Purification and characterization of two oxidoreductases involved in the enantioselective reduction of 3-oxo, 4-oxo and 5-oxo esters in baker's yeast

Two NADPH-dependent oxidoreductases catalyzing the enantioselective reduction of 3-oxo esters to (S)- and (R)-3-hydroxy acid esters, [hereafter called (S)- and (R)-enzymes] have been purified 121- and 332-fold, respectively, from cell extracts of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, Sephadex G-150 filtration, Sepharose 6B filtration and hydroxyapatite chromatography. The relative molecular mass Mr, of the (S)-enzyme was estimated to be 48,000-50,000 on Sephadex G-150 column chromatography and 48,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.9 and reduced 3-oxo esters, 4-oxo and 5-oxo acids and esters enantioselectively to (S)- hydroxy compounds in the presence of NADPH. The Km values for ethyl 3-oxobutyrate, ethyl 3-oxohexanoate, 4-oxopentanoic and 5-oxohexanoic acid were determined as 0.9 mM, 5.3 mM, 17.1 mM and 13.1 mM, respectively. The Mr of the (R)-enzyme, estimated by means of column chromatography on Sepharose 6B, was 800,000. Under dissociating conditions of SDS/polyacrylamide gel electrophoresis the enzyme resolved into subunits of Mr 200,000 and 210,000, respectively. The enzyme is optimally active at pH 6.1, catalyzing specifically the reduction of 3-oxo esters to (R)-hydroxy esters, using NADPH for coenzyme. Km values for ethyl 3-oxobutyrate and ethyl 3-oxohexanoate were determined as 17.0 mM and 2.0 mM, respectively. Investigations with purified fatty acid synthase of baker's yeast revealed that the (R)-enzyme was identical with a subunit of this multifunctional complex; intact fatty acid synthase (Mr 2.4 X 10(6)) showed no activity in catalyzing the reduction of 3-oxo esters.     

Propionyl-CoA condensing enzyme from Ascaris muscle mitochondria. I. Isolation and characterization of multiple forms

The condensation of two propionyl-CoA units or a propionyl-CoA with acetyl-CoA is required for the synthesis of 2-methylvalerate or 2-methylbutyrate, respectively, two of the major fermentation products of Ascaris anaerobic muscle metabolism. An enzyme that preferentially catalyzes the condensation of propionyl-CoA rather than acetyl-CoA has been purified from the mitochondria of the parasitic intestinal nematode Ascaris lumbricoides var. suum. The purified enzyme is over 10 times more active with propionyl-CoA than with acetyl-CoA as substrate. It also catalyzes the coenzyme A-dependent hydrolysis of acetoacetyl-CoA at a rate four times higher than the propionyl-CoA condensation reaction. The purified Ascaris condensing enzyme preferentially forms the 2-methyl-branched-chain keto acids rather than the corresponding straight chain compounds. The native molecular weight of the purified enzyme was estimated to be 160,000 by gel filtration chromatography and 158,000 by high pressure liquid chromatography. The enzyme migrated as a single protein band with Mr 40,000 during sodium dodecyl sulfate-polyacrylamide electrophoresis, indicating that the enzyme is composed of four subunits of the same molecular weight. Chromatography on CM-sephadex resulted in the isolation of two separate peaks of activity, designated as A and B. Both A and B had the same molecular weight and subunit composition. However, they differed in their specific activities and isoelectric points. The pIs of condensing enzymes A and B were 7.6 and 8.4, respectively. Propionyl-CoA was the best substrate for the condensation reaction with both enzymes. However, the specific activity of enzyme B for both propionyl-CoA condensation (3.4 mumol/min/mg protein) and acetoacetyl-CoA thiolysis (13.8 mumol/min/mg protein) was 2.4 times higher than that obtained with enzyme A. Similarly, chromatography on phosphocellulose resolved the Ascaris condensing enzyme activity into one minor and two major peaks. All of these components had the same molecular weight and subunit composition, but differed in their specific activities. The two major phosphocellulose peaks cross-reacted immunologically when examined by the Ouchterlony double immunodiffusion technique. In addition, antiserum against the phosphocellulose most active form cross-reacted with forms A and B isolated by chromatography of the enzyme on CM-Sephadex, indicating that all forms were immunochemically related.     

