Supernatant Fraction Of Rat Liver Research Articles

Isolation and Characterization of a Novel Perchloric Acid-soluble Protein Inhibiting Cell-free Protein Synthesis

We found a novel protein in the postmitochondria supernatant fraction of rat liver, which is soluble in 5% perchloric acid and strongly inhibits protein synthesis in a rabbit reticulocyte lysate system. The protein extracted from the supernatant fraction with 5% perchloric acid was purified by ammonium sulfate fractionation and CM-Sephadex chromatography. The protein was shown to consist of two identical subunits with a molecular mass of 14 kDa. By immunoscreening with the rabbit antisera against the protein, a cDNA encoding the protein was cloned and sequenced. The cDNA contained an open reading frame of 411 base pairs encoding a 136-amino acid protein with a molecular mass of 14,149 Da. The deduced amino acid sequence was completely identical with that constructed from all of the above peptides. Interestingly, the perchloric acid-soluble protein inhibited cell-free protein synthesis in the rabbit reticulocyte lysate system in a different manner from RNase A. The protein is likely to inhibit an initiation stage of cell-free protein synthesis. Among the rat tissues tested, the protein was located only in liver and kidney. These findings are the first report on a new inhibitor that may be involved in the regulation of protein synthesis in those tissues.

Enzymic reduction of [6]-gingerol, a major pungent principle of ginger, in the cell-free preparation of rat liver

A reductive metabolism of S-(+)-[6]-gingerol [1-(4'-hydroxy-3'-methoxyphenyl)-5-hydroxydecan-3-one], the major pungent principle of ginger, was investigated in vitro with phenobarbital-induced rat liver 10,000 x g supernatant containing the NADPH-generating system. The ethyl acetate-extractable products were isolated and two metabolites were identified as diastereomers of [6]-gingerdiol by gas chromatrography/mass spectrometry. The ratio of two isomers formed in the above reaction was about 1:5, suggesting the stereospecific reduction of S-(+)-[6]-gingerol by carbonyl reductase activity present in the postmitochondrial supernatant fraction of rat liver. The enzymic reduction of S-(+)-[6]-gingerol thus introduces the second asymmetric carbon center in the molecule with concomitant production of S, S- and R, S-isomers of [6]-gingerdiol in different proportions. This stereospecific reduction of [6]-gingerol may be relevant to the clinical use of the compound.

In vitro O-demethylation from a heterocyclic nitrogen: A novel metabolic reaction?

1. O-Demethylation of 1-methoxyindole-3-carboxylic acid, in vitro, by determination of liberated formaldehyde, has been demonstrated using fortified 15,000 g rat liver supernatant fraction. 2. Results have been compared to those similarly obtained with a standard O-demethylation substrate, 4-nitroanisole, to substantiate and quantify the extent of the reaction. 3. Km values for 4-nitroanisole and methoxyindole-3-carboxylic acid were 8.9 and 6.5 mM, while Vmax values were 4.9, and 5.2 nmol HCHO produced/mg protein per min. 4. The significance of the metabolic O-demethylation reaction for N-methoxyindoles as a novel metabolic pathway is discussed in terms of pharmacological activity and biological reactivity.

