Effects of temperature on the mitochondrial malate dehydrogenase of adult muscle of Toxocara canis

Purified mitochondrial malate dehydrogenase isoenzyme (m-MDH) of Toxocara canis muscle presented maximum activity at 48 degrees C. A clear change in slope of the Arrhenius plot was observed. The energy of activation calculated for the catalytic process showed values of 3.2 kcal/mol and 10.5 kcal/mol. Thermal inactivation of m-MDH showed that it is more thermolabile than the s-isoenzyme. The inactivation of the enzyme by heat could be reduced at least in part by the addition of 0.1 mM NADH. The heat denaturation showed to be a first-order process. The rate constant (k) was calculated as being of the order of 5.28 X 10(-4) s-1 at 40 degrees C. The activation energy for the heat inactivation process was 16.45 kcal/mol between 30 degrees C and 40 degrees C and 13.79 kcal/mol between 40 degrees C and 48 degrees C.     

Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus Talaromyces emersonii.

Mitochondrial malate dehydrogenase (m-MDH; EC 1.1.1.37), from mycelial extracts of the thermophilic, aerobic fungus Talaromyces emersonii, was purified to homogeneity by sequential hydrophobic interaction and biospecific affinity chromatography steps. Native m-MDH was a dimer with an apparent monomer mass of 35 kDa and was most active at pH 7.5 and 52 degrees C in the oxaloacetate reductase direction. Substrate specificity and kinetic studies demonstrated the strict specificity of this enzyme, and its closer similarity to vertebrate m-MDHs than homologs from invertebrate or mesophilic fungal sources. The full-length m-MDH gene and its corresponding cDNA were cloned using degenerate primers derived from the N-terminal amino acid sequence of the native protein and multiple sequence alignments from conserved regions of other m-MDH genes. The m-MDH gene is the first oxidoreductase gene cloned from T. emersonii and is the first full-length m-MDH gene isolated from a filamentous fungal species and a thermophilic eukaryote. Recombinant m-MDH was expressed in Escherichia coli, as a His-tagged protein and was purified to apparent homogeneity by metal chelate chromatography on an Ni2+-nitrilotriacetic acid matrix, at a yield of 250 mg pure protein per liter of culture. The recombinant enzyme behaved as a dimer under nondenaturing conditions. Expression of the recombinant protein was confirmed by Western blot analysis using an antibody against the His-tag. Thermal stability studies were performed with the recombinant protein to investigate if results were consistent with those obtained for the native enzyme.     

Purification and properties of mitochondrial malate dehydrogenase from unfertilized eggs of the sea urchin, Anthocidaris crassispina

Purification and characterization of mitochondrial malate dehydrogenase [EC 1.1.1.37] from unfertilized eggs of the sea urchin, Anthocidaris crassispina, are described. The purification method consisted of dextran sulfate fractionation, Blue Dextran Sepharose chromatography, Phenyl-Sepharose hydrophobic chromatography and DEAE-cellulose chromatography. The enzyme was purified 771-fold with a 7% yield from the crude extract. The purified enzyme appeared homogeneous on polyacrylamide gel electrophoresis under both native and denatured conditions. After incubation at 45 degrees C for 50 min, the enzyme lost about 90% of its activity. In the presence of NADH, however, the enzyme was protected against the heat denaturation. The native enzyme had a molecular weight of about 65,000 and probably consisted of two identical subunits. In the reduction of oxaloacetate with NADH, a broad optimum pH ranging from 8.2 to 9.4 was found with 50 mM Tris-HCl and glycine-NaOH buffers. Sodium phosphate buffer apparently activated the enzyme. The apparent Km values for oxaloacetate and NADH were 19 microM and 30 microM, respectively. The optimum pH for malate oxidation with NAD+ was 10.2 in 50 mM NaHCO3-Na2CO3 buffer. The apparent Km values for malate and NAD+ were 7.0 mM and 0.6 mM, respectively. Zinc ion, sulfite ion, p-chloromercuriphenylsulfonate and adenine nucleotides strongly inhibited the enzyme.     

