beta-Glucuronidase in the bull seminal plasma and reproductive organs

The biochemical distribution of beta-glucuronidase activity was studied in different reproductive organs, seminal plasma and spermatozoa of the bull. The highest specific activity was found in the epididymis, where the activity seemed to be mostly in nonsecretory and only partly in secretory form. A molecular weight of 340 X 10(3) to 360 X 10(3) was recorded for beta-glucuronidase in the bull seminal plasma and different reproductive organs with gel filtration on Sepharose 6B. In chromatofocusing four activity areas (CF-1 to CF-4) were usually obtained for beta-glucuronidase in the bull seminal plasma. The major peak CF-2 (also in the different reproductive organs) had a pI value of 5.6-5.3 and the two minor activity areas CF-1 and CF-3 had pI values of 6.0-5.8 and 5.2-4.5, respectively. Peak CF-4 eluted with a NaCl gradient after the Polybuffer elution and possibly represents an enzyme form incompletely detached from negatively charged cellular material. Isoelectric focusing on polyacrylamide gel confirmed the heterogeneity of beta-glucuronidase, since several activity bands were detected in the secretion of the different parts of the epididymis. beta-Glucuronidase activities CF-1, CF-2 and CF-3 had similar pH activity profiles (pH optimum around pH 3.0-4.0) and response to thermal inactivation at 50 degrees C. The multiple beta-glucuronidase activities of the bull seminal plasma are proposed to derive mainly from the secretion of the cauda epididymidis.     

Purification and characterization of human seminal plasma aminopeptidase

A 50.4-fold purification of aminopeptidase is achieved by alcohol precipitation, DEAE-cellulose, CM-cellulose and finally Sephadex G-200 chromatography. On polyacrylamide gel electrophoresis of the purified enzyme after molecular sieving on Sephadex G-200, only one band was obtained, suggesting that the enzyme preparation was obtained almost homogeneous by three steps of column chromatography. Aminopeptidase showed highest activity at pH 7.0, using a buffer system, of 70 mM Na-phosphate. The enzyme was found to be active at 40 degrees C, even at 60 degrees C (80% activity), suggesting that the human seminal plasma enzyme is fairly thermostable. Amongst the various aminoacyl derivatives evaluated as substrates in the present study, L-alanine beta-naphthylamide hydrochloride was found to have the highest rate of hydrolysis. Ovalbumin showed effective cleavage in comparison to that of other natural substrates. The Km value for the purified seminal plasma aminopeptidase towards L-alanine beta-naphthylamide hydrochloride was 4 x 10(-4) M. Hg+2 showed highest inhibitory effect than other metal ions tested in the present study. Concentration causing 50% inhibition of the enzyme (I50) by Hg2+ was 4.7 x 10(-6) M. Inhibition by EDTA at 1 mM concentration in the incubation system was higher than by EGTA and sodium azide, suggesting that the enzyme contains a metallo group at the active site. A 50% inhibition of the enzyme by EDTA was obtained at 5.11 x 10(-3) M. The Ackerman and Potter plot for EDTA inhibition suggests that EDTA is a reversible inhibitor of seminal plasma aminopeptidase. A single molecular form of aminopeptidase was found to be present in human seminal plasma as shown by polyacrylamide activity gel electrophoresis.     

Purification and characterization of human seminal plasma acid phosphatase

A 81-fold purification of human seminal plasma acid phosphatase was obtained by a three-step procedure, involving ammonium sulfate precipitation, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. Homogeneity of the preparation during purification steps was tested by polyacrylamide gel electrophoresis and only one major band was obtained after the final step. The pH optimum for the activity of the purified enzyme was 5.6 and thermal stability was obtained even up to 40 degrees C. PNPP was the most specific synthetic substrate. The Km of purified seminal acid phosphatase towards PNPP was 1.5 X 10(-3) M. Among the metal ions tested, Hg+2 showed an I50 value of 4.2 X 10(-7) M. Studies with PCMB, PMSF and EDTA did not show any inhibition, whereas NaF and L(+)tartrate, at 1 mM concentration, inhibited the enzyme by 95% and 85%, respectively.     