Mutagenicity of 3‐chlorodibenzofuran and its metabolic activation

3-Chlorodibenzofuran was the only markedly mutagenic isomer among the four monochlorodibenzofurans. Although it was mutagenic even in the absence of 9,000g supernatant fraction (S9) of rat liver, it was further activated by the addition of S9. Metabolic activation of this compound in mutagenicity was studied using liver S9s and cell fractions which were prepared from rats treated with two inducers. 1,1-Dichloro-2,2-bis(p-chlorophenyl) ethylene (DDE) was used as an inducer of phenobarbital inducible cytochrome P-450, and beta-naphthoflavone (beta NF) was used as an inducer of 3-methylcholanthrene inducible cytochrome P-448. S9, microsomal, mitochondrial, and cytosolic fractions were obtained from four groups of rats, i.e., untreated, DDE treated, beta NF treated, and DDE and beta NF treated groups. Mutagenicity was tested using Salmonella typhimurium tester strain TA98, because this strain is more sensitive to 3-chlorodibenzofuran than strain TA100. This experiment showed that 3-chlorodibenzofuran was activated most highly by beta NF-induced microsomes. However, it was also activated by the cytosolic fraction. Moreover, it was highly activated in rat livers which were not treated with inducers. The activity of aryl hydrocarbon hydroxylase (AHH) of each fraction was measured. AHH did not always become an index of the metabolic activation of 3-chlorodibenzofuran. This study showed that 3-chlorodibenzofuran is activated not only by cytochrome P-448, which is induced by 3-methylcholanthrene type inducers, but also by the enzymes existing in normal rat liver. This result suggests a risk of manifestation of its toxicity to normal animals.

Identity of a cytosolic neutral cholesterol esterase in rat liver with the bile salt stimulated cholesterol esterase in pancreas

A neutral cholesterol esterase has been purified to homogeneity from the cytosolic fraction of rat liver. The 105 000 × g supernatant fraction of rat liver was applied to a DEAE-cellulose column to isolate a partially purified fraction of hepatic cholesterol esterase. Immunoblot analysis of the partially purified liver fraction with the anti-porcine pancreatic cholesterol esterase IgG demonstrated a single band with a molecular weight of 67 000. The hepatic protein was then isolated by immunoaffinity chromatography technique using a column constructed with antibodies prepared against the pancreatic cholesterol esterase. Characterization of the hepatic cholesterol esterase revealed that the hepatic enzyme shared antigenic epitopes with the pancreatic cholesterol esterase and was similarly activated by addition of bile salt such as taurocholate. Moreover, amino-terminal sequencing analysis of the hepatic cholesterol esterase showed an identical sequence with the pancreatic enzyme. Taken together, these results showed that the cholesterol esterases in the liver and the pancreas are very similar and possibly identical proteins.

A selective inhibitor of N8-acetylspermidine deacetylation in mice and hela cells without effects on histone deacetylation

The inhibitory effects of 7-[ N-(3-aminopropyl)amino]heptan-2-one (APAH) on N 8-acetylspermidine deacetylation were studied. In in vitro studies, APAH produced inhibition (apparent K i of 0.18 μ m) of N 8-acetylspermidine deacetylation by the 100,000 g supernatant fraction of rat liver. This apparent K i was 60-fold less than the apparent K m (11 μ m) for deacetylation of the substrate, N 8-acetylspermidine, suggesting that APAH could be a potent, effective inhibitor in vivo. APAH was administered to mice by intraperitoneal injection at a dose of 200 mg/kg, and polyamine and acetylpolyamine levels in liver and spleen were measured. In tissues of control mice, N 8-acetylspermidine was not detectable but increased to detectable levels 30–360 min after APAH treatment. These data are consistent with inhibition of the deacetylase by APAH. Increases in putrescine and N 1-acetylspermidine levels occurred in liver after APAH treatment with increases in N 1-acetylspermidine levels observed in spleen. In HeLa cells, a significant increase in N 8-acetylspermidine was observed following 24 h exposure to 10 μ m APAH while no change occurred in the acetylation level of HeLa cell histones. In contrast, 24 h exposure to 10 m m sodium butyrate produced no change in N 8-acetylspermidine levels and an increase in the acetylation level of histones H4 and H2B. These results suggest that APAH has a relatively selective inhibitory effect on N 8-acetylspermidine but not histone deacetylation. This is the first report of significant levels of N 8-acetylspermidine in animal tissues and of the effects of in vivo inhibition of N 8-acetylspermidine deacetylase.

Metabolism of bromobenzene. Analytical chemical and structural problems associated with studies of the metabolism of a model aromatic compound.

