S-adenosylhomocysteine hydrolase from hamster liver: Purification and kinetic properties

3-Deazaadenosine is both an inhibitor of and a substrate for S-adenosylhomocysteine hydrolase. Its administration to rats results in the accumulation of both S-adenosylhomocysteine and 3-deazaadenosylhomocysteine in the liver and other tissues. In hamsters, however, the administration of 3-deazaadenosine results only in the accumulation of 3-deazaadenosylhomocysteine (P. K. Chiang and G. L. Cantoni (1979) Biochem. Pharmacol. 28, 1897). In order to investigate the possible reasons for this difference, S-adenosylhomocysteine hydrolase from hamster liver has been purified to homogeneity and some of its kinetic and physical parameters have been determined. The molecular weight of the native enzyme is 200,000 with a subunit molecular weight of 48,000. The K_m's for adenosine and 3-deazaadenosine are about 1.0 μm, and the V_max's are also similar. The K_m for S-adenosylhomocysteine is 1.0 μm, or more than 10 times smaller than the K_m of the rat liver enzyme. This difference in K_m value may explain the differences in the response of rat and hamster liver to the administration of 3-deazaadenosine. S-Adenosylhomocysteine hydrolase from hamster liver exhibits an interesting kinetic property in that its activity can be affected bimodally by either adenosine or adenosine Anal.ogs. At very low concentrations of these analogs, the activity of S-adenosylhomocysteine hydrolase can be stimulated by 10–30%, and at higher concentrations these same analogs become competitive inhibitors.     

Purification and characterization of a protein inhibitor of calcium-dependent proteases from rat liver

Soluble extracts of rat liver contain a protein inhibitor of calcium-dependent proteases. The inhibitor has an apparent M_r = 250,000 and is separated from the calcium-dependent proteases by gel-filtration chromatography in the presence of EGTA. The inhibitor has been purified by affinity chromatography using a calcium-dependent protease covalently linked to Affi-Gel 15. The inhibitor specifically binds to this affinity resin in a calcium-dependent manner and elutes in the presence of EDTA or EGTA. The purified inhibitor appears as a single protein with M_r = 125,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Presumably it is a dimer under nondenaturing conditions. The inhibitor inhibits each of two calcium-dependent proteases from rat liver and from other tissues and species. However, it has no effect on any other protease tested.     

Purification and characterization of the individual glutathione S-transferases from sheep liver

The glutathione S-transferases (EC 2.5.1.18) have been purified to electrophoretic homogeneity from 105,000g supernatant of sheep liver homogenate by employing a combination of gel filtration on Sephadex G-150 and affinity chromatography on S-hexylglutathione-linked Sepharose-6B columns. Approximately 70% of the original glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide could be recovered by this purification method. Of particular importance in developing this procedure was the fact that the enzyme preparation obtained after affinity column chromatography represented all the isozymes of sheep liver glutathione S-transferases. Further purification by CM-cellulose and DEAE-cellulose column chromatography resolved the glutathione S-transferases into seven distinct cationic isozymes designated C-1, C-2, C-3, C-4, C-5, C-6, and C-7 and five overlapping anionic transferases designated A-1, A-2, A-3, A-4, and A-5, respectively, in the order of their elution from the ion-exchange columns. The sodium dodecyl sulfate SDS-gel electrophoretic data on subunit composition revealed that cationic enzymes are composed of two subunits with an identical Mr of 24,000 whereas a predominant subunit with Mr of 26,000 was observed in all anionic isozyme peaks except A-1. Cationic isozymes accounted for approximately 98% of the total peroxidase activity associated with the glutathione S-transferase whereas only A-1 of the anionic isozymes displayed some peroxidase activity. Isozyme C-4 was found to be the most abundant glutathione S-transferase in the sheep liver. Characterization of the individual transferases by their specificity toward a number of selected substrates, subunit composition, and isoelectric points showed some similarities to those patterns for human liver glutathione S-transferases.     

