Isolation and characterization of glycolic acid dehydrogenase from human liver.

Glycolic acid dehydrogenase has been purified over 800-fold from human liver by (NH4)2SO4 fractionation and column chromatography with DEAE-cellulose and hydroxyapatite. The enzyme catalyzes the direct oxidation of glycolate to oxalate without forming glyoxylate as a free intermediate. Activity is found only in the liver in the soluble fraction. The enzyme is specific for glycolate and inhibits no activity towards glycine or glyoxylate. Glyoxylate and DL-phenyllactate exhibit the enzyme. Optimum activity occurs sharply at pH 6.1 and the Michaelis constant for glycolate was 6.3.10(-5)M. Molecular oxygen does not appear to be the electron acceptor and no requirement for cofactors has been demonstrated, althoug flavin mononucleotide, ascorbate and cytochrome c stimulate activity. The isolation of this enzyme which may account for a significant part of the normal oxalate excretion in man, provides a more complete understanding of the pathways of oxalate biosynthesis and must be taken into account when considering possible methods for controlling disorders of oxalate metabolism.     

Isolation and characterization of dihydropteridine reductase from human liver.

Dihydropteridine reductase (EC 1.6.99.7) was purified from human liver obtained at autopsy by a three-step chromatographic procedure with the use of (1) a naphthoquinone affinity adsorbent, (2) DEAE-Sephadex and (3) CM-Sephadex. The enzyme was typically purified 1000-fold with a yield of 25%. It gave a single band on non-denaturing and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and showed one spot on two-dimensional gel electrophoresis. The molecular weight of the enzyme was determined to be 50000 by sedimentation-equilibrium analysis and 47500 by gel filtration. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, a single subunit with mol.wt. 26000 was observed. A complex of dihydropteridine reductase with NADH was observed on gel electrophoresis. The isoelectric point of the enzyme was estimated to be pH 7.0. Amino acid analysis showed a residue composition similar to that seen for the sheep and bovine liver enzymes. The enzyme showed anomalous migration in polyacrylamide-gel electrophoresis. A Ferguson plot indicated that this behaviour is due to a low net charge/size ratio of the enzyme under the electrophoretic conditions used. The kinetic properties of the enzyme with tetrahydrobiopterin, 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, NADH and NADPH are compared, and the effects of pH, temperature and a number of different compounds on catalytic activity are presented.     

Isolation and characterization of a mitochondrial D-amino acid oxidase from Neurospora crassa.

D-Amino acid oxidase (EC 1.4.3.3) activity in homogenates of Neurospora crassa strain SY7A was found to sediment with the mitochondrial fraction. Digitonin fractionation studies on purified mitochondria have indicated a matrix localization of the enzyme. Additionally, a peroxidase (EC 1.11.1.7) activity, which may remove hydrogen peroxide formed as a product of D-amino acid oxidation, was also found in the mitochondrial matrix. Partial purification (20- to 30-fold) of the mitochondrial D-amino acid oxidase was achieved. The enzyme exhibited a pH optimum between 9.0 and 9.2, temperature optimum between 20 and 30 degrees C, and a molecular weight of 118 000 +/- 6000 as determined by gel electrophoresis and 125 000 as determined by gel chromatography.     

Xanthine oxidase from human liver: purification and characterization

Xanthine oxidase [EC 1.2.3.2] was purified 2000-fold from human liver. The last step of the procedure involved affinity chromatography. The resulting preparation showed two closely migrating bands of enzyme activity after gel electrophoresis under nondenaturing conditions. No other proteins were detected on these gels. The average particle mass of the enzyme was 300 kDa as determined by size-exclusion chromatography. This together with results of gel electrophoresis under denaturing conditions suggested that the native enzyme was composed of two subunits of approximately 150 kDa each. The electrophoretic patterns also indicated that a portion of these subunits had undergone partial proteolysis. The substrate specificity of the purified human enzyme was studied using an assay in which phenazine ethosulfate coupled the transfer of electrons from the reduced enzyme to cytochrome c. Hypoxanthine, 2-hydroxypurine, xanthine, 2-aminopurine, and adenine were among the most efficient purine substrates studied. Most purine nucleosides tested were oxidized at detectable rates, but with relatively high Km values. The 2'-deoxyribonucleosides were more efficient substrates than were the corresponding ribonucleosides or arabinonucleosides. In a direct comparison with xanthine oxidase from bovine milk, the human enzyme showed a similar specificity toward purine substrates. However, considerable differences between the bovine and human enzymes were observed with nucleoside substrates. With xanthine as the substrate for the human enzyme, 20% of the total electron flow was univalently transferred to oxygen to produce superoxide radicals.     

