Isolation and biochemical characteristics of a molecular form of epididymal acid phosphatase of boar seminal plasma

The fluid of boar epididymis is characterized by a high activity of acid phosphatase (AcP), which occurs in three molecular forms. An efficient procedure was developed for the purification of a molecular form of epididymal acid phosphatase from boar seminal plasma. We focused on the epididymal molecular form, which displayed the highest electrophoretic mobility. The purification procedure (dialysis, ion exchange chromatography, affinity chromatography and hydroxyapatite chromatography) used in this study gave more than 7000-fold purification of the enzyme with a yield of 50%. The purified enzyme was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified molecular form of the enzyme is a thermostable 50kDa glycoprotein, with a pI value of 7.1 and was highly resistant to inhibitors of acid phosphatase when p-nitrophenyl phosphate was used as the substrate. Hydrolysis of p-nitrophenyl phosphate by the purified enzyme was maximally active at pH of 4.3; however, high catalytic activity of the enzyme was within the pH range of 3.5-7.0. Kinetic analysis revealed that the purified enzyme exhibited affinity for phosphotyrosine (K(m)=2.1x10(-3)M) and was inhibited, to some extent, by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor. The N-terminal amino acid sequence of boar epididymal acid phosphatase is ELRFVTLVFR, which showed 90% homology with the sequence of human, mouse or rat prostatic acid phosphatase. The purification procedure described allows the identification of the specific biochemical properties of a molecular form of epididymal acid phosphatase, which plays an important role in the boar epididymis.     

Purification and characterization of a protein tyrosine acid phosphatase from boar seminal vesicle glands

A protein tyrosine phosphatase (PTPase) with acid phosphatase activity was purified (500-fold) from the fluid of boar seminal vesicles. Preparative purification was performed with a 3-step procedure, employing FPLC S-Sepharose Fast Flow, Mono Q and Superdex 75 column. Protein tyrosine acid phosphatase (PTAPase) was homogeneous by polyacrylamide gel electrophoresis (PAGE, SDS-PAGE). PTAPase is a glycoprotein which has a molecular weight of about 41-42 kDa. This enzyme was maximally active at pH 5.5, and its thermostability was less than 80 degrees C. The K(m) value for p-nitrophenylphosphate, a specific synthetic substrate, was 0.87 x 10(-3)M, however, higher substrate specificity was shown when phosphotyrosine (K(m)=0.37 x 10(-3)M) and protein fragments, such as gastrin (K(m)=0.0032 x 10(-3)M) and hirudin (K(m)=0.0075 x 10(-3)M), were used as substrates. Activity of PTAPase was inhibited by dephostatin, molybdate and orthovanadate by 100, 95 and 70%, respectively, when phosphotyrosine was used as the substrate. Immunofluorescence study has shown that the seminal vesicles are the only source of PTAPase in boar seminal plasma.     

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 properties of phospholipase A2 from human seminal plasma

The soluble Ca2+-dependent phospholipase A2 (EC 3.1.1.4) was purified 6500-fold with a yield of about 20% from human seminal plasma. The successive purification steps comprised gel filtration, affinity chromatographies and micropartition. The final preparation consisted of two proteins in about equal quantities with molecular weights of 12000 and 14000, according to SDS-polyacrylamide slab gel electrophoresis. As yet these two proteins can not be separated without complete loss of activity. Apparent kinetic parameters have been determined for the purified preparation with different substrates (Vmax = 494 U/mg, and Km = 1.25 X 10(-4) M long-chain phosphatidylethanolamine; Vmax = 7.4 U/mg, and Km = 2.5 X 10(-5) M long-chain phosphatidylcholine; Vmax = 7196 U/mg and Km = 8.32 X 10(-4) M dioctanoylphosphatidylcholine). The enzymatic activity was not affected by diisopropylfluorophosphate and thiol reagents but it was inhibited by higher concentrations of nonionic and ionic (except taurocholate) detergents and by the alkylating reagent p-bromophenacyl bromide. Although the seminal enzyme functionally strongly resembles the pancreatic phospholipase A2, no immunochemical relationship was observed; anti-pancreatic phospholipase A2 IgGs did not inhibit seminal phospholipase A2. Similarly, partially purified phospholipase A2 from horse seminal fluid was not affected by antibodies raised against horse pancreatic phospholipase A2.     

