Supernatant Fraction Of Liver Research Articles

The role of bound thiamine pyrophosphate in the synthesis of thiamine triphosphate in rat liver

Thiamine pyrophosphate-ATP phosphoryltransferase, the enzyme that catalyzes the synthesis of thiamine triphosphate, has been found in the supernatant fraction of rat liver. The substrate for the enzyme is endogenous, bound thiamine pyrophosphate, since the addition of exogenous thiamine pyrophosphate had no effect. Thus, when a rat liver supernatant was incubated with γlabelled [ 32P] ATP, thiamine [ 32P] triphosphate was formed whereas the incubation of thiamine [ 32P] pyrophosphate with ATP did not produce thiamine [ 32P]. triphosphate. The endogenous thiamine pyrophosphate was found to be bound to a high molecular weight protein which comes out in the void volume of Sephadex G-75, and is not dialyzable. The activity that catalyzes the formation of thiamine triphosphate has an optimum pH between 6 and 6.5, a linear time course of thiamine triphosphate synthesis up to 30 min, and is not affected by Ca 2+, cyclic GMP and sulfhydryl reagents.

Resolution and partial characterization of two aldehyde reductases of mammalian liver.

Investigation of NADP-dependent aldehyde reductase activity in mouse liver led to the finding that two distinct reductases are separable by DE52 ion exchange chromatography. Aldehyde reductase I (AR I) appears in the effluent, while aldehyde reductase II (AR II) is eluted with a salt gradient. By several procedures AR II was purified over 1100-fold from liver supernatant fraction, but AR I could be pruified only 107-fold because of its instability. The two enzymes are different in regard to pH optimum, substrate specificity, response to inhibitors, and reactivity with antibody to AR II. While both enzymes utilize aromatic aldehydes well, only AR II ACTS ON D-glucuronate, indicating that it is the aldyhyde reductase recently reported to be identical to NADP-L-gulonate dehydrogenase. The presence of two NADP-linked aldehyde reductases in liver has apparently not heretofore been reported.

Distribution of Vitamin E among Tissues and Subcellular Fractions in Sublethally Irradiated Mice

The distribution of ${}^{3}{\rm H}\text{-activity}$ among blood plasma, lung, spleen, liver and gut as well as among nuclear, mitochondrial, microsomal, and soluble fractions of the liver has been measured at 48 hr following intraperitoneal injection of 10 μCi of $[{}^{3}{\rm H}]d\text{-}\alpha \text{-tocopherol}$ . Lung and spleen were the most highly labeled tissues and nuclei, mitochondria and microsomes were more highly labeled than the soluble supernatant. Immediately following exposure to 500 R of 260 kVp X-radiation, the specific activity of plasma, lung, and spleen were significantly reduced. In addition, liver nuclear fraction was also reduced. The distribution of label administered 24 hr postirradiation was significantly altered, insofar as increased label was detected in gut and liver. In liver, microsomal, and soluble supernatant fractions were also greatly increased.