Purification and characterization of the malate dehydrogenase from Streptomyces aureofaciens

The malate dehydrogenase (MDH) from Streptomyces aureofaciens was purified to homogeneity and its physical and biochemical properties were studied. Its amino-terminal sequence perfectly matched the amino-terminal sequence of the MDH from Streptomyces atratus whose biochemical characteristics have never been determined. The molecular mass of the native enzyme, estimated by size-exclusion chromatography, was 70 kDa. The protein was a homodimer, with a 38-kDa subunit molecular mass. It showed a strong specificity for NADH and was much more efficient for the reduction of oxaloacetate than for the oxidation of malate, with a pH optimum of 8. Unlike MDHs from other sources, it was not inhibited by excess oxaloacetate. This first complete functional characterization of an MDH from Streptomyces shows that the enzyme is very similar in many respects to other bacterial MDHs with the notable exception of a lack of inhibition by excess substrate.     

Purification and properties of a NAD-linked 1,2-propanediol dehydrogenase from propane-grown Pseudomonas fluorescens NRRL B-1244

NAD-dependent 1,2-propanediol dehydrogenase (EC 1.1.1.4) activity was detected in cell-free crude extracts of various propane-grown bacteria. The enzyme activity was much lower in 1-propanol-grown cells than in propane-grown cells of Pseudomonas fluorescens NRRL B-1244, indicating that the enzyme may be inducible by metabolites of propane subterminal oxidation. 1,2-Propanediol dehydrogenase was purified from propane-grown Ps. fluorescens NRRL B-1244. The purified enzyme fraction shows a single-protein band upon acrylamide gel electrophoresis and has a molecular weight of 760,000. It consists of 10 subunits of identical molecular weight (77,600). It oxidizes diols that possess either two adjacent hydroxy groups, or a hydroxy group with an adjacent carbonyl group. Primary and secondary alcohols are not oxidized. The pH and temperature optima for 1,2-propanediol dehydrogenase are 8.5 and 20-25 degrees C, respectively. The activation energy calculated is 5.76 kcal/mol. 1,2-Propanediol dehydrogenase does not catalyze the reduction of acetol or acetoin in the presence of NADH (reverse reaction). The Km values at 25 degrees C, pH 7.0, buffer solution for 1,2-propan1,2-propanediol dehydrogenase are 8.5 and 20-25 degrees C, respectively. The activation energy calculated is 5.76 kcal/mol. 1,2-Propanediol dehydrogenase does not catalyze the reduction of acetol or acetoin in the presence of NADH (reverse reaction). The Km values at 25 degrees C, pH 7.0, buffer solution for 1,2-propan1,2-propanediol dehydrogenase are 8.5 and 20-25 degrees C, respectively. The activation energy calculated is 5.76 kcal/mol. 1,2-Propanediol dehydrogenase does not catalyze the reduction of acetol or acetoin in the presence of NADH (reverse reaction). The Km values at 25 degrees C, pH 7.0, buffer solution for 1,2-propanediol and NAD are 2 X 10(-2) and 9 X 10(-5) M, respectively. The 1,2-propanediol dehydrogenase activity was inhibited by strong thiol reagents, but not by metal-chelating agents. The amino acid composition of the purified enzyme was determined. Antisera prepared against purified 1,2-propanediol dehydrogenase from propane-grown Ps. fluorescens NRRL B-1244 formed homologous precipitin bands with isofunctional enzymes derived from propane-grown Arthrobacter sp. NRRL B-11315, Nocardia paraffinica ATCC 21198, and Mycobacterium sp. P2y, but not from propane-grown Pseudomonas multivorans ATCC 17616 and Brevibacterium sp. ATCC 14649, or 1-propanol-grown Ps. fluorescens NRRL B-1244. Isofunctional enzymes derived from methane-grown methylotrophs also showed different immunological and catalytic properties.     

