An inhibitor of phosphoinositol kinase from ungerminated mung bean seeds

A protein inhibitor of phosphoinositol kinase has been detected in the later stages of ripening of mung bean seeds. This has been isolated and purified from the ungerminated seeds. It migrated as a single protein band when subjected to polyacrylamide gel electrophoresis. The MW of the inhibitor is approx. 86 000. The phosphoinositol kinase inhibition has been found to be dependent on the protein concentration of the purified inhibitor. It seems that 1 molecule of the inhibitor is necessary to inhibit 1 molecule of enzyme. The nature of the inhibition has been found to be non-competitive, the K_i of which is around 1·47 × 10^?6 M. The enzyme inhibitor complex dissociates on gel electrophoresis without any loss of enzyme activity.     

Purification and characterization of β-(pyrazol-1-yl)-l-alanine synthase from Citrullus vulgaris

From seedlings of Citrullus vulgaris the enzyme β-(pyrazol-1-yl)-l-alanine synthase was purified 200-fold, when it showed electrophoretic homogeneity (MW 58 000) and could be dissociated into identical subunits (MW 32 000) each containing one molecule of pyridoxal 5′-phosphate. The K_m value was 2.5 × 10^?3 M for O-acetyl-l-serine and 7.4 × 10^?2 M for pyrazole. The enzyme did not catalyse the formation of related β-substituted alanines, such as l-mimosine and l-quisqualic acid, and significant differences were found between the β-(pyrazol-1-yl)-l-alanine synthase and β-substituted alanine syntheses and cysteine synthase from other sources.     

Purification and properties of a D-fructose 1,6-bisphosphatase from Saccharomyces cerevisiae.

Fructose 1,6-bisphosphatase (EC 3.1.3.11) from Saccharomyces cerevisiae has been purified to homogeneity. A molecular weight of 115,000 has been obtained by gel filtration. The enzyme appears to be a dimer with identical subunits. The apparent K_m for fructose bisphosphatase varies with the Mg²⁺ concentration of the enzyme, being 1 × 10^?6m at 10 mm Mg²⁺ and 1 × 10^?5m at 2 mm Mg²⁺. Other phosphorylated compounds are not significantly hydrolyzed by the enzyme. An optimum pH of 8.0 is exhibited by the enzyme. This optimum is not changed by addition of EDTA. AMP inhibits the enzyme with a K_i of 8.0 × 10^?5m at 25 °C. The inhibition is temperature dependent, the value of K_i increasing with raising temperature. 2-Deoxy-AMP is also inhibitory with a K_i value at 25 °C of 1.6 × 10^?4m. An ordered uni-bi mechanism has been deduced for the reaction with phosphate leaving the enzyme as the first product and the fructose 6-phosphate as the second one.     

Purification and characterization of L-mimosine synthase from Leucaena leucocephala

L-Mimosine synthase has been isolated from Leucaena leucocephala seedlings and purified 280-fold by heat treatment, ammonium sulphate fractionation, gel filtration and ion-exchange chromatography. The enzyme was shown to be homogeneous by gel electrophoresis (MW 64 000±2000) and to consist of two identical subunits with MWs of 32 000±2000. The purified enzyme has a K_m value of 6.25 x 10^?3 M for O-acetyl-L-serine and 5.0 x 10^?3 M for 3,4-dihydroxypyridine. In these and other properties, the enzyme differs from β-(pyrazol-1-yl)-L-alanine synthase from Citrullus vulgaris seedlings.     

Purification and characterization of diamine oxidase from rice embryos

Diamine oxidase of rice seedlings has been purified 1800-fold to homogeneity. The MW of the enzyme as determined by Sephadex G-100 gel filtration was 12.3 × 10⁴ and the enzyme contained two identical subunits each with a MW of 6.12 × 10⁴. The optimal temperature and pH for the enzyme were 30° and 7.5 respectively and the enzyme followed typical Michaelis kinetics with a K_m of 10^?5 M. Each enzyme molecule contained four molecules of FAD.     

