Purification of ornithine transcarbamylase from rat liver by affinity chromatography with immobilized transition-state analog

Ornithine transcarbamylase (EC 2.1.3.3) was purified to homogeneity from rat liver. The basis of the method is the chromatography of a high-speed supernatant fraction of a homogenized rat liver on an affinity column consisting of the transition-state analog of ornithine transcarbamylase, δ-N-(phosphonacetyl)-l-ornithine, immobilized on epoxy-activated Sepharose 6B through the α-amino group. The enzyme was eluted from the column using a gradient of the substrate, carbamyl phosphate, and further purified by gel filtration. The enzyme elutes with a constant specific activity of 250 to 260 μmol min^?1 mg^?1 at pH 8.5, 37°C, and is free of contaminating proteins on sodium dodecyl sulfate gel electrophoresis. Determination of the molecular weight of the purified enzyme by centrifugation (98,000) and by gel electrophoresis in the presence of sodium dodecyl sulfate (35,300) indicates that the enzyme from rat liver is a trimer. The enzyme exhibits conventional Michaelis-Menten kinetics at pH 7.4 and in this respect differs from the enzyme prepared by other methods.     

Isolation and characterization of argininosuccinate synthetase from human liver

This communication describes the purification and characterization of argininosuccinate synthetase from human liver. By numerous criteria including electrophoresis in sodium dodecyl sulfate containing gels, electrophoresis in nondissociating gels, and analytical ultracentrifugation, the protein is homogeneous at a specific activity of 4.2 mumol/(min mg) assayed at 37 degrees C in the direction of argininosuccinate synthesis. The enzyme has a molecular weight of 183,000, as determined by gel filtration. Electrophoresis in the presence of sodium dodecyl sulfate yielded a single band migrating with an Rf corresponding to 43,000 daltons. Thus, the enzyme is considered to contain four subunits of identical molecular weight. The s20,w of the enzyme is 8.2 S. Antibodies were prepared in rabbits directed against the purified protein. These antibodies react specifically with argininosuccinate synthetase, as determined by electrophoretic analysis of the immunoadsorbed product from crude extracts of human liver. The human enzyme has very similar properties to those published for the beef and rat liver enzymes.     

Purification of human liver microsomal epoxide hydrase. Differences in the properties of the human and rat enzymes.

Human liver microsomal epoxide hydrase has been highly purified to a specific activity (570 to 620 nmol/min/mg of protein) comparable to that of the rat enzyme using styrene oxide as substrate. Like the purified rat liver microsomal epoxide hydrase, the human enzyme has a minimum molecular weight of 49,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and exhibits broad substrate specificity toward a variety of alkene and arene oxides. Despite these similarities, the human and rat enzymes are different proteins as judged by their immunochemical properties as well as their relative catalytic activities toward certain substrates.     

Characterization of rat and human steroid sulfatases

Rat and human steroid sulfatases were purified from liver and placenta, respectively, by the same procedure. The rat and human enzymes were solubilized with Triton X-100, and purified by immunoaffinity chromatography with a monoclonal antibody showing high binding activities to both the enzymes. They were further purified by high-pressure anion-exchange chromatography to compare their structural and catalytic properties. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that both enzymes had a molecular weight of 62,000. The enzymes had similar amino acid compositions and amino-terminal amino acid sequences. Significant differences of the optimum pH, Michaelis constant and maximum velocity were observed between these enzymes. The optimum pH of each enzyme varied from 6.0 to 8.0, depending on substrates and with or without Triton X-100. In detergent-free media, steroid sulfates competitively inhibited the ability of these enzymes to hydrolyze 4-nitrophenyl sulfate. In media containing Triton X-100, on the other hand, the inhibition types of the steroid sulfates on the hydrolyzing activities of the rat and human enzymes were noncompetitive- and mixed-types, respectively.     