A membrane enzyme from Staphylococcus aureus which catalyzes transpeptidase, carboxypeptidase, and penicillinase activities

Staphylococcus aureus H membranes were found to contain four major binding components: Mr = 115,000; Mr = 100,000 doublet; and Mr = 46,000. The low molecular weight protein bound penicillin reversibly and was purified by prebinding membranes with penicillin prior to affinity chromatography. The purified protein catalyzed transpeptidase and carboxypeptidase reactions using di[14C]acetyl-L-lysyl-D-alanyl-D-alanine as the substrate and glycine and hydroxylamine as the acceptors. In addition, the enzyme catalyzed a penicillinase reaction. Kinetic analysis of these reactions revealed similar Vmax values suggesting that, if there is a single active site, the rate-determining steps (i.e. deacetylation) are similar. Rapid denaturation of the enzyme.substrate complex resulted in the detection of covalent penicilloyl- and diacetyl-L-lysyl-D-alanyl.enzyme complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Nonoxidative ethanol metabolism in rabbit myocardium: purification to homogeneity of fatty acyl ethyl ester synthase

Fatty acyl ethyl esters, previously identified in our laboratory as metabolites of ethanol in human and rabbit myocardium, arise from an esterification of free fatty acids with ethanol in the absence of ATP and coenzyme A. This study was designed to isolate and purify the enzyme(s) in rabbit myocardium that catalyze(s) this reaction. Enzyme activity in homogenates of rabbit myocardium, as assayed by the rate of synthesis of ethyl [14C]oleate from 0.4 mM [14C]oleic acid and 0.2 M ethanol, was 31 nmol/(g.h), and all of it was recovered in the 48400g supernatant. This soluble ethyl ester synthase activity bound to DEAE-cellulose at pH 8, and elution with a NaCl gradient (0-0.25 M) separated two enzyme activities accounting for 13 and 87% of recovered synthase activity. The major enzyme activity was then purified over 5000-fold to homogeneity by sequential gel permeation, hydrophobic interaction, and anti-albumin affinity chromatographies with an overall yield of 40%. Up to 45 micrograms of enzyme was present per g of myocardium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single polypeptide with Mr 26 000, and gel permeation chromatography under nondenaturing conditions indicated a Mr of 50 000 for the active enzyme. Kinetic analyses using the purified enzyme indicated that greatest rates of ethyl ester synthesis were observed with unsaturated octadecanoic fatty acid substrates [Vmax = 1.9 and 1.5 nmol/(mg.s) for linoleate and oleate, respectively], with lesser rates associated with palmitate, stearate, and arachidonate substrates [0.14, 0.03, and 0.35 nmol/(mg.s), respectively].(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of the 2-methyl branched-chain Acyl-CoA dehydrogenase, an enzyme involved in NADH-dependent enoyl-CoA reduction in anaerobic mitochondria of the nematode, Ascaris suum