Phosphatidate Phosphohydrolase and the Regulation of Glycerolipid Biosynthesis

Phosphatidate is an intermediate in the biosynthesis of diacylglycerol, triacylglycerol, phosphatidylethanolamine, phosphatidylcholine and CDP-diacylglycerol. In addition, phosphatidate may be subject to degradation by particulate phospholipases. The factors regulating the disposition of phosphatidate have interested numerous investigators. We have studied the reactions of phosphatidate metabolism in several tissues and under various environmental conditions. Phosphatidate phosphohydrolase activity is found primarily in the microsomal and supernatant fractions of liver, adipose tissue and intestine. Presumably the physiologically important substrate for this reaction is the particulate-bound phosphatidate and methods for the preparation of this substrate have been developed (Fallon et al., 1975). Radioactively labelled phosphatidate bound to microsomal particles is prepared from sn-[1 ,3-14C]glycerol 3-phosphate. A wide range of microsomal phosphatidate concentrations may be prepared by this technique, although the usual microsomal concentration is approx. 5 nmol/mg of microsomal protein. This substrate has been used in most of our studies of phosphatidate phosphohydrolase, CDP-diacylglycerol formation and phospholipase A. There are several lines of evidence which support an important role for phosphatidate phosphohydrolase in the regulation of neutral-lipid formation by liver or adipose-tissue particulate preparations. (1) The reaction rate of phosphatidate phosphohydrolase under optimum conditions in vitro is the lowest in the overall synthetic pathway in liver microsomal fractions (Lamb & Fallon, 1974a). The rate of phosphohydrolase activity was determined as 0.6-0.8 nmol/min per mg of microsomal protein when radiolabelled particulate-bound phosphatidate was used as substrate. The overall rate of glycerolipid formation by these microsomal preparations was approx. l.Onmol/min per mg of microsomal protein. The optimum rate of microsomal sn-glycerol 3-phosphate acyltransferase was 5-7-fold higher. In the presence of added rat liver supernatant fraction the rate of microsomal phosphatidate phosphohydrolase is increased 5-20-fold. However, at ratios of microsomal protein to supernatant protein estimated for the intact cell, the increase in phosphohydrolase activity is much more modest. (2) Induction of increased liver triacylglycerol formation by administration of a high-fructose diet is accompanied by an equivalent rise in the activity of the phosphohydrolase, as shown in Table 1. These changes in lipid metabolism occur simultaneously over a 10-day period (Lamb & Fallon, 19746). Further, the phosphatidate content of liver microsomal preparation is diminished by approx. 15 %, and diacylglycerol increased by approx. 90 % under these dietary conditions. (3) The increase in liver triacylglycerol content after partial hepatectomy is accompanied by a corresponding increase in phosphatidate phosphohydrolase activity (Mangiapane et al., 1973). (4) Several drugs which cause substantial inhibition of phosphatidate phosphohydrolase activity in vitro also result in inhibition of neutral-lipid formation when administered to the intact animal (Brindley & Bowley, 1975; Lamb et al., 1977). A series of 1,3-bis(substituted phenoxy)-2-propanones inhibited microsomal phosphatidate phosphohydrolase activity as measured in vitro. The inhibition ranged from 8 to 92% total activity. In animals fed on these agents in laboratory chow the incorporation of [14C]glycerol into hepatic triacylglycerol was decreased by 3-75 %. Agents which caused a 50 % or greater decrease in phosphatidate phosphohydrolase activity in vitro also lowered

Acetylation of spermidine and spermine by rat liver and kidney chromatin

The in vitro enzymatic acetylation of the polyamines, spermidine and spermine, is described. The reaction is catalyzed by chromatin preparations from rat liver and kidney and is dependent on acetyl-CoA. Spermidine, spermine, and putrescine are each converted to the corresponding monoacetyl derivatives. s 0.5 values of 0.5 ± 0.1, 1.0 ± 0.1, and 2.6 ± 0.7 m m (mean ± standard deviation) were obtained for spermidine, spermine, and putrescine, respectively. These values for s 0.5 are similar to the concentrations of polyamines reported for tissues, and therefore, suggest the occurrence of polyamine acetylation in vivo. Evidence is also presented for the metabolism of acetylated polyamines by the 100,000 g supernatant fraction of rat liver. The physiological function of polyamine acetylation is unknown, but the possibility of an effect on the association of polyamines with nucleic acids is discussed.

Liver homogenate-mediated mutagenesis in Chinese hamster V79 cells by polycyclic aromatic hydrocarbons and aflatoxins

Several chemical carcinogens that require metabolic activation have been examined for their cytotoxic and mutagenic activity in Chinese hamster V79 cells. Mutagenic activity was measured as the induced frequency of 6-thioguanine-resistant colonies. Metabolic activation was provided by the 9000 g supernatant fraction of rat liver plus cofactors. The cytotoxicity and mutagenicity of aflatoxin B 1 and B 2, benzo(a)pyrene, 3-Methylcholanthrene, 7, 12-dimethylbenzo(a)anthracene, dibenz(a,h)-anthracene, dibenz(a,c)anthracene, and benz(a)anthracene were examined as functions of concentration. Except for the two isomers of dibenzanthracene, the mutagenic activity in general paralleled the carcinogenic activity. An assay of this type may be useful as a prescreen for environmental chemicals that require metabolic activation.

