Purification and characterization of homogeneous sunflower seed acid phosphatase

Acid phosphatase (EC 3.1.3.2) from sunflower seed was purified 1800-fold to homogeneity using both conventional and affinity chromatographic methods. The purified enzyme was a mixture of two enzyme forms distinguishable by polyacrylamide gel electrophoresis (PAGE). Gel exclusion chromatography, which did not distinguish between the two forms, gave an apparent M, of 103 000. Preparative PAGE permitted the separation of the two forms, and SDS-PAGE showed that they contained equivalent peptide subunits of apparent M, 56 000 and 52 000. Amino acid analysis indicated that both enzyme forms have similar amino acid compositions. Data on substrate specificity and pH dependence is presented. The kinetic constants for hydrolysis of p-nitrophenyl phosphate as catalysed by sunflower seed acid phosphatase were independent of pH in the range 3-5. The enzyme was competitively inhibited by inorganic phosphate and non-competitively inhibited by phosphomycin.     

Purification and characterization of a thermally stable yellow laccase from <Emphasis Type="Italic">Daedalea flavida</Emphasis> MTCC-145 with higher catalytic performance towards selective synthesis of substituted benzaldehydes

A laccase from the culture filtrate of white rot fungus Daedalea flavida MTCC-145 has been purified and characterized. The method involved concentration of the culture filtrate by ultrafiltration and an anion exchange chromatography on diethylaminoethyl (DEAE) cellulose. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (native PAGE) both gave single protein bands indicating that the enzyme preparation was pure. The molecular mass of the enzyme determined from SDS-PAGE analysis was 75.0 kDa. Purification fold was 21.5 while recovery of the enzyme activity was 11.52%. Using 2,6-dimethoxyphenol, diammonium salt of 2,2'-[azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)] and 3,5-dimethoxy-4-hydroxybenzaldehyde azine as substrates, the Km, kcat, and k _cat/K _m values of the laccase were found to be 440 µM, 6.45 s^–1, 1.47 × 10⁴ M^–1 s^–1; 366 µM, 6.45 s^–1, 1.76 × 10⁴ M^–1 s^–1; and 226 µM, 6.45 s^–1, 2.85 × 10⁴ M^–1 s^–1, respectively. The pH and temperature optima were 4.5 and 50°C, respectively. The enzyme was most stable at pH 5.0 when exposed for 1 h. The purified laccase has yellow color and shows no absorption band around 610 nm characteristic of blue laccases. The enzyme transforms toluene and substituted toluenes to corresponding benzaldehyde and substituted benzaldehydes in the absence of mediator molecules with higher catalytic efficiency as compared to other known laccases.     

Purification and properties of malic dehydrogenase from Trichomonas gallinae

The malic dehydrogenase (MDH², l-malate: NAD oxidoreductase, E.C. 1,1.1.37) of Trichomonas gallinae was purified 215-fold and characterized. The molecular weight was found to be 72,000 and the enzyme protein contained essential cations and sulfhydryl groups. Polyacrylamide gel electrophoresis before and after extensive purification yielded a single band of malic dehydrogenase activity strongly suggesting only one molecular form of the enzyme. Analysis of kinetic data yielded the following K_m values: oxalocetate, 16 μM; malate, 200 μM; NADH 11 μM; and NAD, 70 μM. The enzyme was absolutely specific for l-malic acid, NAD, and NADH. The enzyme exhibited a broad band of heat stability with an optimum of 51 C. The pH optimum in the direction of oxalacetate reduction was 9.0. The pH optima in the reverse direction were 9.0 and 10.5 A role for this enzyme in T. gallinae metabolism is discussed.     

Characterization of a latent cysteine proteinase from ascitic fluid as a high molecular weight form of cathepsin B

The latent cysteine proteinase present in ascitic fluid of patients with neoplasia and released from ascites cells in culture has been partially purified and the enzyme after pepsin activation was shown to be immunologically related to the lysosomal proteinase, cathepsin B. The latent form was characterized as a single chain of M_r 40 000 as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions followed by Western blotting and immune staining with an antiserum to human cathepsin B. Using the same techniques the enzyme after pepsin activation gave a single band of M_r 33 000. Analysis by isoelectric focusing showed that the latent enzyme before and after pepsin treatment is composed of several acidic isoenzymes. These findings suggest that this latent proteinase represents a precursor form of cathepsin B which is released extracellularly rather than being processed and directed to the lysosome.     

Purification and characterization of a lysosomal aspartic protease with cathepsin D activity from the mosquito

A lysosomal aspartic protease with cathepsin D activity, from the mosquito, Aedes aegypti, was purified and characterized. Its isolation involved ammonium sulfate (30–50%) and acid (pH 2.5) precipitations of protein extracts from whole previtellogenic mosquitoes followed by cation exchange chromatography. Purity of the enzyme was monitored by SDS-PAGE and silver staining of the gels. The native molecular weight of the purified enzyme as determined by polyacrylamide gel electrophoresis under nondenaturing conditions was 80,000. SDS-PAGE resolved the enzyme into a single polypeptide with M_r = 40,000 suggesting that it exists as a homodimer in its non-denatured state. The pI of the purified enzyme was 5.4 as determined by isoelectric focusing gel electrophoresis. The purified enzyme exhibits properties characteristic of cathepsin D. It utilizes hemoglobin as a substrate and its activity is completely inhibited by pepstatin-A and 6M urea but not by 10 mM KCN. Optimal activity of the purified mosquito aspartic protease was obtained at pH 3.0 and 45°C. With hemoglobin as a substrate the enzyme had an apparent K_m of 4.2 μ M. Polyclonal antibodies to the purified enzyme were raised in rabbits. The specificity of the antibodies to the enzyme was verified by immunoblot analysis of crude mosquito extracts and the enzyme separated by both non-denaturing and SDS-PAGE. Density gradient centrifugation of organelles followed by enzymatic and immunoblot analyses demonstrated the lysosomal nature of the purified enzyme. The N-terminal amino acid sequence of the purified mosquito lysosomal protease (19 amino acids) has 74% identity with N-terminal amino acid sequence of porcine and human cathepsins D.     

Purification and characterization of an exo-polygalacturonase secreted by Rhizopus oryzae MTCC 1987 and its role in retting of Crotalaria juncea fibre

An acidic polygalacturonase (PG) secreted by Rhizopus oryzae MTCC-1987 in submerged fermentation condition has been purified to electrophoretic homogeneity using ammonium sulphate fractionation and anion exchange chromatography on diethylaminoethyl cellulose. The purified enzyme gave a single protein band in sodium dodecyl sulphatepolyacrylamide gel electrophoresis analysis with a molecular mass corresponding to 75.5 kDa. The K _m and k _cat values of the PG were 2.7 mg/mL and 2.23 × 10³ s^?1, respectively, using citrus polygalacturonic acid as the substrate. The optimum pH of the purified PG was 5.0 and it does not loose activity appreciably if left for 24 hours in the pH range from 5.0 to 12.0. The optimum temperature of purified enzyme was 50°C and the enzyme does not loose activity below 30°C if exposed for two hours. The purified enzyme showed complete inhibition with 1 mM Ag⁺, Hg²⁺ and KMnO₄, while it was stimulated to some extent by Co²⁺. The purified PG exhibited retting of Crotalaria juncea fibre in absence of ethylenediaminetetraacetic acid.     

Synthesis and biological activity of hydroxylated derivatives of linoleic acid and conjugated linoleic acids

Allylic hydroxylated derivatives of the C18 unsaturated fatty acids were prepared from linoleic acid (LA) and conjugated linoleic acids (CLAs). The reaction of LA methyl ester with selenium dioxide (SeO₂) gave mono-hydroxylated derivatives, 13-hydroxy-9Z,11E-octadecadienoic acid, 13-hydroxy-9E,11E-octadecadienoic acid, 9-hydroxy-10E,12Z-octadecadienoic acid and 9-hydroxy-10E,12E-octadecadienoic acid methyl esters. In contrast, the reaction of CLA methyl ester with SeO₂ gave di-hydroxylated derivatives as novel products including, erythro-12,13-dihydroxy-10E-octadecenoic acid, erythro-11,12-dihydroxy-9E-octadecenoic acid, erythro-10,11-dihydroxy-12E-octadecenoic acid and erythro-9,10-dihydroxy-11E-octadecenoic acid methyl esters. These products were purified by normal-phase short column vacuum chromatography followed by high-performance liquid chromatography (HPLC). Their chemical structures were characterized by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR). The allylic hydroxylated derivatives of LA and CLA exhibited moderate in vitro cytotoxicity against a panel of human cancer cell lines including chronic myelogenous leukemia K562, myeloma RPMI8226, hepatocellular carcinoma HepG2 and breast adenocarcinoma MCF-7 cells (IC₅₀ 10-75 μM). The allylic hydroxylated derivatives of LA and CLA also showed toxicity to brine shrimp with LD₅₀ values in the range of 2.30-13.8 μM. However these compounds showed insignificant toxicity to honeybee at doses up to 100 μg/bee.     

Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeniety from livers of thioacetamide- and dl-α-hydrazino-δ-aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5·10⁵-fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10⁶ nmol CO₂/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105 000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50 000 on SDS-polyacrylmide gels. The isoelectric point of the enzyme was pH 4.1.     

Alfalfa Root Nodule Carbon Dioxide Fixation : II. Partial Purification and Characterization of Root Nodule Phosphoenolpyruvate Carboxylase

A nonphotosynthetic phosphoenolpyruvate carboxylase (EC 4.1.1.31) was partially purified from the cytosol of root nodules of alfalfa. The enzyme was purified 86-fold by ammonium sulfate fractionation, DEAE-cellulose, hydroxylapatite chromatography, and reactive agarose with a final yield of 32%. The enzyme exhibited a pH optimum of 7.5 with apparent K_m values for phosphoenolpyruvate and magnesium of 210 and 100 micromolar, respectively. Two isozymes were resolved by nondenaturing polyacrylamide disc gel electrophoresis. Subsequent electrophoresis of these isozymes in a second dimension by sodium dodecyl sulfate slab gel electrophoresis yielded identical protein patterns for the isozymes with one major protein band at molecular weight 97,000. Malate and AMP were slightly inhibitory (about 20%) to the partially purified enzyme. Phosphoenolpyruvate carboxylase comprised approximately 1 to 2% of the total soluble protein in actively N₂-fixing alfalfa nodules.     

Flavour biogenesis. Partial purification and properties of a fatty acid hydroperoxide cleaving enzyme from fruits of cucumber

A membrane-bound enzyme, which catalyses the cleavage of fatty acid hydroperoxides to carbonyl fragments, has been partially purified from cucumber fruit. The isomeric 9- and 13-hydroperoxydienes (but not the hydroxydienes) derived from both linoleic and linolenic acids are cleaved by the enzyme but a mixture of 9- and 10-hydroperoxymonoenoic derivatives of oleic acid was not attacked. No evidence was obtained for free intermediates between fatty acid hydroperoxides and the cleavage products. Major volatile products were: cis-3-nonenal and hexanal (from 9- and 13-hydroperoxides of linoleic acid respectively) or cis-3,cis-6-nonadienal and cis-3-hexenal (from 9- and 13-hydroperoxides of linolenic acid). The increase in the ratio of cis-3- to trans-2-enal products with enzyme purification indicated that cis-3-enals are the immediate cleavage products and that the trans-2- forms are produced by subsequent isomerization.     

ACTIVATION OF GUANYLATE CYCLASE IN SYNAPTIC PLASMA MEMBRANES OF CEREBRAL CORTEX BY FREE FATTY ACIDS

Guanylate cyclase (EC 4.6.1.2) of synaptic plasma membranes of rat cerebral cortex was stimulated about 6-fold by several unsaturated fatty acids (arachidonic, linolenic, linoleic, oleic, palmitoleic and myristoleic acid). Ricinoleic acid (12-hydroxyoleic acid) was much less effective. Saturated fatty acids (C₁₀ and C₁₄-C₂₀) and the methylester of linoleic acid were ineffective. Stimulation by linoleic acid was influenced by the concentration of enzyme protein. At 480 μg/ml of protein 0.6 mm -linoleic acid produced maximal activation of 6-fold_ Activity stimulated by linoleic acid examined with 1.0 mm -GTP was maximal at pH 7.8-7.9 and with 2 mm -MnCl₂, whereas basal activity showed broad optimal pH and Mn²⁺-concentration dependence. Activation of the enzyme by linoleic acid was only partially reversed by washing. Particulate guanylate cyclase of heart, small intestine, adrenal medulla, liver and lung was also activated by linoleic acid. The extents of activation (1.5-14.7-fold) by linoleic acid and the concentrations (0.2-1.0 mat) required for maximal activation depended on the tissues.     

Purification and Properties of Arginase from Soybean, Glycine max, Axes

Arginase (EC 3.5.3.1) was purified to homogeneity from cytosol of soybean, Glycine max, axes by chromatographic separations on Sephadex G-200, DEAE-sephacel, hydroxyapatite, and arginine-affinity columns. The molecular weight of the enzyme estimated by pore gradient gel electrophoresis was 240,000, while sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a single band at the molecular weight of 60,000. The optimal pH for activity was 9.5 and the K_m value was 83 millimolar. The enzyme was stimulated by polyamines such as putrescine.     

Biochemical properties of lipoxygenase from opium poppy chloroplasts

Lipoxygenase (LOX) from opium poppy (Papaver somniferum L.) chloroplasts was isolated and 126.1-fold purified to electrophoretic homogeneity by combination of ion-exchange chromatography on HA-Ultragel column and affinity chromatography on a linoleyl-aminopropyl agarose column. The relative molecular mass of the LOX determined by SDS-PAGE was 92 kDa. Kinetic properties of purified LOX were determined in spectrophotometric assay by using of linoleic acid (K_M = 1.78 mM and V_max = 11.4 μmol mg⁻¹ min⁻¹) and linolenic acid (K_M = 1.27 mM and V_max = 10.2 μmol mg⁻¹ min⁻¹). The optimum pH was 6.0 for both linoleic and linolenic acid dioxygenation catalyzed by LOX. HPLC analysis of the products revealed a dual positional specificity of linoleic acid dioxygenation at pH 6.0 with ratio of 9- and 13-hydroperoxide products being about 1:1. The activity of purified LOX was stimulated by Mg²⁺ and Ca²⁺.     

Lipoxygenase in Vicia faba minor

Lipoxygenase activity was demonstrated in partially purified preparations from small faba beans. The enzyme was shown to possess a pH optimum of 6·5 and was inactivated by exposure to 70° for 15 min. The K_m value for linoleic acid was calculated to be 0·57 mM. Ammonium sulphate fractionation yielded two highly active preparations, which were both active towards linoleic and linolenic acids. Neither fraction was inhibited by either cyanide or p-chloromercuribenzoate. The two fractions showed markedly differing responses to calcium ions, suggesting the presence of two lipoxygenases in faba beans. Activation of the enzyme by calcium ions was eliminated by the addition of EDTA.     

Fast protein liquid chromatographic purification and some properties of a partially O-methylated flavonol glucoside 2′-/5′-O-methyltransferase

A flavonol O-methyltransferase was partially purified from Chrysosplenium americanum by fractional precipitation with ammonium sulphate followed by gel filtration and chromatofocusing using an FPLC system. The enzyme which was purified 420-fold catalysed the transfer of the methyl group of SAM to the 2′- or 5′-positions of partially methylated flavonol glucosides, the two terminal methylation steps in the biosynthesis of Chrysosplenium flavonoids. The enzyme had a pH optimum of 7 in Pi buffer, a pI of < 5, an M, of 57 000, no Mg²⁺ requirement and was inhibited by both N-ethylmaleimide and phenylmercuriacetate. The K_m value for the flavonol substrate was 2 μM and that for SAM was 100 μM. The role of this enzyme is discussed in relation to the biosynthesis of polymethylated flavonols in this tissue.     

1,3-β-d-Glucan synthase ofNeurospora crassa: Partial purification and characterization of solubilized enzyme activity

1,3-β-d-Glucan synthase activity ofNeurospora crassa was rendered soluble by treatment of crude protoplast lysates with 0.1% 3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer containing 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium fluoride, 1 mM dithiothreitol, 200 mM inorganic phosphate, 10 μM GTP, and 600 mM glycerol. Approximately 50% of enzyme activity was solubilized; soluble enzyme activity was purified 5.6-fold with a net 38% recovery by sucrose gradient density sedimentation. Partially purified enzyme activity had a half-life of 60 ± 10 h at 4°C, aK_m,app of 0.75 ± 0.05 mM, and a V_max,app of 35 ± 1 enzyme units/mg protein.     

Characterization of β-galactosidases from the germinating seeds of Vigna sinensis

Four forms of β-galactosidase from the germinating seeds of Vigna sinensis were separated and partially purified by ammonium sulphate precipitation, ion exchange chromatography (DE-52) and gel filtration to more than 50% purity as judged by PAGE. The pH and temperature optima, stability, M_r, kinetic parameters and energy of activation of each enzyme have been determined. The four forms differed in their M,_s and ionic charges.     

