Studies on the characterization of the sodium-potassium transport adenosine triphosphatase. Purification and properties of the enzyme from the electric organ of Electrophorus electricus

An unspecific carboxylesterase was purified 180-fold from acid-precipitated human liver microsomes. The final preparation was homogeneous on disc electrophoresis and polyacrylamide gel electrophoresis in the presence of 6.25 M urea at pH 3.2. A single symmetrical peak was also found on gel filtration and on velocity sedimentation in the analytical ultracentrifuge, whereas slight heterogeneity was observed on isoelectric focusing.The amino acid composition of the purified enzyme is presented. From the results the partial specific volume (0.745 ml × g^?1) and the minimal molecular weight (60,000) could be calculated. Fingerprint maps of tryptic peptides from the carboxymethylated enzyme are shown.The molecular weight as determined by gel filtration, disc electrophoresis, and analytical ultracentrifugation is in the range of 181,000–186,000. For the molecular weight of the subunits a value of 61,500 has been obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The equivalent weight of the enzyme has been estimated to be 62,500 from stoichiometry of its reaction with diethyl-p-nitrophenyl-phosphate. Partial cross-linking of the subunits with dimethyl suberimidate and subsequent sodium dodecylsulfate polyacrylamide gel electrophoresis yielded three bands with molecular weights of 60,000, 120,000, and 180,000.From these results it is concluded that human liver esterase is a trimeric protein. It is composed of three subunits of equal size, and there is one active site per subunit.     

不吸水链霉菌葡萄糖异构酶的物化表征

葡萄糖异构酶是一种催化葡萄糖异构为果糖的酶。本文用紫外吸收光谱、红外光谱、氨基酸组分分析、聚丙烯酰胺凝胶梯度电泳、SDS-聚丙烯酰胺凝胶电泳、超薄 层聚丙烯酰胺凝胶等电聚焦电泳技术研究了不吸水链霉菌嗜热亚种M1033菌株产生的葡萄糖异构酶的一些物化性质。结果表明由本实验室制备的均一葡萄糖异构酶的A₂₈₀与A₂₆₀的比值是1.76。它是由一个亚单位组成的酶分子。最小分子量是49000。pI值是5.2。氨基酸组分与其它来源的葡萄糖异构酶的氨基酸组分相比较有一些差异,其中Glu,Gly,ALa和Leu的含量都此其它异构酶的高,而Met,Trp,Asp,Thr则比其它葡萄糖异构酶的低。     

Purification and Some Properties of Active β-Amylase in Rice

An active β-amylase was purified from germinated rice seeds by precipitation with ammonium sulfate, acid treatment, chromatographies on DEAE-cellulose and DEAE-Sephadex A-50, and gel filiations on Sephadex G-75. The purified enzyme was homogeneous in disc electrophoretic analysis.

The molecular weight was estimated to be approximately 53,000 by thin-layer gel filtration and polyacrylamide gel electrophoresis. The isoelectric point was found to be pH 5.0 by disc electrofocusing.

The optimum pH was found to be in the pH range of 5.5 to 6.5. The Km value for soluble starch was 3 mg/ml. The enzyme was inhibited by sulfhydryl reagents or heavy metal ions.

The active β-amylase was oxidatively dimerized by treatment with 0.3 m ferricyanide in 3 m urea. The dimerized enzyme was thought to be one of inert β-amylases in ungerminated rice seeds.     

Purification and Properties of Acid Carboxypeptidase II from Aspergillus oryzae

Acid carboxypeptidase II from Aspergillus oryzae was purified from the rivanol non-precipitated fraction. The purified enzyme was homogeneous on polyacrylamide gel disc electrophoresis. The optimum activity of the enzyme lay at pH 3.0 for carbobenzoxy-L-glutamyl-l-tyrosine. The enzyme was inhibited by diisopropylphosphorofluoridate and SH reagents such as p-chloromercuribenzoate and monoiodoacetate, but not by such metal chelating agents as ethylenediaminetetraacetate, α, α′-dipyridyl and o-phenanthroline. The molecular weight of the enzyme was estimated to be about 105,000.     