Purification and immunochemical characterization of a low-pI form of UDP glucuronosyltransferase from mouse liver

A liver UDP glucuronosyltransferase (GT) enzyme from either phenobarbital- or 3-methylcholanthrene-treated C57BL/6N mice was isolated by phenyl-Sepharose, DEAE-ion exchange, and UDP hexanolamine chromatographic steps. This enzyme had a broad substrate specificity and was mainly responsible for the microsomal capacity to glucuronidate testosterone, 1-naphthol, and morphine. This UDP glucuronosyltransferase ( GTM1 ) appeared to be at least 95% homogeneous and had a subunit molecular weight of 51,000 using sodium dodecyl sulfate-polyacrylamide gel and two-dimensional gel electrophoreses. Antibodies prepared against the purified protein developed a single immunoprecipitin line by double-diffusion analysis with purified antigen and with solubilized microsomes from both control and drug-induced C57BL/6N and DBA/2N mice. A precipitin line was also observed with microsomal proteins which isoelectrofocused at approximately pH 6.7, but not with those which isoelectrofocused at approximately pH 8.5. GTM1 was, therefore, designated at low-pI form. Immunopurified antibody preferentially inhibited and immunoprecipitated GT activities toward testosterone, 1-naphthol, and morphine. To a lesser extent, activities toward phenolphthalein, 3-hydroxybenzo[a]pyrene, and estrone were inhibited while activities toward 4-nitrophenol and 4-methylumbelliferone were not affected. All activities, however, were immunoadsorbed in the presence of protein A-Sepharose. This observation can be explained by the following results. Immunoprecipitates from labeled microsomes contained primarily a 51,000-Da protein. When the immune complexes were adsorbed with protein A-Sepharose, a 54,000-Da protein as well as the expected 51,000-Da GTM1 was detected. This 54,000-Da protein was associated with the glucuronidation of 3-hydroxybenzo[a]pyrene and 4-nitrophenol, and was designated GTM2 .     

Purification and characterization of myosin from bovine retina

Myosin has been isolated from bovine retinae and characterised by its ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, its mobility in sodium dodecyl sulphate polyacrylamide gels and by electron microscopy. The purified myosin shows high ATPase activity in the presence of EDTA or Ca²⁺ and a low activity in the presence of Mg²⁺. The Mg²⁺-dependent ATPase activity is stimulated by rabbit skeletal muscle actin. The presumptive retinal myosin possesses a major component which has a mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis similar to that of the heavy chain of bovine skeletal mucle myosin. Electron microscopy showed retinal myosin to form bipolar filaments in 0.1 M KCl. It is concluded that the retina possesses a protein with enzymic and structural properties similar to those of muscle myosin.     

Purification and characterization of glycolate oxidase from pumpkin cotyledons

Glycolate oxidase was purified and crystallized from cotyledons of germinating pumpkin seedlings. The molecular weight of the enzyme was determined to be 280,000-320,000, consisting of 8 identical subunits with molecular weight of 38,000. There are two absorption peaks at 340 and 450 nm, indicating the glycolate oxidase is a flavin protein. Several kinetic parameters were determined, Km (glycolate) 0.33 mM and Km (O2) 76.2 microM at pH 8.0. Oxalate and oxalacetate were found to be potent competitive inhibitors against glycolate; the Ki values for oxalate and oxalacetate were 4.5 and 7.8 mM, respectively. Fatty acids such as linoleic acid inhibited the enzyme noncompetitively; the Km for linoleic acid was 0.63 mM. The regulation of glycolate oxidase in the glycolate pathway occurring in leaf peroxisomes is discussed.     