A major phosphotyrosyl-protein phosphatase from bovine heart is associated with a low-molecular-weight acid phosphatase

The phosphotyrosyl [Tyr(P)]-immunoglobulin G (IgG) phosphatase activity in the extracts of bovine heart, bovine brain, human kidney, and rabbit liver can be separated by DEAE-cellulose at neutral pH into two fractions. The unbound fraction exhibits a higher activity at acidic than neutral pH while the reverse is true for the bound fraction. Of all tissues examined, the Tyr(P)-IgG phosphatase activity in the unbound fraction measured at pH 5.0 is higher than that in the bound fraction measured at pH 7.2. The acid Tyr(P)-IgG phosphatase activity has been extensively purified from bovine heart. It copurified with an acid phosphatase activity (p-nitrophenyl phosphate (PNPP) as a substrate) throughout the purification procedure. These two activities coelute from various ion-exchange and gel filtration chromatographies and comigrate on polyacrylamide gel electrophoresis, indicating that they reside on the same protein molecule. The phosphatase has a Mr = 15,000 by gel filtration and exhibits an optimum between pH 5.0 and 6.0 when either Tyr(P)-IgG-casein or PNPP is the substrate. It is highly specific for Tyr(P)-protein with little activities toward phosphoseryl [Ser(P)]- or phosphothreonyl [Thr(P)]-protein. The enzyme activities toward Tyr(P)-casein and PNPP are strongly inhibited by microM molybdate and vanadate but insensitive to inhibition by L(+)-tartrate, NaF, or Zn2+. The molecular and catalytic properties of the acid Tyr(P)-protein phosphatase purified from bovine heart are very similar to those of the low-molecular-weight acid phosphatases of Mr = 14,000 previously identified and purified from the cytosolic fraction of human liver, placenta, and other animal tissues.     

Purification and characterization of phosphoglycerate mutase from methanol-grown Hyphomicrobium X and Pseudomonas AM1.

Phosphoglycerate mutase has been purified from methanol-grown Hyphomicrobium X and Pseudomonas AMI by acid precipitation, heat treatment, ammonium sulphate fractionation, Sephadex G-50 gel filtration and DEAE-cellulose column chromatography. The purification attained using the Hyphomicrobium X extract was 72-fold, and using the Pseudomonas AMI extract, 140-fold. The enzyme purity, as shown by analytical polyacrylamide gel electrophoresis, was 50% from Hyphomicrobium X and 40% from Pseudomonas AMI. The enzyme activity was associated with one band. The purified preparations did not contain detectable amounts of phosphoglycerate kinase, phosphopyruvate hydratase, phosphoglycerate dehydrogenase or glycerate kinase activity. The molecular weight of the enzymic preparation was 32000 +/- 3000. The enzyme from both organisms was stable at low temperatures and, in the presence of 2,3-diphosphoglyceric acid, could withstand exposure to high temperatures. The enzyme from Pseudomonas AMI has a broad pH optimum at 7-0 to 7-6 whilst the enzyme from Hyphomicrobium X has an optimal activity at pH 7-3. The cofactor 2,3-diphosphoglyceric acid was required for maximum enzyme activity and high concentrations of 2-phosphoglyceric acid were inhibitory. The Km values for the Hyphomicrobium X enzyme were: 3-phosphoglyceric acid, 6-0 X 10(-3) M: 2-phosphoglyceric acid, 6-9 X 10(-4) M; 2,3-diphosphoglyceric acid, 8-0 X 10(-6) M; and for the Pseudomonas AMI ENzyme: 3-4 X 10(-3) M, 3-7 X 10(-4) M and 10 X 10(-6) M respectively. The equilibrium constant for the reaction was 11-3 +/- 2-5 in the direction of 2-phosphoglyceric acid to 3-phosphoglyceric acid and 0-09 +/- 0-02 in the reverse direction. The standard free energy for the reaction proceeding from 2-phosphoglyceric acid to 3-phosphoglyceric acid was -5-84 kJ mol(-1) and in the reverse direction +5-81 kJ mol(-1).     

