Phenylalanyl-tRNA synthetase of wheat embryos. Purification, molecular weight, structure, properties]

From wheat germ, a phenylalanyl-tRNA synthetase (E.C.6.1.1.20) has been isolated and purified 187 fold by means of ammonium sulfate fractionation (40-50 per cent) followed by Sephadex G-200 gel filtration, chromatographies on DEAE-cellulose and hydroxyapatite. The enzyme appears to be homogeneous on Sephadex G-200 molecular filtration and polyacrylamide gel electrophoresis. Molecular weight determinations by sucrose gradient centrifugation, gel filtration and gel electrophoresis give an average of 250 00 daltons. The enzyme is dissociated in 1 per cent sodium dodecyl sulfate into two different equimolar components of 80 000 and 50 000 daltons ; this result suggests that the phenylalanyl-tRNA synthetase has a subunit structure : alpha2 beta2. Dissociation with sodium dodecyl sulfate and dithiothreitol gives four other components, probably resulting from the breakdown of the subunits. Optima values of pH, Mg2+ and K+ concentrations, effect of SH-compnents, kinetic parameters have been determined in the aminoacylation reaction. Physical and catalytic properties of wheat germ phenylalanyl-tRNA synthetase appear very similar to those of the yeast and E. coli enzymes.     

Purification of N-hydroxy-2-acetylaminofluorene reductase from rabbit liver cytosol

N-Hydroxy-2-acetylaminofluorene reductase was purified from rabbit liver cytosol by fractionation with ammonium sulfate, and chromatography with DEAE-cellulose, Sephadex G-200 and hydroxylapatite. The purified enzyme was homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 34,000 by the electrophoresis and by gel filtration on Sephadex G-200. The enzyme required cysteine, glutathione, dithiothreitol, 2-mercaptoethanol, NADPH or NADH as an electron donor. The enzyme activity was inhibited by p-chloromercuribenzoic acid, N-ethylmaleimide, cupric sulfate or disulfiram, but little by oxygen.     

Isolation and various properties of thiamine-binding protein from synaptosomes in the rat brain

A thiamine-binding protein (ThBP) with a specific activity of 8.21 nmoles/mg protein was isolated from rat brain synaptosomes by affinity chromatography and gel filtration on Sephadex G-200. The protein was purified 746-fold with a 40.5% yield. ThBP was homogeneous during sodium dodecyl sulfate gel electrophoresis; its molecular mass was determined by gel filtration on Sephadex G-200 and by sodium dodecyl sulfate gel electrophoresis and was equal to 107 and 103 kD, respectively. The pH optimum for the binding is 8.35. When the ability of ThBP to bind thiamine phosphates was tested, the latter decreased in the following order: thiamine monophosphate greater than thiamine triphosphate greater than greater than thiamine diphosphate.     

A comparative study of horseradish peroxidase conjugates prepared with a one-step and a two-step method.

In this study we compared horseradish peroxidase (HRP)-labeled rabbit antihuman immunoglobulin G (IgG) conjugates, prepared by a one-step and a two-step method. Glutaraldehyde was used as a cross-linking agent. Two methods were used for removing unconjugated HRP: Sephadex G-200 gel chromatography and ammonium sulfate precipitation. The conjugates were characterized immunologically, immunochemically and enzymatically. The immunohistoenzymic properties of the conjugates were tested on unfixed cryostat sections of the skin of patients with chronic discoid lupus erythematosus. The influence of the presence of unconjugated HRP and unconjugated IgG was studied. Optimal results were obtained with conjugates prepared by a two-step method. Removing unconjugated HRPimproved the immunohistoenzymic properties of the conjugates. Conjugated and unconjugated IgG could be separated by Sephadex G-200 gel chromatography.     

韭菜线粒体锰超氧化物歧化酶纯化及性质研究

经硫酸铵沉淀、ＤＥＡＥ－Ｓｅｐｈａｃｅｌ层析和Ｓｅｐｈａｄｅｘ Ｇ－２００凝胶过滤,将韭菜线粒体ＳＯＤ纯化到均一程度。从６０００ｇ韭菜叶片线粒体中纯化得到２．５ｍｇ酶,酶比活力达１２００Ｕ／ｍｇ蛋白。该酶对ＫＣＮ和Ｈ２Ｏ２都不敏感,热稳定性弱 外光区吸收峰在２８０ｎｍ,凝胶过滤法测得其分子量为８２００Ｄ,ＳＯＳ－ＰＡＧＥ法测定其亚基分子量的２２０００Ｄ,ＤＮＳ法测得其Ｎ－末端氨基酸为缬氨酸。上述结     

