failed to disclose the formation of detectable quantities of galactonic acid. Thus, the existence of a galactose dehydrogenase in rat liver could not be confirmed.

9- 14C]fluorene hydroperoxide as a possible intermediate in the hydroxylation of [9- 3C]fluorene by rat liver homogenate

1. 1 [9- 14C]-fluorene was chosen as substrate to study the mechanism of biological oxygenation. Both fluorene and fluorene hydroperoxide were converted to fluorenol by rat liver supernatant fraction obtained at 10 000 × g. When the liver enzyme was bolied, it no longer converted fluorene to fluorenol; however, it converted fluorene hydroperoxide to fluorenol at 36% of the activity of actiev unheated enzyme. 2. 2. The pretreatment of rats with fluorene doubled the enzymatic activity for the conversion of fluorene to fluorenol, whereas phenobarbital increased activity to 5-fold as compared to control. 3. 3. Radioactive fluorene hydroperoxide has been identified in reaction mixtures hydroxylating [9- 14C]fluorene. An incorporation yield of 4.8% of [9- 14C]fluorene hydroperoxide from [9- 14C]fluorene was obtained. The radioactive fluorene hydroperoxide was isolated, converted into fluorenone and the phenylhydrazone. The identity and purity was confirmed by thin-layer chromatography, by ultraviolet spectra and by specific determination. 4. 4 The findings indicated that [9- 14C]fluorene hydroperoxide can be an intermediate in the hydroxylation of [9- 14C]fluorene by rat liver homogenate.

Purification of rat liver RNase inhibitor and its effect on polyribosome integrity

The RNase inhibitor from 100,000 g rat liver supernatant fraction has been purified 3000–4000-fold by ammonium sulfate fractionation, DEAE-Sephadex A-50 chromatography, and gel filtration on Sephadex G-100. Purification of the inhibitor is paralleled by a concomitant purification of the factor which stabilizes the polysomal profile on sucrose gradients. This is also reflected by amino acid incorporation ability of the polyribosomes. Increasing purity of the factor is associated with increasing lability.

Mechanism of methionine biosynthesis in rat liver.

Synthesis of methionine in rat liver by the B12‐independent N5‐methyltetrahydropterolyltri‐glutamate: homocysteine methyltransferase reported by Wang, Koch and Stokstad has been investigated. In agreement with their results it was found that methionine was formed by crude rat liver supernatant fraction and dialyzed mitochondrial extract on aerobic incubation with N5‐methyltetrahydropteroyltriglutamate, homocysteine and magnesium chloride. However, the folate derivative could not have been involved in this process since N5‐[14C]methyl‐tetrahydropteroyltriglutamate did not incorporate 14C into the methionine. In the supernatant fraction, methionine was formed by the transmethylation of endogenous betaine with added homocysteine. Rat liver supernatant fraction was found to contain 250 nmoles/ml betaine. In the mitochondrial extract, methionine formation occurred by proteolysis. It is concluded that a B12‐independent N5‐methyltetrahydropteroyltriglutamate: homocysteine methyltransferase does not occur in rat liver.

Mechanism of aliphatic hydroxylation tetralin hydroperoxide as an intermediate in the hydroxylation of tetralin in rat-liver homogenate

