Relative distribution of arylsulphatases A and B in rat liver parenchymal and other cells

The relative activities of arylsulphatases A and B were measured in rat liver parenchymal and non-parenchymal cells, in peritoneal macrophages and in a number of rat tissues. Although absolute values cannot be obtained, it was shown that the arylsulphatase B/arylsulphatase A activity ratio is much higher in non-parenchymal cells than in parenchymal cells. The ratios in adrenals, brain and testis are very similar to each other but differ from those found in spleen, kidney and liver. These ratio variations may be caused by alterations in the activity of the B enzyme rather than the A enzyme. The relatively high B enzyme/A enzyme ratios in all rat tissues explains why the method devised for the independent assay of human arylsulphatases A and B cannot be employed with rat tissues.     

Acetoacetyl-CoA synthetase specific activity and concentration in rat tissues

An antibody against acetoacetyl-CoA synthetase purified from rat liver was raised in rabbits. Utilizing the binding of antibody-antigen complexes to a nitrocellulose membrane, a sensitive enzyme-linked immunosorbent assay was developed to estimate the enzyme concentration in rat tissues. The enzyme concentration (microgram immunoreactive protein/mg protein) in rat liver cytosol was increased about 3-, 1.8- and 7-fold by feeding rats diets containing 5% cholestyramine, 0.2% ML-236B (compactin), and 5% cholestyramine plus 0.2% ML-236B for 4 days, respectively, and decreased about 1.8-fold by fasting the animals or 1.3-fold by feeding them a diet containing 5% cholesterol. Changes in the enzyme activity were almost parallel to those in the enzyme concentration, suggesting the physiological role of this enzyme in cholesterol biosynthesis. Immunoblotting of the hepatic cytosol also confirmed that the increase in enzyme concentration on cholestyramine and/or ML-236B feeding was due to an increase in an enzyme protein the same as the purified enzyme and not the isozymic protein. Among various rat tissues examined, the concentrations of immunologically crossreactive enzyme were higher in lipogenic tissues, such as brain, adipose tissue and liver, than in other tissues. The enzymes in these three tissues were identical in molecular weight determined by gel filtration and immunoblotting.     

Comparison of the multiple deoxyribonucleic acid-dependent ribonucleic acid polymerase forms of whole rat liver and a minimal-deviation rat hepatoma cell line

To investigate the possibility that the pattern of multiple DNA-dependent RNA polymerases of an animal cell exerts a controlling influence on its nature, the activities of these enzymes were compared in differentiated rat liver and in a rapidly growing minimal-deviation rat hepatoma cell line by using established techniques of enzyme extraction, separation and determination. Relative to the DNA content of the tissues, RNA polymerase activities of forms AI, AII and B were approx. ninefold, twofold and twofold higher respectively in the cell line than in the liver. Tests indicated that these results could not be explained by differences in extraction efficiency or by the presence of unbound inhibitors or stimulators of polymerase activity in the final enzyme preparations. New forms of the enzyme were not detected in either tissue. The significance of these findings with respect to the possible role of multiple RNA polymerases in the control of cellular activities is discussed.     

The presence of desulfo xanthine dehydrogenase in purified and crude enzyme preparations from rat liver

Crude and purified xanthine dehydrogenase preparations from rat liver were examined for the existence of a naturally occurring inactive form. Reduction of the purified enzyme by xanthine under anaerobic conditions proceeded in two phases. The enzyme was inactivated by cyanide, which caused the release of a sulfur atom from the molybdenum center as thiocyanate. The amount of thiocyanate released was almost in parallel with the initial specific activity. The active and inactive enzymes could be resolved by affinity chromatography on Sepharose 4B/folate gel. These results provided evidence that the purified enzyme preparation from rat liver contained an inactive form. A method for the determination of the active and inactive enzymes in crude enzyme preparations from rat liver was devised based on the fact that only active enzyme could react with [14C]allopurinol and both active and inactive enzymes could be immunoprecipitated quantitatively by excess specific antibody to xanthine dehydrogenase. The amount of [14C]alloxanthine (derived from [14C]allopurinol) bound to the active sulfo enzyme in crude rat liver extracts was about 0.5 mol/mol of FAD. As this content is closely similar to that in the purified enzyme, these results suggest the existence of an inactive desulfo form in vivo.     

