Inosine nucleosidase from Azotobacter vinelandii

An enzyme catalyzing the hydrolysis of purine nucleosides was found to occur in the extract of Azotobacter vinelandii, strain 0, and was highly purified by ammonium sulfate fractionation, DEAE-cellulose chromatography, hydroxylapatite chromatography and gel filtration on Sephadex G-150. A strict substrate specificity of the purified enzyme was shown with respect to the base components. The enzyme specifically attacked the nucleosides without amino groups in the purine moiety: inosine gave the maximum rate of hydrolysis and xanthosine was hydrolyzed to a lesser extent. The pH optimum of inosine hydrolysis was observed from pH 7 to 9, while xanthosine was hydrolyzed maximally at pH 7. The K _m values of the enzyme for inosine were 0.65 and 0.85 mM at pH 7.1 and 9.0, respectively, and the value for xanthosine was 1.2 mM at pH 7.1.Several nucleotides inhibited the enzyme: the phosphate portions of the nucleotides were suggested to be responsible for the inhibition by nucleotides. Although the inhibition of the enzyme by nucleotides was apparently non-competitive type with respect to inosine, allosteric (cooperative) binding of the substrate was suggested in the presence of the inhibitor. The physiological significance of the enzyme was discussed in connection with the degradation and salvage pathways of purine nucleotides.     

Purification, stability and kinetic properties of highly purified adenosine deaminase from Bacillus cereus NCIB 8122

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) from Bacillus cereus NCIB 8122 has been purified to electrophoretic homogeneity by ammonium sulfate precipitation, gel filtration through Sephadex G-100, DEAE-Sephadex A-50 chromatography and ion-exchange HPLC on DEAE-Polyol. The enzyme activity is stabilized (at temperatures from 0 degrees C to 40 degrees C) by 50 mM NH4+ or K+, while it is irreversibly lost in the absence of these or a few other monovalent cations. Glycerol (24% by volume) helps the cation in stabilizing the enzyme activity above 40 degrees C, but also exerts per se a noticeable protecting effect at room temperature. B. cereus adenosine deaminase displays the following properties: Mr on Sephadex G-200, 68,000; Mr in SDS-polyacrylamide gel electrophoresis, 53,700; optimal pH-stability (in the presence of 50 mM KCl) over the range 8-11 at 4 degrees C, and maximal catalytic activity at 30 degrees C between pH 7 and 10; Km for adenosine around 50 microM over the same pH range and Km for 2'-deoxyadenosine around 400 microM.     

Adenosine deaminase from pig thyroid gland purification and some properties

Adenosine deaminase was isolated from the pig thyroid gland and purified over 900-fold using DEAE Sephadex A-50 column chromatography, Sephadex G-100 gel filtration and DEAE Sephadex A-50 rechromatography. The enzyme was specific towards adenosine. The Michaelis constant based on the Lineweaver-Burk plot was 5 × 10^?5M. The optimum pH was about 7.0, and molecular weight 44 700.     

Purification and some properties of an α-amylase glucoamylase fusion protein from Saccharomyces cerevisiae

A fusion gene containing the Bacillus subtilis -amylase gene and Aspergillus awamori glucoamylase cDNA was expressed in Saccharomyces cerevisiae. The resulting bifunctional fusion protein having both -amylase and glucoamylase activities secreted into the culture medium was purified to apparent homogeneity by affinity chromatography and gel filtration on Sephadex G-100. The enzyme had an apparent molecular mass of 150 kDa and showed an optimum pH and temperature of 6.0 and 60 °C, respectively. The main hydrolysis products from soluble starch were glucose and maltose.     

Purification and Some Properties of Adenosine (Phosphate) Deaminase from the Liver of the Squid Todarodes pacificus

An enzyme that catalyzed the deamination of adenosine 3′-phenylphosphonate was purified from squid liver to homogeneity as judged by SDS-PAGE. The molecular weight of the enzyme was estimated to be 60,000 by SDS-PAGE and 140,000 by Sephadex G-150 gel filtration. The enzyme deaminated adenosine, 2′-deoxyadenosine, 3′-AMP, and 2′,3′-cyclic AMP, but not adenine, 5′-AMP, 3′,5′-cyclic AMP, ADP, or ATP. The apparent K_m and V_max at pH 4.0 for these substrates were comparable (0.11-0.34mM and 179-295 μmol min^?1 mg^?1, respectively). The enzyme had maximum activity at pH 3.5-4.0 for adenosine 3′-phenylphosphonate, at pH 5.5 for adenosine and 2′-deoxyadenosine, and at pH 4.0 for 2′,3′-cyclic AMP and 3′-AMP when the compounds were at concentration of 0.1 mM. The K_m at 4.0 and 5.5 for each substrate varied, but the Vmax were invariant. These results indicated that the squid enzyme was a novel adenosine (phosphate) deaminase with a unique substrate specificity.     