Chymotrypsin inhibitor from potatoes: interaction with target enzymes

The interactions of chymotrypsin, subtilisin and trypsin with a low MW proteinase inhibitor from potatoes were investigated. The K_i value calculated for the binding of inhibitor to chymotrypsin was 1.6 ± 0.9 × 10^?10M, while the second-order rate constant for association was 6 × 10⁵ M^?1/sec. Although binding was not observed to chymotrypsin which had been treated with diisopropyl fluorophosphate or with l-tosylamide-2-phenylethyl chloromethyl ketone, the 3-methylhistidine-57 derivative bound inhibitor with a K_i value of 9.6 × 10^?9 M. The inhibitor also exhibited a tight association with subtilisin (K_i < 4 × 10^?9 M). In contrast, little inhibition of trypsin was observed, and this was believed to be due to low levels of a contaminant in our preparations. No evidence for reactive site cleavage was observed after incubation of the inhibitor with catalytic amounts of chymotrypsin or subtilisin at acid pH.     

Purification and characterization of extracellular acid phosphatase of Tetrahymena pyriformis

An extracellular acid phosphatase secreted into the medium during growth of Tetrahymena pryiformis strain W was purified about 900-fold by (NH₄)₂SO₄ precipitation, gel filtration and ion exchange chromatography. The purified acid phosphatase was homogenous as judged by polycrylamide gel electrophoresis and was found to be a glycoprotein. Its carbohydrate content was about 10% of the total protein content. The native enzyme has a molecular weight of 120 000 as determined by gel filtration and 61 000 as determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis. The acid phosphatase thus appears to consist of two subunits of equal size. The amino acid analysis revealed a relatively high content of asparic acid, glutamic acid and leucine. The purified acid phosphatase from Tetrahymena had a rather broad substrate specificity; it hydrolyzed organic phosphates, nucleotide phosphates and hexose phosphates, but had no diesterase activity. The K_m values determined with p-nitrophenyl phosphate, adenosine 5′-phosphate and glucose 6-phosphate were 3.1·10^?4 M, 3.9·10^?4 M and 1.6·10^?3 M, respectively. The optima pH for hydrolysis of three substrates were similar (pH 4.6). Hg²⁺ and Fe³⁺ at 5 mM were inhibitory for the purified acid phosphatase, and fluoride, L-(+)-tartaric acid and molybdate also inhibited its cavity at low concentrations. The enzyme was competitively inhibited by NaF (K_i=5.6·10^?4 M) and by L-(+)-tartaric acid (K_i = 8.5·10^?5 M), while it was inhibited noncompetitively by molybdate K_i = 5.0·10^?6 M). The extracellular acid phosphatase purified from Tetrahymena was indistinguishable from the intracellular enzyme in optimum pH, K_m, thermal stability and inhibition by NaF.     

Inhibition by indomethacin and aspirin of 15-hydroxy-prostaglandin dehydrogenase in vitro

15-Hydroxyprostaglandin dehydrogenase from bovine lung was purified 7.4 times to a specific activity of 1.4 mU/mg of protein. The isoelectric point was estimated to 5.4 and the molecular weight by gelfiltration to 40,000. K_m for prostaglandin E₁ and for NAD⁺ were found to be 3.4 μM and 1.1 × 10^?4M respectively. The enzyme was inhibited by indomethacin and aspirin. The indomethacin inhibition was found to be non-competitive to prostaglandin E₁ having a K_i=1.4 × 10^?4M and a K_i^′=1.6 × 10^?5M.     

Electron transport systems of Candida utilis. Purification and properties of the respiratory chain-linked external NADH dehydrogenase+

The respiratory chain-linked external NADH dehydrogenase has been isolated from Candida utilis in highly purified form. The enzyme is soluble and has a molecular weight of approx. 1.5 · 10⁶. The enzyme contains two moles of FMN per mole of enzyme and is composed of two large subunits of mol. wt. 270 000 and eight smaller subunits of mol. wt. 135 000. Iron and copper are present in the preparations, but appear to be contaminants. The enzyme catalyzes the oxidation of NADH and NADPH at nearly equal rates and reacts readily with 2,6-dichlorophenolindophenol, CoQ₆ and CoQ₁ derivatives as acceptors. Rotenone (10^?5 M) and seconal (10^?3 M) do not inhibit enzymatic activity.     