Our studies of bromobenzene metabolism have shown that the 3,4-oxide is metabolized to 3- and 4-bromophenol through an extended glutathione pathway. The mechanism of sulfur elimination from a dihydrobromobenzene metabolite is not known, although it is known that the aromatization reaction will occur in a 9000-g supernatant fraction of rat liver. The hepatotoxic and nephrotoxic metabolites of bromobenzene are most likely bromthiocatechols and a bromothiopyrogallol, respectively.

Metabolism of bromobenzene : Analytical chemical and structural problems associated with studies of the metabolism of a model aromatic compound

Our studies of bromobenzene metabolism have shown that the 3,4-oxide is metabolized to 3- and 4-bromophenol through an extended glutathione pathway. The mechanism of sulfur elimination from a dihydrobromobenzene metabolite is not known, although it is known that the aromatization reaction will occur in a 9000- g supernatant fraction of rat liver. The hepatotoxic and nephrotoxic metabolites of bromobenzene are most likely bromothiocatechols and a bromothiopyrogallol, respectively.

The presence and activity in normal and regenerating rat liver postmicrosomal supernatant fraction of an enzyme with properties similar to those of membrane-bound 5'-nucleotidase.

An alkaline 5'-nucleotidase with properties similar to those of membrane-bound 5'-nucleotidase was recovered in soluble form in the postmicrosomal supernatant fraction (cytosol) of rat liver. The enzyme seems to constitute a quantitatively distinct fraction, since the activity in postmicrosomal supernatants was increased by a further 10% by additional homogenization of livers. Lysosomal acid phosphatase activity increased similarly, whereas other membrane-bound marker enzymes alkaline phosphatase, phosphodiesterase I and glucose-6-phosphatase showed no increase when homogenization of liver tissue was continued. Gel-permeation chromatography and pH-dependence studies indicated that enzyme activity in the supernatant fraction with 0.3 mM-UMP or -AMP as substrate at pH 8.1 was about 85 or 100% specific respectively. In regenerating liver the enzyme recovered in soluble form showed decreased specific activity, in contrast with alkaline phosphatase measured for comparison. The nucleotidase activity per mg of cytosolic protein was 2.1 nmol/min with AMP as substrate. The total activity measured in the postmicrosomal supernatant was 1.5% of the homogenate activity measured in the presence of detergent.

Physiological changes in the activities of extramitochondrial acetyl-CoA hydrolase in the liver of rats under various metabolic conditions

Significant increase in the activity of an acetyl-CoA hydrolase (ATP-stimulated, ADP-inhibited enzyme) in the supernatant fraction of rat liver was observed after 44-68 h of starvation (about 2-fold), and in the early stage of diabetes (about 1.6-fold), but not in the chronic stage of diabetes. The increased enzymatic activity in starved rats returned to the control level within 20 h when the animals were given laboratory chow, but not when they were given fat-free diet with a high carbohydrate content, and the enzyme activity was increased by the latter diet containing 1% thyroid powder. A single intraperitoneal injection of 3,3'5-triiodo-L-thyronine or 3,3',5,5'-tetraiodo-L-thyronine resulted in twice the normal enzyme activity two days later, and conversely 7 days after thyroidectomy, the enzyme activity was about 60% of the control level. A single subcutaneous injection of alpha-(p-chlorophenoxy)isobutyric acid, a hypolipidemic drug, doubled the enzyme activity in euthyroid rats, but not in thyroidectomized rats. Of the various tissues tested besides the liver, only the kidney had detectable ATP-stimulated and ADP-inhibited enzyme activity (5% of the activity in liver cytosol). The kidney enzyme had similar kinetic and immunochemical properties to the liver enzyme. Changes in the enzyme activity in the liver in various states were closely related to the amount of enzyme present, judging from results obtained by enzyme-linked immunosorbent assay. The physiological role of this enzyme (which hydrolyzes acetyl-CoA to acetate and CoASH) may be in maintenance of the cytosolic acetyl-CoA concentration and CoASH pool for both fatty acid synthesis and oxidation.

Oxidative inactivation of an extramitochondrial acetyl-CoA hydrolase by autoxidation of L-ascorbic acid.

The activity of acetyl-CoA hydrolase (dimeric form) purified from the supernatant fraction of rat liver was shown to have a half-life (t1/2) of 3 min at 0 degree C, but to stable at 37 degrees C (t1/2 = 34 h) [Isohashi, F., Nakanishi, Y. & Sakamoto, Y. (1983) Biochemistry 22, 584-590]. Incubation of the purified enzyme with L-ascorbic acid (AsA) at 37 degrees C resulted in inactivation of the enzyme (t1/2 = 90 min at 2 mM AsA). The extent of inactivation was greatly enhanced by addition of transition metal ions (Cu2+, Fe2+, and Fe3+). Thiol reducing agents, such as reduced glutathione and DL-dithiothreitol, protected the hydrolase from inactivation by AsA. However, these materials did not restore the catalytic activity of the enzyme inactivated by AsA. When AsA solution containing Cu2+ was preincubated under aerobic conditions at 37 degrees C for various times in the absence of enzyme, and then aliquots were incubated with the enzyme solution for 20 min, remaining activity was found to decrease with increase in the preincubation time, reaching a minimum at 60 min. However, further preincubation reduced the potential for inactivation. Catalase, a hydrogen peroxide (H2O2) scavenger, almost completely prevented inactivation of the enzyme by AsA plus Cu2+. Superoxide dismutase and tiron, which are both superoxide (O2-) scavengers, also prevented inactivation of the enzyme. A high concentration of mannitol, a hydroxyl radical (OH) scavenger, partially protected the enzyme from inactivation. These results suggest that inactivation of the enzyme by AsA in the presence of Cu2+ was due to the effect of active oxygen species (H2O2, O2-, OH) that are known to be autoxidation products of AsA. Valeryl-CoA, a competitive inhibitor of acetyl-CoA hydrolase, greatly protected the enzyme from inactivation by AsA plus Cu2+, but ATP and ADP, which are both effectors of this enzyme, had only slight protective effects. These results suggest that inactivation of this enzyme by addition of AsA plus Cu2+ was mainly due to attack on its active site.

Post-translational import of fatty acyl-CoA oxidase and catalase into peroxisomes of rat liver in vitro.

Total polysomal RNA of rat liver was translated in vitro in a rabbit reticulocyte lysate system. The translation products were mixed with a postnuclear supernatant fraction of rat liver and incubated post-translationally at 26 degrees C for 15-60 min. The import assay mixture was separated into a particulate fraction and supernatant by centrifugation, both of which were analyzed by immunoprecipitation with a goat antibody against rat liver peroxisomal proteins, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography. One peroxisomal translation product (Mr 72,000) appeared in the particulate fraction, was partly proteinase K-resistant, and addition of detergents prior to proteolysis abolished this resistance. In isopycnic centrifugation of the uptake assay mixture, the protease-resistant 35S-polypeptide of Mr 72,000 cosedimented with the peroxisomes. This translation product was identified immunochemically as fatty acyl-CoA oxidase; both before and after import it was indistinguishable in size from subunit A of the purified enzyme by prolonged sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the cell-free translation products were incubated with highly purified peroxisomes, 35S-catalase entered peroxisomes (by the criterion of protease resistance), and its entry was stimulated by the addition of a high speed supernatant (cytosolic) fraction of rat liver. These results demonstrate the post-translational import into peroxisomes in vitro of at least two cell-free translation products.

The glutathione S-transferases in selenium and vitamin E deficiency.

Preliminary experiments confirmed the work of others showing that the total glutathione peroxidase (GSH-px) activity of rat liver supernatant fraction may be resolved into two peaks of activity (peaks I and II) by gel filtration, and that peak I is the selenium-containing enzyme and peak II is another peroxidase indistinguishable from glutathione S-transferase (GST). In selenium and vitamin E deficiency, the total activity of the GSH-px became very low, and the total activity of GST with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate was enhanced. Study of the time course of these changes as deficiency progressed indicated that the stimulus for the rise in GST (CDNB) activity was the fall in GSH-px activity which preceded it. The peroxidase activity of GST was found to reside only in the GST AA, B and B2 forms of the enzyme, which were shown to be respectively a homodimer of the Yc subunit, a homodimer of the Ya subunit and a heterodimer of the YaYc subunit. As vitamin E and selenium deficiency progressed, the B2 and AA forms of the enzyme showed enhanced activity, which was interpreted as implying that the Yc subunit of the enzyme becomes enriched as a consequence of the withdrawal of selenium from the animal's diet. Densitometric measurements of the Yc and Ya subunits confirmed that the amount of the Yc subunit was nearly doubled in selenium deficiency, relative to the Ya subunit.

83] Kynurenine aminotransferase from kidney supernatant

This chapter provides an overview of the kynurenine aminotransferase from kidney supernatant. L-kynurenine: α-ketoglutarate aminotransferase (cyclizing) activity of rat kidney supernatant, an enzyme in tryptophan catabolism, copurifies with the activity of α-aminoadipate: α-ketoglutarate aminotransferase. The presence of aminoadipate (kynurenine) aminotransferase in kidney represents an enzyme associated with lysine catabolism rather than with tryptophan degradation. α-aminoadipate (kynurenine) aminotransferase also catalyzes aminotransfer of 3,5-diiodotyrosine (halogenated tyrosine aminotransferase) suggesting a possible role in thyroid hormone metabolism. The reactions catalyzed by the kynurenine and halogenated tyrosine aminotransferase activities are presented diagrammatically. α-aminoadipate (kynurenine):α-ketoglutarate aminotransferase activity is also present in rat kidney mitochondria and the supernatant and mitochondrial fractions of rat liver. The activity of the L-α-aminoadipate aminotransferase is measured by the rate of disappearance or appearance of α-ketoglutarate with glutamate dehydrogenase. The chapter also reviews the kynurenine aminotransferase assay. The activity of kynurenine aminotransferase is measured spectrophotometrically by following the appearance of kynurenate.

Thyroid hormone binding in rat liver cytosol.

Binding of 3,5,3'-tri-iodothyronine (T3) and thyroxine (T4) to components of perfused rat liver supernatant fraction and isolated liver cell cytosol was studied. Of the four binding fractions in supernatant (X, A, Y and Z) separable by gel chromatography, both T3 and T4 bound preferentially to the A-fraction, which was shown to contain albumin as the major binding protein. When cytosol prepared from isolated cells was examined, T4 was again bound mainly in the A-fraction; however, T3 was observed to bind predominantly in the Y-region. Hormone binding to soluble protein in the latter system is thought to reflect the pattern in vivo, better than does binding in supernatant, although the possibility exists that the concentration of albumin observed in cytosol may be artificially high due to transfer of membrane-bound albumin during cell disruption. Nevertheless, albumin (possibly derived from more than one intracellular source) is capable of binding T4 in vivo. The presence of this protein within the hepatocyte may thus contribute to the high T4 binding capacity of the liver compared to other tissues.

A cold-labile acetyl-coenzyme-A hydrolase from the supernatant fraction of rat liver. Reactivation and reconstitution of the active species from the inactive monomer.