Enthalpy of acetylcholine hydrolysis by acetylcholinesterase

Direct microcalorimetric measurements were made of the reaction between acetylcholine chloride and acetylcholinesterase (EC 3.1.1.7) that was extracted from electric eel (Electrophorus electricus) and purified by affinity chromatography. Tris-HCl, sodium phosphate and potassium phosphate were used as buffers and sources of ions for the reaction. At pH 7.2 and in 0.1-0.2 M phosphate buffer, the delta H for acetylcholine hydrolysis was found to be -0.107 kcal/mol (under buffered conditions) and -0.931 kcal/mol under unbuffered conditions (water). At pH 8.0 in 0.1 M Tris-HCl buffer, values greater than -2.5 kcal/mol were obtained, with the highest value of -9.2 kcal/mol being seen with bovine erythrocyte acetylcholinesterase. Tris-HCl buffer at 4 X 10(-2) M enhanced the reaction velocity by 51.2% over that of 4 X 10(-3) M buffer. Enzyme purity, pH and ionic milieu of reaction mixture, and substrate concentration affected the measured delta H value.     

Properties and function of malate enzyme from Dicentrarchus labrax L. liver

Malate enzyme (L-malate:NADP+ oxidoreductase (oxaloacetate decarboxylating, EC 1.1.1.40) has been purified from Dicentrarchus labrax liver to 99% homogeneity by gel filtration, anion exchange and affinity chromatographies. The apparent molecular weight was estimated by gel filtration chromatography to be 148,000. Analysis of the enzyme on sodium dodecyl sulphate polyacrylamide disc gel electrophoresis was shown to be a tetrameric protein. The purified enzyme showed a pH optimum 8.5 (Tris-HCl buffer) and required bivalent cations for catalysis. The temperature-activity relationship for the enzyme showed broken Arrhenius plots with inflexions at 15 and 40 degrees C. Kinetic properties and the effects of some metabolites related to L-malate are studied.     

Kinetics of the reduction of oxaloacetate catalyzed by mitochondrial malate dehydrogenase of Toxocara canis muscle

1. The reaction of reduction of oxaloacetate to L-malate in the presence of NADH catalyzed by mitochondrial malate dehydrogenase (EC 1.1.1.37) of Toxocara canis muscle has been studied. 2. The data obtained in initial velocity experiments as well as those involving product inhibition suggest that the reaction mechanism is of the sequential type with a kinetically significant ternary complex and in which the coenzymes bind to the free enzyme. 3. The kinetic parameters, including the inhibition constant for NADH were estimated by non-linear regression analysis using the appropriate rate equations.     

Heat-stable protease from Pseudomonas fluorescens T16: purification by affinity column chromatography and characterization

A heat-stable extracellular protease from Pseudomonas fluorescens T16, a psychrotroph, was purified by affinity column chromatography on a carbobenzoxy-D-phenylalanine-triethylene tetramine-Sepharose-4B column. The purified enzyme is a monomer with a molecular weight of 38,905 +/- 2,000. In an analytical ultracentrifuge, the Schlieren profile revealed a single symmetrical peak. The sedimentation coefficient was estimated to be 3.93S. Alpha-casein was the preferred substrate, with a Km of 0.05 mM. Heating crude enzyme and purified enzyme in buffer at 50, 90, and 120 degrees C resulted in a rapid initial loss of more than 50% of the initial activity followed by a gradual inactivation which exhibited first-order kinetics. The activation energy for the hydrolysis of casein was calculated to be 3.2 kcal/mol (13.4 kJ/mol).     

Heat-stable protease from Pseudomonas fluorescens T16: purification by affinity column chromatography and characterization.