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 and immunochemical characterization of monoamine oxidase from rat and human liver.

1. Monoamine oxidase from rat and human liver was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 2. The enzyme activity was extracted from mitochondrial preparations by Triton X-100. The enzyme was purified by (NH4)2SO4 fractionation followed by chromatography on DEAE-cellulose, Sepharose 6B, spheroidal hydroxyapatite, and finally chromatography on diazo-coupled tyramine-Sepharose. 3. Distinct differences occur in the chromatographic behaviour of the two enzymes on both DEAE-cellulose and spheroidal hydroxyapatite. 4. It is unlikely that the purification of the enzymes on tyramine-Sepharose is due to affinity chromatography and reasons for this are discussed. 5. The purified enzymes did not oxidize-5-hydroxytryptamine and the relative activities of the enzymes with benzylamine were increased approx. 1.25-fold compared with the enzyme activities of mitochondrial preparations. 6. Immunotitration of enzyme activity in extracts of mitochondrial preparations from rat liver was carried out with 5-hydroxytryptamine, tyramine and benzylamine. The enzyme activities were completely immunoprecipitated by the same volume of antiserum. Similar results were obtained with the antiserum to the enzyme from human liver.     

Isolation and characterization of human heart cytochromec oxidase

Cytochromec oxidase was isolated from human hearts and separated by SDS gel electrophoresis. The identity of polypeptide bands with known subunits was demonstrated by immunoblotting with monospecific antisera to rat liver cytochromec oxidase subunits. The polarographically determined kinetics of cytochromec oxidation were similar to those reported for the bovine heart enzyme.     

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 and characterization of peroxisomal L-pipecolic acid oxidase from monkey liver

L-Pipecolic acid oxidase has been purified to near homogeneity from Rhesus monkey liver. The protein, a yellow monomer, has a molecular weight of 46,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 8.9. It contains a covalently bound flavin with absorption maxima at 457 and 383 nm and a shoulder at 480 nm. The purified enzyme is most reactive toward L-pipecolic acid, with lesser reactivities toward L-proline and sarcosine. The enzyme has no significant reactivity toward the D-enantiomer of pipecolic acid or toward any other amino acid tested. Benzoic acid is a competitive inhibitor of the enzyme with a Ki of 750 microM. The Km of the purified enzyme is 3.7 mM for L-pipecolic acid. With less purified preparations, the reaction product is alpha-aminodipic acid. The purified enzyme, however, produces an intermediate which reacts with ortho-aminobenzaldehyde to form an alpha-aminoadipic acid semialdehyde adduct. Thus, the formation of alpha-aminoadipic acid requires at least two enzymes.     

Isolation and characterization of UDP-glucose dolichyl-phosphate glucosyltransferase from human liver

The enzyme UDP-glucose dolichyl-phosphate glucosyltransferase has been purified to near homogeneity from human liver microsomes. A 1100-fold enrichment over starting microsomal membranes was achieved by selective solubilization followed by anion- and cation-exchange chromatography, 5-HgUDP-thiopropyl-Sepharose affinity chromatography, butylagarose chromatography and hydroxyapatite chromatography. The glucosyltransferase was shown to be separated from other dolichyl-phosphate-dependent glycosyltransferases catalyzing the formation of dolichyl diphospho-N-acetylglucosamine and dolichyl phosphomannose. Sodium dodecyl sulfate/polyacrylamide gradient gel electrophoresis of the purified enzyme under reducing conditions revealed a protein band of Mr 36,000. Protection of the solubilized enzyme against rapid inactivation was achieved by its competitive inhibitor uridine. The purified glucosyltransferase activity exhibited a specific requirement for the presence of phospholipids. Phosphatidylethanolamine was the most effective activator of enzyme activity.     

