Latent form of glutathione reductase in the rat liver

The existence of membrane-bound forms of glutathione reductase in rat liver and transplantable hepatoma G-27 was demonstrated, using differential centrifugation techniques. The activity of the sedimentable form of the liver enzyme was detected only in the presence of detergents. Conditions for the manifestation of the latent glutathione reductase activity in whole liver homogenates and in the 105000 g pellet were determined. Solubilization of the latent form of the enzyme in the presence of sodium deoxycholate increases 2-fold the glutathione reductase activity in liver homogenates (but not in hepatoma). Simultaneous determination of the disulfidereductase, nonspecific NADPH-oxidase and gamma-glutamyltransferase (membrane-bound enzyme of glutathione metabolism) activities was performed.     

Isolation from rat liver of a peroxisomal enzyme which converts molecular form 1 of biliverdin reductase into molecular form 3

Cobaltous chloride induced in rat liver an enzyme which converted biliverdin reductase molecular form 1 into the molecular form 3. This conversion involves the oxidation of two sulfhydryl groups of form 1 giving rise to a disulfide bond in form 3. The converting enzyme was isolated from the liver peroxisomal fraction (which was devoid of biliverdin reductase activity), and was absent in liver peroxisomes of control rats. The enzyme was solubilized by treatment of the peroxisomes with 0.1% sodium deoxycholate, and partially purified by DEAE-cellulose and Sephadex G-100 filtration. It is a NAD⁺ dependent enzyme which was inactivated by trypsing and heat treatments. It did not oxidize either reduced glutathione or cysteine. The converting enzyme had a molecular weight of about 54,000 daltons. The oxidation of biliverdin reductase molecular form 1 mediated by the converting enzyme did not affect the latter's molecular weight or activity.     

Endogenous factor in rat liver that converts tyrosine aminotransferase form III to form I: a rapid assay of this converting factor.

After induction by cortisol, tyrosine aminotransferase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) present in rat liver homogenates can be resolved into three peaks of activity by CM-Sephadex chromatography. Based on differential elution of these forms by a linear KCl gradient, a three-tube assay was developed that quantitates the amount of form III relative to total enzyme. The assay was used to determine the presence of a factor in the liver that converts tyrosine aminotransferase form III to form I. Definitive evidence for the liberation of such a factor is presented.     

Possible processing of mitochondria-bindable hexokinase to the nonbindable form by a lysosomal protease in rat liver

As a possible mechanism for the absence of mitochondria-bindable hexokinase in the liver, the presence of a protease similar in action to chymotrypsin, which specifically eliminates the binding ability of the bindable hexokinase without changing its catalytic properties, was investigated in rat liver. The lysosomal fraction prepared from the liver converted the bindable hexokinase prepared from rat brain to the nonbindable form with little change in catalytic activity. The activity of such a "processing protease" was much lower in rat brain, where the bindable form is predominant. The processing activity cosedimented with lysosomal marker enzyme activities in the subcellular fractionation of livers from normal and Triton WR-1339-injected rats. A fair portion of the activity was detected in the lysosomes without disruption. The activity was maximal at pH 6.0-7.0, inactivated almost completely by tosylphenylalanine chloromethyl ketone, tosyllysine chloromethyl ketone, leupeptin, antipain, and chymostatin, and dependent on dithiothreitol and mercaptoethanol. These results suggest that a protease, properties of which are fairly similar to those of cathepsin M, may be involved in the post-translational processing of original bindable hexokinase to the nonbindable form in rat liver.     

pH-dependent conversion of liver-membranous alkaline phosphatase to a serum-soluble form by n-butanol extraction

Alkaline phosphatase released from rat liver plasma membrane under usual conditions was electrophoretically not identical with a soluble form in serum which was derived from the liver. The liver-membranous alkaline phosphatase, however, was converted to the serum-soluble form when the liver plasma membrane was treated with n-butanol under the acidic conditions lower than pH 6.5. Such pH-dependent conversion of the enzyme was not observed in plasma membrane of rat ascites hepatoma AH-130 cells. The converting activity for alkaline phosphatase was detected not only in plasma membrane but also in lysosomal membrane of rat liver.     