Purification and Characterization of Amidase which Participates in Nitrile Degradation

Amidase was purified from the cell-free extract of acetonitrile-grown Arthrobacter sp. J-1 by a procedure involving protamine sulfate precipitation, ammonium sulfate fractionation, and column chromatographies on DEAE-cellulose, hydroxyapatite and Sephadex G-200. The overall purification was 47-fold. The purified enzyme was homogeneous as judged by ultracentrifugal analysis and disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 300,000 and 320,000 by disc gel electrophoresis and gel filtration, respectively. The enzyme was possibly composed of eight identical subunits of a molecular weight of 39,000. The isoelectric point was 3.8. The enzyme catalyzed the stoichiometric hydrolysis of acetamide to form acetic acid and ammonia. The enzyme was active toward acetamide, acrylamide and propionamide and the Km values were 0.97, 23.3 and 8.05 mm, respectively. The enzyme showed acyltransferase activity.     

Ferredoxin-dependent Nitrite Reductase from Spinach Leaves

A ferredoxin-dependent nitrite reductase from Spinacea oleracea was purified approximately 180-fold, with a specific activity of 285 units/mg protein. This purified enzyme also had methyl viologen-dependent nitrite reductase activity, with a specific activity of 164 units/mg protein. After disc electrophoresis with polyacrylamide gel, the purified enzyme showed one major and one minor protein band.

The molecular weight of the enzyme was estimated to be 86,000 from Ultrogel filtration. This purified enzyme in oxidized form had absorption peaks at 278, 390, 573 and 690 nm. The absorbance ratios, A₃₉₀: A₂₇₈ and A₆₇₃: A₃₉₀ were 0.61 and 0.37, respectively.

By applying the purified enzyme to DEAE-Sephadex A–50 column chromatography, the ferredoxin-dependent nitrite reductase activity was selectively decreased. However, the methyl viologen-dependent nitrite reductase activity was increased, with a specific activity of 391 units/mg protein. This modified enzyme was homogeneous by disc electrophoresis with polyacrylamide gel.     

Purification and Properties of β-Glucosidase from Humicola insolens YH-8

The thermophilic fungus, Humicola insolens YH-8 exhibited high β-glucosidase activity when grown in solid wheat bran medium. The β-glucosidase was purified from the culture extract by consecutive column chromatographies and found to be homogeneous on polyacrylamide gel disc electrophoresis. The molecular weight was estimated to be 250,000 by SDS-gel electrophoresis, and the isoelectric point was at pH 4.23. The enzyme had an optimum pH of 5.0, an optimum temperature of 50°C, and showed significant resistance to urea, dimethyl sulfoxide and ethyl alcohol.     

Isolation of Hydroxyanthrones from the Roots of Rumex acetosa Linn

Microorganisms capable of producing high amounts of α-acetolactate decarboxylase (ALDC; EC 4.1.1.5) were screened for with stock type cultures. Brevibacterium acetylicum had the most potent enzyme activity among the strains tested. The productivity of ALDC by B. acetylicum was elevated by adding Zn²⁺ to the medium. ALDC was purified from the cell-free extract of B. acetylicum by a procedure involving ammonium sulfate fractionation, Sephadex G-100 gel filtration, and DEAE-cellulose and FPLC-MonoQ column chromatographies. The purified enzyme was homogeneous by polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was 62,000 by TSK-gel filtration and the subunit molecular weight was 31,000 by SDS polyacrylamide gel electrophoresis. The enzyme activity was inhibited by metal chelators such as diethyldithiocarbamate, 8-oxyquinoline, and o-phenanthroline. Analysis by atomic absorption spectrophotometry showed that zinc atoms were involved in the purified enzyme preparation.     