Isolation and characterization of hyodeoxycholic-acid: UDP-glucuronosyltransferase from human liver

The enzyme hyodeoxycholic-acid: UDP-glucuronosyltransferase was purified about 230-fold from a solubilized human liver microsomal preparation utilizing anion-exchange chromatography, ampholyte-displacement chromatography and UDP-hexanolamine--Sepharose affinity chromatography. The homogeneity of the final enzyme preparation was judged by two criteria: the appearance of a single band of Mr 52000 in SDS/PAGE; the elution of a single peak in reversed-phase FPLC. The isolated enzyme catalyzed the glucuronidation of the 6 alpha-hydroxy bile acids hyodeoxycholic and hyocholic acids, and of the steroid hormone estriol, with a ratio of relative reaction rates of 13:1:2.7. UDP-glucuronosyltransferase activities toward the 3 alpha-hydroxy bile acid lithocholic acid, androsterone, testosterone, bilirubin and p-nitrophenol were not detectable in the pure enzyme preparation and were shown to be separated from enzyme activity toward hyodeoxycholic acid during ampholyte-displacement chromatography and/or UDP-hexanolamine--Sepharose affinity chromatography. Two-substrate kinetic analysis of hyodeoxycholic-acid-conjugating activity gave a sequential mechanism with apparent Km values of 12 microM and 4 microM for hyodeoxycholic acid and UDP-glucuronic acid, respectively. Phospholipids were required for reconstitution of maximal activity toward hyodeoxycholic acid. Phosphatidylcholine was the most effective activator of enzyme activity.     

Isolation and characterization of a homogeneous acid phosphatase from catfish liver

A homogeneous, tartrate-inhibitable acid phosphatase (AcPase) was obtained from the liver of channel catfish (Ictalurus punctatus) by the use of Affi Gel-10-coupled aminohexyltartramic acid affinity chromatography. The enzyme has a molecular weight of 82,500 and is a dimer consisting of two apparently equivalent subunits with subunit weights of 35,000 +/- 3000. Amino acid composition data are presented and compared with those of mammalian acid phosphatases. Data suggest that the enzyme is a metalloacid phosphatase. Catfish liver AcPase exhibits two molecular forms with pI 5.66 and 5.37 which were separated by chromatofocusing. A spontaneous conversion of the less acidic form to a more acidic form was observed and this conversion was accompanied by a decreased sensitivity towards tartrate inhibition.     

Isolation and characterization of human liver mRNA

Poly(A)-RNA isolated from human liver shows a predominant size in the 18 S region. It can be translated into a broad range of proteins up to molecular weights of more than 100 000 with predominant translation products in the 45 000-55 000 region. Full length cDNA has been transcribed and used for a complexity analysis of this mRNA giving a total sequence complexity of about 7 000 different mRNA species.     

Purification and characterization of recombinant human liver glycolate oxidase

Glycolate oxidase, an FMN-dependent peroxisomal oxidase, plays an important role in plants, related to photorespiration, and in animals, where it can contribute to the production of oxalate with formation of kidney stones. The best studied plant glycolate oxidase is that of spinach; it has been expressed as a recombinant enzyme, and its crystal structure is known. With respect to animals, the enzyme purified from pig liver has been characterized in detail in terms of activity and inhibition, the enzyme from human liver in less detail. We describe here the purification and initial characterization of the recombinant human glycolate oxidase. Its substrate specificity and the inhibitory effects of a number of anions are in agreement with the properties expected from previous work on glycolate oxidases from diverse sources. The recombinant enzyme presents an inhibition by excess glycolate and by excess DCIP, which has not been documented before. These inhibitions suggest that glycolate binds to the active site of the reduced enzyme, and that DCIP also has affinity for the oxidized enzyme. Glycolate oxidase belongs to a family of l-2-hydroxy-acid-oxidizing flavoenzymes, with strongly conserved active-site residues. A comparison of some of the present results with studies dealing with other family members suggests that residues outside the active site influence the binding of a number of ligands, in particular sulfite.     