Comparison of blood biochemical indices in rats exposed to lead in macrodispersed form and nanoform

It is shown that the increasing content of lead in blood (6.3 and 3.7 times) and liver (30.1 and 4.6 times) in rats after 14-days per os exposure both to lead acetate (macrodispersed form) and lead nanoparticles (nanoform) in a dose of 7 mg/100 g of body weight leads to the increased of activity of blood enzymes: alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, lactate dehydrogenase and a decrease of creatinine level. Lead in nanoform with slight accumulation, due to the greater elimination, expressed higher biological activity and reactivity as compared to macrodispersed form. Thus the exposure to lead in different dispersed form suggests metabolic disorders in rats, and accumulation of lead and biochemical changes are more expressed in the liver.     

Intermediate dehydrogenase-oxidase form of xanthine oxidoreductase in rat liver.

A spectrophotometric method for the determination of three forms of xanthine oxidoreductase, namely dehydrogenase (D), dehydrogenase-oxidase (D/O) and oxidase (O), is described. Enzymic fractions obtained from rat liver were found to contain either all three forms, or (under special conditions of preparation) only two forms, D and D/O. The conversion of form D leads to form D/O leads to form O in the presence of Cu2+ ions was shown. Form D/O acted with NAD+ as well as with O2 as electron acceptors, it exhibited greater affinity to NAD+ than to O2, and NAD+ abolished the oxidase activity of this form. Moreover, oxidase activity of form D/O was inhibited by NADH. These facts indicate that NAD+ and O2 compete for the same active site on the enzyme molecule.     

Purification and properties of the native form of rabbit liver aldolase. Evidence for proteolytic modification after tissue extraction.

Aldolase was purified from rabbit liver by affinity-elution chromatography. By taking precautions to avoid rupture of lysosomes during the isolation procedure, a stable form of liver aldolase was obtained. The stable form of the enzyme had a specific activity with respect to fructose 1,6-bisphosphate cleavage of 20-28 mumol/min per mg of protein and a fructose 1,6-bisphosphate cleavage of 20-28mumol/min per mg of protein and a frutose 1,6-bisphosphate/fructose 1-phosphate activity ratio of 4. It was distinguishable from rabbit muscle aldolase, as previously isolated, on the basis of its electrophoretic mobility and N-terminal analysis. Muscle and liver aldolases were immunologically distinct. The stable liver aldolase was degraded with a lysosomal extract to a form with catalytic properties resembling those reported for aldolase B4. It is postulated that liver aldolase prepared by previously described methods has been modified by proteolysis and does not constitute the native form of the enzyme.     

Purification and characterization of a lysosomal form and a variant form of beta-glucuronidase from the rat basophil leukaemia tumour.

Two isoenzyme of beta-glucuronidase from a rat basophil leukaemia tumour were co-purified 4067-fold by (NH4)2SO4 precipitation and sequential chromatography on concanavalin A--Sepharose, Sephadex G-200, DEAE-cellulose, CM-cellulose and phosphocellulose. The purity of the mixture was established by the coincidence of the peaks of enzyme activity and protein at a molecular weight of 300 000 on Bio-Gel P-300, the presence of only two protein bands, both of them enzymically active, in polyacrylamide gels after electrophoresis under non-denaturing conditions, and the presence of a single subunit species, of mol.wt. 75 000, after electrophoresis in polyacrylamide gels under a denaturing conditioning. The major isoenzyme co-migrated with the L form from rat liver during electrophoresis in alkaline polyacrylamide gels, whereas the minor isoenzyme migrated more rapidly than either the lysosomal form or the rat liver microsomal form and was designated the tumour (T) isoenzyme. A mixture of the purified isoenzymes from two preparations had an average specific activity of 1389 units/mg for phenolphthalein beta-D-glycopyranosiduronic acid. The L and T isoenzymes, which had pI5.9 and 5.7 respectively, could be obtained free of cross-contamination by isoelectric focusing and had similar specific activities. Although the T isoenzyme could be a catabolic product of the M or the L form, it could also be a unique tumour product, because it was not detected in extracts of normal rat tissues.     

Identification of a precursor form of cathepsin D in microsomal lumen: characterization of enzymatic activation and proteolytic

A precursor form of cathepsin D with 45 kDa was demonstrated in the rat liver microsomal lumen by immunoblotting analysis. The microsomal fraction containing procathepsin D which passed through a pepstatin-Sepharose resin showed no appreciable activity of cathepsin D. The in vitro incubation of this fraction at pH 3.0 resulted in a gradual increase of proteolytic activity toward hemoglobin as substrate and also, the proteolytic conversion of procathepsin D to the mature form was concomitantly observed. The proteolytic processing step was sensitive to pepstatin. These results suggest that procathepsin D is inactive in the endoplasmic reticulum and may be converted to the active forms by autoproteolytic processing mechanism at acidic pH during biosynthesis.     

A biochemically distinct form of cytochrome oxidase (COX) deficiency in the Saguenay-Lac-Saint-Jean region of Quebec.

We report the results of biochemical and molecular investigations on a group of patients from the Saguenay-Lac-Saint-Jean region of Quebec who have an unusual form of cytochrome oxidase deficiency and Leigh disease. This group can be distinguished from the classical presentation of cytochrome oxidase deficiency with Leigh disease, by the severity of the biochemical defect in different tissues. The activity in skin fibroblasts, amniocytes, and skeletal muscle of cytochrome oxidase is 50% of normal, while in kidney and heart it is close to normal values. Brain and liver, on the other hand, have very low activities. The defect in activity appears to result from a failure of assembly of the cytochrome oxidase complex in liver, but levels of mRNA for both mitochondrially encoded and nuclear-encoded subunits in liver and skin fibroblasts were found to be the same as those in controls. The cDNA sequence of the liver-specific cytochrome oxidase subunits VIa and VIIa were determined in samples from patient liver and skin fibroblasts and showed normal coding sequence.     

Characteristics of the dephosphorylated form of phosphorylase purified from rat liver and measurement of its activity in crude liver preparations.

The phosphorylated form of liver glycogen phosphorylase (alpha-1,4-glucan : orthophosphate alpha-glucosyl-transferase, EC 2.4.1.1) (phosphorylase a) is active and easily measured while the dephosphorylated form (phosphorylase b), in contrast to the muscle enzyme, has been reported to be essentially inactive even in the presence of AMP. We have purified both forms of phosphorylase from rat liver and studied the characteristics of each. Phosphorylase b activity can be measured with our assay conditions. The phosphorylase b we obtained was stimulated by high concentrations of sulfate, and was a substrate for muscle phosphorylase kinase whereas phosphorylase a was inhibited by sulfate, and was a substrate for liver phosphorylase phosphatase. Substrate binding to phosphorylase b was poor (KM glycogen = 2.5 mM, glucose-1-P = 250 mM) compared to phosphorylase a (KM glycogen = 1.8 mM, KM glucose-1-P = 0.7 mM). Liver phosphorylase b was active in the absence of AMP. However, AMP lowered the KM for glucose-1-P to 80 mM for purified phosphorylase b and to 60 mM for the enzyme in crude extract (Ka = 0.5 mM). Using appropriate substrate, buffer and AMP concentrations, assay conditions have been developed which allow determination of phosphorylase a and 90% of the phosphorylase b activity in liver extracts. Interconversion of the two forms can be demonstrated in vivo (under acute stimulation) and in vitro with little change in total activity. A decrease in total phosphorylase activity has been observed after prolonged starvation and in diabetes.     

The existence of an inactive form of transglutaminase within metastasising tumours

Separation by anion exchange chromatography of detergent extracts from a poorly metastatic HSV-2-induced hamster fibrosarcoma, its highly metastatic variant and a highly metastatic rat fibrosarcoma indicated the presence of an inactive form of transglutaminase antigen, when eluent fractions were assayed for transglutaminase activity and antigen. This inactive antigenic transglutaminase was clearly separable from the particulate and cytosolic forms of the transglutaminase enzyme. Unlike tumours, its presence could not be demonstrated in extracts from normal rat liver. Measurement of activity levels during tumour growth indicated that the progression of the two highly metastatic tumours was accompanied by a decrease in cytosolic transglutaminase activity, whilst the activity of this enzyme form remained constant in the poorly metastatic tumour. Measurement of antigen levels indicated an inverse relationship between the level of inactive transglutaminase and the level of cytosolic transglutaminase activity, suggesting that the two forms are inter-related. Gel filtration indicated the molecular weight of the inactive form to be greater than both the particulate and cytosolic forms, and it was estimated to be 120,000. Partial proteolysis of the semi-purified inactive form, by either trypsin or thrombin, led to its activation and to the appearance of a transglutaminase similar in molecular weight and ionic mobility, both by anion-exchange chromatography and electrophoresis, to the cytosolic transglutaminase.     

Alteration of tyrosine aminotransferase multiple form patterns by antisickling agents and a decomposition product of antitumor agents

Antineoplastic agents of the nitrosourea class also have antisickling properties. It has been shown that compounds such as BCNU (1,3-bis-(2-chloroethyl)-1-nitrosourea) decompose under physiological conditions and give rise to substituted isocyanates. The latter are carbamylating agents and, presumably, through such activity have antisickling and antineoplastic activity. In fresh liver homogenates (at 0–4°C) cyanate and 2-chloroethyl isocyanate caused conversion of tyrosine aminotransferase form I to form III. This did not occur with the parent compound BCNU. However, liver homogenates prepared from rats pretreated with BCNU contained significantly more tyrosine aminotransferase form III than controls. Considering the effects that cyanate and isocyanate have on hemoglobin and tyrosine aminotransferase, it is possible that the antineoplastic activity of the nitrosoureas is due to carbamylation of specific regulators of cell division.     

Characterization of a Mr 25,000 basic fibroblast growth factor form in adult, regenerating, and fetal rat liver

A heparin-binding Mr 25,000 immunoreactive bFGF-like protein (ir-bFGF) is recognized in adult rat liver extract by affinity-purified polyclonal anti-human placental bFGF antibodies. Hepatic levels of this protein increase 4-fold in regenerating rat liver during the first 48 h after partial hepatectomy. Also, they appear to be higher in embryonic than in newborn or in adult rat liver. Mr 25,000 ir-bFGF from regenerating rat liver, partially purified by heparin-affinity chromatography, induces plasminogen activator activity and cell proliferation in transformed fetal bovine aortic endothelial GM 7373 cells and competes with Mr 18,000 [125I]bFGF for the binding to high affinity bFGF receptors. The data indicate the presence in rat liver of a high molecular weight form of bFGF whose expression is modulated during embryonic development and liver regeneration.     

Changes in the proportion of acetyl-CoA carboxylase in the active form in rat liver. Effect of starvation, lactation and weaning

1. The activity of acetyl-CoA carboxylase (EC 6.4.1.2) in extracts of freeze-clamped liver samples from fed or 24 h-starved virgin, pregnant, lactating and weaned rats was measured (i) immediately after preparation of extracts (;I activity'), (ii) after incubation of extracts with partially purified preparations of either rabbit muscle protein phosphatase 1 [Antoniw, Nimmo, Yeaman & Cohen (1977) Biochem. J.162, 423-433] or rabbit liver phosphatase [Brandt, Capulong & Lee (1975) J. Biol. Chem.250, 8038-8044] (;A activity') and (iii) after incubation with 20mm-potassium citrate before or after incubation with phosphatases (;C activity'). 2. Incubation of liver extracts at 30 degrees C without any additions resulted in activation of acetyl-CoA carboxylase that was shown to be due to dephosphorylation of the enzyme by endogenous protein phosphatase activity. This latter activity was not stimulated by Ca(2+) and/or Mg(2+) but was stimulated by 1 mm-Mn(2+). Incubation of extracts with either of the partially purified phosphatases (0.2-0.5 unit) resulted in faster dephosphorylation and activation. The activity achieved after incubation with either of the exogenously added phosphatases was similar. 3. The A and C activities increased during late pregnancy, were lower than in the virgin rat liver during early lactation and increased by 2-fold in liver of mid-lactating rats. Weaning of mid-lactating rats for 24 h resulted in no change in A and C activities but after 48 h weaning they were significantly lower than those in livers from suckled mothers. 4. The I activity followed a similar pattern of changes as the A and C activities during pregnancy and lactation such that, although the I/A and I/C activity ratios tended to be lower during late pregnancy and early lactation, there were no significant changes in I/A and I/C ratios between lactating and virgin animals. However, these ratios were significantly higher in liver from fed 24 h-weaned animals. 5. Starvation (24 h) resulted in a marked decrease in I activity for all animals studied except early-lactating rats. This was due to a combination of a decrease in the concentration of acetyl-CoA carboxylase in liver of starved animals (A and C activities) and a decrease in the fraction of the enzyme in the active form (lower I/C and I/A ratios). The relative importance of the two forms of regulation in mediating the starvation-induced fall in I activity was about equal in livers of virgin, pregnant and lactating animals. However, the decrease in I/A and I/C ratios was of dominating importance in livers of weaned animals. The A/C activity ratios were the same for livers from all animals studied. 6. The maximal activity of fatty acid synthase was also measured in livers and was highly and positively correlated with the A and C activities of acetyl-CoA carboxylase, suggesting that the concentrations of the two enzymes in the liver were controlled coordinately. 7. It is suggested that the lack of correlation between plasma insulin levels and rates of lipogenesis in the transition from the virgin to the lactating state may be explained by different effects of insulin and prolactin on the concentration of acetyl-CoA carboxylase in the liver and on the fraction of the enzyme in the active form.     