Isolation and biochemical characteristics of a molecular form of epididymal acid phosphatase of boar seminal plasma

The fluid of boar epididymis is characterized by a high activity of acid phosphatase (AcP), which occurs in three molecular forms. An efficient procedure was developed for the purification of a molecular form of epididymal acid phosphatase from boar seminal plasma. We focused on the epididymal molecular form, which displayed the highest electrophoretic mobility. The purification procedure (dialysis, ion exchange chromatography, affinity chromatography and hydroxyapatite chromatography) used in this study gave more than 7000-fold purification of the enzyme with a yield of 50%. The purified enzyme was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified molecular form of the enzyme is a thermostable 50 kDa glycoprotein, with a pI value of 7.1 and was highly resistant to inhibitors of acid phosphatase when p-nitrophenyl phosphate was used as the substrate. Hydrolysis of p-nitrophenyl phosphate by the purified enzyme was maximally active at pH of 4.3; however, high catalytic activity of the enzyme was within the pH range of 3.5–7.0. Kinetic analysis revealed that the purified enzyme exhibited affinity for phosphotyrosine (K_m = 2.1 × 10⁻³ M) and was inhibited, to some extent, by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor. The N-terminal amino acid sequence of boar epididymal acid phosphatase is ELRFVTLVFR, which showed 90% homology with the sequence of human, mouse or rat prostatic acid phosphatase.The purification procedure described allows the identification of the specific biochemical properties of a molecular form of epididymal acid phosphatase, which plays an important role in the boar epididymis.     

Partial purification and characterization of phosphotyrosyl-protein phosphatase from Ehrlich ascites tumor cells

We have previously described a phosphotyrosylprotein phosphatase in membrane vesicles from human epidermoid carcinoma A431 cells which is inhibited by micromolar concentration of Zn2+ and is insensitive to ethylenediaminetetraacetic acid (EDTA) and NaF [Brautigan, D. L., Bornstein, P., & Gallis, B. (1981) J. Biol. Chem. 256, 6519-6522]. Here we present the identification and partial purification of a similar enzyme from lysates of Ehrlich ascites tumor cells. the enzyme was purified by using diethylaminoethyl-Sephadex, Zn2+ affinity, and Sephadex G-75 chromatography. During purification, the phosphatase was separated into at least three fractions, all of which exhibited very similar properties and an apparent molecular weight of 40 000 upon gel filtration. The enzyme dephosphorylated phosphotyrosine (P-Tyr)-containing carboxymethylated and succinylated (CM-SC) phosphorylase with an apparent Km of 0.8 microM, as well as P-Tyr containing casein and epidermal growth factor (EGF) receptor kinase, but did not dephosphorylate P-Ser-phosphorylase. The phosphatase was inhibited by Zn2+ at micromolar concentrations (K0.5 with EGF receptor kinase = 5 X 10(-6) M; with CM-SC phosphorylase = 3.3 X 10(-5) M) but not by millimolar concentrations of EDTA and NaF. No inhibition was seen with 1 mM tetramisole, a specific inhibitor of alkaline phosphatases. P-Tyr inhibited the enzyme by 50% at 0.4 X 10(-3) M, while Tyr, Pi, PPi, and p-nitrophenyl phosphate, an excellent substrate for alkaline phosphatases and structurally very similar to P-Tyr, exerted partial inhibition at concentrations above 10(-3) M. The pH optimum was found to be 6.5-7, depending on the substrate used. Very little activity was seen below pH 5 and above pH 8.5. These properties clearly distinguish this enzyme from alkaline phosphatases, as well as the neutral and acidic protein phosphatases so far described, and therefore define it as a new enzyme of the phosphatase family--a phosphotyrosyl-protein phosphatase.     