Subunit structures of AMP deaminase isozymes in rat

AMP deaminases A and B have been purified to apparent homogeneity from rat muscle and liver, respectively. The molecular weights of 286,000 and 351,000 were obtained for the native muscle and liver enzymes, respectively, by sedimentation equilibrium studies. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the muscle preparation exhibited a single polypeptide band with a molecular weight of 72,000; the liver preparation, a molecular weight of 85,000. The data indicate that each enzyme has a tetrameric structure.     

A comparison of ornithine aminotransferase from human and rat sources

Ornithine aminotransferase was purified from human liver, rat liver and rat kidney. Sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated a subunit molecular weight of 45,000 in all three cases. Estimations of the native molecular weights of ornithine aminotransferase were determined by Sephadex G-200 chromatography in the presence and absence of 0.1% (w/v) Triton X-100. Human and rat enzymes were tetrameric in the presence of detergent but the rat subunits aggregated further in its absence. Characterisation of ornithine aminotransferase from the two rat sources indicated that they were the same protein. The human and rat enzymes were similar but not identical.     

Purification and NH2-terminal amino acid sequence of human thymidylate synthase in an overproducing transformant of mouse FM3A cells

Human thymidylate synthase [EC 2.1.1.45] was purified to homogeneity and its NH2-terminal amino acid sequence was determined taking advantage of the following facts: i) The source of the enzyme was a transformant of mouse FM3A mutant cells which lacks mouse thymidylate synthase but overproduces human thymidylate synthase. ii) The enzyme could be purified on two kinds of affinity column, Cibacron blue dye-bound agarose and methotrexate-bound Sepharose. iii) The enzyme could finally be separated from a trace of impurities by electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate. The purified human thymidylate synthase had a subunit with a molecular weight of 33,000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was subjected to Edman degradation and the NH2-terminal 24 amino acids were sequenced by successive use of a high-sensitivity gas-phase protein sequencer and high performance liquid chromatography to be as follows: Pro-Val-Ala-Gly-Ser-Glu-Leu-Pro-Arg-Arg-Pro-Leu-Pro-Pro-Ala-Ala-Gln-Glu- Arg-Asp -Ala-Glu-Pro-Arg-.     

Purine nucleoside phosphorylases purified from rat liver and Novikoff hepatoma cells.

Purine nucleoside phosphorylase (PNP) was purified from rat hepatoma cells and normal liver tissue utilizing the techniques of ammonium sulfate fractionation, heat treatment, ion-exchange and molecular exclusion chromatography, and polyacrylamide gel electrophoresis. Homogeneity was established by disc gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Purified rat hepatoma and liver PNPs appeared to be identical with respect to subunit and native molecular weight, substrate specificity, heat stability, kinetics and antigenic identity. A native molecular weight of 84,000 was determined by gel filtration. A subunit molecular weight of 29,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point was observed at pH 5.8, and the pH optimum was 7.5. Inosine, guanosine, xanthosine, and 6-mercaptopurine riboside were substrates for the enzymes. The apparent K_m for both inosine and guanosine was about 1.0 × 10^?4m and for phosphate was 4.2 × 10^?4m. Hepatoma and liver PNP showed complete cross-reactivity using antiserum prepared against the liver enzyme.     

Phenol sulfotransferases.

Two phenol sulfotransferases have been purified from rat liver by conventional techniques coupled with affinity chromatography on Affi-Gel blue and ATP-agarose. Both enzymes are homogeneous by the criterion of sodium dodecyl sulfate gel electrophoresis. Each enzyme has a molecular weight of approximately 65,000 and consists of two subunits of apparently equal size. The enzymes are also similar in specificity and in their kinetic parameters but differ in amino acid composition and in their elution from DEAE-cellulose. With adenosine 3'-phosphate 5'-phosphosulfate as donor, a large variety of phenolic compounds serve as sulfate acceptor; sterols, simple alcohols, bile acids, and hydroxamates do not serve as substrates. The transferases may be considered as detoxification enzymes which catalyze the conjugation of xenobiotics containing a phenol group or of phenolic compounds generated by endogenous oxidation. The enzymes act on 3-hydroxyindole to yield indican, suggesting that their in vivo function may include the production of this normal tryptophan metabolite.     