限制性核酸内切酶Bsp 63Ⅰ的纯化及性质研究

用Ｂａｃｉｌｌｕｓｓｐｈａｅｒｉｃｕｓ６３菌为材料,经ＤＮＡ－Ｓｅｐｈａｒｏｓｅ和ＣｉｂａｃｒｏｎＢｌｕｅＦ３ＧＡ－Ｓｅｐｈａｒｏｓｅ两步亲和层析,将Ｂｓｐ６３Ⅰ纯化到均一程度。酶比活力达６１４００Ｕ／ｍｇ蛋白。用凝胶过滤法测得该酶分子量为１１３８００。该酶样品在ＳＤＳ－ＰＡＧＥ中呈现为一条蛋白带,并测得其亚基分子量为５６８００。用ＤＮＳ－Ｃｌ法测得该酶Ｎ－末端氨基酸为丙氨酸。上述结果表明该酶分子是由两个相同亚基组成。     

Peptidoglutaminase-I and II: Isolation in Homogeneous Form

Peptidoglutaminase-I and II that catalyzed the hydrolysis of the γ-amide of peptidebound glutamine, were purified from the cell-free extracts of Bacillus circulans by streptomycin sulfate precipitation, ammonium sulfate fractionation, DEAE-Sephadex, Sephadex G-200, QAE-Sephadex, hydroxylapatite-cellulose column chromatography, and finally preparative polyacrylantide gel disc electrophoresis. The purification steps resultd in a 714-fold increase in specific activity for peptidoglutaminase-I and in a 223-fold for peptidoglutaminase-II over the original extracts. The both enzymes were homogeneous in disc electrophoresis in polyacrylamide gel, immunoelectrophoresis in agar gel, and sedimentation analysis. Using gel filtration, the molecular weights of peptidoglutaminases I and II were estimated to be 90,000 and 125,000. However, during the purification steps, the both enzymes were observed to cause the dissociation and aggregation reaction which did not so much affect on their enzyme activities.     

PURIFICATION OF BOVINE PINEAL HYDROXYINDOLE O-methylTRANSFERASE BY IMMUNOADSORPTION CHROMATOGRAPHY

A procedure is described for the use of immunoadsorption chromatography of hydroxyindole O-methyltransferase (HIOMT). HIOMT was purified from bovine pineal extract by affinity chromatography on immunoglobulins (Ig)-Sepharose. The overall purification was about 45-fold; the yield was 84%. This enzyme constitutes about 2.0% of the soluble proteins in the pineal gland. The enzyme represented a single precipitin line on Ouchterlony double diffusion plate and immunoelectrophoresis. Ultracentrifugation analysis indicated the existence of molecular aggregates of enzyme and disc gel electrophoresis showed one main protein band and several minor bands. However sodium dodecyl sulphate (SDS) gel electrophoresis showed a single protein band with subunit molecular weight 38,000 demonstrating bovine pineal HIOMT to be polymer enzyme of a single subunit. The properties of the purified enzyme including disc gel electrophoretic pattern, the effect of pH, chemicals and substrates and immunological properties were identical with those of the crude enzyme.     

Purification and Properties of Extracellular Acid Phosphatase from Zizania latifolia-pavasite,stilago esculenta

An extracellular acid phosphatase from Ustilago esculenta was purified to homogeneity on the basis of polyacrylamide gel electrophoresis. It was a glycoprotein with an isoelectric point of 4.7. The molecular weight of the enzyme was estimated to be about 343,000 by gel filtration on Sephadex G-200, whereas on SDS-polyacrylamide gel electrophoresis, the enzyme gave a single protein band with a molecular weight of 116,000. This result suggests that the enzyme consists of three identical subunits. The enzyme showed an optimum activity at pH 4.5, retained 90% of its activity for 10 min at 55°C and had a Km value of 0.25 mm for p-nitrophenylphosphate. No definite substrate specificity of the enzyme was observed.     