The 2-methyl branched-chain acyl-CoA dehydrogenase was purified to homogeneity from mitochondria of the parasitic nematode, Ascaris suum. The native molecular weight of the enzyme was estimated to be 170,000 by gel filtration. The enzyme migrated as a single protein band with Mr = 42,500 during sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting that the enzyme is a tetramer composed of identical subunits. The enzyme exhibited absorbance maxima at 272, 375, and 452 with a ratio 7.9:0.8:1.0, respectively. FAD content was estimated to be 0.9 mol/mol of subunit and the absorption coefficient of FAD at 452 nm was 14.1 mM-1 cm-1. The purified enzyme dehydrogenated both 2-methylbutyryl-CoA and 2-methylvaleryl-CoA with apparent Km and Vmax values of 18 microM and 1.62 mumol/min/mg and 21 microM and 1.58 mumol/min/mg, respectively. This enzyme also appeared to dehydrogenate butyryl-CoA, valeryl-CoA, and octanoyl-CoA but at a much lower rate. The enzyme did not dehydrogenate propionyl-CoA, isobutyryl-CoA, isovaleryl-CoA, and palmitoyl-CoA. Tiglyl-CoA and 2-methyl-2-pentenoyl-CoA were identified as reaction products from 2-methylbutyryl- and 2-methylvaleryl-CoA, respectively. Dehydrogenating activity with both substrates was inhibited by tiglyl-CoA, acetoacetyl-CoA, and straight chain acyl CoAs of increasing chain length. N-Ethylmaleimide and p-hydroxymercuribenzoate had little effect on dehydrogenating activity but the heavy metals Hg2+ and Ag2+ were potent inhibitors. Physiologically, the dehydrogenase functions as a branched-chain enoyl-CoA reductase. Incubations of A. suum submitochondrial particles, NADH, tiglyl-CoA, purified A. suum electron-transfer flavoprotein, and the 2-methyl branched-chain acyl-CoA dehydrogenase resulted in the rotenone-sensitive, dehydrogenase-dependent formation of 2-methylbutyryl-CoA.     

Purification of a soluble, sodium-nitroprusside-stimulated guanylate cyclase from bovine lung

A soluble, sodium-nitroprusside-stimulated guanylate cyclase as been purified from bovine lung by DEAE-cellulose chromatography, ammonium sulfate precipitation, chromatography on Blue Sepharose CL-6B and preparative gel electrophoresis. Apparent homogeneity was obtained after at least 7000-fold purification with a yield of 3%. A single stained band (Mr 72000) was observed after gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme migrated as one band also under non-denaturing conditions in acrylamide gels (5-12%). The mobility of this band corresponded to an Mr of 145000. The enzyme sedimented on sucrose gradients with an S20, w of 7.0 S. Gel filtration yielded a Stokes' radius of 4.6 nm. These data suggest that the enzyme has an Mr of approximately 150000 and consists of two, presumably identical, subunits of Mr 72000. Sodium nitroprusside stimulated the purified enzyme 15-fold and 140-fold to specific activities of 8.5 and 15.7 mumol of cGMP formed min-1 mg-1 in the presence of Mn2+ and Mg2+, respectively. Formation of cGMP was proportional to the incubation time and to the amount of enzyme added. The stimulatory effect of sodium nitroprusside was half-maximal at about 2 microM, was observed immediately after addition and could be reversed either by dilution or by removal of sodium nitroprusside on a Sephadex G-25 column. The purified enzyme in the absence of catalase was stimulated by sodium nitroprusside, N-methyl-N'-nitro-N-nitrosoguanidine and 3-morpholino-sydnonimine and in the presence of catalase by sodium nitrite and sodium azide. In the presence of Mn2+ and sodium nitroprusside, the purified enzyme catalyzed the formation of cAMP from ATP at a rate of 0.6 mumol min-1 mg-1.     

Purification and properties of the bovine liver mitochondrial dihydroorotate dehydrogenase