Purification and properties of methylmalonyl coenzyme A mutase from human liver

Methylmalonyl coenzyme A (CoA) mutase has been purified to apparent homogeneity from human liver by a procedure involving column chromatography on DEAE-cellulose, Matrex-Gel Blue A, hydroxylapatite, and Sephadex G-150. The overall purification achieved is 500- to 600-fold, yield 3–5%. Electrophoresis of the native purified protein on nondenaturing polyacrylamide gels shows a single diffuse band coincident with the enzyme activity; dodecyl sulfate/polyacrylamide gels show a single protein band with an apparent molecular weight of 77,500. The native protein has a molecular weight of approximately 150,000 by Sephadex G-150 chromatography, suggesting that it is composed of two identical subunits. The activity of the purified enzyme is stimulated only slightly (10–20%) by the addition of its cofactor, adenosylcobalamin, indicating that the purified enzyme is largely saturated with coenzyme. The spectrum of the enzyme is consistent with the presence of about 1 mole of adenosylcobalamin per mole of subunit. The enzyme displays complex kinetics with respect to dl-methylmalonyl CoA; substrate inhibition by l-methylmalonyl CoA appears to occur. The enzyme activity is stimulated by polyvalent anions (PO₄^3? > SO₄^2? > Cl^?); monovalent cations are without effect, but high concentrations of divalent cations are inhibitory. The enzyme activity is insensitive to N-ethylmaleimide, is rapidly destroyed at temperatures > 50 °C, and shows a broad pH optimum around pH 7.5.     

Isolation and characterization of liver glycogen synthase from diabetic rats

NAD-specific pig heart isocitrate dehydrogenase is composed of three distinct types of subunits: α, β, and γ, which have molecular weights of about 40,000 but differ in amino acid composition and in isoelectric points. When the native enzyme is subjected to polyacrylamide gel electrophoresis under nondenaturing conditions, two major protein bands with M_r values of about 360,000 (band 1) and 100,000 (band 2) and two minor bands (bands 3 and 4) with M_r values of about 40,000 are consistently present. Enzymatic activity, as detected from NADH fluorescence, is distributed throughout the protein-staining region. Analytical isoelectric focusing in urea reveals that band 1 is composed of all three subunits in roughly the normal ratio of 2α:1β:1γ, and is probably an octamer, band 2 of an equal amount of α and β and is probably dimer, while bands 3 and 4 each consist of only the monomeric α subunit. The highest enzymatic specific activity is associated with a region intermediate between octamer and dimer, which includes the 160,000 tetramer. The protein pattern resulting from isoelectric focusing under nondenaturing conditions consists of protein bands comparable in pattern to those in the presence of urea along with bands of intermediate pI values, many of which are associated with enzymatic activity. Analysis of the subunit composition of these bands supports the activity of the α species in isolation and establishes the activity of the separated β component. No activity of the isolated γ subunit species has thus far been demonstrated. However, the highest apparent specific activity is observed when at least two types of subunits are present. These studies indicate that a range of oligomeric species of the enzyme are enzymatically active and that at least three of the four subunit chains comprising the minimum complete enzyme molecule (2α:1β:1γ) possess an active site.     

Purification and characterization of glucose-6-phosphate dehydrogenase from Aspergillus parasiticus

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was purified from mycelium of Aspergillus parasiticus (1-11-105 Whl). The enzyme had a molecular weight of 1.8 × 10⁵ and was composed of four subunits of apparently equal size. The substrate specificity was very strict, only glucose 6-phosphate and glucose being oxidized by NADP or thio-NADP. Zinc ion was a powerful inhibitor of the enzyme, inhibition being competitive with respect to glucose 6-phosphate, with K_i about 2.5 μm. Other divalent metal ions which also serve as inhibitors are nickel, cadmium, and cobalt. It is proposed that the stimulation of polyketide synthesis by zinc ion may be mediated in part by inhibition of glucose-6-phosphate dehydrogenase.     