Tissue and species distribution of liver type and tumor type nuclear poly(A) polymerases

Previous studies in this laboratory have identified two distinct nuclear poly(A) polymerases, a 48 kDA tumor type enzyme and a 36-38 kDA liver type enzyme. To investigate the tissue and species specificity of these enzymes, nuclear extracts were prepared from various rat tissues, pig brain and two human cell lines. These as well as whole cell extract from yeast were probed for the two enzymes by immunoblot analysis using polyclonal anti-tumor poly(A) polymerase antibodies or autoimmune sera which contain antibodies specific for the liver type enzyme. Results indicate that both tumor and liver type enzymes are conserved across species ranging from rat to human. The yeast enzyme does not appear to be immunologically related to the liver or the tumor type poly(A) polymerase. The liver type enzyme appears to be specific for normal tissues whereas the tumor type enzyme is detected only in tissues in a "tumorigenic" state or cell lines originating from tumor tissues.     

A one-step procedure for the determination of protein kinase activities and a protein kinase inhibitor in nuclear extract

An isoelectric focusing procedure has been developed that achieves the complete separation of an inhibitor protein from its target enzymes, the nuclear protein kinases, in a one-step operation. The method thus enables one to estimate the amount of inhibitory activity in nuclear extracts, an activity that can not be measured in the presence of the protein kinases. Using this procedure, we have determined the apparent and the actual protein kinase activities in nuclear extracts of rat liver and of two transplantable rat hepatomas by two independent measurements: (a) the increase in protein kinase activity upon separation of the enzyme(s) from the inhibitor, relative to the activity in the crude extracts which was taken as 100%; (b) the inhibitory activity of the pooled focused inhibitor fractions. The amount of cryptic enzyme activity closely correlated with the amount of inhibitor present in extracts from different tissues. Considerably less cryptic kinase activity was found in the tumor nuclei than in liver which may account, at least in part, for the higher phosphorylating capacity previously observed in hepatoma.     

Comparison of D-beta-hydroxybutyrate dehydrogenase from rat liver and brain mitochondria

The properties of D-beta-hydroxybutyrate dehydrogenase (BDH) from rat liver and brain mitochondria were compared to determine if isozymes of this enzyme exist in these tissues. The BDHs from these tissues behaved similarly during the purification process. The enzymes were indistinguishable by sodium dodecyl sulfate-polyacrylamide or acid-urea-polyacrylamide gel electrophoresis and they had identical isoelectric points. The BDHs from rat liver and brain were also quite similar in functional parameters determined by kinetic analysis and phospholipid activation of apo-BDH (i.e., the lipid-free enzyme). Antiserum against rat liver BDH inhibited both enzymes to an equivalent extent in a titration assay. The enzymes had similar patterns of peptide mapping by partial digestion with Staphylococcus aureus V8 protease, followed by immunoblotting using antiserum against the liver enzyme. These results suggest that the BDHs in rat liver and brain are very similar and possibly identical.     

Isolation and properties of cytochrome c oxidase from rat liver and quantification of immunological differences between isozymes from various rat tissues with subunit-specific antisera

Cytochrome c oxidase was isolated from rat liver either by affinity chromatography on cytochrome-c--Sepharose 4B or by chromatography on DEAE-Sepharose. Dodecyl sulfate gel electrophoresis of both preparations showed the same subunit pattern consisting of 13 different polypeptides. Kinetic analysis of the two preparations gave a higher Vmax for the enzyme isolated by chromatography on DEAE-Sephacel. Specific antisera were raised in rabbits against nine of the ten nuclear endoded subunits. A monospecific reaction of each antiserum with its corresponding subunit was obtained by Western blot analysis, thus excluding artificial bands in the gel electrophoretic pattern of the isolated enzyme due to proteolysis, aggregation or conformational modification of subunits. With an antiserum against rat liver holocytochrome c oxidase a different reactivity was found by Western blot analysis for subunits VIa and VIII between isolated cytochrome c oxidases from pig liver or kidney and heart or skeletal muscle. For a quantitative analysis of immunological differences a nitrocellulose enzyme-linked immunosorbent assay was developed. Monospecific antisera against 12 of the 13 subunits of rat liver cytochrome c oxidase were titrated with increasing amounts of total mitochondrial proteins from different rat tissues dissolved in dodecyl sulfate and dotted on nitrocellulose. The absorbance of a soluble dye developed by the second peroxidase-conjugated antibody was measured. From the data the following conclusions were obtained: (a) The mitochondrial encoded catalytic subunits I-III of cytochrome c oxidase are probably identical in all rat tissues. (b) All nine investigated nuclear encoded subunits of cytochrome c oxidase showed immunological differences between two or more tissues. Large immunological differences were found between liver, kidney or brain and heart or skeletal muscle. Minor but significant differences were observed for some subunits between heart and skeletal muscle and between liver, kidney and brain. (c) Between corresponding nuclear encoded subunits of cytochrome c oxidase from fetal and adult tissues of liver, heart and skeletal muscle apparent immunological differences were observed. The data could explain cases of fatal infantile myopathy due to cytochrome c oxidase deficiency.     