Development of radioimmunoassay for thromboxane B2

A simple method for the preparation of rat liver urate oxidase is described. The enzyme was purified from rat liver homogenate by cell fractionation, detergent treatment, alkali treatment, and affinity chromatography on 8-aminoxanthine-bound Sepharose 4B. This enzyme preparation had a specific activity of 9.1 U/mg of protein and was purified about 1000-fold from the liver homogenate. After sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by staining with Coomassie brilliant blue, this preparation yielded one protein band at a position corresponding to a molecular weight of 33,000.     

Biochemical and immunological studies of purified mouse beta-galactosidase.

Beta-Galactosidase (EC 3.2.1.23) has been purified from the livers of C57BL/6J mice. The enzyme migrated as a single band of protein on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of the denatured and reduced enzyme was 63,000. The native form of beta-galactosidase appeared to be a tetramer of 240,000 at pH 5.0, which was reversibly dissociated at alkaline pH to a dimer with apparent molecular weight of 113,000. Multiple charge isomers of beta-galactosidase were resolved by polyacrylamide gel electrophoresis and ion exchange chromatography. Treatment of beta-galactosidase with neuraminidase markedly reduced its electrophoretic mobility. Purified enzyme as well as crude liver extract hydrolyzed p-nitrophenyl-beta-D-fucoside at one-tenth the rate of hydrolysis of the beta-galactoside. Antiserum to the purified enzyme precipitated the major portion of beta-galactosidase activity of mouse liver, brain, and kidney. This antiserum cross-reacts with beta-galactosidases from rat and Chinese hamster, but not with human, porcine, or bovine beta-galactosidase.     

Structural and immunological similarities between high molecular weight zinc ion-dependent p-nitrophenylphosphatase and fructose-1,6-bisphosphate aldolase from bovine liver

High molecular weight zinc ion-dependent acid p-nitrophenylphosphatase (HMW-ZnAPase) was purified from bovine liver to homogeneity as judged by native and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The partial sequence of the purified enzyme electroblotted on PVDF membrane reveals a 95% sequence homology with human and bovine liver fructose-1,6-bisphosphate aldolase isozyme B (FALD B). FALD B was isolated from bovine liver using an affinity elution from phosphocellulose column. FALD B from bovine liver shows a native and subunit molecular weight that is indistinguishable from that of HMW-ZnAPase. In addition, an affinity purified antiserum raised in rabbits against purified HMW-ZnAPase cross-reacts with bovine liver FALD B and rabbit muscle isozymes. Despite these similarities, HMW-ZnAPase does not show FALD activity and bovine liver FALD does not display any zinc ion-p-nitrophenylphosphatase activity. These results suggested the existence of structural and immunological similarities between bovine liver HMW-ZnAPase and FALD B. Differences in some amino acid residues in enzyme activity indicate that they may be involved in different biochemical functions.     

Liver microsomal expoxide hydrase. Solubilization, purification, and characterization.

Epoxide hydrase was solubilized from liver microsomes of phenobarbital-treated rats by treatment with cholate and purified to apparent homogeneity by ammonium sulfate fractionation and column chromatography in the presence of the nonionic detergent Emulgen 911 on DEAE-cellulose and hydroxylapatite. The purified enzyme preparation had a single major band with a molecular weight of 53,000 to 54,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Other studies indicated that in the absence of sodium dodecyl sulfate, purified epoxide hydrase exists as high molecular weight aggregates. The preparation was essentially free of heme and flavin, but still contained small amounts of lipids and Emulgen 911.     