Purification and characterization of a xylobiose- and xylose-producing endo-xylanase from Aspergillus niger

A xylanase from a commercial Aspergillus niger pentoglycanase was purified to homogeneity by column chromatography on Ultrogel AcA 54, SP-Sephadex, Sephadex G-50, and SP-Sephadex. The enzyme hydrolyzed xylotriose slowly to xylose and xylobiose, and xylotetraose and higher xylo-oligosaccharides rapidly to mixtures of smaller xylo-oligosaccharides, with xylobiose and xylose being the preponderant final products. The anomeric configuration of the products was inverted, in contrast to the behavior of most other carbohydrases that initially produce mixtures of oligosaccharides. This enzyme is a glycoprotein having an amino acid composition high in acidic residues. Its molecular weight is 20,800 and its isoelectric point is at pH 6.7. Optimal pH values for activity and stability are between 4 and 6 and, in a 20-min assay, maximal activity is attained at 55°.     

Functional characterization of single-chain factor X from rat liver

¹⁴C-Labeled single-chain factor X prepared by vitamin K-dependent carboxylation in vitro was partially purified by adsorption to BaSO₄ and chromatography on DEAE-Sephacel. Known activators of factor X were analyzed for their effect on the single-chain molecule. ¹⁴C-Labeled factor X antigens were recovered immunochemically from incubation mixtures and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation with trypsin resulted in the generation of factor Xa clotting activity, and the ¹⁴C-labeled product migrated after reduction with an apparent molecular weight of 22,500 ± 1500 (mean ± 1 SD). The light chain produced by factor Xa was similar to that produced by trypsin (M_r 24,500 ± 1500; mean ± 1 SD). Incubation of single-chain factor X with factor VII and thromboplastin, factor IXa, or the factor X activating enzyme from Russell's viper venom gave a reducible product with a light chain of higher apparent molecular weight (M_r 37,000–38,000). Incubation with factor VII and thromboplastin also resulted in the generation of factor Xa clotting activity. Incubation of single-chain factor X with platelets resulted in the binding of about 20% of the ¹⁴C. The bound ¹⁴C-labeled factor X antigen released by freezing and thawing in the presence of EDTA was reduced to give a ¹⁴C-labeled polypeptide with M_r 31,000. Walker 256 tumor cells bound about 30% of the ¹⁴C. The bound material, after reduction, gave a ¹⁴C-labeled polypeptide with M_r 23,000.     

Purification and molecular characterization of human fibroblast interferon

Human fibroblast interferon was purified from serum-containing culture medium by a combination of concanavalin A or Blue Dextran Sepharose affinity chromatography with high-performance liquid chromatography to material exhibiting a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The interferon could be chromatographed and purified at acidic pH in volatile buffers on RP-8, RP-18, cyclohexyl, phenylalkyl, diphenyl, cyanopropyl, and diol supports. A specific activity averaging around 4 × 10⁸ units/mg was found for the pure material with a molecular weight of 20,000–21,000 after 20,000- to 50,000-fold purifications. In some preparations, low activity levels were also found at positions corresponding to 10,000, 17,000–18,000, 35,000, and 40,000 daltons. Amino acid and amino sugar analysis, partial NH₂- and COOH-terminal sequences, and tryptic peptide patterns determined at the picomole level are reported for the purified interferon.     

Purification and characterization of the soluble form of mitochondrial adenosine triphosphatase from sweet potato

The soluble form of mitochondrial adenosine triphosphatase was purified in an electrophoretically and immunologically pure form from sweet potato root tissue. The enzyme consisted of six kinds of subunits with different molecular weights (52,500, 51,500, 35,500, 26,000, 23,000, and 12,000), and its molecular weight was about 370,000. Adenosine triphosphatase associated with the submitochondrial particles was oligomycin-sensitive and heat-labile, whereas the soluble form of the enzyme was oligomycin-insensitive and cold-labile. The enzyme in either the membrane-bound or the soluble form showed negative cooperativity. Both experiments with polyacrylamide gel electrophoresis and immunological methods suggest that some of the subunits, probably those with molecular weights of 52,500 and 51,500, are dissociated from the enzyme protein during storage of the enzyme preparations.     