Isolation and characteristics of benzoyl-DL-arginl-p-nitroanilide-hydrolysing enzyme (BAPAase) from vetch seedlings]

The enzyme hydrolysing N-benzoyl-D,L-arginine-p-nitroanilide (BAPA). is isolated from vetch seedlings and 1600-fold purified by means of chromatography on DEAE-cellulose, hdroxyapatite and gel filtration through Sephadex G-100. The preparation is chromatographically homogenous, but disc electrophoresis in polyacrylamide gel revealed an insignificant contamination by inactive proteins. The data of disc electrophoresis in polyacrylamide gel in the presence of sodium dodecylsulphate have shown that BAPAase has a quaternary structure containing, probably, four subunits identical in their molecular weight. BAPAase has a narrow substrate specificity: it hydrolyses BAPA, benzoyl-D,L,-argininenaphtylamide, benzoyl-L-arginyglycine CBZ-L-arginylglycine histones and protamine, but does not attack L-arginyl-p-nitroanilide benzoyl-L-arginineamide, tosyl-L-arginine methyl ester and casein.     

Purification and properties of GM1 ganglioside beta-galactosidases from bovine brain

Two GM1-beta-galactosidases, beta-galactosidases I, and II, have been highly purified from bovine brain by procedures including acetone and butanol treatments, and chromatographies on Con A-Sepharose, PATG-Sepharose, and Sephadex G-200. beta-Galactosidase I was purified 30,000-fold and beta-galactosidase II 19,000-fold. Both enzymes appeared to be homogeneous, as judged from the results of polyacrylamide disc gel electrophoresis. Enzyme I had a molecular weight of 600,000-700,000 and enzyme II one of 68,000, as determined on gel filtration. On sodium dodecyl sulfate polyacrylamide slab gel electrophoresis under denaturing conditions, enzyme II gave a single band with a molecular weight of 62,000, while enzyme I gave two minor bands with molecular weights of 32,000 and 20,000 in addition to the major band at 62,000. Both enzymes liberated the terminal galactose from GM1 ganglioside and lactosylceramide but not from galactosylceramide. Enzyme I showed a pH optimum of 4.0 and was heat stable, while enzyme II showed a pH optimum of 5.0 and lost 50% of its activity in 15 min at 45 degrees C. Enzyme I showed a pI of 4.2 and enzyme II one of 5.9.     

Purification and some properties of trehalase from Chaetomium aureum MS-27

The trehalase of Chaetomium aureum was purified about 196-fold with a yield of 51% from the culture filtrate by ammonium sulfate fractionation, DEAE-cellulose column chromatography, acetone fractionation, and Sephadex G-100 gel filtration. The enzyme preparation was homogeneous on disc electrophoresis. The enzyme was most active at pH 4.0 and 50°C. The enzyme was stable from pH 4.0 to 9.0 on 12 h incubation at 37°C. The molecular weight of the enzyme was estimated to be 450,000 by gel filtration on a column of Sepharose 6B, and 115,000 by SDS polyacrylamide gel electrophoresis. This indicated that the enzyme might consist of 4 subunits. The isoelectric point of the enzyme was pH 4.0. The enzyme was active specifically on trehalose and not active on the other disaccharides tested.     

Methyl Viologen-linked Nitrite Reductase from Bean Roots

Methyl viologen-linked nitrite reductase (EC 1.7.7.1), an enzyme which catalyzes the 6-electron reduction of nitrite to ammonia, was isolated from bean roots. The isolated enzyme was homogeneous by disc electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 62,000 by SDS-polyacrylamide gel electrophoresis. In the oxidized form, the enzyme had absorption maxima at 280, 397 (Soret band), 535, and 573 nm (α band), indicating that siroheme is directly involved in the catalysis of nitrite reduction. The absorbance ratios, A₃₉₇ : A₂₈₀ and A₅₇₃ : A₃₉₇, were 0.3 and 0.39, respectively. Antiserum to spinach leaf nitrite reductase failed to give a positive Ouchterlony result with bean root nitrite reductase, but this antiserum did inhibit the activity of the latter enzyme.     