Purification and properties of phenylalanyl-tRNA-synthetase from Escherichia coli MRE-600

A preparative scale method for isolation of highly purified phenylalanyl-tRNA synthetase from E. coli MRE-600 was developed. It consists of cell destroying, nucleic acid precipitation with streptomycine sulfate, fractionation with ammonium sulfate followed by chromatography on different carriers (Sephadex G-200, DEAE-cellulose, DEAE-Sephadex A-50, and hydroxyapatite). The mode of cell destroying was found to affect the process of the further enzyme purification. The phenylalanyl-tRNA synthetase was purified 540-fold, with recovery being 20.6% and the specific activity - 540 units per mg protein. The enzyme content in the purified preparation was 80-90% judging by electrophoresis in PAAG. The molecular weights of the subunits determined by electrophoresis under denaturative conditions were found to be 102,000 +/- 4000 (beta) and 42,000 +/- 2000 (alpha). The molecular weight of the native enzyme determined by gel filtration through Sephadex G-200 and electrophoresis at varied concentrations of polyacrylamide was found to be 340,000 +/- 20,000. The Km values for tRNA, ATP and phenylalanine in the aminoacylation reaction are equal to 5.4 X 10(-7) M, 1,9 X 10(-4) M, and 3.7 X 10(-6) M, respectively.     

Polyamine-mediated turnover of ornithine decarboxylase in Chinese-hamster ovary cells.

The major secreted isoenzyme of human prostatic acid phosphatase (PAcP) (EC 3.1.3.2), which catalyses p-nitrophenyl phosphate (PNPP) hydrolysis at acid pH values, was found to have phosphotyrosyl protein phosphatase activity since it dephosphorylated three different phosphotyrosine-containing protein substrates. Several lines of evidence are presented to show that the phosphotyrosyl phosphatase and PAcP are the same enzyme. A highly purified PAcP enzyme preparation which contains a single N-terminal peptide sequence was used to test for the phosphotyrosyl phosphatase activity. Both activities comigrated during gel filtration by high performance liquid chromatography. Phosphotyrosyl phosphatase activity and PNPP acid phosphatase activity exhibited similar sensitivities to different effectors. Both phosphatase activities showed the same thermal stability. Specific anti-PAcP antibody reacted to the same extent with both phosphatase activities. PNPP acid phosphatase activity was competitively inhibited by the phosphotyrosyl phosphatase substrate. To characterize further the phosphotyrosyl phosphatase activity, the Km values using different phosphoprotein substrates were determined. The apparent Km values for phosphorylated angiotensin II, anti-pp60src immunoglobulin G and casein were in the nM range for phosphotyrosine residues, which was about 50-fold lower than the Km for phosphoserine residues in casein.     

Purification and characterization of acid phosphatase from cotyledons of germinating soybean seeds

Soybean acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) was completely separated from phytase (EC 3.1.3.8) isolated from cotyledons of germinating seeds and purified to homogeneity. A four-step purification regimen consisting of ammonium sulfate fractionation, and ion-exchange, affinity, and chromatofocusing gel chromatographies was employed to achieve a homogeneous preparation. Acid phosphatase activity appeared as a major band of the three forms of acid phosphatase identified on native gels. The purified enzyme had a molecular weight of 53,000 when electrophoresed on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a molecular weight of 53,000 from its mobility in a Fracto-gel TSK HW-50F gel permeation column. The molar extinction coefficient of the enzyme at 278 nm was estimated to be 4.2 X 10(4) M-1 cm-1. The isoelectric point of the protein, as revealed by chromatofocusing, was about 6.7. The optimal pH for activity, like other plant acid phosphatases, was 5.0. While the enzyme failed to accommodate phytate as a substrate, the enzyme did exhibit a broad substrate selectivity. The affinity of the enzyme for p-nitrophenyl phosphate was high (Km = 70 microM), and activity was competitively inhibited by orthophosphate (Ki = 280 microM). The estimated catalytic turnover number (Kcat) of the enzyme for p-nitrophenyl phosphate was about 430 per second. Although the purified enzyme was stable at 0 degrees C and exhibited maximum catalytic activity at 60 degrees C, thermal inactivation studies indicated that the enzyme lost 100% activity after treatment at 68 degrees C for 10 min.     