Dihydroorotate dehydrogenase has been purified 6,000-fold from bovine liver mitochondria to apparent homogeneity in six steps. Electrophoretic migration of the homogeneous enzyme on sodium dodecyl sulfate-polyacrylamide gels reveals a subunit Mr of 42,000. By contrast to the well-characterized, cytosolic dihydroorotate oxidases (EC 1.3.3.1), the purified bovine dehydrogenase is a dihydroorotate:ubiquinone oxidoreductase. Maximal rates of orotate formation are obtained using coenzymes Q6 or Q7 as cosubstrate electron acceptors. Concomitant with substrate oxidation, the enzyme will reduce simple quinones, such as benzoquinone, but at significantly lower rates (10-15%) than that obtained for reduction of coenzyme Q6. Enzyme-catalyzed substrate oxidation is not supported by molecular oxygen. The specificity of the purified enzyme for dihydropyrimidine substrates has also been explored. The methyl-, ethyl-, t-butyl-, and benzyl-S-dihydroorotates are substrates, but 1- and 3-methyl and 1,3-dimethyl methyl-S-dihydroorotates are not. Competitive inhibitors include product orotate, 5-methyl orotate, and racemic cis-5-methyl dihydroorotate.     

Purification and properties of the 5,10-methenyltetrahydromethanopterin cyclohydrolase from Methanobacterium thermoautotrophicum.

The 5,10-methenyltetrahydromethanopterin cyclohydrolase of Methanobacterium thermoautotrophicum was purified 128-fold to homogeneity. The enzyme had a subunit Mr of 41,000 as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From high-performance size exclusion chromatography of the native protein, an Mr of 82,000 was determined, suggesting a dimer of identical subunits. The enzyme was inhibited by 10-formyltetrahydromethanopterin and stimulated by Mg2+. Evaluation of the reaction equilibrium indicated that the methenyl derivative was favored over 5-formyltetrahydromethanopterin, with a much higher equilibrium constant than for the analogous reaction of tetrahydrofolate derivatives. Folate derivatives did not serve as substrates for this enzyme.     

Purification and characterization of a plasminogen activator secreted by a pig kidney cell line

The plasminogen activator secreted by calcitonin-treated pig kidney cells was purified, characterized and compared with human urinary urokinase. The purification procedure was based on the following steps: sulphopropyl-Sephadex chromatography, p-aminobenzamidine-Sepharose chromatography, preparative sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectrofocusing. The purified enzyme was obtained from the conditioned medium with a yield of 13% and a purification factor of 390-fold. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under non-reducing conditions showed one closely spaced doublet with an Mr of 50 000; in the presence of reducing agents, two additional bands of Mr 30 000 and 20 000 appeared. The purified enzyme resembles the 53 000-Mr components of human urinary urokinase in amino acid composition and two-dimensional tryptic peptide maps and in its catalytic properties, and the two enzymes cross-react immunologically with rabbit antibodies raised against either. The enzyme appears to be different from tissue plasminogen activator secreted by HeLa cells.     

3-keto-5 alpha-steroid-delta 4-dehydrogenase from Nocardia corallina: purification and characterization

The inducible 3-keto-5 alpha-steroid-delta 4-dehydrogenase of Nocardia corallina was purified to homogeneity using affinity chromatography on 19-nortestosterone-17-acetoxyaminoethyl Sepharose 4B. SDS-polyacrylamide gel electrophoresis, gel filtration and spectral analysis of flavin suggest that the purified dehydrogenase is a monomeric protein of Mr 60,000 containing one flavin. It has a typical absorption spectrum of flavoprotein with maxima at 457, 375, and 277 nm. The values shifted to 470 and 395 nm on binding of 19-nortestosterone. The enzyme catalyzed the dehydrogenation of 3-keto-5 alpha-steroid at the 4- and 5-position, e.g. the conversion of 5 alpha-androst-1-ene-3,17-dione to 1,4-androstadiene-3,17-dione with the reduction of phenazine methosulfate. The substrate 3-ketosteroid has essentially the 5 alpha-configuration. The enzyme did not reduce potassium ferricyanide but did reduce cytochrome c at a moderate rate, and exhibited only a weak steroid oxidase activity. Stereochemical study demonstrated that the enzyme abstracts the 4 beta, 5 alpha-hydrogens of the substrate as a hydrogen ion through a protein-based reaction and as a hydride ion by transfer to FAD, respectively. The enzyme oxidizes a wide variety of 3-keto-5 alpha-steroids but not 3 beta-hydroxysteroid. The dehydrogenase also catalyzed steroid transhydrogenation between 3-keto-5 alpha-steroid and 3-keto-1,4-diene-steroid. The properties of this enzyme are compared with those of 3-keto-steroid-delta 1-dehydrogenase.     