Purification of plasminogen activators from human seminal plasma.

Two plasminogen activators (1 and 2) were isolated from human seminal plasma by hiigh-speed centrifugation, Sephadex-gel filtration and ion-exchange chromatography. The activators were shown to be homogeneous by polyacrylamide-disc -gel electrophoresis at pH 8.3 and 4.5, and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weights of activators 1 and 2 were estimated as 69 000 and 74 000. Their amino acid compositions are very similar, both being high in aspartic acid, glutamic acid, serine, glycine and leucine, and low in methionine, tryptophan, tyrosine, isoleucine and histidine. Activators 1 and 2 each possess 16 cysteine residues. Both activators have isoelectric points of approx. 7.0, are stable over a wide pH range at temperatures up to 60 degrees C, but lose activity at higher temperatures, particularly under very basic or acidic conditions. They are not inhibited by EDTA, Mg2+ and Ca2+ at 10 mM concentrations, but their activity decreases on addition of 10 mM-cysteine or Fe2+ and 6-aminohexanoate or sera from pregnant women. The precipitin band formed between urokinase and its antiserum is continuous with the precipitin bands formed between the seminal plasminogen activators and the urokinase antiserum. Antisera to urokinase inhibit both the activity of urokinase and the seminal plasminogen activators.     

Purification and characterisation of a beta-glucosidase (cellobiase) from a mushroom Termitomyces clypeatus

A beta-glucosidase with cellobiase activity was purified to homogeneity from the culture filtrate of the mushroom Termtomyces clypeatus. The enzyme had optimum activity at pH 5.0 and temperature 65 degrees C and was stable up to 60 degrees C and within pH 2-10. Among the substrates tested, p-nitrophenyl-beta-D-glucopyranoside and cellobiose were hydrolysed best by the enzyme. Km and Vm values for these substrates were 0.5, 1.25 mM and 95, 91 mumol/min per mg, respectively. The enzyme had low activity towards gentiobiose, salicin and beta-methyl-D-glucoside. Glucose and cellobiose inhibited the beta-D-glucosidase (PNPGase) activity competitively with Ki of 1.7 and 1.9 mM, respectively. Molecular mass of the native enzyme was approximated to be 450 kDa by HPLC, whereas sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated a molecular mass of 110 kDa. The high molecular weight enzyme protein was present both intracellularly and extracellularly from the very early growth phase. The enzyme had a pI of 4.5 and appeared to be a glycoprotein.     

Purification and properties of a novel thermostable galacto-oligosaccharide-producing beta-galactosidase from Sterigmatomyces elviae CBS8119.

A thermostable beta-galactosidase which catalyzed the production of galacto-oligosaccharide from lactose was solubilized from a cell wall preparation of Sterigmatomyces elviae CBS8119. The enzyme was purified to homogeneity by means of chromatography on DEAE-Toyopearl, Butyl-Toyopearl, Chromatofocusing, and p-aminobenzyl 1-thio-beta-D-galactopyranoside agarose columns. The molecular weight of the purified enzyme was estimated to be about 170,000 by gel filtration with a Highload-Superdex 200pg column and 86,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its isoelectric point, determined by polyacrylamide gel electrofocusing, was 4.1. The optimal temperature for enzyme activity was 85 degrees C. It was stable at temperatures up to 80 degrees C for 1 h. The optimal pH range for the enzyme was 4.5 to 5.0, it was stable at pH 2.5 to 7.0, and its activity was inhibited by Hg2+. The Km values for o-nitrophenyl-beta-D-galactopyranoside and lactose were 9.5 and 2.4 mM, respectively, and the maximum velocities for these substrates were 96 and 240 mumol/min per mg of protein, respectively. In addition, this enzyme possessed a high level of transgalactosylation activity. Galacto-oligosaccharides, including tri- and tetrasaccharides, were produced with a yield, by weight, of 39% from 200-mg/ml lactose.     