Isolation and characterization of human liver guanine deaminase

Guanine deaminase (EC 3.5.4.3, guanine aminohydrolase [GAH]) was purified 3248-fold from human liver to homogeneity with a specific activity of 21.5. A combination of ammonium sulfate fractionation, and DEAE-cellulose, hydroxylapatite, and affinity chromatography with guanine triphosphate ligand were used to purify the enzyme. The enzyme was a dimer protein of a molecular weight of 120,000 with each subunit of 59,000 as determined by gel filtration and sodium dodecyl sulfate-gel electrophoresis. Isoelectric focusing gave a pI of 4.76. It was found to be an acidic protein, as evidenced by the amino acid analysis, enriched with glutamate, aspartate, alanine and glycine. It showed a sharp pH optimum of 8.0. The apparent Km for guanine was determined to be 1.53 X 10(-5) M at pH 6.0 and 2 X 10(-4) M for 8-azaguanine as a substrate at pH 6.0. The enzyme was found to be sensitive to p-hydroxymercuribenzoate inhibition with a Ki of 1.53 X 10(-5) M and a Ki of 5 X 10(-5) M with 5-aminoimidazole-4-carboxamide as an inhibitor. The inhibition with iodoacetic acid showed only a 7% loss in the activity at 1 X 10(-4) M and a 24% loss at 1 X 10(-3) M after 30 min of incubation, whereas p-hydroxymercuribenzoate incubation for 30 min resulted in a 91% loss of activity at a concentration of 1 X 10(-4) M. Guanine was the substrate for all of the inhibition studies. The enzyme was observed to be stable up to 40 degrees C, with a loss of almost all activity at 65 degrees C with 30 min incubation. Two pKa values were obtained at 5.85 and 8.0. Analysis of the N-terminal amino acid proved to be valine while the C-terminal residue was identified as alanine.     

Tryptic cleavage of rat liver sulfite oxidase. Isolation and characterization of molybdenum and heme domains.

Treatment of rat liver sulfite oxidase with trypsin leads to loss of ability to oxidize sulfite in the presence of cytochrome c as electron acceptor. Ability to oxidize sulfite with ferricyanide as acceptor is undiminished, while sulfite leads to O2 activity is partially retained. Gel filtration of the proteolytic products has led to the isolation of two major fragments of dissimilar size derived from sulfite oxidase. The smaller fragment has a molecular weight of 9500 and appears to be monomeric when detached from sulfite oxidase. It contains the heme in its cytochrome b5 structure, has no sulfite oxidase activity, and is reducible with dithionite but not with sulfite. The heme fragment can mediate electron transfer between pig liver microsomal NADH cytochrome b5 reductase and cytochrome c. The larger fragment has a molecular weight of 47,400 under denaturing conditions but elutes from Sephadex G-200 as a dimer. It contains no heme but retains all of the molybdenum and the modified sulfite-oxidizing capacity present in the proteolytic mixture. All of the EPR properties of the molybdenum center of native sulfite oxidase are retained in the molybdenum fragment. The molybdenum center is a weak chromophore with an absorption sectrum suggestive of coordination with sulfur ligands. Reduction by sulfite generates a spectrum attributable to molybdenum (V). Spectra of oxidized and sulfite-reduced preparations are sensitive to anions and pH. NH2-terminal analysis of native sulfite oxidase and the two tryptic fragments has permitted the conclusion that the sequence represented by the heme fragment is the NH2 terminus of native enzyme. These studies have demonstrated that the two cofactor moieties of sulfite oxidase are contained in distinct domains which are covalently held in contiguity by means of an exposed hinge region. Isolation of functional heme and molybdenum domains of sulfite oxidase after tryptic cleavage has demonstrated conclusively that the cytochrome b5 region of the molecule is required for electron transfer to the physiological acceptor, cytochrome c.