Purification of 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase from mouse epidermis

Ornithine decarboxylase was purified at least 1500-fold from mouse epidermis pretreated with five consecutive doses of 12-O-tetradecanoylphorbol-13-acetate and 3-isobutyl-1-methylxanthine at 3- to 4-day intervals. Following DEAE-cellulose chromatography and ammonium sulfate precipitation, ornithine decarboxylase was purified further by affinity chromatography. Ornithine decarboxylase was then radioactively labeled by covalently binding [3H]-alpha-difluromethylornithine to the enzyme following polyacrylamide gel electrophoresis under non-denaturing conditions. Following sodium dodecyl sulfate polyacrylamide gel electrophoresis and silver staining of protein, a band was identified that corresponded to a molecular weight of approx. 56,000, coincident with a peak of radioactivity. This is the first study to purify ornithine decarboxylase from mouse epidermis.     

Isolation and characterization of ornithine transcarbamylase from normal human liver.

We report experiments describing the isolation and characterization of ornithine transcarbamylase from normal human liver. Our preparative procedure employs initial centrifugation and heat steps, intermediate batch-wise adsorption and desorption from ion exchange resins and column chromatographic elution from hydroxylapatite, and final purification by gel filtration chromatography and glycerol density gradient centrifugation. The enzyme, purified 580-fold in this way, is homogeneous as judged by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Human ornithine transcarbamylase has a molecular weight of 114,000 and is a trimer of identical 38,000 molecular weight subunits. It focuses at pH 6.8 as a single band on polyacrylamide gel, has a COOH-terminal phenylalanine, an NH2-terminal glycine, an apparent Km for L-ornithine of 0.4 mM and for carbamyl phosphate of 0.16 mM, and a pH optimum of 7.7. The enzyme is quite stable over a temperature range from -50 degrees to +60 degrees C and over the pH range from 5.8 to 8.2. The quaternary structure and amino acid composition of the human enzyme are very similar to those of its bovine homologue.     

Purification of N-hydroxy-2-acetylaminofluorene reductase from rabbit liver cytosol

N-Hydroxy-2-acetylaminofluorene reductase was purified from rabbit liver cytosol by fractionation with ammonium sulfate, and chromatography with DEAE-cellulose, Sephadex G-200 and hydroxylapatite. The purified enzyme was homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 34,000 by the electrophoresis and by gel filtration on Sephadex G-200. The enzyme required cysteine, glutathione, dithiothreitol, 2-mercaptoethanol, NADPH or NADH as an electron donor. The enzyme activity was inhibited by p-chloromercuribenzoic acid, N-ethylmaleimide, cupric sulfate or disulfiram, but little by oxygen.     

Purification and Characterization of Aromatic L-Amino Acid Decarboxylase from Rat Kidney and Monoclonal Antibody to the Enzyme

Aromatic L-amino acid decarboxylase was purified from rat kidney to homogeneity, as judged by polyacrylamide gel electrophoresis, in the presence and absence of sodium dodecyl sulfate (SDS). The final preparation showed an activity of 3,4-dihydroxyphenylalanine (dopa) decarboxylation of approximately 11,000 nmol/min/mg of protein at 37 degrees C. The purified enzyme also catalyzed the decarboxylation of 5-hydroxytryptophan, tyrosine, tryptophan, and phenylalanine. The enzyme appeared to be composed of two identical subunits, each possessing a molecular weight of 48,000. The isoelectric point of the enzyme was estimated to be 6.7 in the presence of 8 M urea and 5.60-5.85 in its absence. To examine the identity of aromatic L-amino acid decarboxylase from various tissues, a monoclonal antibody directed against the enzyme from rat kidney was prepared. Immunotitration and analysis by antibody-affinity chromatography followed by SDS-polyacrylamide gel electrophoresis revealed that the enzymes from the striatum, adrenal medulla, pineal gland, liver, and kidney were indistinguishable with respect to immunological cross-reactivity and molecular size.     

Production of monospecific antibodies to rat liver ornithine decarboxylase and their use in turnover studies.