The fractional composition of immunoglobulin preparations studied by gel filtration on different carriers and by high-pressure liquid chromatography]

The fractional composition of immunoglobulin preparations produced by different manufacturing enterprises of this country has been studied by gel chromatography in columns packed with different carriers (Sephadex G-200 and ultragel AcA-34) and by high-performance liquid chromatography (HPLC). This study has revealed the nonstandard character of immunoglobulin preparations produced according to the same technological procedure (modified Cohn's method). The fractionation of immunoglobulins on different carriers with the use of different methods has yielded similar results confirmed by the statistical processing of the data. The results obtained in the study of the fractional composition of immunoglobulin preparations evidence that gel filtration with the use of ultragel and HPLC have greater resolving capacity in comparison with the method of gel filtration on traditionally used Sephadex G-200.     

Isolation and some properties of NAD+ reductase of the green photosynthetic bacterium Prosthecochloris aestuarii.

NAD+ reductase of the green photosynthetic bacterium Prosthecochloris aestuarii was isolated and purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. This enzyme is an FAD-containing flavoprotein and has absorption maxima at 485 (shoulder0 452, 411, and 385 nm (the 411 nm band is due to cytochrome). The molecular weight of the enzyme as determined by gel filtration using Sephadex G-200 is 119,000. The enzyme catalyzes the reduction of NAD+ and NADP+ by photoreduced spinach ferredoxin or reduced benzyl viologen...     

Staphylococcal L-asparaginase: purification and properties of enzymic protein

Staphylococcal L-asparaginase has been purified 400-fold with 40% recovery. The procedure involves ammonium sulphate precipitation and a column chromatography on Sephadex G-200 gel filtration). The enzyme is composed of not identical subunits. protein (pI 4.4) with the approximate molecular weight of 125,000 (estimated by Sephadex G-200 gel filtration). The enzyme is composed of not identical subunits. The polyacrylamide-SDS gel electrophoresis indicated two subunits with molecular weight 18,000 and 22,000.     

Starch phosphorylase from tapioca leaves: Absence of pyridoxal phosphate

Starch phosphorylase from tapioca leaves has been purified to homogeneity, using the technique of ammonium sulfate fractionation, heat treatment, DEAE-cellulose chromatography, filtration through Sephadex G-100 and Sephadex G-200, and DEAE-Sephadex chromatography. The enzyme has a molecular weight of 450,000, as determined by gel filtration through Sephadex G-200 and contains 22 sulfhydryl groups per mole of the enzyme protein. Several types of evidence indicate the absence of pyridoxal 5′-phosphate as a prosthetic group of the enzyme. The kinetic data show a sequential type of the reaction mechanism. The enzyme activity is inhibited by tyrosine (K_i = 2.15 mm).     

Some properties of 3-phosphoglycerate phosphatase from developing rice grain

Some properties of 3-P-glycerate phosphatase from developing caryopsis of rice (Oryza sativa L., variety IR26) were studied. The enzyme was found to be soluble and not bound to starch, and concentrated mainly in the pericarp-aleurone layer; its maximum activity was at 12 to 14 days after flowering. Contents of 3-P-glycerate and chlorophyll were highest in the grain at 7 to 8 days after flowering when starch synthesis was at a maximum. The enzyme was purified about 100-fold by precipitation with 50 to 80% ammonium sulfate, followed by chromatography through Sephadex G-200 and CM-Sephadex C-50. The pH optimum was from 5.7 to 6 and no cation was required for activity. The purified preparation had an apparent Km of 2.85 mm and was inhibited by Cu(2+), Hg(2+), Zn(2+), Fe(3+), molybdate, and F(-). The enzyme also exhibited high activity toward UTP, ATP, and p-nitrophenyl phosphate; moderate activity toward other phosphates; but no activity toward phytate. A molecular weight of about 23,000 was obtained for the 3-P-glycerate peak during gel filtration on Sephadex G-200, which corresponded to a value of 26,000 for the major protein fraction by thin layer gel filtration on Sephadex G-150. Zymograms of the whole extract and semipurified preparations showed two phosphatase bands with 3-P-glycerate as substrate.     