Appearance of magnesium guanylate cyclase activity in rat liver with sodium azide activation

Native soluble and particulate guanylate cyclase from several rat tissues preferred Mn2+ to Mg2+ as the sole cation cofactor. Wtih 4mM cation, activities with Mg2+ were less than 25% of the activities with Mn2+. The 1 mM NaN3 markedly increased the activity of soluble and particulate preparations from rat liver. Wtih NaN3 activation guanylate cyclase activities wite similar with Mn2+ and Mg2+. Co2+ was partially effective as a cofactor in the presence of NaN3, while Ca2+ was a poor cation with or without NaN3. Activities with Ba, Cu2+, or Zn2+ were not detectable without or with 1 mM NaN3. With soluble liver enzyme both manganese and magnesium activities were dependent upon excess Mn2+ or Mg2+ at a fixed MnGTP or MgGTP concentration of 0.4 mm; apparent Km values for excess Mn2+ and Mg2+ were 0.3 and 0.24 mM, respectively. After NaN3 activation, the activity was less dependent upon free Mn2+ and retained its dependence for free Mg2+, at 0.4 mM MgGTP the apparent Km for excess Mg2+ was 0.3 mM. The activity of soluble liver guanylate cyclase assayed with Mn2+ or Mg2+ was increased with Ca2+. After NaN3 activiation, Ca2+ had no effect or was somewhat inhibitory with either Mn2+. After NaN activation, Ca2+ had no effect or was somewhat inhibitory with either Mn2+ or Mg2+. The stimulatory effect of NaN2 on Mn2+-and Mg2+-dependent guanylate cyclase activity from liver or cerebral cortex supernatant fractions required the presence of the sodium azide-activator factor. With partially purified soluble liver guanylate cyclase and azide-activator factor, the concentration (1 mjM) of NaN3 that gave half-maximal activation with Mn2+ or Mg2+ was imilar. Thus, under some conditions guanylate cyclase can effectively use Mg2+ as a sole cation cofactor.

Adenosine 3':5'-cyclic monophosphate-binding proteins in bovine and rat tissues.

1. At least two classes of high-affinity cyclic AMP-binding proteins have been identified: those derived from cyclic AMP-dependent protein kinases (regulatory subunits) and those that bind a wide range of adenine analogues (adenine analogue-binding proteins). 2. In fresh-tissue extracts, regulatory subunits could be further subdivided into 'type I or 'type II' depending on whether they were derived from 'type I' or 'type II' protein kinase [see Corbin et al. (1975) J. Biol. Chem. 250, 218-225]. 3. The adenine analogue-binding protein was detected in crude tissue supernatant fractions of bovine and rat liver. It differed from the regulatory subunit of cyclic AMP-dependent protein kinase in many of its properties. Under the conditions of assay used, the protein accounted for about 45% of the binding of cyclic AMP to bovine liver supernatants. 4. The adenine analogue-binding protein from bovine liver was partially purified by DEAE-cellulose and Sepharose 6B chromatography. It had mol.wt. 185000 and was trypsin-sensitive. As shown by competition and direct binding experiments, it bound adenosine and AMP in addition to cyclic AMP. At intracellular concentrations of adenine nucleotides, binding of cyclic AMP was essentially completely inhibited in vitro. Adenosine binding was inhibited by only 30% under similar conditions. 5. Rat tissues were examined for the presence of the adenine analogue-binding protein, and, of those examined (adipose tissue, heart, brain, testis, kidney and liver), significant amounts were only found in the liver. The possible physiological role of the adenine analogue-binding protein is discussed. 6. Because the adenine analogue-binding protein or other cyclic AMP-binding proteins in tissues may be products of partial proteolysis of the regulatory subunit of cyclic AMP-dependent protein kinase, the effects of trypsin and aging on partially purified protein kinase and its regulatory subunit from bovine liver were investigated. In all studies, the effects of trypsin and aging were similar. 7. In fresh preparations, the cyclic AMP-dependent protein kinase had mol.wt. 150000. Trypsin treatment converted it into a form of mol.wt 79500. 8. The regulatory subunit of the protein kinase had mol.wt. 87000. It would reassociate with and inhibit the catalytic subunit of the enzyme. Trypsin treatment of the regulatory subunit produced a species of mol.wt. 35500 which bound cyclic AMP but did not reassociate with the catalytic subunit. Trypsin treatment of the protein kinase and dissociation of the product by cyclic AMP produced a regulatory subunit of mol.wt. 46500 which reassociated with the catalytic subunit. 9. These results may be explained by at least two trypsin-sensitive sites on the regulatory subunit. A model for the effects of trypsin is described.

Purification of a soluble rat liver protein that stimulates microsomal 4-methyl sterol oxidase activity.

A soluble rat liver protein that stimulates microsomal methyl sterol oxidase activity has been isolated and purified to homogeneity by salt fractionation, differential heat inactivation, two calcium phosphate gel association, and Sephadex filtration. The protein, as isolated, may be dissociated with detergent into subunits with a molecular weight of approximately 10,300, as determined electrophoretically. In addition to this stimulatory protein, postmicrosomal supernatant fraction of rat liver contains a low molecular weight methyl sterol oxidase inhibitor, possibly cholesterol ester, which is removed during protein purification. Purified soluble protein enhances the observed rate of oxidative attack of the methyl sterol substrates, 4, 4-dimethyl-5alpha-cholest-7-en-3beta-ol and 4alpha-methyl-5alpha-cholest-7-en-3beta-ol. Addition of increasing amounts of either partially purified or homogeneous soluble protein yields hyperbolic stimulation, from which a K'm of about 7 muM has been calculated (based on a monomeric molecular weight of 10,300). Sequential additions of the soluble protein yield equal increments of stimulation. These results are consistent with the suggestion that the soluble protein may be a reactant in the oxidative process. Methyl sterol oxidase is inhitited in vitro by cholesterol, several oxygenated sterols, and cholesterol esters. The extent of inhibition is much greater when the soluble protein is present in the incubation. The inhibition is competitive with respect to methyl sterol substrate; cholesterol succinate, a water-soluble ester, is strongly inhibitory, K'i (ester)/K'm(substrate) approximately 0.2. Since end product inhibition of methyl sterol oxidase may be produced by accumulation of cholesterol or cholesterol metabolites, the soluble protein may participate in regulation of the activity of some microsomal enzymes of cholesterol biosynthesis.

Some Properties of Phospholipid Exchange Proteins from Rat Liver

The phospholipid exchange proteins of rat liver that catalyze the transfer of phosphatidylcholine and phosphatidylinositol from rat liver microsomes to liposomes, have been purified and characterized. Two proteins were detected with dual specificities catalyzing the transfer of both phosphatidylinositol and phosphatidylcholine. Both proteins showed a strong preference for phosphatidylinositol transferring 8 to 9 times as much of the microsomal phosphatidylinositol pool as the microsomal phosphatidylcholine pool. The two proteins had iso-electric points of 5.1 and 5.3 and were purified 300-fold and 500-fold, respectively. A protein that catalyzed specifically the transfer of phosphatidylcholine, was purified 7000-fold. This protein had an iso-electric point of 8.4 and a molecular weight of approximately 16000 calculated from Sephadex G-50 chromatography and sodium dodecylsulfate-polyacrylamide gel electrophoresis; the amino acid composition was determined. An antiserum against this protein was raised in rabbits. Treatment of a rat liver supernatant fraction with the antiserum immunoglobulin fraction demonstrated that 60% of the phosphatidylcholine transfer activity is due to this protein.

Streptozotocin: its excretion and metabolism in the rat.