Purification and crystallization of rat liver fatty acid synthetase

A purification procedure for rat liver fatty acid synthetase has been developed using polyethylene glycol. This procedure results in high yields of the enzyme which is essentially free of endogenous proteolytic nicking and also free of any contaminating proteases. The fatty acid synthetase obtained has a specific activity range of 1.8–2.1 measured at 25 °C and is stable at 4 °C for a few weeks and indefinitely when frozen. Approximately 1 mg of enzyme can be obtained per gram of induced rat liver. The enzyme is pure as determined by sodium dodecyl sulfate-gel electrophoresis, sedimentation velocity, and immunoelectrophoresis. The first crystallization of rat liver fatty acid synthetase is also reported.     

Purification and characterization of a vanadate-sensitive nucleotide tri- and diphosphatase with acid pH optimum from rat bone

Rat bone was extracted with KCl and Triton X-100, and a tartrate-resistant acid phosphatase activity was purified by protamine sulfate precipitation, ion-exchange chromatography (CM-cellulose), and gel filtration on Sephadex G-200 according to previously described procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining demonstrated a major band with an apparent monomer molecular size of approximately 14,000 Da. The enzyme is active with p-nitrophenylphosphate (p-NPP) but exhibits a 5- to 10-fold higher affinity towards several nucleotides of which ATP and ADP are the most readily hydrolyzed substrates based on kinetic studies. Based on sensitivity towards proteolytic treatment and detergent removal, as well as pH-optimum studies, a single enzyme was found to be responsible for activity towards nucleotide phosphates as well as p-NPP. This nucleotide tri- and diphosphatase constitutes around 15% of the total acid phosphatase activity in rat bone. The activity with ATP as substrate in contrast to that with p-NPP was inhibited in a noncompetitive fashion by MgCl2, sodium metavanadate, and p-chloromercuribenzoate. Enzyme activity with p-NPP and ATP is dependent on the presence of KCl and detergent and is activated by Fe3+ and ascorbate. The reported characteristics of the enzyme suggest that it functions as a unique membrane acid ATPase.     

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.     

Purification and chemical characterization of polysaccharides obtained from Lampteromyces japonicus by concanavalin A-sepharose affinity chromatography

Two classes of neutral polysaccharide which could not be separated from each other by conventional methods were isolated from the fungus, Lampteromyces japonicus, by affinity chromatography using concanavalin A-Sepharose. The polysaccharide retained on the concanavalin A-Sepharose column was eluted with 0.05 M methyl α-d-mannopyranoside and appeared to be α-mannan, while that which passed through the column was virtually all β-glucan.Both polysaccharides were subjected to Smith-type degradation, methylation, acetolysis and glucosidase treatment. The results indicated that the α-mannan contained predominantly α-(1 → 2)-linked side chains branching from an α-(1 → 6)-linked backbone at the (1 → 2,6)-linked mannopyranosyl residues. Galactose was attached to approximately one-quarter of the non-reducing mannose terminals. The β-glucan seemed to contain mainly (1 → 6)-linked side chains branching from a (1 → 3)-linked backbone at the (1 → 3,6)-linked glucopyranosyl residues.     

Purification and properties of hog liver 4-hydroxyphenylpyruvate dioxygenase

The purification of hog liver 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27), and the determination of some of its characteristics, are reported. The enzyme was purified 330-fold in 22% yield from an acetone powder extract by ammonium sulfate fractionation, chromatography twice using sulfopropyl Sephadex under carefully controlled pH conditions (once at pH 5.36 and a second time with a pH gradient from 5.25 to 5.80), and a final chromatography on DEAE-cellulose. The purified enzyme was found to be homogeneous by several standard criteria, but activity measurements indicated that a small amount (less than 5%) of a carboxylesterase (EC 3.1.1.1) isoenzyme is present as a minor impurity. On long-term storage at ?20 °C the enzyme forms polymers but this can be reversed with thiols. The molecular weight of the freshly prepared or depolymerized enzyme was estimated to be 89,000 ± 2000 by equilibrium ultracentrifugation, and 50,000 to 54,000 by gel filtration. Sodium dodecyl sulfate-gel electrophoresis experiments, performed in the presence and absence of mercaptoethanol, indicate that the enzyme is composed of two nonidentical subunits with similar molecular weights (44,000 ± 2000). The enzyme gives a typical protein ultraviolet absorption spectrum with no noticeable peaks above 300 nm, it has no detectable carbohydrate content, and it contains 0.9 atom iron and 0.4 atom copper/89,000 daltons. Added iron and copper salts activate the enzyme to some extent but by less than a factor of 2. The enzymatic reaction has a large temperature coefficient (the rate increases ca. fivefold for each 10 °C rise) and is markedly stimulated (up to sixfold) by the presence of some organic solvents in concentrations up to 10% of the medium. These results suggest that a protein conformation change, possibly aided by binding of the organic solvent, is involved in the rate-determining step of the reaction. The similarities and differences of this 4-hydroxyphenylpyruvate dioxygenase to those from other sources, and to prolyl hydroxylase, are discussed.     