Purification and biochemical characterization of phytocystatin from Brassica alba

Phytocystatins belong to the family of cysteine proteinases inhibitors. They are ubiquitously found in plants and carry out various significant physiological functions. These plant derived inhibitors are gaining wide consideration as potential candidate in engineering transgenic crops and in drug designing. Hence it is crucial to identify these inhibitors from various plant sources. In the present study a phytocystatin has been isolated and purified by a simple two‐step procedure using ammonium sulfate saturation and gel filtration chromatography on Sephacryl S‐100HR from Brassica alba seeds (yellow mustard seeds).The protein was purified to homogeneity with 60.3% yield and 180‐fold of purification. The molecular mass of the mustard seed cystatin was estimated to be nearly 26 000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis as well as by gel filtration chromatography. The stokes radius and diffusion coefficient of the mustard cystatin were found to be 23A° and 9.4 × 10^?7 cm²s^?1 respectively. The isolated phytocystatin was found to be stable in the pH range of 6–8 and is thermostable up to 60 °C. Kinetic analysis revealed that the phytocystatin exhibited non‐competitive type of inhibition and inhibited papain more efficiently (K_i = 3 × 10^?7 M) than ficin (K_i = 6.6 × 10^?7 M) and bromelain (Ki = 7.7 × 10^?7 M respectively). CD spectral analysis shows that it possesses 17.11% alpha helical content. Copyright © 2016 John Wiley & Sons, Ltd.     

Coconut α-d-galactosidase isoenzymes: isolation,purification and characterization

α-d-Galactosidases (α-d-galactoside galactohydrolase, EC 3.2.1.22) from normal coconut endosperm were isolated and partially purified by a combination of ammonium sulfate fractionation, SP-Sephadex C50–120 ion-exchange chromatography and Sephadex G-200 and G-100 gel filtration. Two molecular forms of the enzyme, designated as A and B, were eluted after SP-Sephadex C50–120 ion-exchange chromatography. α-d-Galactosidase A, which is the major isoenzyme, was partially purified 43-fold on Sephadex G-200 and has a MW of about 23 000 whereas α-d-galactosidase B was partially purified 23-fold on Sephadex G-100 and has a similar MW of about 26 600. Both isoenzymes exhibited optimum activity at pH 7.5. The apparent K_m and V_max of α-d-galactosidase A were obtained at 3.46 × 10^?4M and 1.38 × 10^?3 M p-nitrophenyl α-<d-galactoside, respectively. A distinct substrate inhibition was noted. The enzyme was inhibited strongly by d-galactose and to a lesser extent by myo-inositol, d-glucose-6-phosphate, l-arabinose, melibiose and iodoacetic acid. Similarly, makapuno α-d-galactosidase was localized in the 40–70 % (NH₄)₂SO₄ cut but its optimum activity at pH 7.5 was considerably lower as compared to the normal. Its K_m was obtained at 6.75 × 10^?4 M p-nitrophenyl α-d-galactoside while the V_max was noted at 5.28 × 10^?3 M p-nitrophenyl α-d-galactoside. Based on the above kinetic data, the possible cause(s) of the deficiency of α-d-galactosidase activity in makapuno is discussed.     

UFEIII, a fibrinolytic protease from the marine invertebrate, Urechis unicinctus

A new serine protease with fibrinolytic activity from a marine invertebrate, Urechis unicinctus, was purified to electrophoretic homogeneity using column chromatography. SDS-PAGE of the purified enzyme showed a single polypeptide chain with MW ~20.8 kDa. Its N-terminal sequence was IIGGSQAAITSY. The purified enzyme, UFEIII, was stable at pH 6–10 below 60 °C with an optimum pH of 8.5 at approx. 55 °C. The enzyme activity was significantly inhibited by PMSF and SBTI suggesting that it was a serine protease. In fibrin plate assays, UFEIII was contained 1.46 × 10³ U (urokinase units) mg^?1 total fibrinolytic activity, which consisted of 692 U mg^?1 direct fibrinolytic activity and 769 U mg^?1 plasminogen-activator activity. K_m and V_max values for azocasein were 1 mg ml^?1 and 43 μg min^?1 ml^?1, respectively.     

Partial purification and some properties of shikimate dehydrogenase from tomatoes

The partial purification of shikimate dehydrogenase (SDH) from tomato fruit was achieved by precipitation with ammonium sulphate, and chromatography on DEAE-cellulose and hydroxyapatite. The enzyme has a MW of 73000, shows an optimum at pH 9.1 and K_m values of 3.8 × 10^?5 M and 1.0 × 10^?5 M with shikimic acid and NADP as substrates. NADP could not be replaced by NAD. The tomato enzyme is competitively inhibited by protocatechuic acid with a K_i value of 7.7 × 10^?5 M. On the other hand, cinnamic acid derivatives and 2-hydroxybenzoic acid were ineffective. At 50° for 5 min the SDH is inactivated by 85%. The activity was inhibited by pCMB and N-ethylmaleimide, suggesting a requirement for SH groups. The inactivation plot of oxidation by pCMB was biphasic, and NADP decreased the reactivity of sulphydryl groups to the reagent. The activation energy was found to be 14.2kcal/mol. The properties of the SDH are discussed in relation to the enzymes from other sources.     