Purification of the secretor-type beta-galactoside alpha 1----2-fucosyltransferase from human serum.

The secretor-type beta-galactoside alpha 1----2-fucosyltransferase from human serum was purified by hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, and affinity chromatography on GDP-hexanolamine-Sepharose. Final purification of the enzyme was achieved by high pressure liquid chromatography gel filtration and resulted in a homogeneous protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the radiolabeled protein. The native enzyme appears as a molecule of apparent Mr 150,000 as determined by gel filtration high pressure liquid chromatography. The apparent Mr of the enzyme resolved in the presence of beta-mercaptoethanol by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined to be 50,000, indicating a multisubunit structure of the enzyme. Secretor-type alpha 1----2-fucosyltransferase is a glycoprotein as determined by WGA binding properties. A comparison of the Mr of the native blood group H gene encoded with the secretor-type beta-galactoside alpha 1----2-fucosyltransferases as well as comparison of subunit Mr for both enzymes suggests structural similarity. The alpha 1----2 linkage formed between alpha-L-fucose and terminal beta-D-galactose by the purified H- and secretor-type alpha 1----2-fucosyltransferases was determined by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide products. The substrate specificity and Km values calculated from the initial rate using various oligosaccharide acceptors showed that purified enzymes differ primarily in affinity for phenyl-beta-D-galactopyranoside and GDP-fucose as well as type 1 (Gal beta 1----3GlcNAc), 2 (Gal beta 1----4GlcNAc), and 3 (Gal beta 1----3GalNAc) oligosaccharide acceptors. The secretor-type alpha 1----2-fucosyltransferase shows significantly lower affinity than the H enzyme for phenyl-beta-D-galactopyranoside and GDP-fucose as well as for type 2 oligosaccharide acceptors. On the contrary, type 1 and 3 oligosaccharide acceptors are preferentially utilized by the secretor-type enzyme as compared with the H enzyme. The enzymes also differ in several physicochemical properties, implying nonidentity of the two enzymes (Sarnesto, A., K?hlin, T., Thurin, J., and Blaszczyk-Thurin, M. (1990) J. Biol. Chem. 265, 15067-15075).     

Isolation to homogeneity and partial characterization of a histo-blood group A defined Fuc alpha 1----2Gal alpha 1----3-N-acetylgalactosaminyltransferase from human lung tissue

The soluble histo-blood group A glycosyltransferase (Fuc alpha 1----Gal alpha 1----3-N-acetylgalactosaminyltransferase) was purified approximately 600,000-fold to homogeneity from human lung tissue. The enzyme was solubilized in 1% Triton X-100, partially purified by affinity chromatography on Sepharose 4B, and eluted with UDP. Final purification was obtained by twice repeated fast protein liquid chromatography ion exchange (Mono STM) with NaCl gradient elution and reverse-phase chromatography (proRPC) with acetonitrile gradient elution. Identity of the purified protein was established by (i) demonstration of the putative A transferase protein only in affinity-purified extracts of A but not O individuals, and (ii) specific immunoprecipitation of enzyme activity and putative protein with monoclonal antibodies. Sodium dodecyl sulfate electrophoresis revealed a single protein band with apparent Mr of approximately 40,000 under both reducing and nonreducing conditions. Digestion with N-glycanase yielded a reduction in Mr of approximately 6,000 (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), suggesting that the A transferase is a glycoprotein with N-linked carbohydrate chains. Amino acid composition and N-terminal amino acid sequence of the intact transferase, as well as of peptides released by endolysyl peptidase digest or cyanogen bromide cleavage, are presented.     