The excretion of radioisotope following the administration of three specifically 14C-labelled forms of streptozotocin was investigated in the rat using ureter and bile duct cannulation techniques. The urine collected during the first hour following the administration of the drug contained the highest proportion of injected radioactivity (approximately 34% with (3'-methyl-14C)-streptozotocin and approximately 40% each with (1-14C)-and (2'-14C)-streptozotocin. Over the entire experimental period (6 hours), approximately 70% of the injected radioactivity of (1-14C)- and (2'-14C)-streptozotocin appeared in the urine. With (3'-methyl-14C)-streptozotocin, only 53% of the injected radioactivity appeared in the urine over the same period. In contrast to the high urinary excretion, less than 3% of the injected radioactivity from all three radiolabelled streptozotocin samples appeared in the bile. The in vivo and in vitro metabolism of streptozotocin was also investigated. In addition to substantial amounts of unchanged drug, three radiolabelled metabolites (two major and one minor) were detected in the urine during the 6 hour collection period following the administration of (1-14C)- and (2'-14C)-streptozotocin. In contrast, only unchanged (3'-methyl-14C)-streptozotocin was detected in the urine collected over the same period following the administration of the methyl labelled drug. The two major metabolites were also produced when (1-14C)-and (2'-14C)-streptozotocin were incubated with a rat liver supernatant fraction (100,000 X g). The liver was further demonstrated to be the major site of metabolism in isolated liver perfusion studies in which both (1-14C)- and (2'-14C)-streptozotocin were quantitatively converted to the two major metabolites. The two major metabolites of (1-14C)-streptozotocin, whether produced in vivo or in vitro, were chromatographically homogenous with the two major metabolites formed from (2'-14C)-streptozotocin. Nicotinamide pretreatment had no apparent effect on the urinary excretion of streptozotocin and its metabolites.

Species differences in the conjugation of 4-hydroxy-3-methoxyphenylethanol with glucuronic acid and sulphuric acid.

The biosynthesis of the glucuronide and sulphate conjugates of 4-hydroxy-3-methoxyphenylethanol was demonstrated in vitro by using the high-speed supernatant and microsomal fractions of liver respectively. These two conjugates were also produced simultaneously by using the post-mitochondrial fraction of rat, rabbit or guinea-pig liver. In contrast only the glucuronide was synthesized by human liver and only the sulphate by mouse and cat livers. Neither of these conjugates was formed by the kidney or the small or large intestine of the rat. A high sulphate-conjugating activity was observed in mouse kidney; the rate of sulphation of 4-hydroxy-3-methoxyphenylethanol with kidney homogenate and high-speed supernatant preparations was 1.8 times greater than with liver preparations. The sulpho-conjugates of 4-hydroxy-3-methoxyphenylethanol and 4-hydroxy-3-methoxy-phenylglycol were also formed by enzyme preparations of rabbit adrenal and rat brain; the glycol was the better substrate in the latter system. Mouse brain did not possess any sulphotransferase activity. For the conjugation of 4-hydroxy-3-methoxyphenylethanol by rabbit liver, the Km for UDP-glucuronic acid was 0.22 mM and that for Na2SO4 was 3.45 mM. The sulphotransferase has a greater affinity for 4-hydroxy-3-methoxyphenyl-ethanol than has glucuronyltransferase, as indicated by their respective Km values of 0.036 and 1.3 mM. It was concluded that sulphate conjugation of 4-hydroxy-3-methoxyphenylethanol predominates in most species of animals.

Rat and mouse tissue-mediated mutagenicity of ring-substituted 3,3-dimethyl-1-phenyltriazenes in Salmonella typhimurium

3,3-Dimethyl-1-phenyltriazene and a series of ring-substituted derivatives X4ØN=N( CH 3) 3: X = substituent(s); ø = phenyl) were tested for their mutagenic and toxic action upon Salmonella typhimurium G-46 in a liquid incubation system containing 9000 g tissue supernatants and an NADPH-generating system. The compounds could be grouped into four classes according to their toxicity and mutagenicity after 1 h incubation at 37°C at a concentration of 5 mM in the presence of liver supernatant fractions from phenobarbitone-pretreated mice. When a liver supernatant from untreated mice was compared with one from phenobarbitone-pretreated animals, the mutagenic effect of a series of triazenes (with X = H; 4-chloro; 4-chloro; 4-bromo; 2,4,6-trichloro) in vitro was enhanced twice to ten times. The toxicity of triazenes with X= 4-methoxy or 4-acetamido was strongly decreased by a liver fraction from phenobarbitone-pretreated mice in the presence of an NADPH-generating system. With 3,3-dimethyl-1-phenyl-triazene, rat liver fractions caused a lower enzyme-mediated mutagenicity in S. typhimurium G-46 than those of mouse liver,, whereas a 9000 g supernatant from brain, a major target organ for the carcinogenic action of certain triazenes, was unable, in either species, to generate metabolites mutagenic for S. typhimurium G-46.