Complete release of adenosine deaminase from mouse lymphocytes stabilized by low-pH acetate

Complete release of adenosine deaminase from mouse lymphocytes takes place when intact cells are stabilized by low-pH acetate buffer. Both the low pH and the acetate affect the enzyme extraction markedly. At pH 5.0 all the adenosine deaminase activity detectable in the whole cell homogenates is released into the acetate buffer in very few minutes, with a total amount of 2% protein being extracted. The complete extraction of the enzyme activity is never observed when, at pH 5.0, the acetate is replaced by glutamate, citrate, succinate or maleate and only 45% and 15% of the adenosine deaminase activity is extracted by the acetate at pH 6.0 and 7.0, respectively. The breakdown of adenosine by the enzyme activity extracted from the stabilized cells is due to deamination alone, since inosine is the only product of the catalyzed reaction and its formation is completely inhibited by coformycin, a selective inhibitor of adenosine deaminase. The enzyme extracted shows a specific activity 50-times higher than that found in the crude homogenates, and a substantial purification of the enzyme extracted is achieved by a single Sephadex G-100 gel filtration.     

Purification of multiple forms of adenosine deaminase from rabbit intestine.

Two forms of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), differing in molecular size, have been purified and obtained in homogeneous form from rabbit intestine. The purification procedures involved extraction with acetate buffer, pH 5.5, precipitation and fractional reextraction with (NH4)2SO4, ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-75 and Sephadex G-200. Gel filtrations analysis gave molecular weight estimates of 265 000 and 32 000 for the large and small deaminases respectively. The two enzymes forms had similar pH optima and pH stability ranges.     

Purification and characterisation of the extracellular maltase ofPaecilomyces varioti CMI 16196

Summary The extracellular maltase of the fungusPaecilomyces varioti CMI 16196 was purified by acetone precipitation and Sephadex G-150 gel filtration. The enzyme functioned optimally at pH 3.5–4.0 and 60°C. The maltase hydrolysed maltose and maltotriose preferentially but was inactive on both the aryl-and alkyl--D-glucosides. The Km for maltose was 1.31 mM. End product configuration was maintained and transferase activity was observed.     

Rapid and efficient separation and identification of the two molecular forms of human adenosine deaminase by thin-layer gel filtration chromatography

Two molecular forms of adenosine deaminase have been found in human tissues. The column gel filtration method has been used for the separation of the two enzyme forms. Routine separation and analysis of the enzyme forms based on the molecular size difference can be achieved by thin-layer gel filtration on Sephadex G-200 superfine gel. The thin-layer method has been found to be more rapid and efficient than the column method. Enzymes in crude preparations can be studied effectively with the thin-layer method.     

Partial purification and characterization of a soluble protein phosphatase fromLeishmania donovani promastigotes

A soluble protein phosphatase from the promastigote form of the parasitic protozoanLeishmania donovani was partially purified using Sephadex G-100, DEAE-cellulose and again Sephadex G-100 columns. The partially purified enzyme showed a native molecular weight of about 42, 000 in both Sephadex G-100 and sucrose density gradient centrifugation. The sedimentation constant, stokes radius and frictional ratio were found to be 3.43S, 2.8 nm and 1.20 respectively. The enzyme preferentially utilized phosphohistone as the best exogenous substrate. Mg²⁺ ions were essential for enzyme activity; among other metal ions Mn²⁺ can replace Mg²⁺ to a certain extent whereas Ca²⁺, Co²⁺ and Zn²⁺ could not substitute for Mg²⁺. The pH optimum of the enzyme was 6.5–7.5 and the temperature optimum 37°C. The apparent Km for phosphohistone was 7.14 M. ATP, ADP, inorganic phosphate and pyrophosphate had inhibitory effect on the enzyme activity whereas no inhibition was observed with sodium tartrate and okadaic acid. These results suggest thatL. donovani promastigotes possess a protein phosphatase which has similar characteristics with the mammalian protein phosphatase 2C.Abbreviations PMSF phenylmethylsulfonyl fluoride - DTT dithiothreitol - TCA trichloroacetic acid - BSA bovine serum albumin - EDTA ethylenediamine tetraacetic acid - ATP adenosine triphosphate - ADP adenosine diphosphate - AMP adenosine monophosphate - EGTA Ethyleneglycol-bis-(-aminoethyl ether) N,N,N,N-tetraacetic acid     