L-fucose metabolism in mammals. Purification of pork liver 2-keto-3-deoxy-L-fuconate:NAD+ oxidoreductase by NAD+-Agarose affinity chromatography.

Pork liver has previously been reported to contain a soluble enzymatic pathway which converts L-fucose to 2-keto-3-deoxy-L-fuconate and D-arabinose to 2-keto-3-deoxy-D-arabonate. We now report the isolation from pork liver of a soluble NAD+-dependent dehydrogenase which acts on both 2-keto-3-deoxy-L-fuconate and 2-keto-3-deoxy-D-arabonate. This enzyme has been purified to homogeneity by a five-step procedure; the final step involved affinity chromatography on NAD+-agarose. A purification factor of about 3000-fold was achieved with a yield of over 20%. The enzyme was homogeneous on polyacrylamide gel electrophoresis at pH 9.1 and 7.0 and on the basis of sedimentation equilibrium analysis with the ultracentrifuge. The molecular weight of the native enzyme is about 100,000 while disc gel electrophoresis in the presence of sodium dodecyl sulfate and thiol showed the presence of a polypeptide of molecular weight 26,800; these results suggest that the enzyme is a tetramer. The enzyme has an isoelectric point of 5.4. The enzyme is unstable in the dilute state and in the absence of thiol but can be kept for 2 years at -70 degrees at a protein concentration of 4 mg per ml and in the presence of 1 mM dithiothreitol.     

Purification and characterization of peptidylarginine deiminase from rabbit skeletal muscle

The preceding paper described the identification and some properties of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle, kidney, brain, and lung. In the present work we purified peptidylarginine deiminase from rabbit skeletal muscle with a 16% yield by 7 steps. The purification involved ion-exchange chromatography on DEAE-Sephacel, gel filtration on Bio-Gel A-0.5 m, and affinity chromatography on soybean trypsin inhibitor-Sepharose 4B and aminohexyl-Sepharose 4B. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be about 83,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 130,000-140,000 by gel filtration on Sephadex G-200. The isoelectric point was 5.3 and the amino acid composition was also determined. The enzyme preferably catalyzed the formation of citrulline derivatives from arginine derivatives in which both the amino and carboxyl groups were substituted and showed the highest activity towards Bz-L-Arg-O-Et among the arginine derivatives tested. The Km value for Bz-L-Arg-O-Et was found to be 0.50 X 10(-3) M. The enzyme also showed marked activities towards native protein substrates, such as protamine sulfate, soybean trypsin inhibitor, histone and bovine serum albumin.     

Solubilization and purification of rat liver 5''-nucleotidase by use of a zwitterionic detergent and a monoclonal-antibody immunoadsorbent.

1. A variety of detergents were used to solubilize 5'-nucleotidase from rat liver plasma membranes. 2. The zwitterionic detergent Sulphobetaine 14 gave optimal solubilization by the criteria of release into a high-speed-centrifugation supernatant and the formation of the smallest and least polydisperse active enzyme observed on polyacrylamide-gel electrophoresis. 3. The Sulphobetaine 14-solubilized enzyme from rat liver was purified by using the conventional techniques of ion-exchange chromatography and gel filtration, or by an immunoaffinity step with a monoclonal antibody immunoadsorbent. 4. 5'-Nucleotidase was purified at least 12 000-fold relative to liver homogenate by the immunoaffinity purification scheme and had a specific activity in the range 285-340 mumol/min per mg of protein. The yield was in the range 9-16%. 5. The purified enzyme shows a major polypeptide band of apparent Mr 70 000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and a minor band of apparent Mr 38 000. 6. A rational approach to the general problem of the purification of minor intrinsic membrane proteins is discussed, with the use of polyacrylamide-gel electrophoresis to determine the most appropriate detergent and monoclonal antibodies in subsequent immunoaffinity purification.     

Characterization of affinity-purified juvenile hormone esterase from Trichoplusia ni

Juvenile hormone (JH) esterase was purified greater than 1000-fold in one step from hemolymph and whole larval homogenates from the last larval instar of Trichoplusia ni to give a single diffuse band that migrates at Mr = 64,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purification was based on an affinity chromatography procedure that employs trifluoromethyl ketone ligands. Isoelectric focusing of the purified preparations resulted in multiple bands that coincided to all significant hydrolysis of juvenile hormone detected in this manner. Kinetic experiments using optically pure enantiomers of JH II as substrates showed the two main electromorphs of JH esterase from the hemolymph to have apparently identical kinetic parameters as well as a similar capability to distinguish between substrates that differ in the orientation of the epoxide moiety of JH. However, the enzyme could hydrolyze esters lacking the JH structure. The proteins were shown to be monomers and to have asparagine-linked oligosaccharides, most likely of hybrid structure. Immunochemical and other evidence showed that the affinity-purified proteins were responsible for all significant JH esterase activity during periods of rapid esterolysis in vivo.     

Purification and characterization of a multicatalytic high-molecular-mass proteinase from rat skeletal muscle.

A proteolytic enzyme was purified from the post-myofibrillar fraction of rat skeletal muscle. The purification procedure consisted of fractionation of the muscle extract by (NH4)2SO4, chromatography on DEAE-Sephacel, fast protein liquid chromatography on Mono Q and gel filtration on Sepharose 6B. The enzyme preparation appeared to be homogeneous as judged by disc electrophoresis in polyacrylamide gels and by immunoelectrophoresis. The isoelectric point of the proteinase is at 5.1-5.2. The enzyme has an Mr of about 650 000 and dissociates into eight subunits of Mr 25 000-32 000 when subjected to electrophoresis in sodium dodecyl sulphate/polyacrylamide gels. The proteinase contains hydrolytic activity against N-blocked tripeptide 4-methyl-7-coumarylamide substrates with an arginine or phenylalanine residue adjacent to the leaving group. Maximum activity with the first group of substrates was at pH 10.5, and this activity was inhibited by leupeptin, chymostatin and Ca2+. Maximum activity with the latter group of substrates was at pH 7.5, and was also inhibited by the two microbial inhibitors, but was activated by Ca2+ ions. By using [14C]methylcasein as a substrate, maximum activity was observed at pH9.0, and this proteolytic activity was not affected by leupeptin, was enhanced by chymostatin and inhibited by Ca2+. Similar effects were observed when benzyloxycarbonyl-Leu-Leu-Glu 2-naphthylamide was used as a substrate. These enzymic activities were abolished by p-hydroxymercuribenzenesulphonic acid or mersalyl acid, whereas a small activation was observed with cysteine or dithiothreitol.     

Comparative purification and characterization of invertebrate muscle glycogen synthase from the porcine parasite Ascaris suum

Glycogen synthase has been purified from the obliquely striated muscle of the swine parasite Ascaris suum. The muscle contains a concentration of glycogen synthase and glycogen which is 20-fold and 15-fold, respectively, greater than rabbit skeletal muscle. The enzyme could not be solubilized with salivary amylase, but partial solubilization was achieved by activation of endogenous phosphorylase. The enzyme was purified to 85-90% homogeneity (specific activity = 4.3 units/mg) by DEAE-cellulose, Sepharose 4B, and glucosamine 6-phosphate chromatography. The purified glycogen synthase was substantially similar to rabbit skeletal muscle enzyme with respect to Mr (gel electrophoresis and gel filtration), pH dependence, aggregation properties, temperature dependence, and kinetic constants for substrates and activators. Glycogen synthase I was converted to glycogen synthase D by the cyclic AMP-dependent protein kinase. The cyclic AMP-dependent protein kinase catalyzed the incorporation of 1.3 mol of phosphate into each glycogen synthase I subunit and the concomitant interconversion to glycogen synthase D. Since glycogen is the sole fuel utilized by this organism during nonfeeding periods of the host, the characterization of this enzyme provides further insight into the regulatory mechanisms which determine glycogen turnover.     