Purification and characterization of acid trehalase from the yeast suc2 mutant

Acid trehalase was purified from the yeast suc2 deletion mutant. After hydrophobic interaction chromatography, the enzyme could be purified to a single band or peak by a further step of either polyacrylamide gel electrophoresis, gel filtration, or isoelectric focusing. An apparent molecular mass of 218,000 Da was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate suggested a molecular mass of 216,000 Da. Endoglycosidase H digestion of the purified enzyme resulted after sodium dodecyl sulfate gel electrophoresis in one distinct band at 41,000 Da, representing the mannose-free protein moiety of acid trehalase. The carbohydrate content of the enzyme was 86%. Amino acid analysis indicated 354 residues/molecule of enzyme including 9 cysteine moieties and only 1 methionine. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7. The catalytic activity showed a maximum at pH 4.5. The activity of the enzyme was not inhibited by 10 mM each of HgCl2, EDTA, iodoacetic acid, phenanthrolinium chloride or phenylmethylsulfonyl fluoride. There was no activation by divalent metal ions. The acid trehalase exhibited an apparent Km for trehalose of 4.7 +/- 0.1 mM and a Vmax of 99 mumol of trehalose min-1 X mg-1 at 37 degrees C and pH 4.5. The acid trehalase is located in the vacuoles. The rabbit antiserum raised against acid trehalase exhibited strong cross-reaction with purified invertase. These cross-reactions were removed by affinity chromatography using invertase coupled to CNBr-activated Sepharose 4B. Precipitation of acid trehalase activity was observed with the purified antiserum.     

Purification and characterization of glutamate decarboxylase from Solanum tuberosum

Glutamate decarboxylase has been purified from potato tubers. The final preparation was homogeneous as judged from native and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Gel filtration on Sephadex G-200 gave a relative molecular mass Mr, of 91 000 for the native enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a subunit Mr of 43 000. Thus the enzyme appears to be a dimer of identical subunits. It has 2 mol pyridoxal 5'-phosphate/mol protein, which could not be removed by exhaustive dialysis or gel filtration on Sephadex G-25. The enzyme has an absorption maximum at 370 nm in sodium phosphate buffer, pH 5.8. Reduction of the enzyme with sodium borohydride abolished the absorption maximum at 370 nm with attendant loss of catalytic activity. The enzyme exhibited pH-dependent spectral changes. The enzyme was specific for L-glutamate and could not decarboxylate other amino acids tested. The enzyme was maximally active at pH 5.8 and a temperature of 37 degrees C. Isoelectric focussing gave a pI of 4.7 Km values for L-glutamate and pyridoxal 5'-phosphate were 5.6 mM and 2 microM respectively. Thiol-directed reagents and heavy metal ions inhibited the enzyme, indicating that an -SH group is required for activity. The nature of the functional groups at the active site of the enzyme was inferred from competitive inhibition studies. L-Glutamate promoted inactivation of the enzyme caused by decarboxylation-dependent transamination was demonstrated. The characteristics of potato enzyme were compared with enzyme from other sources.     

Isolation and characterization of acylneuraminate cytidylyltransferase from frog liver

Frog liver (Rana esculenta) is a rich source of acylneuraminate cytidylyltransferase. The soluble enzyme was purified 250-fold almost to purity with 25% yield and a specific activity of 9 mkat/kg protein (0.54 U/mg protein) using DEAE Sephadex and Sepharose 6B chromatography, followed by preparative polyacrylamide gel electrophoresis. The molecular weight of the cytidylyltransferase was determined to be 163 000 with the aid of Sepharose 6B chromatography and gel electrophoresis, with or without dodecyl sulphate or urea. No subunits were found. The isoelectric point of the enzyme is at pH 6. Optimum reaction rate was observed at pH 9, 37 degrees C, 50mM Mg2 or Ca2 and ImM mercaptoethanol. The Km values for N-acetylneuraminic acid, N-glycoloylneuraminic acid and CTP are 1.6mM, 2.3 mM and 0.6mM, respectively. O-Acetylated sialic acids are inactive with the cytidylyltransferase from frog liver. Enzyme activity can be inhibited by SH reagents and CMP (Ki = 0.5mM).     

Purification and properties of haloalkane dehalogenase from Corynebacterium sp. strain m15-3

A haloalkane dehalogenase was purified to electrophoretic homogeneity from cell extracts of a 1-chlorobutane-utilizing strain, m15-3, which was identified as a Corynebacterium sp. The enzyme hydrolyzed C2 to C12 mono- and dihalogenated alkanes, some haloalcohols, and haloacids. The Km value of the enzyme for 1-chlorobutane was 0.18 mM. Its molecular weight was estimated to be 36,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 33,000 by gel filtration. The isoelectric point was pH 4.5. The optimum pH for enzyme activity was found to be 9.4, and the optimum temperature was 30 to 35 degrees C. The enzyme was stable for 1 h at temperatures ranging from 4 to 30 degrees C but was progressively less stable at 40 and 50 degrees C.     

Partial purification and properties of a beta-glucosidase from Erwinia herbicola Y46.

A constitutive beta-glucosidase of Erwinia herbicola Y46 was studied as a prerequisite to an assessment of its significance in the release of bacteriotoxic aglycones from plant beta-glucosides, and the possible effects of the aglycones on the course of such plant diseases as "fire-blight". The enzyme was purified 86.5-fold from crude extracts of cells grown on yeast beef broth. Ammonium sulfate precipitation, DEAE-cellulose fractionation, and gel filtration through Sephadex G-100 resulted in a preparation having one peak of activity on isoelectrofocussing, on gel filtration through Sephadex G-200, and on polyacrylamide gel electrophoresis. The latter techniques demonstrated, in addition to the major protein band associated with activity, a single minor impurity. The enzyme was active against p-nitrophenyl-beta-glucoside (p-NPG) and phloridzin, but showed only very slight activity against salicin and arbutin, and no detectable activity against beta-methyl-D-glucoside, cellobiose, lactose, and esculin. The production of beta-glucosidase was maximum at the late log phase of growth on yeast beef broth medium and declined somewhat thereafter. The incorporation of inducers (carbohydrates) in defined basal medium resulted in only small variations in specific activity in the resulting cells; The activity (p-NPG substrate) was not inhibited by D-glucose, phloretin, esculin, salicin, arbutin, lactose, or cellobiose, but was slightly inhibited by 1.0 mM phloridzin. Slight inhibition was observed in the presence of sulfhydryl reagents (iodoacetamide, p-chloromercuribenzoate), but sodium azide, ethylene-diaminetetraacetic acid, Cu2+, and Zn2+ ions produced no effect. The activity was stable, in both crude and purified preparations, over the pH ranges 6.0-7.5 (100% activity) and 4.5-greater than 8.5 (50% activity). The enzyme retained 80% activity after 30 min at 50 degrees C, but only 25% after 30 min at 60 degrees C. The enzyme had a mean K-m value (phloridzin) of 1.35 times 10-4 M, an isoelectric point of 4.75, a molecular weight, determined by Sephadex G-200 gel filtration, of about 122 000, and an optimum pH for activity of 6.5-7.0.     

Purification and characterization of a novel mannitol dehydrogenase from Lactobacillus intermedius

Mannitol 2-dehydrogenase (MDH) catalyzes the pyridine nucleotide dependent reduction of fructose to mannitol. Lactobacillus intermedius (NRRL B-3693), a heterofermentative lactic acid bacterium (LAB), was found to be an excellent producer of mannitol. The MDH from this bacterium was purified from the cell extract to homogeneity by DEAE Bio-Gel column chromatography, gel filtration on Bio-Gel A-0.5m gel, octyl-Sepharose hydrophobic interaction chromatography, and Bio-Gel Hydroxyapatite HTP column chromatography. The purified enzyme (specific activity, 331 U/mg protein) was a heterotetrameric protein with a native molecular weight (MW) of about 170 000 and subunit MWs of 43 000 and 34 500. The isoelectric point of the enzyme was at pH 4.7. Both subunits had the same N-terminal amino acid sequence. The optimum temperature for the reductive action of the purified MDH was at 35 degrees C with 44% activity at 50 degrees C and only 15% activity at 60 degrees C. The enzyme was optimally active at pH 5.5 with 50% activity at pH 6.5 and only 35% activity at pH 5.0 for reduction of fructose. The optimum pH for the oxidation of mannitol to fructose was 7.0. The purified enzyme was quite stable at pH 4.5-8.0 and temperature up to 35 degrees C. The K(m) and V(max) values of the enzyme for the reduction of fructose to mannitol were 20 mM and 396 micromol/min/mg protein, respectively. It did not have any reductive activity on glucose, xylose, and arabinose. The activity of the enzyme on fructose was 4.27 times greater with NADPH than NADH as cofactor. This is the first highly NADPH-dependent MDH (EC 1.1.1.138) from a LAB. Comparative properties of the enzyme with other microbial MDHs are presented.     

Responses of seminal vesicle and testicular beta-glucuronidase and beta-N-acetylglucosaminidase to testosterone and some metabolites in Heteropneustes fossilis (Bloch).

Intraperitoneal administration of testosterone for 20 days produced differential effects on beta-glucuronidase and beta-N-acetylglucosaminidase (beta-Glc) activity in seminal vesicle (SV) and testis of the catfish Heteropneustes fossilis in preparatory phase (March). The lower dosages of 0.25 and 0.5 microg/g body weight (BW) of the steroid did not alter enzyme activity, and the higher dosages (1.0 and 2.0 microg/g BW) inhibited it significantly. Under in vitro conditions, addition of ascorbate and fructose (0.5-100 mM) to the incubation medium influenced enzyme activity differentially. At concentrations 0.5 and 1.0 mM, both fructose and ascorbate were ineffective except for the inhibition of testicular beta-Glc activity in the 1.0 mM ascorbate group. At higher concentrations (10, 50, and 100 mM), ascorbate inhibited enzyme activity in a concentration-dependent manner. At 10 mM concentration of fructose, only testicular beta-Glc activity was inhibited, but at higher concentrations (50 and 100 mM), activities of both enzymes decreased uniformly in a concentration-dependent manner. The addition of glucose had no significant effect on the enzyme activity at any of the concentrations tested. The results suggest that the inhibitory effect of testosterone on enzyme activity may be mediated through androgen-dependent metabolites, such as fructose and ascorbate.     

Purification and characterization of alkaline phosphatase from plasma membranes of rat ascites hepatoma.

Alkaline phosphatase was purified from plasma membranes of rat ascites hepatoma AH-130, the homogenate of which had 50-fold higher specific activity than that found in the liver homogenate. The presence of Triton X-100, 0.5%, was essential to avoid its aggregation and to stabilize its activity. The purified enzyme, a glycoprotien, was homogeneous in polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated a protein molecular weight of 140,000. The addition of beta-mercaptoethanol caused the dissociation of the alkaline phosphatase into two subunits of identical molecular weight, 72,000. Isoelectric focusing revealed that the pI of this enzyme is 4.7. The pH optimum for the purified enzyme was 10.5 or higher with p-nitrophenylphosphate, and slightly lower pH values (pH 9.5--10.2) were obtained when other substrates were used. Of the substrates tested, p-nitrophenylphosphate (Km-0.3 mM) was most rapidly hydrolyzed. Vmax values of other substrates relative to that of p-nitrophenylphosphate were as follows; beta-glycerophosphate, 76%; 5'-TMP, 82%; 5'-AMP, 62%; 5'-IMP, 43%; glucose-6-phosphate, 39%; ADP, 36% and ATP, 15%. More than 90% of the activity of the purified enzyme was irreversibly lost when it was heated at 55 degrees C for 30 min, or exposed either to 10 mM beta-mercaptoethanol for 10 min to 3 M urea for 30 min, or to an acidic pH below pH 5.0 for 2 h. Of the effects by divalent cations, Mg2+ activated the enzyme by 20% whereas Zn2+ strongly inhibited it by 95% at 0.5 mM. EDTA at higher than 1 mM inactivated the enzyme irreversibly, although the effect of EDTA at lower than 0.1 mM was reversible by the addition of divalent cations, particularly by Mg2+. The enzyme was most strongly inhibited by L-histidine among the amino acids tested, and also strongly inhibited by imidazole. These results suggest that alkaline phosphatase of rat hepatoma AH-130 is very similar to that of rat liver in most of the properties reported so far.     