NADPH-cytochrome c reductase in outer membrane of kidney mitochondria. Purification and properties.

NADPH-cytochrome c reductase of vitamin D₃-deficient chick kidney mitochondria has been purified approximately 1100-fold to a specific activity of 788 nmol cytochrome c reduced/min/mg protein. Analytical gel electrophoresis of the purified enzyme revealed two bands when stained for protein, but only the more anodic band demonstrated NADPH-tetrazolium reductase activity. The relative ease of solubilization of the reductase without the use of lipases, proteases, or detergents was the first line of evidence that suggested a novel mitochondrial localization for this previously unreported NADPH-linked cytochrome c reductase. The apparent properties of the reductase suggest that the enzyme is a component of kidney mitochondrial outer membrane. The kinetic determination of Michaelis constants with respect to NADPH, cytochrome c, and NADH gave the following values: K_mNADPH = 1.7 μM, K_mcytc = 3.4 μM, and K_mNADH = 20 mM. These constants were different from those of the intact kidney microsomal reductase: K_mNADPH = 5.5 μM, K_mcytc = 10.5 μM, and K_mNADH = 13.3 μM. The mitochondrial as well as the intact microsomal reductases exhibited a ping-pong kinetic mechanism for NADPH-mediated cytochrome c reduction. Spectrofluorometric measurements demonstrated the presence of equimolar amounts of FAD and FMN. The oxidized enzyme has absorption maxima at 280 and 450 nm with a shoulder at 415 nm. Upon reduction with NADPH a distinct loss in the 450-nm absorption was observed. Ouchterlony immunodiffusion studies with rabbit antiserum to chick renal mitochondrial ferredoxin did not reveal cross-reactivity when the purified reductase was the antigen. This excludes the involvement of a ferredoxin-type iron-sulfur protein in the NADPH-mediated reduction of cytochrome c by the purified reductase.     

Characterization of purified guanine aminohydrolase.

Guanine aminohydrolase (GAH) (E.C. 3.5.4.3) was purified by affinity chromatography on 9-(p-β-aminoethoxyphenyl)guanine-Sepharose to a specific activity of 35.5 units/mg. The molecular weight of the enzyme was estimated to be 110,000 by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) showed that the enzyme was composed of subunits with molecular weights of approximately 52,000. Data from SDS-gel electrophoresis in a discontinuous buffer system and from isoelectric focusing in the presence of 8-m urea indicated that more than one type of subunit were present. This was consistent with multiple forms of the native enzyme seen by electrophoresis and isoelectric focusing in polyacrylamide gels. The isoelectric points for the different forms of GAH were in the range of 4.65–4.85. Amino acid analyses showed cysteine to be the minimum amino acid and gave a calculated molecular weight for GAH of 53,016 when the assumption that there were four cysteines per subunit was made. Guanine, 8-azaguanine, and 6-thioguanine served as substrates for the enzyme but 3-deazaguanine, a potent competitive inhibitor of GAH, did not. Fluoride ion inhibited the enzyme in a noncompetitive manner, and this inhibition decreased as pH increased. Variation of the kinetic parameters with pH suggested that hydroxide ion might be the second substrate and that a functional group on the enzyme with a pK_a near 5.6 was involved in the reaction. The enzyme was inactivated by treatment with p-hydroxymercurobenzoate and by photooxidation in the presence of rose bengal. Two plausible mechanisms are proposed for the reaction catalyzed by GAH.     

Purification and characterization of a novel exopolygalacturonase from Fusarium oxysporum f.sp. lycopersici

A novel exopolygalacturonase (EC 3.2.1.15) was purified to apparent homogeneity from cultures of Fusarium oxysporum f.sp. lycopersici on synthetic medium supplemented with polygalacturonic acid, using two steps of purification: preparative isoelectric focusing and cationic exchange chromatography. The enzyme designated PG3 had an apparent M_r of 63 000±3000 Da upon SDS-PAGE and a pI of 7.0. PG3 was active within a broad range of pH from 3.5 to 9. The temperature optimum was 55°C. PG3 hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by analysis of degradation products. The enzyme was N-glycosylated. The production of PG3 was constitutive at low levels, and synthesis was increased following induction by PGA and partially repressed by glucose.