Purification and properties of NADP-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from the green alga Chlamydomonas reinhardtii

NADP-dependent non-phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9), previously described in higher plants, has been now found to be present in eukaryotic green algae, but in neither cyanobacteria nor non-photosynthetic microorganisms. The enzyme from the unicellular green alga Chlamydomonas reinhardtii, strain 6145c, has been purified to apparent electrophoretic homogeneity. The non-phosphorylating enzyme was effectively separated from the NADP-dependent phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) dye-ligand chromatography on Reactive Red-120 agarose. The purified enzyme exhibited an optimum pH in the 8.5–9.0 range and a specific activity of approx. 8 μmol·(mg protein)⁻¹·min⁻¹. The native protein was characterized as a homotetramer with a molecular weight of 190 000, a Stokes radius of 5.2 mn, and an isoelectric point of 6.9. From kinetic studies, K_m-values of 9.8 and 51 μM were calculated for NADP and D-glyceraldehyde 3-phosphate, respectively, an absolute specificity for both substrates being observed. L-Glyceraldehyde 3-phosphate was a potent non-competitive inhibior (K_i, 48 μM). The reaction products NADPH and D-3-phosphoglycerate inhibited enzyme activity in a competitive manner with respect to NADP (K_i, 78 μM) and D-glyceraldehyde 3-phosphate (K_i, 1.2 mM), respectively. Thermal inactivation occurred above 45°C and was effectively prevented by either substrate. The presence of essential vicinal thiol groups is suggested by the inactivation produced by diamide, with D-glyceraldehyde 3-phosphate, but not NADP, behaving as a protective agent. The enzyme's possible physiological role in photosynthetic metabolism is discussed briefly.     

S-adenosylmethionine: Protein-carboxyl methyltransferase from erythrocyte

Protein methylase II (S-adenosylmethionine:protein—carboxyl methyltrans-ferase), which modifies free carboxyl residues of protein, was purified from both rat and human blood, and properties of the enzymes were studied. The pH optima for the reaction were dependent on the substrate proteins used; pH 7.0 was found with endogenous substrate, 6.1 with plasma, 6.5 with γ-globulin, and 6.0 with fibrinogen. The molecular weight of the enzymes from both rat and human erythrocytes were identical (25,000 daltons) determined by Sephadex G-75 chromatography. Partially purified enzyme from rat erythrocytes showed three peaks on electrofocusing column at pH 4.9, 5.5 and 6.0. The K_m values of the enzymes from rat and human erythrocytes showed 3.1 × 10^?6m and 1.92 × 10^?6m at pH 6.0, 1.96 × 10^?6m and 1.78 × 10^?6m at pH 7.2, respectively, for S-adenosyl-l-methionine. It is also found that S-adenosyl-l-homocysteine is a competitive inhibitor for protein methylase II with K_i value of 1.6 × 10^?6m.     

Isolation and characterization of wheat ribulose-1,5-diphosphate carboxylase

Wheat ribulose-1,5-diphosphate carboxylase purified to homogeneity had a MW of 540 000, sedimentation coefficient (S_{20, W}) of 18.5 S, apparent diffusion constant (D_app) of 3.07 × 10^?7 cm²/sec, Stoke's radius 5.44 nm, and fractional ratio of 1.17. Electron microscopy revealed particles of 10–12 nm diameter. The enzyme was dissociated by sodium dodecyl sulphate into two subunits of MW 53 000 (S_{20, W} = 3.0 S) and 13 500 (S_{20, W} = 1.7 S). The total amino acid residues in the large and small subunits were 481 and 117, respectively. Tryptic peptide maps of the two subunits confirmed the estimated numbers of Arg and Lys residues. Although the amino acid pattern of the large subunit closely resembled that from barley, rather than that for spinach, beet or tobacco, the pattern of the small subunit was markedly different from those of all the other species.     