Purification of human tissue plasminogen activator with Erythrina trypsin inhibitor

Seeds of the legume Erythrina latissima contain a 20,000-dalton, single-chain protein that has been shown to inhibit the amidolytic activity of trypsin and tissue plasminogen activator. It had no comparable effect on urokinase. IC50 values of 1.1 X 10(-7) M for tissue plasminogen activator and 6.9 X 10(-10) M for trypsin were determined by titration. When coupled to agarose, the Erythrina inhibitor provided an effective reagent for affinity purification of tissue plasminogen activator from melanoma cell-conditioned tissue culture medium. Using this as a single-step procedure, 270-fold purified enzyme was reproducibly obtained with yields of 90% or greater. Both one- and two-chain forms of tissue plasminogen activator were purified. The enzyme migrated, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as a predominant 72,000-dalton doublet with lesser amounts of immunochemically similar, 115,000- and 68,000-dalton components.     

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.     

Purification and characterization of phytase from cotyledons of germinating soybean seeds

Soybean phytase (myo-inositol-hexakisphosphate phosphohydrolase; EC 3.1.3.8) was purified from 10-day-old germinating cotyledons using a four-step purification scheme. Phytase was separable from the major acid phosphatase present, and stained as a minor band of the three acid phosphatases detectable by activity staining after gel electrophoresis. The purified enzyme exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 59 and 60 KDa. The molar extinction coefficient of the enzyme at 280 nm was estimated to be 7.5 X 10(4) M-1 cm-1. The isoelectric point of phytase, as judged by the elution profile on chromatofocusing, was about 5.5. The enzyme was totally absorbed to a Procion Red HE3B column and eluted as a single protein component at a salt concentration of 250-300 mM. The enzyme possessed a high affinity for phytic acid (apparent Km = 48 microM), and was strongly inhibited by phosphate (apparent Ki = 18 microM), vanadate, and fluoride. Characteristic of other plant phytases, the pH and temperature optima were 4.5-4.8 and 55 degrees C, respectively.     

Purification and characterization of hemolymph juvenile hormone esterase from the cricket, Gryllus assimilis.

Juvenile hormone esterase (JHE) from the serum of the cricket, Gryllus assimilis, was purified to homogeneity in a four-step procedure involving polyethylene glycol precipitation, hydrophobic interaction FPLC, and ion exchange FPLC. This procedure could be completed in 4 days and resulted in a greater than 900-fold purification with greater than 30% recovery. The purified enzyme exhibited a single band on a silver-stained SDS PAGE gel and had an apparent subunit molecular mass of 52 kDa. The native subunit molecular mass, determined by gel permeation FPLC, was 98 kDa, indicating that JHE from Gryllus assimilis is a dimer of two identical or similar subunits. The turnover number of the purified enzyme (1.41 s(-1)), K(M(JH-III)) (84 +/- 12 nM) of nearly-purified enzyme, and k(cat)/K(M) (1.67 x 10(7) s(-1) M(-1)) were similar to values reported for other well-established lepidopteran and dipteran JHEs. JHE from Gryllus assimilis was strongly inhibited by the JHE transition-state analogue OTFP (octylthio-1,1,1-trifluoro-2-propanone; I(50) = 10(-7) M) and by DFP (diisopropyl fluorophosphate; I(50) = 10(-7) M). The shapes of the inhibition profiles suggest the existence of multiple binding sites for these inhibitors or multiple JHEs that differ in inhibition. Isoelectric focusing separated the purified protein into 4 isoforms with pIs ranging from 4.7-4.9. N-terminal amino acid sequences (11-20 amino acids) of the isoforms differed from each other in 1-4 positions, suggesting that the isoforms are products of the same or similar genes. Homogeneously purified JHE hydrolyzed alpha-napthyl esters, did not exhibit any detectable acetylcholinesterase, acid phosphatase, or aminopeptidase activity, and exhibited only very weak alkaline phosphatase activity. JHE exhibited a low (11 microM) K(M) for long-chain alpha-naphthyl esters, indicating that JHE may have physiological roles other than the hydrolysis of JH-III. Purification of JHE represents a key step in our attempts to identify the molecular causes of genetically-based variation in JHE activity in G. assimilis. This represents the first homogeneous purification of JHE from a hemimetabolous insect.     