PURIFICATION OF L-GLUTAMIC ACID DECARBOXYLASE FROM CATFISH BRAIN

—L-Glutamic acid decarboxylase (GAD) from brain of the channel catfish (Ictalurus punctatus) has been purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, calcium phosphate gel and preparative polyacrylamide gel electrophoresis. The purity of the enzyme preparation was established by showing that on both 7.5% regular and 3.7–15% gradient polyacrylamide gel electrophoresis the enzyme migrated as a single protein band which contained all the enzyme activity. The molecular weight of the purified GAD was estimated by gel filtration and gradient polyacrylamide gel to be 84,000 ± 2000 and 90,000 ± 4000, respectively. SDS-polyacrylamide gel electrophoresis revealed three major proteins with molecular weights of 22,000 ± 2000, 40,000 ± 5000 and 90, 000 ± 6000 which may represent a monomer, dimer, and tetramer. Antibodies against the purified enzyme were obtained from rabbit after four biweekly injections with a total of 80 μg of the enzyme. A double immunodiffusion test using these antibodies and a crude extract from catfish brains showed only a single, sharp precipitin band which still retained the enzyme activity, suggesting that the precipitin band was indeed a GAD-anti-GAD complex. In an enzyme inhibition study, a maximum inhibition of 60–70% was obtained at a ratio of GAD protein/anti-GAD serum of about 1:1.6. Furthermore, the precipitate from the GAD-anti-GAD incubation mixture also contained the enzyme activity, suggesting that the antibody was specific to GAD and that the antigen used was homogeneous. Advantages and drawbacks of the purification procedures described here and those used for mouse brain preparations are also discussed.     

Immunochemical purification and characterization of ovine alpha-fetoprotein.

Ovine alpha-fetoprotein was successfully isolated from fetal sheep serum by using rabbit anti-ovine alpha-fetoprotein linked to an agarose immunoadsorbent column. Antibody used in this affinity chromatography column was produced by immunizing a rabbit with highly purified alpha-fetoprotein-antibody complex to yield a monospecific antiserum to ovine alpha-fetoprotein. Following affinity chromatography, alpha-fetoprotein was further purified by preparative polyacrylamide disc gel electrophoresis ultimately yielding a 105-fold purification. The purified alpha-fetoprotein was homogeneous on analytical polyacrylamide disc gel electrophoresis. Ovine alpha-fetoprotein was found to be immunochemically related to human alpha-fetoprotein and to exhibit a molecular weight and amino acid composition similar to other mammalian alpha-fetoproteins.     

Purine nucleoside phosphorylases purified from rat liver and Novikoff hepatoma cells.

Purine nucleoside phosphorylase (PNP) was purified from rat hepatoma cells and normal liver tissue utilizing the techniques of ammonium sulfate fractionation, heat treatment, ion-exchange and molecular exclusion chromatography, and polyacrylamide gel electrophoresis. Homogeneity was established by disc gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Purified rat hepatoma and liver PNPs appeared to be identical with respect to subunit and native molecular weight, substrate specificity, heat stability, kinetics and antigenic identity. A native molecular weight of 84,000 was determined by gel filtration. A subunit molecular weight of 29,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point was observed at pH 5.8, and the pH optimum was 7.5. Inosine, guanosine, xanthosine, and 6-mercaptopurine riboside were substrates for the enzymes. The apparent K_m for both inosine and guanosine was about 1.0 × 10^?4m and for phosphate was 4.2 × 10^?4m. Hepatoma and liver PNP showed complete cross-reactivity using antiserum prepared against the liver enzyme.     

Selection of High Ubiquinone 10-Producing Strain of Tobacco Cultured Cells by Cell Cloning Technique

A novel enzyme, which was named N^α-benzyloxycarbonyl amino acid urethane hydrolase, was purified from a cell-free extract of Streptococcus faecalis R ATCC 8043, using N^α-benzyloxycarbonyl glycine as substrate. The enzyme was purified 1300-fold with an activity yield of 8%. The purified enzyme was homogeneous by disc electrophoresis. The molecular weight of the native enzyme is about 220,000 by gel filtration, and a molecular weight of 32,000 was determined for the reduced and denatured enzyme by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point was 4.48. The enzyme was inhibited by p-chloromercuribenzoate. The presence of divalent cations (i.e., Co²⁺ or Zn²⁺) is essential for its activity.