Purification of 6-phosphogluconate dehydrogenase from chicken liver and investigation of some kinetic properties

6-phosphogluconate (6PG) dehydrogenase (EC 1.1.1.44; 6PGD) was purified from chicken liver; some kinetic and characteristic properties of the enzyme were investigated. The purification procedure consisted of four steps: preparation of the hemolysate, ammonium sulfate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Thanks to the four consecutive procedures, product having a specific activity of 61 U (mg proteins)(-1), was purified 344-fold with a yield of 5.57%. Optimum pH, stable pH, optimum temperature, and KM and Vmax values for NADP+ and 6PG substrates were determined for the enzyme. Molecular weight of the enzyme was also determined by Sephadex G-200 gel filtration chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, Ki values and inhibition types were estimated by means of Lineweaver-Burk graphs obtained for NADPH and CO2 products.     

Preparation from keratin sulfate of substrates for the measurement of 2-acetamido-2-deoxy-D-glucose 6-sulfate sulfatase and (1 goes to 3)-N-acetyl-beta-D-glucosaminidase

An extract of bacterial cells Pseudomonas sp. IFO-13309 grown on medium containing 0.1% bovine cornea keratan sulfate of low sulfate content degraded exhaustively bovine cornea keratan sulfate to give 2-acetamido-2-deoxy-beta-D-gluco-pyranosyl 6-sulfate-(1 goes to 3)-D-galactose, isolated by gel filtration on Sephadex G-25 and purified by preparative paper chromatography. This was reduced with sodium borotritide to give 2-acetamido-2-deoxy-beta-D-glucopyranosyl 6-sulfate-(1 goes to 3)-D-[1-3H]galactitol, purified by gel filtration on Sephadex G-15, which was an excellent substrate for the measurement of 2-acetamido-2-deoxy-D-glucose 6-sulfate sulfatase. The reduced, radioactive monosulfated disaccharide was desulfated with methanolic 70mM hydrogen chloride and purified by gel filtration on Sephadex G-15 to give O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1 goes to 3)-D-[1-3H]galactitol, which allowed the measurement of (1 goes to 3)-N-acetyl-beta-D-glucosaminidase. This enzyme may participate in the normal degradation of keratan sulfate.     

Purification of a High Molecular Weight Kininogen from Rat Plasma

A high molecular weight kininogen has been isolated from rat plasma and purified. At each preparative step the kininogen concentration and purity were monitored by assay on the perfused isolated rat uterus in terms of bradykinin equivalents formed per mg protein following incubation of the plasma fractions with rodent acid protease for 24 hours at 37 and pH 4.0. Kinin formation by crystalline trypsin and human pancreatic kallikrein also was compared. Citrated rat plasma first was precipitated with 43% ammonium sulfate. The kininogen fractions then were subjected to a series of gel filtration ion exchange chromatographic columns that included G-200 Sephadex, G-200: G-100 Sephadex interconnected columns, DEAE-A50 Sephadex, and hydroxylapatite. The kininogen fractions finally were subjected to preparative polyacrylamide gel electrophoresis, resulting in a final purification of 92.9-fold compared to the initial rat plasma. A single major kininogen protein band and a minor band of protein impurity were obtained on disc gel electrophoresis. Only the pancreatic kallikrein did not form kinin from this purified kininogen. The apparent molecular weight was estimated by SDS polyacrylamide gel technique to be 110,000.     

Isolation and characterization of a sulfated glycoprotein from rabbit uterus.

Crude glycoproteins were extracted with 0.15 M NaCl from the pooled endometrial scrapings of rabbit uteri after treatment with estrogen. The crude glycoproteins were fractionated with ammonium sulfate, followed by DEAE-cellulose column chromatography, treatment with CM-Sephadex C-25 and gel filtration on Sephadex G-200. Subsequently, purification of an acidic glycoprotein was carried out by gel filtration on Sephadex G-200 and then Sepharose 4B. The results of electrophoresis and enzymatic digestion, together with analytical data and the infrared spectrum indicated that the acidic glycoprotein was a sulfated glycoprotein.     

Purification and characterization of a plasminogen activator secreted by cultured human pancreatic carcinoma cells.

A plasminogen activator secreted by cultured human pancreatic carcinoma (Mia PaCa-2) cells has been purified to apparent homogeneity by procedures including Sepharose-L-arginine methyl ester affinity chromatography, Sephadex G-200 gel filtration, isoelectric focusing, and sodium dodecyl sulfate gel electrophoresis. The plasminogen activator shares many properties with urokinase including: molecular weight (55 000), isoelectric point (8.7), heat stability (60 degrees C, 30 min), PH stability (1.5-10), and its mode of activation of plasminogen. The intracellular enzyme is membrane bound and can be solubilized by detergent. Solubilized activator has a molecular weight similar to that of the secreted enzyme as determined by sodium dodecyl sulfate gel electrophoresis. The production of plasminogen activator by Mia PaCa-2 cells is totally inhibited by actinomycin D and cycloheximide.     