A heat-stable extracellular protease from Pseudomonas fluorescens T16, a psychrotroph, was purified by affinity column chromatography on a carbobenzoxy-D-phenylalanine-triethylene tetramine-Sepharose-4B column. The purified enzyme is a monomer with a molecular weight of 38,905 +/- 2,000. In an analytical ultracentrifuge, the Schlieren profile revealed a single symmetrical peak. The sedimentation coefficient was estimated to be 3.93S. Alpha-casein was the preferred substrate, with a Km of 0.05 mM. Heating crude enzyme and purified enzyme in buffer at 50, 90, and 120 degrees C resulted in a rapid initial loss of more than 50% of the initial activity followed by a gradual inactivation which exhibited first-order kinetics. The activation energy for the hydrolysis of casein was calculated to be 3.2 kcal/mol (13.4 kJ/mol).     

Purification,catalytic and regulatory properties of malate dehydrogenase from the foot of Patella caerulea (L.)

• 1.1. Malate dehydrogenase has been purified from the foot muscle of Patella caerulea by ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Blue Agarose and gel filtration on Sephadex G-150.
• 2.2. The yield was 23.5% of the initial activity with a final specific activity of 257 U/mg of protein.
• 3.3. The apparent mol. wt of the native enzyme is approx. 75,000 and it consists of two subunits of mol. wts in the range of 36,000–39,000.
• 4.4. The enzyme exhibits hyperbolic kinetics with respect to oxaloacetate, NADH and l-malate. The K_m values were determined to be 0.055 mM for oxaloacetate, 0.010 mM for NADH and 0.37 mM for l-malate. The pH optima are around 8.4 for the reduction of oxaloacetate and 9.2–9.6 for the reduction of oxaloacetate and 9.2–9.6 for the l-malate oxidation. V_max and K_m values for oxaloacetate change in an opposite manner with respect to pH values.
• 5.5. Of the various compounds tested, only α-ketoglutarate, citrate and adenylate phosphates were found to inhibit the enzyme activity.
• 6.6. From the above properties it appears that the reaction of cytoplasmic malate dehydrogenase of P. caerulea foot muscle is a key reaction in the anaerobic pathway and it occurs with the production of malate.

     

Purification and some kinetic properties of carbonic anhydrase from rainbow trout (Oncorhynchus mykiss) liver and metal inhibition

In the present study, carbonic anhydrase (CA) enzyme was purified from rainbow trout (RT) liver with a specific activity of 4318 EUxmg(-1) and a yield of 38% using Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. The overall purification was approximately 2260-fold. To check the purity and determine subunit molecular weight of enzyme, SDS-polyacrylamide gel electrophoresis was performed, which showed a single band and MW of approx. 29.4 kDa. The molecular weight of native enzyme was estimated to be approx. 31 kDa by Sephadex-G 200 gel filtration chromatography. Optimum and stable pH were determined as 9.0 in 1 M Tris-SO(4) buffer and 8.5 in 1 M Tris-SO(4) buffer at 4 degrees C, respectively. The optimum temperature, activation energy (E(a)), activation enthalpy ((DeltaH) and Q(10) from Arrhenius plot for the RT liver CA were 40 degrees C, 2.88 kcal/mol, 2.288 kcal/mol and 1.53, respectively. The purified enzyme had an apparent K(m) and V(max) of 0.66 mM and 0.126 micromol x min(-1) for 4-nitrophenylacetate, respectively. K(cat) of the CA was found to be 32.8 s(-1). The inhibitory effects of low concentrations of different metals (Co(II), Cu(II), Zn(II) and Ag(I)) on CA activity were determined using the esterase method under in vitro conditions. The obtained IC(50) values, 50% inhibition of in vitro enzyme activity, were 0.03 mM for cobalt, 30 mM for copper, 47.1 mM for zinc and 0.01 mM for silver. K(i) values for these substances were also calculated from Linewaever-Burk plots as 0.050 mM for cobalt, 1.950 mM for copper, 7.035 mM for zinc and 2.190 mM for silver respectively and determined that cobalt and zinc inhibit the enzyme a competitive manner and copper and silver inhibit the enzyme in an uncompetitive manner.     