An acetyl-coenzyme-A hydrolase from the supernatant fraction of rat liver is known to be rapidly inactivated at low temperature. Loss of catalytic activity is accompanied by apparent dissociation of tetrameric and dimeric forms of the enzyme into monomers. It was found that rewarming under appropriate conditions almost completely reversed the cold-induced inactivation and dissociation of the enzyme: At a protein concentration of 14 micrograms/ml, simple rewarming only partially restored the enzyme activity (less than 3% of the original activity), but at a higher concentration of the enzyme or in the presence of 1 mg/ml bovine serum albumin, the reactivation by warming was greater. Warming at 37 degrees C appeared to be optimal for reactivation; warming at 25 degrees C or at 43 degrees C was less effective. Longer exposure to cold did not affect reactivation on rewarming, but on repeated inactivation and reactivation the reactivation decreased to some extent, especially at lower concentrations of enzyme protein. Among various nucleotides tested, ATP greatly enhanced the restoration of the activity, while ITP, UTP and ADP were less effective and AMP, GTP, TTP and CTP had little effect. At an enzyme-protein concentration of 14 micrograms/ml, 2 mM ATP restored the enzyme activity to about 70% of that before cold treatment, while acetyl-CoA (0.5 mM) restored the activity about 50%. High concentrations of phosphate (0.92 M) and pyrophosphate (0.45 M) restored about 80% and 95%, respectively, of the original activity. Sucrose density gradient centrifugation of the active dimer at high enzyme concentration at 4 degrees C for 20 h produced a monomeric form without catalytic activity. Gel filtration showed that simple rewarming mostly converted the monomeric enzyme obtained in this way to the dimeric form, whereas on rewarming with ATP the monomer was mostly converted to a tetrameric form. The dimeric and tetrameric forms both had catalytic activity.

Effect of acetaldehyde and cyanamide on the metabolism of formaldehyde by hepatocytes, mitochondria, and soluble supernatant from rat liver

Formaldehyde can be metabolized primarily by two different pathways, one involving oxidation by the low- K m mitochondrial aldehyde dehydrogenase, the other involving a specific, glutathione-dependent, formaldehyde dehydrogenase. To estimate the roles played by each enzyme in formaldehyde metabolism by rat hepatocytes, experiments with acetaldehyde and cyanamide, a potent inhibitor of the low- K m aldehyde dehydrogenase were carried out. The glutathione-dependent oxidation of formaldehyde by 100,000 g rat liver supernatant fractions was not affected by either acetaldehyde or by cyanamide. By contrast, the uptake of formaldehyde by intact mitochondria was inhibited 75 to 90% by cyanamide. Acetaldehyde inhibited the uptake of formaldehyde by mitochondria in a competitive fashion. Formaldehyde was a weak inhibitor of the oxidation of acetaldehyde by mitochondria, suggesting that, relative to formaldehyde, acetaldehyde was a preferred substrate. In isolated hepatocytes, cyanamide, which inhibited the oxidation of acetaldehyde by 75 to 90%, produced only 30 to 50% inhibition of formaldehyde uptake by cells as well as of the production of 14CO 2 and of formate from [ 14C]formaldehyde. The extent of inhibition by cyanamide was the same as that produced by acetaldehyde (30–40%). In the presence of cyanamide, acetaldehyde was no longer inhibitory, suggesting that acetaldehyde and cyanamide may act at the same site(s) and inhibit the same formaldehyde-oxidizing enzyme system. These results suggest that, in rat hepatocytes, formaldehyde is oxidized by cyanamide- and acetaldehydesensitive (low- K m aldehyde dehydrogenase) and insensitive (formaldehyde dehydrogenase) reactions, and that both enzymes appear to contribute about equally toward the overall metabolism of formaldehyde.

Factors affecting the cold inactivation of an acetyl-coenzyme-A hydrolase purified from the supernatant fraction of rat liver.