1. 1. Tetralin and tetralin 1-hydroperoxide were chosen as model to study the mechanism of biological oxygenation. Both tetralin and tetralin hydroperoxide were converted to tetral-1-ol by rat liver supernatant fraction obtained at 10000 × g. Both the conversions of tetralin to tetralol and tetralin hydroperoxide to tetralol required NADPH. 2. 2. For the conversion of tetralin hydroperoxide to tetralol, ferrous sulfate, peroxidase, hemin, catalase and cytochrome c had no catalytic effect, whereas both boiled liver enzyme and hemoglobin had 30% of the activity of liver 10000 × g supernatant fraction. Boiled liver enzyme had no catalytic effect on the conversion of tetralin to tetralol. 3. 3. The optimal pH for the conversion of tetralin to tetralol was found to be between 6.0 and 7.6, while the optimal pH for the conversion of tetralin hydroperoxide to tetralol lay sharply between 6.8 and 7.0. 4. 4. For the conversion of tetralin to tetralol, a K m value of 6·10 −4M, and a v max value of 0.024 μg/min per mg protein were found. For the conversion of tetralin hydroperoxide to tetralol, a K m value of 3·10 −4 M and a v ax value of 0.087 μg/min per mg protein were obtained. 5. 5. Of the inhibitors at the concentrations studied, o-iodosobenzoate and mercuric chloride were the most effective in inhibiting the conversion of tetralin hydroperoxide to tetralol (>90%). They inhibited the conversion of tetralin to tetralol by 65% and 40% respectively. Sodium azide and carbon monoxide inhibited the conversion of tetralin to tetralol by 50%, but had no effect in inhibiting the conversion of tetralin hydroperoxide to tetralol. Other inhibitors had various effects. 6. 6. By the addition of p-chloromercuribenzoate (PCMB) or by the removal of NADPH, tetralin hydroperoxide was accumulated and detected in the hydroxylation of tetralin. 7. 7. These findings indicate that tetralin hydroperoxide can be an intermediate in the hydroxylation of tetralin to tetralol in rat-liver homogenate and eliminate the possibility of a mono-atomic oxygen species as a reactant in this reaction.

Chain extension of ribonucleic acid by enzymes from rat liver cytoplasm.

1. The 105000g supernatant fraction of rat liver catalyses the incorporation of ribonucleotides from ribonucleoside triphosphates into polyribonucleotide material. The reaction requires Mg(2+) ions and is enhanced by the addition of an ATP-generating system and RNA, ATP, UTP and CTP but not GTP are utilized in this reaction. In the case of UTP, the product is predominantly a homopolymer containing 2-3 uridine residues, and there is evidence that these may be added to the 3'-hydroxyl ends of RNA or oligoribonucleotide primers. 2. The microsome fraction of rat liver incorporates ribonucleotides from ATP, GTP, CTP and UTP into polyribonucleotide material. This reaction requires Mg(2+) ions and is enhanced slightly by the addition of an ATP-generating system, and by RNA but not DNA. Supplementation of the reaction mixture with the three complementary ribonucleoside 5'-triphosphates greatly increases the utilization of a single labelled ribonucleoside 5'-triphosphate. The optimum pH is in the range 7.0-8.5, and the reaction is strongly inhibited by inorganic pyrophosphate and to a much smaller degree by inorganic orthophosphate. It is not inhibited by actinomycin D or by deoxyribonuclease. In experiments with [(32)P]UTP in the absence of ATP, GTP and CTP, 80-90% of (32)P was recovered in UMP-2' or -3' after alkaline hydrolysis of the reaction product. When the reaction mixture was supplemented with ATP, GTP and CTP, however, about 40% of the (32)P was recovered in nucleotides other than UMP-2' or -3'. Although the reactions seem to lead predominantly to the synthesis of homopolymers, the possibility of some formation of some heteropolymer is not completely excluded.

The effect of diphenylhydantoin in vitro on the metabolism of testosterone by rat liver subcellular fractions.

ABSTRACT The effect of diphenylhydantoin (DPH) in vitro on the »metabolite profile« of testosterone-4-14C has been studied utilizing incubates of high speed supernatant and microsomal fractions of rat liver. In contrast to earlier experiments with rat liver slices, in which a standard amount of DPH resulted in increased non-polar metabolite production, in the present experiments, when separate subcellular fractions are incubated, no such DPH effect can be detected under the conditions utilized. When certain mixtures of supernatant and microsomal fractions are incubated, however, there is evidence that the DPH augmentation of non-polar metabolite production can again be manifest. An explanation for these results can only be speculated upon at present.