Presence of rhodanese in the cytosolic fraction of the fruit bat (Eidolon helvum) liver

Rhodanese was isolated and purified from the cytosolic fraction of liver tissue homogenate of the fruit bat, Eidolon helvum, by using ammonium sulphate precipitation and CM-Sephadex C-50 ion exchange chromatography. The specific activity was increased 130-fold with a 53% recovery. The K(m) values for KCN and Na(2)S(2)O(3) as substrates were 13.5 +/- 2.2mM and 19.5 +/- 0.7 mM, respectively. The apparent molecular weight was estimated by gel filtration on a Sephadex G-100 column to be 36,000 Da. The optimal activity was found at a high pH (pH 9.0) and the temperature optimum was 35 degrees C. An Arrhenius plot of the heat stability data consisted of two linear segments with a break occurring at 35 degrees C. The apparent activation energy values from these slopes were 11.5 kcal/mol and 76.6 kcal/mol. Inhibition studies on the enzyme with a number of cations showed that Mg(2+), Mn(2+), Ca(2+), and Co(2+) did not affect the activity of the enzyme, but Hg(2+) and Ba(2+) inhibited the enzyme.     

Purification and properties of acyl/alkyl dihydroxyacetone-phosphate reductase from guinea pig liver peroxisomes

The peroxisomal acyl/alkyl dihydroxyacetone-phosphate reductase (EC 1.1.1.101) was solubilized and purified 5500-fold from guinea pig liver. The enzyme could be solubilized by detergents only at high ionic strengths in presence of the cosubstrate NADPH. Peroxisomes, isolated from liver by a Nycodenz step density gradient centrifugation, were first treated with 0.2% Triton X-100 to remove the soluble and a large fraction of the membrane-bound proteins. The enzyme was solubilized from the resulting residue by 0.05% Triton X-100, 1 M KCl, 0.3 mM NADPH, and 2 mM dithiothreitol in Tris-HCl buffer (10 mM) at pH 7.5. The enzyme was further purified after precipitating it by dialyzing out the KCl and then resolubilized with 0.8% octyl glucoside in 1 M KCl (plus NADPH and dithiothreitol). The second solubilized enzyme was purified to homogeneity (370-fold from peroxisomes) by gel filtration in a Sepharose CL-6B column followed by affinity chromatography on an NADPH-agarose gel matrix. NADPH-agarose was prepared by reacting periodate-oxidized NADP+ to adipic acid dihydrazide-agarose and then reducing the immobilized NADP+ with NaBH4. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme showed a single homogeneous band with an apparent molecular weight of 60,000. The molecular weight of the native enzyme was estimated to be 75,000 by size exclusion chromatography. Amino acid analysis of the purified protein showed that hydrophobic amino acid comprised 27% of the molecule. The Km value of the purified enzyme for hexadecyldihydroxyacetone phosphate (DHAP) was 21 microM, and the Vmax value in the presence of 0.07 mM NADPH was 67 mumol/min/mg. The turnover number (Kcat), after correcting for the isotope effect of the cosubstrate NADP3H, was calculated to be 6,000 mol/min/mol of enzyme, assuming the enzyme has a molecular weight of 60,000. The purified enzyme also used palmitoyldihydroxyactone phosphate as a substrate (Km = 15.4 microM, and Vmax = 75 mumol/min/mg). Palmitoyl-DHAP competitively inhibited the reduction of hexadecyl-DHAP, indicating that the same enzyme catalyzes the reduction of both acyl-DHAP and alkyl-DHAP. NADH can substitute for NADPH, but the Km of the enzyme for NADH (1.7 mM) is much higher than that for NADPH (20 microM). The purified enzyme is competitively (against NADPH) inhibited by NADP+ and palmitoyl-CoA. The enzyme is stable on storage at 4 degrees C in the presence of NADPH and dithiothreitol.     

Cationic form of beta-galactosidase in the germinating seeds of Vigna sinensis (Linn) Savi

The cationic form of beta-galactosidase (EC 3.2.1.23) from the germinating seeds of Vigna sinensis has been separated from its other isoforms by DEAE-cellulose (DE-52) column chromatography and further purified by gel filtration and affinity chromatography. Polyacrylamide gel electrophoresis of the purified enzyme imparted a single protein band. The molecular mass of the enzyme as determined by Sephadex G-150 gel filtration is 58,800 Da. The optimum temperature and the optimum pH are 60 degrees C and 4.5, respectively. Most of the metal ions tested were inhibitory to the enzyme activity. The enzyme has Km for p-nitrophenyl beta-D-galactoside and o-nitrophenyl beta-D-galactoside of 0.56 and 2.0 mM, respectively. The Ki values of galactose and lactose are 2.4 and 70.0 mM, respectively. The energy of activation of PNPG for the enzyme is 10.3 kcal/mol.     

Induction, isolation, and some properties of the NADPH-dependent glutamate dehydrogenase from the nonheterocystous cyanobacterium Phormidium laminosum.

The level of the NADPH-dependent glutamate dehydrogenase activity (EC 1.4.1.4) from nitrate-grown cells of the thermophilic non-N2-fixing cyanobacterium Phormidium laminosum OH-1-p.Cl1 could be significantly enhanced by the presence of ammonium or nitrite, as well as by L-methionine-DL-sulfoximine and other sources of organic nitrogen (L-Glu, L-Gln, and methylamine). The enzyme was purified more than 4,400-fold by ultracentrifugation, ion-exchange chromatography, and affinity chromatography, and at 30 degrees C it showed a specific activity of 32.9 mumol of NADPH oxidized per min per mg of protein. The purified enzyme showed no aminotransferase activity and catalyzed the amination of 2-oxoglutarate preferentially to the reverse catabolic reaction. The enzyme was very specific for its substrates 2-oxoglutarate (Km = 1.25 mM) and NADPH (Km = 64 microM), for which hyperbolic kinetics were obtained. However, negative cooperativity (Hill coefficient h = 0.89) and [S]0.5 of 18.2 mM were observed for ammonium. The mechanism of the aminating reaction was of a random type with independent sites. The purified enzyme showed its maximal activity at 60 degrees C (Ea = 5.1 kcal/mol [21.3 kJ/mol]) and optimal pH values of 8.0 and 7.5 when assayed in Tris hydrochloride and potassium phosphate buffers, respectively. The native molecular mass of the enzyme was about 280 kilodaltons. The possible physiological role of the enzyme in ammonia assimilation is discussed.     

Interaction between succinate dehydrogenase and ubiquinone-binding protein from succinate-ubiquinone reductase

Purified ubiquinone-binding protein in succinate-ubiquinone reductase (QPs) reconstitutes with pure soluble succinate dehydrogenase to form succinate-ubiquinone oxidoreductase upon mixing of the two proteins in phosphate buffer at neutral pH. The maximal reconstitution was found with a weight ratio of succinate dehydrogenase to QPs of about 5, which is fairly close to the calculated value of 6.5, a value obtained by assuming one mole of QPs reacts with one mole of succinate dehydrogenase. Succinate-cytochrome c reductase was reconstituted when succinate dehydrogenase and QPs were added to Complex III or cytochrome b-c₁ III complex (a highly purified ubiquinol-cytochrome c reductase). The reconstituted enzyme possessed kinetic parameters which were identical to those of the native enzyme complex. Interaction between QPs and succinate dehydrogenase resulted in the disappearance of low K_m ferricyanide reductase activity from the latter. Unlike soluble succinate dehydrogenase, the reconstituted enzyme, as well as native succinate-cytochrome c reductase, reduced low concentration ferricyanide only in the presence of excess ubiquinone. The apparent K_m for ubiquinone was 6 μM for reduction of ferricyanide (300 μM) by succinate, which is similar to the K_m when ubiquinone was used as electron acceptor. When 2,6-dichlorophenolindophenol was used as electron acceptor for reconstitution of succinate-ubiquinone reductase very little or no exogeneous ubiquinone was needed to show the maximal activity with QPs made by Method II, indicating that the bound ubiquinone in QPs is enough for enzymatic activity. In addition to restoring the succinate-ubiquinone reductase activity the interaction between QPs and succinate dehydrogenase not only stabilized succinate dehydrogenase but also partially deaggregated QPs. The reconstituted succinate-ubiquinone reductase had a minimal molecular weight of 120000 when the reconstituted system was dispersed in 0.2% Triton X-100. The maximal reconstitution was observed at neutral pH in phosphate buffer, Tris-acetate or Tris-phosphate buffer. Tris-HCl buffer, however, produced a less efficient reconstitution. These results indicate that the interaction between QPs and succinate dehydrogenase may involve some cationic group which has a high affinity for Cl^?. Primary amino groups of QPs are not directly involved in the interaction as the reconstitution showed no significant difference when the amino groups of QPs were alkylated with fluorescamine. The Arrhenius plots of reconstituted succinate-ubiquinone reductase show that the enzyme catalyzes the reaction with an activation energy of 19.7 kcal/mol and 26.6 kcal/mol at temperatures above and below 26°C, respectively. These activation energies are similar to those obtained with native enzyme. The Arrhenius plots of the interaction between QPs and succinate dehydrogenase also have a break point at 26°C. The activation energy for this interaction was calculated to be 11.2 kcal/mol and 6.9 kcal/mol for the temperatures above and below the break-point. The significance of the difference in activation energies between the enzymatic reaction and the reconstitution reaction are further explored in the discussion.     

Peroxidase from Ipomoea batatas seedlings: Purification and properties

A peroxidase has been purified to homogeneity from Ipomoea batatas seedlings using ammonium sulphate precipitation and chromatography on DEAE-cellulose and SP-Sephadex columns. The pH optimum of the enzyme was found to be dependent on the buffer and substrate used. The isoelectric point is 7.3. The activation energy was estimated to be 14 kcal/mole. The prosthetic group was shown to be ferriprotoporphyrin IX. Gel chromatography and PAGE indicate that the purified protein is composed of a single polypeptide of MW 42 000. The amino acid composition appears to be similar to those reported for other plant peroxidases.     

Purification and Some Properties of α-Galactosidase from Tubers of Stachys affinis

An α-galactosidase from tubers of S. affinis was purified about 130 fold by ammonium sulfate fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. The purified enzyme showed a single protein band on disc gel electrophoresis. The molecular weight of the enzyme was determined to be approximately 42,000 by gel filtration and 44,000 by SDS disc gel electrophoresis. The optimum reaction pH was 5.2. The enzyme hydrolyzed raffinose more rapidly than planteose. The activation energy of raffinose and planteose by the enzyme was estimated to be 7.89 and 11.4 kcal/mol, respectively. The enzyme activity was inhibited by various galactosides and structural analogs of d-galactose. Besides hydrolytic activity, the enzyme also catalyzed the transfer reaction of d-galactosyl residue from raffinose to methanol.     

NADP-specific isocitrate dehydrogenase of Mycobacterium phlei ATCC 354: purification and characterization

NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) from Mycobacterium phlei ATCC 354 was purified to homogeneity by ammonium sulphate fractionation, followed by DEAE cellulose and Sephadex G-200 chromatography. The pH optimum of the enzyme was 8.5. The Km values for isocitrate and NADP were 74 and 53 microM, respectively. Mn2+ was essential for enzyme activity. The enzyme lost all activity on incubation at 70 degrees C for 15 min; isocitrate and NADP protected against this thermal inactivation. p-Chloromercuribenzoate inhibited the enzyme; pre-incubation of enzyme with isocitrate + Mn2+ prevented this inhibition. The purified enzyme showed concerted inhibition by glyoxylate + oxaloacetate and was inhibited by oxalomalate.