Appearance of the liver form of pyruvate kinase in differentiating cultured foetal hepatocytes

Abstract. From about the 16th day of gestation three forms of pyruvate kinase are present in foetal rat liver (L, R, and M2). Hepatocytes isolated from 15-day-old foetuses do not possess the liver form of pyruvate kinase, but after three days in culture this enzyme can be detected. No effect on the appearance of the enzyme could be seen by administration of insulin and fructose.
Hepatocytes isolated from 19-day-old foetuses exhibit three forms of the enzyme (L, R, and M2) on day 1 of culture but thereafter only two forms are detectable (L and M2). A decrease in activity of the L form is observed. This could be retarded by administration of insulin and fructose.     

Appearance of the liver form of pyruvate kinase in differentiating cultured foetal hepatocytes

From about the 16th day of gestation three forms of pyruvate kinase are present in foetal rat liver (L, R, and M2). Hepatocytes isolated from 15-day-old foetuses do not possess the liver form of pyruvate kinase, but after three days in culture this enzyme can be detected. No effect on the appearance of the enzyme could be seen by administration of insulin and fructose. Hepatocytes isolated from 19-day-old foetuses exhibit three forms of the enzyme (L, R, and M2) on day 1 of culture but thereafter only two forms are detectable (L and M2). A decrease in activity of the L form is observed. This could be retarded by administration of insulin and fructose.     

Purification and properties of the intact form of NADH-cytochrome b5 reductase from rabbit liver microsomes.

NADH-cytochrome b5 reductase [EC 1.6.2.2] has been solubilized with Triton X-100 and purified to homogeneity from rabbit liver microsomes. The purified enzyme is essentially free of the detergent and phospholipids and exists in aqueous media as an oligomeric aggregate of about 13 S. Its monomeric molecular weight is about 33,000 and 1 mole of FAD is associated with 1 mole of the monomeric unit. The enzyme catalyzes the reductions by NADH of ferricyanide and 2,6-dichlorophenol indophenol at an activity ratio of 1 : 0.09. Although the intact form of cytochrome b5 is a poorer electron acceptor than its hydrophilic fragment for the purified flavoprotein, electron transfer from the reductase to the intact cytochrome can be markedly stimulated by detergents or phospholipids, which also cause profound enhancement of the NADH-cytochrome c reductase activity reconstituted from the reducatse and cytochrome b5. Upon digestion with trypsin [EC 3.4.21.4], the ability of the reductase to form an active NADH-cytochrome c reductase system with the intact form of cytochrome b5 and Triton X-100 is rapidly lost. This loss of the reconstitution capability can be prevented by preincubation of the reductase with phosphatidylcholine liposomes. Trypsin digestion also results in the cleavage of the reductase molecule to a protein having a molecular weight of about 25,000 and a smaller fragment. The purified flavoprotein can bind to liver microsomes, liver mitochondria, sonicated human erythrocyte ghosts, and phosphatidylcholine liposomes. The reductase solubilized directly from liver microsomes by lysosomal digestion however, is devoid of membrane-binding capacity. It is concluded that the intact form of NADH-cytochrome b5 reductase is an amphipathic protein and its hydrophobic moiety, which is removable by lysosomal digestion, is responsible for the tight binding of the reductase to microsomes and for its normal functioning in the membrane.     

Purification and characterization of the Novikoff hepatoma glycogen phosphorylase and its relations to a fetal form

The Novikoff hepatoma glycogen phosphorylase b has been purified over 300-fold, free of glycogen synthetase, some of its properties have been studied, and its relationship to fetal forms of rat muscle and liver phosphorylase has been established immunochemically. Its molecular weight is approximately 200,000, and, like the liver but unlike the muscle isozyme, it does not dimerize on conversion to the a form. However, it differs from the liver isozyme in being activated by AMP (K_a = 0.2 mM) and in not being activated by sulfate ion. Antibody to the adult rat muscle phosphorylase did not inhibit the activity of the tumor or liver isozyme. Although antibody to liver or hepatoma phosphorylase had no effect on adult muscle phosphorylase, each of these antibodies partially inhibited the other enzyme. These findings indicate the presence of some liver isozyme in the tumor, and this was confirmed by isoelectric focusing. Rat liver and muscle phosphorylase (and synthetase) were low during embryonal development but rose rapidly at or shortly after birth. Immunochemical studies revealed that both fetal liver and fetal muscle phosphorylases are immunologically identifiable with the tumor enzyme; and the fetal form is also present as a major form in rat kidney and brain.