Further studies on bovine serum amylase. 3. Affinity chromatography

The major bovine serum isoamylases controlled by the AmI locus have been examined by gel filtration. On Sephadex G-200 the isoamylases can be resolved into two classes. The AmI A and AmI B have apparent molecular weights of 307,000 daltons whilst the AmI C isozyme has an apparent molecular weight of 44,400 daltons. The separation of the isozymes into two classes according to their elution behaviour on Sephadex G-200 has been shown to be an affinity separation. All three AmI isozymes are eluted from a non-dextran media (BioGel A1.5m) with apparent molecular weights of 417,000 daltons. The affinity separation on Sephadex G-200 has been shown to be inhibited by the addition of 1% (w/v) maltose to the elution buffer. In the presence of 1% (w/v) maltose all three AmI isozymes are coeluted from Sephadex G-200 with apparent molecular weights of 321,000 daltons. The maltase and amylase activities of the AmI isozymes were eluted coincidentally under all the conditions studied.     

Further studies on bovine serum amylase. 3. Affinity chromatography

The major bovine serum isoamylases controlled by the AmI locus have been examined by gel filtration. On Sephadex G-200 the isoamylases can be resolved into two classes. The AmI A and AmI B have apparent molecular weights of 307 000 daltons whilst the AmI C isozyme has an apparent molecular weight of 44 400 daltons. The separation of the isozymes into two classes according to their elution behaviour on Sephadex G-200 has been shown to be an affinity separation. All three AmI isozymes are eluted from a non-dextran media (BioGel A1.5m) with apparent molecular weights of 417 000 daltons. The affinity separation on Sephadex G-200 has been shown to be inhibited by the addition of 1% (w/v) maltose to the elution buffer. In the presence of 1 % (w/v) maltose all three AmI isozymes are coeluted from Sephadex G-200 with apparent molecular weights of 321000 daltons. The maltase and amylase activities of the AmI isozymes were eluted coincidentally under all the conditions studied.     

Purification and Properties of Wall-bound α-Glucosidase from Suspension-cultured Rice Cells

Wall-bound α-glucosidase (EC 3.2.1.20) has been solubilized from suspension-cultured rice cells with Sumyzyme C and Pectolyase Y-23 and isolated by a procedure including fractionation with ammonium sulfate, Sephadex G-100 column chromatography, CM-cellulose column chroma-tography, Sephadex G-200 column chromatography, and preparative disc gel electrophoresis. The molecular weight of the enzyme was 64,000. The enzyme readily hydrolyzed maltose, maltotriose, and amylose, but hydrolyzed isomaltose and soluble starch more slowly. The Michaelis constant for maltose of the enzyme was estimated to be 0.272 mm. The enzyme produced panose as the main α- glucosyltransferred product from maltose.     

Purification and characterization of glutathione reductase from bovine erythrocytes

Glutathione reductase (E.C.1.8.1.7; GR) was purified from bovine erythrocytes and some characteristics properties of the enzyme were investigated. The purification procedure was composed of preparation of the hemolysate, ammonium sulfate fractionation, affinity chromatography on 2',5'-ADP Sepharose 4B, and gel filtration chromatography on Sephadex G-200. As a result of four consecutive procedures, the enzyme was purified 31,250-fold with a yield of 11.39%. Specific activity at the final step was 62.5 U (mg proteins)(-1). For the enzyme, optimum pH, optimum temperature, optimum ionic strength, and stable pH were found to be 7.3, 55 degrees C, 435 mM, 7.3, respectively. The molecular weight of the enzyme was found to be 118 kDa by Sephadex G-200 gel filtration chromatography and the subunit molecular weight was found to be 58 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, Km and Vmax values were determined for glutathione disulfide (GSSG) and NADPH. Ki constants and inhibition types were established for glutathione (GSH) and NADP+. Also, effects of NADPH and GSSG were investigated on the enzyme activities.     

四种土壤微生物总DNA的纯化方法的比较

比较了4种从土壤中直接抽提的微生物总DNA的纯化方法,实验结果表明1 %的琼脂糖凝胶电泳纯化方法及葡聚糖凝胶G 2 0 0离心层析纯化方法均不能完全纯化从土壤中抽提的微生物总DNA。若将直接抽提的总DNA先经葡聚糖凝胶G 2 0 0离心层析纯化,再用1 %的琼脂糖凝胶电泳纯化,则能取得较好的纯化效果。含2 %PVP的1 %琼脂糖凝胶电泳纯化,用DNA凝胶回收试剂盒回收后没有得到纯化后的土壤微生物总DNA。