Purification and partial characterization of manganese peroxidase from immobilized Phanerochaete chrysosporium

Solid-state culture of the white-rot fungus Phanerochaete chrysosporium BKMF-1767 (ATCC 24725) has been carried out, using an inert support, polystyrene foam. Suitable medium and culture conditions have been chosen to favor the secretion of manganese peroxidase (MnP). The enzyme was isolated and purified from immobilized P. chrysosporium and partially characterized. Partial protein precipitation in crude enzyme was affected using ammonium sulphate, polyethylene glycol, methanol, and ethanol methods. Fractionation of MnP was performed by DEAE-Sepharose ion exchange chromatography followed by Ultragel AcA 54 gel filtration chromatography. This purification attained 23.08% activity yield with a purification factor of 5.8. According to data on gel filtration chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of the enzyme was 45 000±1000 Da. The optimum pH and temperature of purified MnP were 4.5 and 30 °C, respectively. This enzyme was stable in the pH range 4.5–6.0, at 25 °C and also up to 35 °C at pH 4.5 for 1 h incubation period. MnP activity was inhibited by 2 mM NaN₃, ascorbic acid, β-mercaptoethanol and dithreitol. The K_m values of MnP for hydrogen peroxide and 2.6-dimetoxyphenol were 71.4 and 28.57 μM at pH 4.5, respectively. The effects of possible inhibitors and activators of enzyme activity were investigated.     

Metabolism of 2-ketoaldehydes in mold: purification and characterization of glyoxalase I from Aspergillus niger

Glyoxalase I catalyzing the conversion of methylglyoxal into S-lactoylglutathione in the presence of glutathione was purified approximately 1,400-fold with 2.9% activity yield from mold, Aspergillus niger. The enzyme consisted of a single polypeptide chain with a relative molecular weight of 36,000 on both SDS-polyacrylamide gel electrophoresis and Sephadex G-150 gel filtration. The enzyme was most active at pH 7.0, 35-37 degrees C. Among the various aldehydes tested, the enzyme was active on methylglyoxal and 4,5-dioxovalerate with Km values of 1.25 and 0.87 mM, respectively. The activity of the enzyme was completely inhibited by Zn2+ at 0.5 mM. An equimolar amount of EDTA (0.5 mM) protected the enzyme from inactivation by Zn2+. EDTA competitively (K1 = 1.3 mM) inhibited the activity of the enzyme. Fe2+ was a potent activator for the enzyme, the activation being approximately 2.4-fold at 0.5 mM.     

Purification, crystallization and properties of triacylglycerol lipase from Pseudomonas fluorescens.

Triacylglycerol lipase of Pseudomonas fluorescens was purified from the crude enzyme by ammonium sulfate precipitation and chromatographies on Sephadex G-75 and DEAE-cellulose. The crystallization of the lipase was successfully carried out. The purified lipase was demonstrated to be homogenous on disc electrophoresis and its molecular weight was calculated to be 32 000 by gel filtration. The optimum pH for hydrolysis of sesame oil was 7.0. The enzyme was stable up to 40 degrees C under the condition of pH 7.0 for 30 min and had more than 80% of the remaining activity between pH 5.0--11.0 at 37 degrees C for 60 min. The lipase was strongly inhibited by iodine and partially inhibited by FeCl3 and N-bromosuccinimide, and showed the most activity on tricaproyglycerol, among the triacylglycerols used.     