A Glutamine-dependent Enzyme for the Synthesis of Carbamyl Phosphate for Pyrimidine Biosynthesis in Fetal Rat Liver

Abstract Experiments with the soluble supernatant fraction of fetal rat liver indicate that it contains a glutamine-dependent carbamyl phosphate synthetase. This enzyme can utilize ammonia or glutamine as substrate, but the Km for glutamine is low (approximately 10-5 m) while the Km for ammonium ion-ammonia is at least 100 times higher. The ATP saturation curve for the enzyme appears to be sigmoidal. The pH optimum is 7.4. Glutamine analogues inhibit the ability of either glutamine or ammonia to serve as substrate. Comparisons of the change in the amounts of the glutamine-dependent carbamyl phosphate synthetase and aspartate transcarbamylase in the soluble fraction of the cell and in the amounts of acetylglutamate-dependent carbamyl phosphate synthetase and ornithine transcarbamylase in the particulate fraction of the cell with development from 4 days prior to gestation to 2 days after birth are reported. They suggest that the new enzyme provides carbamyl phosphate for the pyrimidine pathway. The same enzyme activity has also been found in the soluble fraction of a pigeon liver homogenate.

Incorporation of [ 14C]arginine into rat liver proteins catalysed by soluble enzymes only

Rat liver supernatant fractions which are completely free of microsomes and are either freshly prepared or dialysed and freeze-dried, actively incorporate radioactive arginine into their own proteins. The incorporation requires ATP and magnesium ions and is inhibited by iodoacetate, p-chloromercuribenzoate, ribonuclease and, slightly, by deoxyribonuclease. It differs from microsomal protein synthesis in that it is hardly affected by the presence of GTP, of phosphocreatine plus creatine kinase or of a mixture of amino acids, and is relatively insensitive to puromycin or to a change of the KCl in the incubation medium for NaCl. The entire arginine molecule is incorporated in such a way that its α amino group is blocked.

Use of a ribonuclease inhibitor from rat liver supernatant fraction in the preparation of polyribosome-like particles from isolated rat liver nuclei

Rat liver supernatant fraction has been used as a ribonuclease inhibitor to prepare polyribosome-like particles from rat liver nuclei disrupted by deoxycholate. The RNA of these nucleus-associated particles was labelled with [14C]-orotic acid and the sedimentation characteristics of acid-insoluble radioactive material as determined by zone centrifugation analysis in a sucrose density-gradient corresponded to the sedimentation of cytoplasmic polyribosomes. The nucleus-associated particles did not incorporate amino acids into protein rapidly. A role in the transfer of messenger RNA from nucleus to cytoplasm is suggested for these polyribosome-like particles.

Nature of a soluble, glutathione-dependent enzyme system active in cleavage of methyl parathion to desmethyl parathion.

The nature of the enzyme, in the supernatant fraction of tissue homogenates of rat liver and insect mid-gut, that cleaves O, O-dimethyl O-p-nitrophenyl phosphorothioate (methyl parathion) to O-methyl O-p-nitrophenyl phosphorothioate (desmethyl parathion) was examined. The enzyme, from both sources, is inactivated by dialysis or gel filtration and its activity is effectively restored by addition of reduced glutathione (GSH). Many rat tissues are active in methyl parathion-demethylation, but highest activity occurs in the liver. Although the addition of GSH increases the activity of insect mid-gut and fat body, these insect enzymes fall short of the degrading activity of rat liver. The degradation product of methyl parathion as formed by the supernatant fraction of rat liver fortified with GSH was identified as the desmethyl derivation. Two metabolites, the desmethyl derivative and phosphoric acid, were detected from the insect mid-gut preparations when fortified with GSH. Rat liver has approximately 5.5 times as much GSH as the mid-gut of larvae of horn beetle, Xylitrupes dichotomus (L.). Cellulose chromatographic separation of the supernatant of rat liver and insect mid-gut homogenates reveals that the nature of these 2 enzyme proteins is different. The metabolic difference in the degradation of organophorphorus insecticides containing the P-O-methyl between mammals and insects is discussed in relation to the selective toxicity of these compounds.