Purification of Cathepsin D by Ah-Sepharose Affinity Chromatography

A rapid and reliable method for coupling the protease in-hibitor pepstatin to AH-Sepharose 4B was developed. The matrix prepared was used to purify cathepsin D from rat liver. The enzyme was eluted in one fraction and proved to be pure by gel electrophoresis, two types of ion exchange chromatography, molecular sieve chromatography, and immunologically homogenous by Immunoelectrophoresis. This method is more rapid and gives a higher yield than previous techniques. The possibility to use this technique for the purification of other enzymes inhibitable by pepstatin should be considered.     

Increased activity of cyclic AMP phosphodiesterase from frozen-thawed rat liver. A role of lysosomal protease in enzyme activation.

The activity of cyclic AMP phosphodiesterase (3':5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) in 105 000 X g supernatant fraction from frozen-thawed rat liver was 2.5 times higher than the corresponding preparation from fresh liver. This increased activity of frozen liver enzyme was accompanied by a decreased sensitivity of the enzyme to known activators such as alpha-tocopheryl phosphate and trypsin. Neither membrane-bound cyclic AMP phosphodiesterase, nor supernatant cyclic GMP phosphodiesterase increased in frozen liver preparation. It is unlikely that the activator protein of phosphodiesterase participated in the observed change of enzyme activity. Among rat tissues so far tested, the increased level of cyclic AMP phosphodiesterase was noted only in tissues rich in lysosome content. In the recombination experiment where phosphodiesterase from fresh liver was incubated with lysosomal fraction, stimulation of the enzyme activity was observed with a concomitant loss of sensitivity to above-mentioned activators. Since the stimulation by lysosomal fraction was effectively inhibited by cathepsin B1 inhibitors, leupeptin and antipain, it was deduced cathepsin-B1 (EC 3.4.12.3) type protease(s) was the main causative of activating the cyclic AMP phosphodiesterase. The freezing-thawing process of rat liver made the lysosomal membrane more permeable, and hence lysosomal proteases were released into soluble fraction during phosphodiesterase preparation. These results provide a warning not to use frozen liver for phosphodiesterase preparation, otherwise altered properties of the enzymes will be seen.     

N-Acetylglutamate synthetase in human liver: Regulation of activity by L-Arginine and N-Acetylglutamate

A sensitive assay for the determination of N-Acetylglutamate synthetase activity in human liver has been developed. The activity was markedly stimulated by L-Arginine and inhibited by its product N-Acetylglutamate. Apparent kinetic properties were similar to those of the rat liver enzyme. This work provides the first evidence for the presence of N-Acetylglutamate synthetase activity in human.     

An h.p.l.c. assay for protoporphyrinogen oxidase activity in rat liver.

An h.p.l.c. method is described for the assay of protoporphyrinogen oxidase activity in rat liver. A relatively pure protoporphyrinogen IX substrate was obtained by selectively removing any protoporphyrin IX unreduced by sodium amalgam on a small disposable cartridge packed with a strong anion-exchanger. The protoporphyrin IX formed was extracted with dimethyl sulphoxide/methanol (3:7, v/v) containing mesoporphyrin as the internal standard for separation and quantification by reversed-phase chromatography. The Km for protoporphyrinogen was 9.5 +/- 1.6 microM, and the enzyme activities were 0.59 +/- 0.11 nmol of protoporphyrin IX produced/min per mg of mitochondrial protein and 33.5 +/- 2.7 nmol protoporphyrin IX produced/min per g of liver tissue homogenate. The method is applicable to the determination of enzyme activity in small amounts of human liver biopsy.     

A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts

Superoxide dismutase (EC 1.15.1.1) has been assayed by a spectrophotometric method based on the inhibition of a superoxide-driven NADH oxidation. The assay consists of a purely chemical reaction sequence which involves EDTA, Mn(II), mercaptoethanol, and molecular oxygen, requiring neither auxiliary enzymes nor sophisticated equipment. The method is very flexible and rapid and is applicable with high sensitivity to the determination of both pure and crude superoxide dismutase preparations. The decrease of the rate of NADH oxidation is a function of enzyme concentration, and saturation levels are attainable. Fifty percent inhibition, corresponding to one unit of the enzyme, is produced by approximately 15 ng of pure superoxide dismutase. Experiments on rat liver cytosol have shown the specificity of the method for superoxide dismutase. Moreover, common cellular components do not interfere with the measurement, except for hemoglobin when present at relatively high concentrations. The assay is performed at physiological pH and is unaffected by catalase.     