Separation, purification and characterization of three isoenzymes of UDP-glucuronyltransferase from rat liver microsomes

Three isoenzymes of UDP-glucuronyltransferase (UDPGT) have been separated and purified from liver microsomes of untreated female rats or female rats pretreated with 3-methylcholanthrene. The UDPGT isoenzymes were purified utilizing Chromatofocusing, column isoelectric focusing, and UDP-hexanolamine Sepharose 4B affinity chromatography. UDPGT activities could also be separated during UDP-hexanolamine affinity chromatography by elution with different UDPGA (UDP-glucuronic acid) concentrations. One isoenzyme exhibits a subunit molecular weight of 56,000 and is capable of conjugating p-nitrophenol, 1-naphthol, and 4-methylumbelliferone. This isoenzyme is inducible by 3-methylcholanthrene treatment and requires high UDPGA concentrations for elution from the UDP-hexanolamine affinity column in contrast to the other UDPGT isoenzymes. A second isoenzyme was purified and displayed a subunit molecular weight of 50,000. This isoenzyme was not induced by 3-methylcholanthrene and was active towards testosterone, the 17-OH position of beta-estradiol, p-nitrophenol, and 1-naphthol. A third isoenzyme was also purified and exhibited a subunit molecular weight of 52,000. This isoenzyme conjugated androsterone and etiocholanolone and was not induced by 3-methylcholanthrene treatment. This study reports the purification of two separate and distinct rat liver UDPGT isoenzymes capable of conjugating p-nitrophenol, only one of which is inducible by 3-methylcholanthrene treatment. Also, this is the first report of the purification of a UDPGT isoenzyme active towards the 3-OH position of androgens.     

Solubilization and characterization of vitamin K epoxide reductase from normal and warfarin-resistant rat liver microsomes

Two procedures have been developed for the solubilization of vitamin K epoxide reductase from rat liver microsomal membranes using the detergent Deriphat 160 at pH 10.8. The methods are applicable to both normal and Warfarin-resistant-strain rat liver microsomes and yield material suitable for further purification. The preparations retain dithiothreitol-dependent vitamin K quinone reductase activity as well as vitamin K epoxide reductase and are free of vitamin K-dependent carboxylase and epoxidase activities. Optimal epoxide reductase activity is obtained at 0.1 M KCl and pH 9 in the presence of sodium cholate. Artifactual formation of vitamin K metabolites was eliminated through the use of mercuric chloride to remove excess dithiothreitol prior to extraction and metabolite assay. Using the solubilized enzyme, valid initial velocities were measured, and reproducible kinetic data was obtained. The substrate initial velocity patterns were determined and are consistent with a ping-pong kinetic mechanism. The kinetic parameters obtained are a function of the cholate concentration, but do not vary drastically from those obtained using intact microsomal membranes. At 0.8% cholate, the enzymes solubilized from normal Warfarin-sensitive- and Warfarin-resistant-strain rat livers exhibit respective values of Vmax = 3 and 0.75 nmol/min/g liver; Km for vitamin K epoxide = 9 and 4 microM; and Km for dithiothreitol of 0.6 and 0.16 mM.