Characterization of 2,3-butanediol-forming and valine-sensitive α-acetolactate synthase of Enterobacter cloacae

-Acetolactate synthase (-ALS) of Enterobacter cloacae ATCC 27613 was purified to homogeneity by ammonium sulphate precipitation, Sephadex G-200 gel filtration and hydroxyapatite affinity chromatography. The molecular weight of the enzyme was found to be 60 kDa by SDS–polyacrylamide gel electrophoresis and 200 kDa by gel filtration through Sephadex G-200, showing that the enzyme is a homotrimer. The K _m and V _max of the enzyme were 20 mM and 200 mol min^–1 mg (protein)^–1 respectively. The enzyme was optimally active at pH 6.0–8.0, 37 °C and showed concentration-dependent sensitivity to cofactors viz. FAD, NADP and NADPH and branched chain amino acids: leucine, isoleucine and valine. Substances like sodium formate, sodium acetate and sodium propionate, sugars and the selected intermediates of glycolytic pathway inhibited the enzyme. Glycerol, BSA and pyruvate-TPP stabilized the -ALS. The enzyme showed the properties of both a catabolic as well as an anabolic -ALS.     

Regulation of threonine biosynthesis in barley seedlings (Hordeum vulgare L.)

Homoserine kinase was purified 700-fold by fractional ammonium sulfate precipitation, heat treatment, CM-Sephadex C-50 and DEAE-Sephadex A-50 ion exchange chromatography, and Sephadex G-100 gel filtration. The reaction products O-phosphohomoserine and ADP were the only compounds which caused considerable inhibition of homoserine kinase activity. Product inhibition studies showed non-competitive inhibition between ATP and O-phosphohomoserine and between homoserine and O-phosphohomoserine, and competitive inhibition between ATP and ADP. ADP showed non-competitive inhibition versus homoserine at suboptimal concentrations of ATP. At saturating concentrations of ATP no effect of ADP was observed. The homoserine kinase activity was negligible in the absence of K⁺ and the K_m value for K⁺ was observed to be 4.3 mmol l^–1. A non-competitive pattern was observed with respect to the substrates homoserine and ATP. Threonine synthase in the first green leaf of 6-day-old barley seedlings was partially purified 15-fold by ammonium sulfate fractionation and Sephadex G-100 gel chromatography. Threonine synthase was shown to require pyridoxal 5-phosphate as coenzyme for optimum activity and the enzyme was strongly activated by S-adenosyl-L-methionine. The optimum pH for threonine synthase activity was 7 to 8.Abbreviations PLP Pyridoxal 5-phosphate - SAM S-adenosyl-L-methionine - HSP O-phosphohomoserine     

人胃腺苷脱氨酶的分离纯化及性质研究

用硫酸铵分级沉淀、ＤＥＡＥ－纤维素离子交换层析、免疫亲和层析、ＳｅｐｈａｄｅｘＧ１００凝胶柱层析从人胃组织中提取出腺苷脱氨酶,酶纯化１９３２４倍,比活力为５７９７Ｕ／ｍｇ蛋白．提取酶液经ＰＡＧＥ、ＳＤＳ－ＰＡＧＥ和等电聚焦只呈现一条区带。测得该酶的分子量为４１．２ｋＤ,等电点为ｐＨ４．８．氨基酸组成分析表明该酶由３８８个氨基酸残基组成,Ｎ端氨基酸为精氨酸。酶的最适ｐＨ为６．５,ｐＨ小于５．０或大于９．０时不稳定;最适温度为３７℃,对热不太稳定,以腺苷及２－脱氧腺苷作为底物,其Ｋｍ分别为８７μｍｏｌ／Ｌ和４１μｍｏｌ／Ｌ。     

Purification and properties of a fibrinolytic enzyme from Bacillus subtilis

A fibrinolytic enzyme obtained from B. subtilis was purified, using DEAE-cellulose column chromatography, and gel filtration on Sephadex G-100. The preparation was homogeneous as tested by gel filtration on Sephadex G-200, and disc electrophoresis. The molecular weight of this enzyme was 29.400 estimated by gel filtration on Sephadex G-100. The optimum pH for enzyme activity was 7.2 Copper ions significantly increased enzyme activity, while Zn++ and Mn++ caused marked inhibition.     