Catalytic activity and substrate specificity of the flavin-containing monooxygenase in microsomal systems: characterization of the hepatic, pulmonary and renal enzymes of the mouse, rabbit, and rat

Inhibitory antibodies against NADPH-cytochrome P-450 reductase, detergent solubilization to dissociate functional interaction between the reductase and cytochrome P-450, and selective trypsin degradation have been used to characterize flavin-containing monooxygenase activity in microsomes from different tissues and species. A comparison of assay methods is reported. The native microsome-bound flavin-containing monooxygenase of mouse, rabbit, and rat liver, lung, and kidney can metabolize compounds containing thiol, sulfide, thioamide, secondary and tertiary amine, hydrazine, and phosphine substituents. Therefore, this enzyme from these common experimental animals has catalytic capabilities similar to those of the well-characterized porcine liver enzyme. True allosteric activation by n-octylamine does not appear to be a property of either the mouse, rabbit, or rat liver enzymes, but is a property of the pig liver and mouse lung enzymes. The microsomal pulmonary flavin-containing monooxygenase of the rabbit has some unique substrate preferences which differ from the mouse lung enzyme. Both the rabbit and mouse pulmonary enzymes have recently been shown to be distinct enzyme forms. However, the rat pulmonary flavin-containing monooxygenase appears to be catalytically identical to the rat liver enzyme, and does not have any of the unusual catalytic properties of either the rabbit or mouse lung enzymes. Enzyme activity of mouse, rabbit, and rat kidney microsomes is qualitatively similar to the hepatic activities. Substrates which saturate the microsome-bound flavin-containing monooxygenase at 1.0 mM, including thiourea, thioacetamide, methimazole, cysteamine, and thiobenzamide, are metabolized at common maximal velocities. This suggests that the kinetic mechanism of the native enzyme is similar to that established for the isolated porcine liver enzyme in that the rate-limiting step of catalysis occurs after substrate binding, and that all substrates capable of saturating the microsomal enzyme should be metabolized at a common maximal velocity.     

Determination of cytochrome P450 1A activities in mammalian liver microsomes by high-performance liquid chromatography with fluorescence detection

A sensitive method for the determination of cytochrome P450 (P450 or CYP) 1A activities such as ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) in liver microsomes from human, monkey, rat and mouse by high-performance liquid chromatography with fluorescence detection is reported. The newly developed method was found to be more sensitive than previous methods using a spectrofluorimeter and fluorescence plate reader. The detection limit for resorufin (signal-to-noise ratio of 3) was 0.80 pmol/assay. Intra-day and inter-day precisions (expressed as relative standard deviation) were less than 6% for both enzyme activities. With this improved sensitivity, the kinetics of EROD and MROD activities in mammalian liver microsomes could be determined more precisely. EROD activities in human and monkey liver microsomes, and MROD activities in liver microsomes from all animal species exhibited a monophasic kinetic pattern, whereas the pattern of EROD activities in rat and mouse liver microsomes was biphasic. In addition, the method could determine the non-inducible and 3-methylcholanthrene-inducible activities of EROD and MROD in rat and mouse liver microsomes under the same assay conditions. Therefore, this method is applicable to in vivo and in vitro studies on the interaction of xenobiotic chemicals with cytochrome CYP1A isoforms in mammals.     

Human,rat and crocodile liver microsomal monooxygenase activities measured using diazepam and nifedipine: effects of CYP3A inhibitors and relationship to immunochemically detected CYP3A apoprotein

Nifedipine oxidase and diazepam C₃-hydroxylase were tested as activities for selectively measuring CYP3A enzymes using liver microsomes from male and female human organ donors, male and female Wistar rats and male and female estuarine crocodiles. The association between CYP3A enzymes and these monooxygenations was confirmed for the human samples. Male rat samples had lower specific contents of CYP3A apoprotein than the human samples but had equivalent (nifedipine) or higher (diazepam) monooxygenase specific activities. CYP3A apoprotein was undetectable in female rat samples which had very low activities towards both substrates. Enzyme inhibition studies showed that diazepam C₃-hydroxylase of male rat liver was attributable to CYP3A but corresponding results for female rats suggested a contribution from non-CYP3A enzyme. Western blotting with immunochemical detection using anti-CYP3A4 IgG suggested the presence of putative CYP3A apoprotein in male and female crocodile liver samples and inhibition studies with diazepam as substrate suggested the presence of CYP3A subfamily monooxygenase activity in these enzyme preparations. Results for nifedipine oxidase with male and female rat liver and male crocodile liver suggested major contributions to catalysis from non-CYP3A enzymes. Inhibition studies suggested that a higher proportion of nifedipine oxidase in female crocodile liver may be attributable to the putative CYP3A enzyme(s) than in male crocodile liver. These results show the need for care in the assessment of CYP3A activity of fractionated tissues when using these substrates in cross-species studies and where gender is a variable.     