Two forms of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) were purified from the livers of rats which had been treated with thioacetamide for 16 h (for details, see miniprint to Obenrader, M.F., and Prouty, W. F. (1977) J. Biol. Chem. 252, 2860-2865). The enzyme was purified over 7,000-fold from liver cytosol with an overall yield of 8%. Enzyme activity was eluted finally in two distinct fractions by chromatography on activated thiol-Sepharose 4B. Both forms appear to be dimeric proteins having molecular weights of approximately 100,000 by equilibrium sedimentation and analysis on a calibrated Sephadex G-200 column. The apparent subunits are approximately 50,000 daltons as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Since electrophoresis in the presence of detergent is the only method used here to indicate subunits, the possibility that conditions of sample preparation resulted in splitting of a labile protein cannot be excluded from consideration. Ornithine decarboxylase has a very broad pH-activity curve with an optimum that shifts from pH 7.0 to pH 7.8 as the enzyme is purified. The apparent Km values for a highly purified mixture of the two forms of enzyme for L-ornithine and pyridoxal 5'-phosphate were determined to be 0.13 mM and 0.25 micronM, respectively. Both sodium and potassium chloride were shown to inhibit enzymatic activity; 50% inhibition occurred at 270 mM for each when Km amounts or ornithine were used. Rat liver ornithine decarboxylase antiserum was prepared in rabbits using Form I of the enzyme as the antigen. The antibody was shown to precipitate quantitatively the ornithine decarboxylase activity isolated from induced rat liver and rat ventral prostate. The specificity of the antiserum was demonstrated by rocket immunoelectrophoresis and by gel electrophoresis in the presence of sodium dodecyl sulfate using immunoprecipitates obtained from enzyme preparations labeled either in vivo, with [3H]leucine, or in vitro, by reductive methylation using formaldehyde and sodium [3H]borohydride. The antibody preparation has been used in a titration method to assess the half-life of antigen in livers of rats induced for ornithine decarboxylase by injection of thioacetamide. In two experiments, the t1/2 of activity at the height of induction, following injection of cycloheximide, was 19 and 24 min, while the t1/2 of disappearance of antigen was 28 and 33 min, respectively. In each experiment the t1/2 for antigen was significantly longer than the t1/2 for loss of enzyme activity. Enzyme levels appear to be modulated primarily by synthesis and degradation of antigen. Furthermore, the observation that enzyme activity is lost with a shorter t1/2 than antigen is consistent with the idea that denaturation is an initial step in the degradation of this enzyme...     

Quaternary structure of delta-aminolevulinic acid synthase from Rhodopseudomonas spheroides.

Delta-Aminolevulinic acid synthase (succinyl-CoA: glycine C-succinyltransferase (decarboxylating) EC 2.3.1.37) was purified from Rhodopseudomonas spheroides. The purity of the enzyme preparation was established by its behavior in disc electrophoresis in the presence and absence of sodium dodecyl sulfate and by analytical ultracentrifugation. The molecular weight of the enzyme as determined by sedimentation equilibrium was found to be about 80,300, a value similar to those obtained by gel filtration, polyacrylamide gel electrophoresis, and sucrose gradient centrifugation. The molecular weight of the enzyme, denatured with either sodium dodecyl sulfate or guanidine hydrochloride, was found to be about 45,000 and 41,000, respectively. The dimeric structure was supported by sedimentation in sucrose gradients. Further evidence for the dimetic nature of the enzyme was obtained by gel electrophoresis of the enzyme treated with dimethylsuberimidate and sodium dodecyl sulfate.     

Purification of NADPH-linked alpha,beta-ketoalkene double bond reductase from rat liver

NADPH-linked alpha,beta-ketoalkene double bond reductase was purified from rat liver cytosol by fractionation with ammonium sulfate, and chromatography with DEAE-cellulose. AF-Blue Toyopearl and hydroxyapatite. The purified enzyme was homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 39,500 by the electrophoresis and by HPLC gel filtration on a TSK gel G3000 SWXL column. The double bond of 2-alkenals was also reduced by the enzyme, but to a lesser extent. The enzyme activity was inhibited by 5,5'-dithiobis(2-nitrobenzoic acid), p-chloromercuribenzoic acid, N-ethylmaleimide, iodoacetamide, dicumarol, quercitrin, and disulfirum. However, the enzyme was insensitive to oxygen.