Production, Purification, and Characterization of alpha-Galactosidase from Monascus pilosus

A Monascus pilosus strain was selected for production of intracellular alpha-galactosidase. Optimum conditions for mycelial growth and enzyme induction were determined. Galactose was one of the best enzyme inducers. The enzyme was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography and was demonstrated to be homogeneous by slab gel electrophoresis. The molecular weight of this enzyme, estimated by gel filtration, was about 150,000. The optimum conditions for the enzyme reaction was pH 4.5 to 5.0 at 55 degrees C. The purified enzyme was stable at 55 degrees C or below and in buffer at pH 3 to 8. The activity was inhibited by mercury, silver, and copper ions. The kinetics of this enzyme, with p-nitrophenyl-alpha-d-galactoside as substrate, was determined: K(m) was about 0.8 mM, and V(max) was 39 mumol/min per mg of protein. Enzymatic hydrolysis of melibiose, raffinose, and stachyose was analyzed by thin-layer chromatography.     

5'-Nucleotidase from rat heart

5'-Nucleotidase has been extracted from rat heart and purified to apparent homogeneity. The enzyme is a glycoprotein. Gel electrophoresis in the presence of sodium dodecyl sulfate indicates that the apparent molecular weight of the subunit is 74 000 at several different gel concentrations. Cross-linking of the native enzyme with dimethylpimelimidate followed by gel electrophoresis shows that the enzyme is a dimer. The enzyme hydrolyzes all nucleoside 5'-monophosphates tested. A comparison of Vmax/Km for 14 different substrates shows that AMP is the best substrate. The enzyme shows lowest Km values for AMPS, AMP, isoAMP, GMP, and IMP. It shows no activity with nucleoside 2'- and 3'-monophosphates, sugar phosphates, and p-nitrophenyl phosphate, even when tested at high enzyme concentrations. The optimum activity of the enzyme occurs at pH 7.5 with AMP as substrate. Above this pH, buffer ions affect the activity in a complex manner, a second optimum being observed under some conditions. Magnesium ions activate the enzyme above pH 7.5 in the presence of some buffer ions but not of others. Magnesium ions show only a slight activation when the reaction is run in diethanolamine buffer, pH 9.5, at 30 degrees C; the activation in this buffer is considerably greater when the reaction is run at 37 degrees C. The enzyme is strongly inhibited by free ADP, maximum inhibition occurring below pH 6. The ADP inhibition is diminished as the pH is raised above 6, becoming negligible above pH9. The enzyme is inhibited by EDTA. The inhibition is partially reversed when the EDTA is removed from the enzyme by gel filtration. This as well as other evidence indicates that the enzyme contains a tightly bound metal ion.     

Metabolism of 2-oxoaldehydes in yeasts. Purification and characterization of lactaldehyde dehydrogenase from Saccharomyces cerevisiae

NAD-dependent lactaldehyde dehydrogenase, catalyzing an oxidation of lactaldehyde to lactate, was purified approximately 70-fold from cell extracts of Saccharomyces cerevisiae with a 28% yield of activity. The enzyme was homogeneous on polyacrylamide gel electrophoresis. The relative molecular mass of the enzyme was estimated to be 40 000 on Sephadex G-150 column chromatography and on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.5, 60 degrees C and specifically oxidized L-lactaldehyde to L-lactate in the presence of NAD. The Km values for L-lactaldehyde and NAD were 10 mM and 2.9 mM, respectively. The purest enzyme was extremely unstable and almost completely inactivated during storage at -20 degrees C, pH 7.5. For the reactivation of the enzyme, halide ions such as Cl-, I- and Br- were required.