N5-(L-1-carboxyethyl)-L-ornithine:NADP+ oxidoreductase from Streptococcus lactis. Purification and partial characterization

N5-(L-1-Carboxyethyl)-L-ornithine:NADP+ oxidoreductase (EC 1.5.1.-) from Streptococcus lactis K1 has been purified 8,000-fold to homogeneity. The NADPH-dependent enzyme mediates the reductive condensation between pyruvic acid and the delta- or epsilon-amino groups of L-ornithine and L-lysine to form N5-(L-1-carboxyethyl)-L-ornithine and N6-(L-1-carboxyethyl)-L-lysine, respectively. The five-step purification procedure involves ion-exchange (DE52 and phosphocellulose P-11), gel filtration (Ultrogel AcA 44), and affinity chromatography (2',5'-ADP-Sepharose 4B). Approximately 100-200 micrograms of purified enzyme of specific activity 40 units/mg were obtained from 60 g of cells, wet weight. Anionic polyacrylamide gel electrophoresis revealed a single enzymatically active protein band, whereas three species (pI 4.8-5.1) were detected by analytical electrofocusing. The purified enzyme is active over a broad pH range of 6.5-9.0 and is stable to heating at 50 degrees C for 10 min. Substrate Km values were determined to be: NADPH, 6.6 microM; pyruvate, 150 microM; ornithine, 3.3 mM; and lysine, 18.2 mM. The oxidoreductase has a relative molecular mass (Mr = 150,000) as estimated by high pressure liquid chromatography exclusion chromatography and by polyacrylamide gradient gel electrophoresis. Conventional gel filtration indicated an Mr = 78,000, and a single protein band of Mr = 38,000 was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is composed of identical subunits of Mr = 38,000, which may associate to yield both dimeric and tetrameric forms. Polyclonal antibody to the purified protein inhibited enzyme activity. The amino acid composition of the enzyme is reported, and the sequence of the first 37 amino acids from the NH2 terminus has been determined by stepwise Edman degradation.     

Purification of the beta-N-acetylglucosaminide alpha 1----3-fucosyltransferase from human serum.

A beta-N-Acetylglucosaminide alpha 1----3-fucosyltransferase was purified from human serum by ammonium sulfate precipitation, hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, affinity chromatography on GDP-hexanolamine-Sepharose, and finally high pressure liquid chromatography gel filtration. Gel filtration chromatography of the native enzyme revealed a Mr of 45,000. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified protein also appeared as a single molecular species of Mr 45,000. In contrast to the multisubunit beta-galactoside alpha 1----2-fucosyltransferases with an apparent Mr of 150,000, present in human serum, the native beta-N-acetylglucosaminide alpha 1----3-fucosyltransferase is a monomer with a Mr of 45,000. The enzyme is glycosylated, as revealed by wheat germ agglutinin binding properties. The alpha 1----3 linkage formed by the enzyme between alpha-L-fucose and the penultimate beta-N-acetylglucosamine by the purified enzyme was confirmed by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide product. The specificity of the purified enzyme is restricted to type 2 structures, as revealed by its reactivity with different substrates and from the Km values calculated from the initial rate data using various oligosaccharide acceptors. The enzyme has the ability to utilize the N-acetyl-beta-lactosamine determinant (Gal beta 1----4GlcNAc) and the sialylated (NeuAc alpha 2----3Gal beta 1----4GlcNAc) and fucosylated (Fuc alpha 1----2Gal beta 1----4GlcNAc) derivatives of N-acetyl-beta-lactosamine and thus is distinct from both the human Lewis gene-encoded enzyme and the alpha 1----3-fucosyltransferase of the myeloid cell type.