Detection and investigation of a protein factor concerned with the stimulation of disulfide reductase activity by cyclic AMP and other effectors

Stimulation of disulfide reductase (DSR) in the supernatant fraction of mouse liver by 3′,5′-AMP (10−7 M), ATP (5·10−5 M), Mg++ (5·10−5 M), EDTA (5·10−4 M), and protamine (5 mg/ml) is mediated by a factor which is readily adsorbed by BaSO4, Al(OH)3, and activated charcoal, and is readily eluted by a tenfold increase in the molarity of the buffer. Barium eluates of the liver and brain mutually and equally restore the effect of 3′,5′-AMP when abolished by BaSO4. The adsorbed factor is evidently a protein, for it does not dialyze, it is very thermolabile, and it is easily inactivated by low concentrations of trypsin. The factor restores activation of DSR when abolished or reduced by protein inhibitor of protein kinase (PIPK). The fact that the relationship between the active quantities of PIPK and protein factor (PF) is linear indicates that the PF is similar or identical to protein kinase. The possible biological role of activation of DSR and the hypothesis that a special protamine-sensitive protein kinase exists are discussed.

Effects of 1,1,1-trichloroethane administered by different routes and in different solvents on barbiturate hypnosis and metabolism in mice.

A 24-hr inhalation of 1,1,1-tricholoroethane (methylchloroform), 3,000 ppm, reduced pentobarbital hypnosis and increased hexobarbitaloxidation by the 9,000 x g liver supernatant fraction in male mice. On the other hand, an ip injection of methylchloroform, l ml/kg, increased the duration of pentobarbital hypnosis and reduced hexobarbital metabolism by the liver microsomal enzymes. The potentioating effect of methylchloroform on pentobarbital hypnosis as diminished when it was diluted with olive oil, but was markedly enhanced when diluted with dimethysusoxide (DMSO) before injection. Three local applications of methylchloroform )1:1 dilution with DMSO) had an effect similar to that of inhalation on pentobarbital hypnosis.

Retinol receptors of the retina and pigment epithelium: Further characterization and species variation

Cytosol (100 000 × g supernatant) fractions of retina, pigment epithelium, liver and brain and also serum samples of diverse animal species demonstrate discrete peaks of bound [ 3H]retinol as assessed by sucrose density gradient ultracentrifugation. Retina cytosol of rat, guinea pig and chick embryo demonstrates only a 2 S binding species (“receptor”) although bovine retina also has a much more rapidly sedimenting peak. Pigment epithelium and liver show 2 S and 5 S species while brain shows little 2 S binding. Saturation of specific binding sites and reversibility of [ 3H]retinol binding in chick retina cytosol are observed by both sucrose gradient and gel filtration analysis. Treatment with DNase, neuraminidase or lipase has little effect on retinol binding to the 2 S receptor. From this and a previous study Wiggert and Chader, 1975 we conclude that a limited number of retinol receptors are present in retina and other tissues which are protein in nature and which bind the vitamin with high affinity and specificity.

Effects of phenobarbital and 3-methylcholanthrene administration on glutathione- S-epoxide transferase activity in rat liver

Glutathione- S-epoxide transferase activity in rat liver was studied with three epoxide substrates. The specific activities in the high-speed liver supernatant fraction for 3MC 11, 12 oxide, styrene oxide and 3,3,3-trichloro-1,2 epoxy propane were 5.7, 86.4 and 165 nmoles/5 min/mg protein, respectively. Phenobarbital (75 mg/kg body wt for 3 days) or 3-methylcholanthrene (20 mg/kg body wt for 2 days) administration to rats resulted in a 40–60% increase in enzyme activity. β-napthoflavone administration on the other hand was without any effect on glutathione- S-epoxide transferase activity.

Purification, characterization and identification of rat liver histidine-pyruvate aminotransferase isoenzymes.