Purification and properties of agmatine iminohydrolase from groundnut cotyledons

Agmatine iminohydrolase was purified ca 375-fold from groundnut cotyledons. The enzyme exhibited an optimum pH between 5.5 and 8.5 and the energy of activation was 22 kcal/mol. The K_m for agmatine was (7.57 ± 0.77) × 10^?4 M. The enzyme was inhibited by tryptamine, putrescine, cadaverine, spermidine and spermine. Inhibition by cadaverine and spermidine was competitive. The K_i values for cadaverine and spermidine were 4.1 × 10^?3 and 7.5 × 10^?4 M, respectively.     

Purification and properties of myo-inositol-1-phosphate dehydrogenase from germinating mung bean seeds

A novel enzyme, myo-inositol-1-phosphate dehydrogenase, which catalyzes the conversion of myo-inositol 1-phosphate to ribulose 5-phosphate has been purified 84-fold from mung bean seedling employing several common techniques. The molecular weight of this purified enzyme has been recorded as 88,500 by Sephadex G-200 column chromatography, and in sodium dodecyl sulfate-polyacrylamide gel electrophoresis one protein band containing three subunits of M_r 32,000 each was discernible. K_m values for NAD⁺ and myo-inositol 1-phosphate have been recorded as 2.8 × 10^?4 and 5.0 × 10^?4m, respectively. Production of NADH in myo-inositol-1-phosphate dehydrogenase reaction has also been evidenced by measurement of NADH fluorescence. Dehydrogenation and decarboxylation of myo-inositol 1-phosphate are mediated by the same enzyme. In fact, the rate of dehydrogenation corroborates with that of decarboxylation. Stoichiometry of this reaction suggests that for the production of 1 mol of ribulose 5-phosphate 2 mol of NAD⁺ are reduced.     

Isolation and characterization of the polyphenoloxidase from senescent leaves of black poplar

The polyphenoloxidase (PPO) from black poplar senescent leaves has been purified to almost complete homogeneity by a combination of ammonium sulphate precipitation, Sephadex G75 filtration and DEAE-cellulose chromatography. The purified enzyme has a MW of 60 000 and is probably a Cu⁺ enzyme. Peroxidase (PO) activity co-purifies with PPO and has the same MW as it. The two enzymes differ in pH optimum and in response to the effect of ionic strength. Natural phenols are either substrates, inhibitors or activators of black poplar PPO. This enzyme is an o-diphenoloxidase which binds substrates with K_m in the millimolar range. With caffeic and chlorogenic acids inhibition by excess substrate is observed. Benzoic acid phenols and cinnamic acid phenols are either competitive or non-competitive inhibitors of PPO. Hydroquinone is a highly potent non-competitive inhibitor of the enzyme (K_i  90 μM). Ferulic acid is a potent activator of the PPO-catalysed oxidation of catechol (K_a  0.34 mM, ν_sat/ν_o  7.7).     

Properties of a repressible alkaline phosphatase secreted by the wild-type strain 74a of neurospora crassa

A repressible extracellular alkaline phosphatase (with activity increasing steadily even up to pH 10.5) was purified from cultures of the wild-type strain 74A of Neurospora crassa, after growth on acetate and under limiting amounts of inorganic phosphate for 72 hr at 30°. The enzyme was homogeneous on polyacrylamide gel electrophoresis (PAGE) with or without sodium dodecyl sulphate (SDS). The MW was ca 172 000 and 82 000 as determined by Sephadex G-200 gel filtration and SDS-PAGE, respectively. The enzyme contained 23.6% neutral sugars, cations were not required for activity, and it was not inactivated by 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) at pH 8. Kinetic data showed Michaelian behaviour for the enzymatic hydrolysis of 4-nitrophenyl disodium orthophosphate (PNP-P) at pH 9 (the K_m value and Hill coefficient were 2.2 × 10^?4 M and 0.95, respectively). It was also shown that, at pH 9, the apparent number of Pi bound per dimer molecule equalled one, with a K_i value of 7.0 × 10^?4 M. The secreted enzyme showed half-lives of 23.5, 49.0 and 23.5 min at, pH 5.4, 7.4 and 9.0, respectively, after thermal inactivation at 60°. At pH 5.4, the half-life value was quite similar, while the others were respectively 2 and 4 times greater than those previously described for the repressible alkaline phosphatase retained by the mycelium at pH 5.6 or secreted by ‘slime’ cells.