Purification and properties of cathepsin D from the mammary glands of lactating rabbits

Cathepsin D was purified from the lactating rabbit mammary gland by a rapid procedure, which included fractionation with (NH4)2SO4, acid precipitation, double affinity chromatography on pepstatin-Sepharose 4B and gel filtration on Sephadex G-100, resulting in approximately 360-fold purification of the enzyme over the homogenate and approximately 16% recovery. After isoelectric focusing, the enzyme dissociated into four (pI 5.8, 6.3, 6.5 and 7.2) multiple forms, but appeared homogeneous on polyacrylamide gel electrophoresis. Cathepsin D has a Mr of 45 kDa as determined by Sephadex G-100 column chromatography. On sodium dodecylsulfate/polyacrylamide gel electrophoresis the enzyme gave a single protein band, corresponding to Mr of 45 kDa. The amino acid composition of the enzyme is similar to that of cathepsins D from other tissues. A single N-terminal amino acid was glycine. Cathepsin D contains 6.4% carbohydrates consisting of mannose, galactose, fucose and glucosamine at a ratio of 3:9:2:2. Cathepsin D is inhibited by pepstatin with Ki of 2.5 X 10(-9) M and irreversibly by N-diazoacetyl-N'-2.4-dinitrophenyl-ethylene diamine. The enzyme hydrolyzes bovine hemoglobin with the maximal activity at pH 3.0 with Km = 10(-5) M and HLeu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe with Km = 4 X 10(-5) M and Rcat = 0.95 s-1. The major cleavage sites were Leu15-Tyr16, Phe24-Phe25 and Phe25-Tyr26 during hydrolysis of the oxidized insulin B-chain by cathepsin D.     

Purification and properties of glutamate dehydrogenase from a thermophilic bacillus.

A 250- to 300-fold purification of a nicotinamide adenine denucleotide phosphate (NADP)-dependent glutamate dehydrogenase (GDH, E.C. 1.4.1.4) with a yield of 60% from a thermophilic bacillus is described. More than one NADP-specific GDH was detected by polyacrylamide gel electrophoresis. The enzyme is of high molecular weight (approximately 2 X 10-6), similar to that of the beef and frog liver GDH. The pI of the thermophilic GDH is at pH 5.24. The enzyme is highly thermostable at the pH range of 5.8 to 9.0. The purified GDH, unlike the crude enzyme, was very labile at subzero temperatures. An unidentified factor(s) from the crude cell-free extract prevented the inactivation of the purified GDH at -70 C. Various reactants of the GDH system and D-glutamate also protected, to some extent, the enzyme from inactivation at -70 C. From the Michaelis constants for glutamate (1.1 X 10-2M), NADP (3 X 10-4M), ammonia (2.1 X 10-2M), alpha-ketoglutarate (1.3 X 10-3M), and reduced NADP (5.3 X 10-5M), it is suggested that the enzyme catalyzes in vivo the formation of glutamate from ammonia and alpha-ketoglutarate. The amination of alpha-ketoglutarate and deamination of glutamate by the thermophilic GDH are optimal at the pH values of 7.2 and 8.4, respectively.     