1. Histidine-pyruvate aminotransferase (isoenzyme 1) was purified to homogeneity from the mitochondrial and supernatant fractions of rat liver, as judged by polyacrylamide-gel electrophoresis and isolectric focusing. Both enzyme preparations were remarkably similar in physical and enzymic properties. Isoenzyme 1 had pI8.0 and a pH optimum of 9.0. The enzyme was active with pyruvate as amino acceptor but not with 2-oxoglutarate, and utilized various aromatic amino acids as amino donors in the following order of activity: phenylalanine greater than tyrosine greater than histidine. Very little activity was found with tryptophan and 5-hydroxytryptophan. The apparent Km values were about 2.6mM for histidine and 2.7 mM for phenylalanine. Km values for pyruvate were about 5.2mM with phenylalanine as amino donor and 1.1mM with histidine. The aminotransferase activity of the enzyme towards phenylalanine was inhibited by the addition of histidine. The mol.wt. determined by gel filtration and sucrose-density-gradient centrifugation was approx. 70000. The mitochondrial and supernatant isoenzyme 1 activities increased approximately 25-fold and 3.2-fold respectively in rats repeatedly injected with glucagon for 2 days. 2. An additional histidine-pyruvate aminotransferase (isoenzyme 2) was partially purified from both the mitochondrial and supernatant fractions of rat liver. Nearly identical properties were observed with both preparations. Isoenzyme 2 had pI5.2 and a pH optimum of 9.3. The enzyme was specific for pyruvate and did not function with 2-oxoglutarate. The order of effectiveness of amino donors was tyrosine = phenylalanine greater than histidine greater than tryptophan greater than 5-hydroxytryptophan. The apparent Km values for histidine and phenylalanine were about 0.51 and 1.8 mM respectively. Km values for pyruvate were about 3.5mM with phenylalanine and 4.7mM with histidine as amino donors. Histidine inhibited phenylalanine aminotransferase activity of the enzyme. Gel filtration and sucrose-density-gradient centrifugation yielded a mol.wt. of approx. 90000. Neither the mitochondrial nor the supernatant isoenzyme 2 activity was elevated by glucagon injection.

Enzyme activities and cytochrome P-450 levels of some Japanese quail tissues with respect to their metabolism of several pesticides

In the Japanese quail, cytochrome P-450, A- and B-esterase, amidase, and glutathione S-aryl transferase were assayed in postmitochondrial centrifugal fractions, in microsomes, and supernatant fractions of liver, lungs, kidneys, and testes. Liver microsomes contained the highest A-esterase activity and P-450 levels. B-esterase was more generally distributed and higher in the microsomal tissue fractions. Microsomal amidase activity was highest in quail lung and kidney, and lowest in the liver (per mg protein). Very little difference in glutathione S-aryl transferase activity was noted among the tissues assayed. In vitro metabolism of carbaryl, phosphamidon, and chlorotoluron by the various centrifugal fractions revealed that the production of 1-naphthyl- N-hydroxymethylcarbamate and 1-naphthol, the major metabolites, was greatest in the postmitochondrial fraction of the liver. The major carbaryl metabolite in all other quail tissue fractions was 1-naphthol. Phosphamidon metabolism in postmitochondrial preparations of quail liver was higher than in the supernatant and microsomes. Chlorotoluron metabolism occurred only in the postmitochondrial fractions of quail liver. The major products were the oxidative metabolites, N-(3-chloro-4-methylphenyl)- N′-methylurea and N-(3-chloro-4-hydroxymethylphenyl)- N′-methylurea.

Evidence for the association of L-asparaginase with cytomembrane components in the guinea pig liver soluble fraction.

Homogenization of guinea pig liver in isotonic sucrose solution followed by the separation of the subcellular fractions by differential centrifugation releases the liver L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) activity into the supernatant fraction. Electron micrographs of the liver L-asparaginase-antibody complexes, precipitated from the clear supernatant phase by addition of L-asparaginase-specific antiserum, show membrane-liek structures and some amorphous material. The attachment of L-asparaginase to the membrane-like structures is indicated by the ferritin-labeled antibody technique. The immunoprecipitates possess low activities of 5'-nucleotidase, alkaline phosphodiesterase I, NADPH cytochrome c reductase, glucose-6-phosphatase, and acid phosphatase. This observation suggests that L-asparaginase found in the liver supernatant fraction is associated with cytomembrane components. Analysis of guinae pig serum L-asparaginase-antibody complexes is polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gives three distinct protein bands. These bands correspond to heavy and light chains of rabbit immunoglobulins and the L-asparaginase subunits. Analysis of the liver L-asparaginase-antibody complexes by the above procedure shows similar but more diffuse protein bands.