An extramitochondrial acetyl-coenzyme-A hydrolase from rat liver is shown to be a cold-labile oligomeric enzyme that undergoes a reversible conformational transition between a dimeric and a tetrameric form in the presence of adenosine 5'-triphosphate or adenosine 5'-diphosphate at 25-37 degrees C, and between a dimeric and a monomeric form at low temperature. The enzymatically active dimer is fairly stable at 25-37 degrees C, but much less stable at low temperature, dissociating into monomer with no activity. At 37 degrees C and low concentrations of enzyme protein (less than or equal to 14 micrograms/ml), the activity decreased rapidly and only 10% of the initial activity remaining after 60 min. Addition of bovine serum albumin or immunoglobulin G to the medium completely prevented inactivation of the dimeric enzyme at low concentration at 37 degrees C, but had little effect on cold inactivation of the enzyme. Cold inactivation of the dimeric enzyme was partially prevented by the presence of various CoA derivatives. The order of potency was acetyl-CoA (substrate) greater than or equal to butyryl-CoA greater than octanoyl-CoA greater than CoA (product) greater than acetoacetyl-CoA. Another enzyme product, acetate, had little effect on cold inactivation. Polyols, such as sucrose, glycerol, and ethylene glycol, and high concentrations of NaCl, KCl, pyrophosphate and phosphate also greatly prevented cold inactivation. Cold inactivation was scarcely affected by pH within the pH range at which the enzyme was stable at 37 degrees C.

Effects of nucleotides on a cold labile acetyl-coenzyme A hydrolase from the supernatant fraction of rat liver

An acetyl-CoA hydrolase that is labile at low temperature was purified to homogeneity from the supernatant fraction of rat liver. The monomeric molecule, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, had a molecular weight of about 63 000, while that of the purified enzyme, estimated by gel filtration, was 135 000. Thus, the enzyme consists of two subunits of identical molecular weight. On addition of adenosine 5'-triphosphate (ATP) or adenosine 5'-diphosphate (ADP) at 25 degrees C, the dimeric form of the enzyme aggregated to tetrameric forms (Mr 242 000 and Mr 230 000, respectively), whereas addition of adenosine 5'-monophosphate had little effect on enzyme association (Mr 145 000). When ATP was removed from the ATP-treated tetrameric enzyme by dialysis, the tetramer was mostly dissociated into the dimeric form. The apparent Km values for acetyl coenzyme A of the dimeric enzyme and tetrameric enzyme, reconstituted from the former in the presence of 2 mM ATP, were 170 microM and 60 microM, respectively. The purified dimeric enzyme was inactivated by exposure to lower temperature, especially below 10 degrees C. The various nucleotides tested partially stabilize the dimeric enzyme at low temperature, ATP being the most effective. Sucrose density gradient centrifugation showed that loss of catalytic activity by cold treatment was accompanied by dissociation of the dimer and tetramer into protomer.

Purification and characterization of cysteine aminotransferase from rat liver cytosol.

Cysteine aminotransferase (L-cysteine: 2-oxoglutarate aminotransferase, EC 2.6.1.3) was purified over 400-fold from the high-speed supernatant fraction of rat liver. The purified enzyme was homogeneous as judged by gel filtration, isoelectric focusing and disc electrophoresis. The molecular weight of the enzyme was about 74,000 by gel filtration and the isoelectric point was 6.2 (4 degrees C). The enzyme catalyzed transamination between L-cysteine and 2-oxoglutarate and the reverse reaction. The optimum pH was 9.7. The Km value for L-cysteine was 22.2 mM, and that for 2-oxoglutaric acid was 0.06 mM. L-Aspartate was a potent inhibitor of the cysteine aminotransferase reaction. The enzyme was very active toward L-alanine 3-sulfinic acid at pH 8.0, and was also very active toward L-aspartic acid (Km = 1.6 mM). Ratios of activities for L-aspartic acid and L-cysteine were essentially constant during the purification of the enzyme. Evidence based on substrate specificity, enzyme inhibition, and physicochemical properties indicates that cytosolic cysteine aminotransferase is identical with cytosolic aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1).