Purification and characterization of, and preparation of an antibody to, transketolase from human red blood cells

Transketolase (sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1) was purified 16 000-fold from human red blood cells, using DEAE-Sephadex A-50, Sephadex G-150, FPLC on Mono P, and Sephadex G-100. The purified enzyme migrated as a single protein band on SDS-polyacrylamide gel electrophoresis. The FPLC step resolved transketolase into three peaks, designated I, II and III. From results of re-FPLC on Mono P, SDS-polyacrylamide gel electrophoresis, gel filtration, catalytic studies, amino acid analysis and immunological studies, it was concluded that I, II and III were originally the same protein, modified during storage and purification. Transketolase had a subunit (Mr 70 000) and appeared to be composed of two identical subunits. 1 mol of subunit contained 0.9 mol of thiamine pyrophosphate. The pH optimum of the reaction lay within the range 7.6-8.0, and the Km values were determined to be 1.5 X 10(-4) M for xylulose 5-phosphate and 4.0 X 10(-4) M for ribose 5-phosphate. Hg2+ and p-chloromercuribenzoate inhibited the enzyme reaction, and the inhibition of the latter disappeared upon the addition of cysteine. Thiamine and its phosphate esters did not, but cysteine (1 X 10(-2) M) and ethanol (10% and 1% v/v) did activate the enzyme reaction. Antibody prepared to II bound all forms of transketolase in the hemolysate, but inhibited the reaction only about 20%.     

Large scale purification of myo-inositol monophosphate phosphatase from porcine brains

myo-Inositol monophosphate phosphatase (IMPase) has been purified 888-fold to apparent homogeneity from procine brains. The purification procedure involves: homogenization, ammonium sulfate fractionation, and a number of ion-exchange and gel-filtration chromatography steps. The purified enzyme exhibited a final specific activity of 932 nmol . min(-1) . mg(-1). The molecular mass of the enzyme was estimated to be 29kDa by SDS poly-acrylamide gel electrophoresis and 58 +/- 5 kDa by HPLC gel filtration in 10mM Tris-HCI, pH 7.4. Kinetic measurements have shown that the apparent K(m) value of the phosphatase for the utilization of inositol-1-phosphate and beta-glycerol phosphate are 3.20 x 10(-4) and 8 x 10(-3) M, respectively. Similar to the same enzyme isolated from bovine brains, the porcine brain enzyme has been shown to be inhibited by lithium. The K(1) was determined to be 6.38 x 10(-4) M and the inhibition is uncompetitive. (c) 1995 John Wiley & Sons, Inc.     

Purfication and properties of a specific isoflavone 7-O-glucoside-6'-malonate malonyestrase from roots of chickpea (Cicer arietinum L.)

Protein extracts from roots of chickpea (Cicer arietinum L.) plants contained high esterase activity hydrolyzing malonate hemiesters of isoflavone 7-O-glucosides. Using 5,7-dihydroxy-4'-methoxyisoflavone (biochanin A) 7-O-glucoside-6"-malonate as a substrate, a specific malonylesterase was purified about 700-fold to near homogeneity. The purified enzyme possesses an extremely low enzyme activity with synthetic esterase substrates. Various putative nonspecific esterases, as tested with alpha-naphthylacetate, were removed during enzyme purification. The malonylesterase demonstrated a very high molecular mass in gel chromatography and in sedimentation analyses with sucrose gradients (greater than or equal to 2 X 10(6)). Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis pointed to a single subunit of 32,000. The catalyzed reaction showed a pH optimum at 7.5 and a temperature optimum between 30 and 35 degrees C. The apparent Km for biochanin A 7-O-glucoside-6"-malonate was (4.2 +/- 1.2) X 10(-4) M. The malonylesterase was insensitive to the esterase inhibitors eserine and neostigmine (10(-3) M) as well as phenylmethylsulfonyl fluoride, paraoxon, and diisopropylfluorophosphate (10(-4) M). On the other hand enzyme activity was totally inhibited by Hg2+ ions (10(-5) M) and p-hydroxymercuribenzoate (10(-4) M), whereas iodoacetamide (10(-6)-10(-4) M) inhibited only partially. Di- and tricarboxylic acids strongly stimulated enzyme activity at 10(-2) M. These properties indicate that the malonylesterase from chickpea roots greatly differs from other known esterases. The possible biological function of the specific malonylesterase is discussed in relation to isoflavone conjugate metabolism in chickpea.