Colorimetric assay of liver and Escherichia coli asparatate transcarbamylases in the presence of 2-mercaptoethanol

A modification of the method of Prescott and Jones (1) for the colorimetric determination of carbamyl aspartate has been developed to permit the assay of aspartate transcarbamylases in the presence of 2-mercaptoethanol. Interference by this compound is eliminated by means of N-ethylmaleimide. The usefulness of the modified method is illustrated by examination of the contrasting properties of the Escherichia coli and rat liver enzymes.     

Selective preparation and characterization of membranous and soluble forms of alkaline phosphatase from rat tissues. A comparison with the serum enzyme

We developed a method for selective preparation of two forms of alkaline phosphatase from rat tissues. The enzyme was extracted by n-butanol treatment at pH 5.5 and pH 8.5 as soluble and aggregated (membranous) forms, respectively. The soluble form prepared from liver was found to be identical with the serum enzyme. Complete solubilization of the membrane-bound enzyme without detergents had a great advantage in its purification. Rat hepatoma AH-130 cells enriched in alkaline phosphatase were first used for purification of the liver-type enzyme. The hepatoma enzyme, purified by chromatographies on concanavalin-A-Sepharose, Sephacryl S-300 and hydroxyapatite was used for production of antibodies specific for the liver-type isozyme. An immunoaffinity column, prepared with anti-(hepatoma-enzyme) IgG was utilized for the enzyme purification from other tissues including the membranous form. Analyses of amino acid composition of the purified enzymes revealed that all the liver-type enzymes from hepatoma, liver, kidney and serum had the same composition, whereas the intestinal type consisted of the composition distinctly different from that in the liver type. In addition, there was no significant difference in amino acid composition between the soluble and membranous forms, suggesting a possible involvement in the membranous form of a hydrophobic component other than its polypeptide domain. The present method for selective preparation of the soluble and membranous forms of alkaline phosphatase will be useful for a further investigation on the interaction of the enzyme with membranes.     

Unique cathepsin D-type proteases in rat thoracic duct lymphocytes and in rat lymphoid tissues.

Two unique cathepsin D-type proteases apparently present only in rat thoracic duct lymphocytes and in rat lymphoid tissues are described. One, termed H enzyme, has an apparent molecular weight of similar to95,000; the other, termed L enzyme, has an apparent molecular weight of similar to45,000, in common with that of most cathepsins D from other tissues and species. Both enzymes differ from cathepsin D, however, by a considerably greater sensitivity to inhibition by pepstatin and by a smaller degree of inhibition by an antiserum which inhibits rat liver cathepsin D. H enzyme is converted to L enzyme by treatment with beta-mercaptoethanol; the relationship between the two enzymes remains unknown. H and L enzyme have been detected in rat lymphoid tissues and in mouse spleen, but they are not present in other rat tissues (liver, kidney, adrenals), rabbit tissues, calf thymus, bovine spleen, or human tonsils. As measured on acid-denatured bovine hemoglobin as substrate, both enzymes have pH activity curves identical with that of rat liver cathepsin D, with optimal activity at pH 3.6. Activity on human serum albumin is much less and also shows an optimum at pH 3.6; hence, neither enzyme has the properties of cathepsin E. Thiol-reactive inhibitiors have no effect on the activity of H and L enzyme; thus they do not belong to the B group of cathepsins. Additional information, discussed in this paper, leads us to conclude that partially purified H and L enzymes are cathepsin D-type proteases.     

Gulonolactone oxidase is missing in teleost fish. The direct spectrophotometric assay

Data in the literature imply that some fish species evolved with the capacity to synthesize ascorbic acid. Gulonolactone oxidase activity has been reported in kidney and/or liver tissues. However, it is shown here that this microsomal enzyme activity is missing in common carp hepatopancreas and kidney, whereas high activity was confirmed in pigeon kidney, rat liver, bovine liver and amphibian (Xenopus) kidney tissues. A new assay using either the whole tissue homogenate or microsomes solubilized by sodium deoxycholate was developed to directly measure the formation of ascorbic acid spectrophotometrically. Identical values were found using this assay as well as the assay in which formed ascorbate was determined by the dinitrophenyl hydrazine (DNPH) method. In some experiments, these results were confirmed by polarographically measured oxygen consumption.