Purification and Properties of Neutral Proteinase I from Aspergillus oryzaePurification and Properties of Neutral Proteinase II from Aspergillus oryzae

Neutral proteinase I (the first peak in DEAE-cellulose chromatogrraphy) was purified from the Amberlite IRC-50 adsorbed fraction by chromatography on DEAE-cellulose and gel filtration through Sephadex G-100. It shows an optimum pH of 7.0 for milk casein. The enzyme was found to be stable in the pH range of 5.5 to 12.0. The molecular weight of the enzyme was estimated to be about 41,000 by gel filtration. The enzyme had neither aminopeptidase nor carboxypeptidase activity, but degraded carbobenzoxy-glycyl-phenyl-alanine amide, poly-l-lysine and poly-l,α-glutamic acid. The enzyme was inhibited by ethylenediaminetetraacetate, but not inhibited by diisopropylphosphorofluoridate and potato inhibitor.     

A Novel Synthesis of (±)-Munduserone via Acetylenic Intermediates

A riboflavin α-glucoside-synthesizing enzyme from the acetone powder of pig liver was purified by a procedure including fractionation with ammonium sulfate, heat treatment, fractionation with acetone, gel filtration on a Sephadex G-150 column, calcium phosphate gel treatment, and isoelectric focusing. A final enzyme preparation was homogeneous on polyacrylamide disc gel electrophoresis and in the ultracentrifuge. The enzyme had a sedimentation coefficient of 9.90 S and an isoelectric point of pH 3.7. The enzyme had a pH optimum at 6.0 with maltose as substrate. The enzyme catalyzed the hydrolysis of diverse kinds of α-glucosidic substrates, and the transfer of α-glucosyl residue from these substrates to riboflavin. The Km value for maltose was 1.20×10^?3m. The enzyme hydrolyzed phenyl α-maltoside to glucose and phenyl α-glucoside. Amylose was almost completely hydrolyzed to glucose by the enzyme. Maltotriose was obtained as the main transfer product after the treatment of maltose with the enzyme. The enzyme also catalyzed the transfer of α-glucosyl residue from maltose to pyridoxine, esculin, rutin, and adenosine. It was recognized that a single enzyme catalyzed not only the hydrolysis of maltose and α-glucosidic substrates but also the transfer of the α-glucosyl residue of these substrates to suitable acceptors.     

Changes in Liver Enzyme Activities of Glycine Metabolism of Rats Fed Excess Glycine Diets

A gram negative bacterium isolated from soil was found to produce a high level of endo-β-N-acetylglucosaminidase in the culture medium. The organism was identified as a Flavobacterium sp. from various bacteriological characteristics. The enzyme from the Flavobacterium sp. was purified to homogeneity from culture broth by fractionation with ammonium sulfate and column chromatographies on DEAE-cellulose, hydroxylapatite, and Sephadex G-150 and G-100. The molecular weight of the enzyme was estimated to be 27,000 and 30,000 by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, and it appeared to consist of a single polypeptide chain. The optimal pH for activity was 5.0 to 6.0 and the stable pH range was 5~7. The Michaelis constant was 0.30 mm with dansyl-Asn-(GlcNAc)₂(Man)₆ as the substrate. The enzyme hydrolyzed oligosaccharides of native ovalbumin, bovine pancreatic ribonuclease B and a yeast invertase.     

Purification and some properties of β-mannanase from Bacillus sp.

-Mannanase produced by Bacillus sp. W-2, isolated from decayed commercial konjak cake, was purified from the culture supernatant by (NH₄)₂ SO₄ precipitation, adsorption to konjak gel, and column chromatography with DEAE-cellulose, Sephadex G-100 and Sephacryl S-200. Its molecular size was estimated by SDS-PAGE as 40 kDa, and by gel filtration as 36 kDa. The enzyme was most active at pH 7 and 70°C and was stable for at least 1 h between pH 5 and 10 and below 60°C. Its activity was completely inhibited by Hg²⁺. The enzyme hydrolysed galactomannan better than glucomannan and mainly produced mannose and mannobiose.The authors are with the Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya University. Utsunomiya, Tochigi 321, Japan     

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.     

Purification and characterization of barley-aleurone xylanase

Xylanase (-1,4-D-xylan xylanohydrolase; EC 3.2.1.8) from aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) was purified and characterized. Purification was by preparative isoelectric focusing and a Sephadex G-200 column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme showed a single protein band with an apparent molecular weight (Mr)=34000 daltons. The isoelectric point of the enzyme was 4.6. The enzyme had maximum activity on xylan at pH 5.5 and at 35° C. It was most stable between pH 5 and 6 and at temperatures between 0 and 4° C. The K_m was 0.86 mg xylan·ml^-1.Abbreviations GA₃ gibberellic acid - kDa kilodalton - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis