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.     

Isolation of alpha-lactalbumin,beta-lactoglobulin,and bovine serum albumin from cow's milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity

The aim of this study was to introduce a simple, reproducible, and less expensive method for isolation of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin from cow's milk while retaining their antigenicity. Whey (lactoserum) was obtained by isolating casein from defatted milk using hydrochloric acid. Globulins were then precipitated from whey by half-saturated ammonium sulfate and beta-lactoglobulin was purified further using Sephadex G-50 gel filtration. The proteins in the supernatant were also fractionated using diethylaminoethyl cellulose chromatography in which beta-lactoglobulin was separated from alpha-lactalbumin and bovine serum albumin. The latter two proteins that co-eluted in anion-exchange chromatography were then gently isolated from each other by Sephadex G-50 gel filtration. Pure beta-lactoglobulin was also obtained by anion-exchange chromatography of the ammonium sulfate-precipitated globulins. Using enzyme-linked immunosorbent assay (ELISA), Western blotting, and ELISA inhibition assay, antigenicity of the purified proteins was evaluated. Our results showed high purity and well-preserved antigenicity of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin thus purified.     

Heterogeneity of streptococcus group A cell wall protein components

Cell wall surface proteins of group A streptococcus (M 29) were isolated by mild chemical extraction with 1 M hydroxylamine pH 6.0 (37 degrees C). The proteins were purified by ammonium sulfate fractionation, gel filtration on Sephadex G-150 and ion-exchange chromatography on DEAE-Trisacryl M. Using two independent methods (disc electrophoresis in 7.5% PAAG pH 8.9 and high pressure gel filtration), it was shown that after chromatography on Sephadex G-150 the original protein fraction contains up to 8 protein components, while SDS-PAAG electrophoresis performed according to Laemmli revealed up to 25 protein components in the same fraction. During SDS-PAAG electrophoresis six protein fractions performed after ion-exchange chromatography were resolved into 40 protein components whose molecular masses vary from 13 to 80 kDa. Possible reasons for the heterogeneity of surface proteins of group A streptococcus cell wall are discussed.     

Purification of the cellulase complex produced byPenicillium camemberti and its partial characterization

Three cellulase components (FP-ase, CMC-ase and cellobiase) were purified by affinity binding on Avicel followed by Sephadex G-25, DEAE-Sepharose, DEAE-cellulose and Sephadex G-100 chromatography from the culture filtrate of the newly isolated strain Penicillium camemberti. The isolated enzymes had the properties of cellobiohydrolase, endo-1,4-beta-D-glucanase and cellobiase and their respective molar masses were 99, 87 and 61 kDa as determined by molecular sieve chromatography on Sephadex G-100. The amino acid composition of each fraction was also determined.     

Purification of the nitrogenase proteins from Clostridium pasteurianum

A method is described for the purification of the nitrogenase proteins from Clostridium pasteurianum by two polyethylene glycol precipitations and chromatography on columns of DEAE-cellulose, Sephadex G-100 and Sephadex G-200. The Mo-Fe protein and the Fe protein have been purified 70–80-fold from the crude extract, and they appear essentially pure when tested by anaerobic polyacrylamide gel electrophoresis.     

Isolation and characterization of twenty-three ribosomal proteins from large subunits of yeast.

The proteins of large ribosomal subunits from Saccharomyces cerevisiae were separated into 25 fractions by chromatography on columns of carboxymethylcellulose (CMC). Twenty-three proteins were then purified from the 12 CMC fractions by filtration through Sephadex G-75, Sephadex G-100, and Sephacryl S-200, and/or by phosphocellulose column chromatography. The isolated proteins are YP 1, YP 2, YP 9, YP 11, YP 13', YP 16, YP 18, YP 26, YP 39, YP 41, YP 42, YP 42', YP 44, YP 45, YP 47', YP 52a, YP 53, YP 55, YP 59, YP 62, YP 68, YP A1, and YP A2. The molecular weight and amino acid composition of these proteins are presented.     

Amino acid sequence of the tryptic peptide containing the catalytic-site thiol group of bovine liver uridine diphosphate glucose dehydrogenase.

The catalytic-site thiol groups of UDP-glucose dehydrogenase from bovine liver were carboxymethylated with iodo[2-14C]acetate or with iodoacetamidofluorescein. After the residual thiol groups were carboxymethylated with iodoacetate, the proteins were digested with trypsin. The 14C-labelled peptide from the carboxymethylated enzyme was purified to homogeneity by successive thick-layer chromatography on silica gel, paper electrophoresis and chromatography, and column chromatography on Bio-Gel P-6. Homogeneous fluoresceincarboxamidomethylated peptide was prepared from a tryptic digest of fluoresceincarboxamidomethylated enzyme by specific adsorption--desorption from Sephadex G-25. The sequences of either peptide determined by the manual Edman dansyl procedure is: Ala-Ser-Val-Gly-Phe-Gly-Gly-Ser-Cys-Phe-Glx-Glx-Gly-Lys.     

条斑紫菜中一种琼胶多糖的分离纯化及鉴定

条斑紫菜(Porphyra yezoensis)的热水提取物,经DE-23和Sephadex G-200柱层析纯化,得到一种含微量硫酸基的琼胶精品(PY1)。此多糖经Sepharose 6B柱层析鉴定为单一组分,分子量为220 kD。紫外光谱显示它不含蛋白质、多肽及核酸。红外光谱揭示它含3,6_内醚_半乳糖的特征吸收以及微量的硫酸基。该多糖的水解产物经气相色谱分析,主要由半乳糖及其衍生物组成,有少量岩藻糖存在。通过高磺酸氧化和Smith除解以及１３C-NMR谱的分析,该多糖主要由琼胶二糖结构单元和它的生物前体组成,以琼胶二糖为主。     

Possible existence of high molecular precursor proteins for substance P biosynthesis in rabbit spinal ganglia

The presence of precursor protein for substance P (SP) was examined. Sephadex G-75 chromatography of extracts from rabbit spinal ganglia incubated with [³⁵S]methionine gave two radioactive peaks. In the lower molecular weight peak SP was identified by radioimmunoassay, Sephadex G-15 and TLC. When higher molecular weight proteins were incubated with spinal ganglia microsomal preparation and applied to Sephadex G-75, G-15, TLC and HPLC, ³⁵S-labeled SP was identified and characterized as authentic by immunoprecipitation followed by Sephadex G-15. The amount of ³⁵S-labeled SP was reduced by prior heating of ganglia homogenates, addition of N-ethylmaleimide or p-chloromercuribenzoic acid but not by cycloheximide. Characterization of higher molecular weight proteins by Sephadex G-200, gel-permeation chromatography and chromatofocusing revealed that the proteins were of approx. 100,000 and 7000 dalton with isoelectric points of 9.0, 8.4 and 7.8. These results suggest that the processing from a precursor protein to SP may involve several steps and our high molecular weight protein of 7000 dalton may be one of these intermediate precursor peptides for SP.     

Purification of a retinol binding protein from the hen-oviduct cytosol and its immunological cross-reactivity with those from the nucleus

Cellular retinol-binding protein was purified from the cytosol of the oviducts of laying hens by ammonium sulphate fractionation and chromatography on Sephadex G-75 and DEAE-Sephadex A-50 columns. Analysis of the purified retinol-binding protein on 10% SDS-polyacrylamide gel revealed the presence of a doublet representing very similar molecular sizes. Antiserum was prepared against the purified cellular retinol-binding protein, and on the basis of (a) immunodiffusion test and (b) immunoneutralization of 3H-labelled retinol-cellular retinol-binding protein complex on a column of Sephadex G-75, the antiserum appeared to be specific. The antiserum showed cross-reactivity with the nucleosol and a 0.4 M NaCl extract of the chromatin of the oviduct nuclei, while it did not react with the major egg-white proteins such as ovalbumin, conalbumin and ovomucoid.     

Purification and characterization of streptomycin 6-kinase, an enzyme implicated in self-protection of a streptomycin-producing micro-organism

Streptomycin 6-kinase of the streptomycin-producing strain Streptomyces griseus HUT 6037 was purified by fractionation with (NH4)2SO4 and chromatography on DEAE-Sephadex A-25, hydroxyapatite and Sephadex G-100. After PAGE of the final fraction, a protein band corresponding to streptomycin 6-kinase was detected, together with a less intense band having no enzyme activity. Molecular weights determined by SDS-PAGE and by Sephadex G-100 chromatography were about 36000 and 38000, respectively, suggesting that the enzyme was a monomer. The isoelectric point of the enzyme was pH 6.6. Among the nucleoside 5'-triphosphates tested, ATP was the preferred phosphoryl donor. The Km values for streptomycin and ATP were 3.5 mM and 0.4 mM, respectively. The enzyme activity was strongly inhibited by EDTA and AgNO3. It was shown by using an in vitro protein-synthesizing system that purified streptomycin 6-kinase could protect polyphenylalanine synthesis of the streptomycin-susceptible S. griseus strain KSN from inhibition by streptomycin.     

Highly acidic proteins from human brain: purification and properties of Glu-50 protein

Three extremely acidic proteins were isolated from human brain and purified to apparent homogeneity. One of them, Glu-50 protein, contained much glutamic acid (about 50% of the total amino acids). Its purification involved ammonium sulfate fractionation, DEAE-Sephadex A-50 chromatography, and gel filtration on Sephadex G-100 and G-75. Its molecular weight was determined to be 11,000 by SDS polyacrylamide gel electrophoresis and 34,000-36,000 by gel filtration on Sephadex G-75, suggesting that it consists of three identical polypeptide chains. Its isoelectric point was pH 3.9. Its N-terminal amino acid sequence was NH2-Asp-Glu-Pro-Pro-Asp-Glu and its C-terminal amino acid was Lys. It contained no detectable carbohydrate.     

Isolation and characterization of a neurophysin from ostrich neurohypophyses

A neurophysin has been isolated from ostrich neurohypophyses using acid acetone extraction, salt fractionation and Sephadex G-75 chromatography. The crude neurophysin eluting from the Sephadex G-75 column was subjected to a) reverse-phase HPLC followed by Sephadex G-75 chromatography, b) DEAE-Sephadex A-50 chromatography or c) isoelectric focusing. The different homogeneous ostrich neurophysin fractions so obtained were compared i.t.o. amino acid composition, spectral properties, N-terminal amino acid residues and PAGE. They all revealed a single N-terminal Ala residue and displayed spectral properties (A280/A260 less than 1) which are typical of mammalian neurophysin-like polypeptides. Ultracentrifugation studies on purified ostrich neurophysin over a range of concentrations revealed a reversible concentration dependent association behaviour characterized by the presence of dimeric complexes at higher concentrations. Partial sequencing from the N-terminus revealed the molecule to be VLDV-like. The purified molecule was also submitted to CNBr fragmentation.     

大鼠精核蛋白的纯化

用大鼠睾丸制备长形精子细胞核及用附睾制备的附睾精子核,用盐酸胍法提取总碱性蛋白（Ｔｏｔａｌ　ｂａｓｉｃ　ｐｒｏｔｅｉｎ,ＴＢＰ）。通过Ｓｅｐｈａｄｅｘ　Ｇ－１００分子筛层析,其第三峰用５％ＴＣＡ沉淀,得到纯大鼠精核蛋白（Ｒａｔ　ｐｒｏｔａｍｉｎｅ,ＲＰ）。ＲＰ也可用ＴＢＰ通过ＨＰＬＣ反相柱得到纯化,ＲＰ位于２６％－２８％乙腈梯度。     

华丽曲霉Z58有机磷农药降解酶的纯化和性质

华丽曲霉(Aspergillus ornatus)Z58有机磷农药降解酶经硫酸铵分级沉淀、Sephadex G100凝胶过滤、DEAE52离子交换层析得到了分离纯化,用聚丙烯酰胺凝胶电泳(PAGE)鉴定为单一组分。凝胶过滤法测得分子量为67 000,提纯倍数为34.2,收率为17.8%。该酶的最适反应温度45℃,最适反应pH72,对热较稳定,并且能在pH6～10范围保持活性。重金属Cu2+对该酶具有明显的促进作用,而SDS对酶具有抑制作用。此酶对所试的有机磷农药都有较好降解作用。     

Characterisation of cathepsin B and collagenolytic cathepsin from human placenta

1. Human placental cathepsin B and collagenolytic cathepsin were separated by chromatography on columns of Amberlite CG-50. Collagenolytic cathepsin was partially purified by chromatography on DEAE-Sephadex (A-50) and Sephadex G-100. Cathepsin B was purified by chromatography on CM-cellulose and Sephadex G-100. 2. Both enzymes required activation by thiol compounds and were bound to organomercurial-Sepharose-4B. Sulphydryl-blocking reagents were inhibitory, which confirmed an essential thiol group to be present. 3. The enzymes degraded soluble calf skin collagen and insoluble bovine tendon collagen in the telopeptide region at pH 3.5 and 28 degrees C to yield mainly alpha-chain components. 4. In contrast to cathepsin B, collagenolytic cathepsin was found not to hydrolyse any of the low-molecular-weight synthetic substrates that were tested. 5. Leupeptin, a structural analogue of arginine-containing synthetic substrates, and antipain, an inhibitor of papain, were strongly inhibitory to both enzymes. 6. The isoelectric points of the enzymes were similar, being 5.4 for cathepsin B and 5.1 for collagenolytic cathepsin. 7. From chromatography on Sephadex G-100 the molecular weight of cathepsin B was calculated to be 24 500 and that of collagenolytic cathepsin to be 34 600.     

Purification and characterization of a collagenase from the mackerel,Scomber japonicus

Collagenase from the internal organs of a mackerel was purified using acetone precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, gel filtration chromatography on a Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel, and gel filtration chromatography on a Sephadex G-75 column. The molecular mass of the purified enzyme was estimated to be 14.8 kDa by gel filtration and SDS-PAGE. The purification and yield were 39.5-fold and 0.1% when compared to those in the starting-crude extract. The optimum pH and temperature for the enzyme activity were around pH 7.5 and 55 degrees, respectively. The K(m) and V(max) of the enzyme for collagen Type I were approximately 1.1mM and 2,343 U, respectively. The purified enzyme was strongly inhibited by Hg2+, Zn2+, PMSF, TLCK, and the soybean-trypsin inhibitor.     

Purification of prealbumin from human serum

A method is presented by which prealbumin (thyroxine-binding prealbumin; tryptophan-rich prealbumin) may be purified to homogeneity from human serum. The method involves precipitation of contaminating proteins with dilute aqueous phenol, ion-exchange chromatography on DEAE-Sephacel, and gel permeation chromatography on Sephadex G-100. The yield is 25-30%, and the prealbumin is homogeneous by polyacrylamide gel electrophoresis at pH 8.9 and pH 3.6.     

Induction of oxidative stress in leukocytes by an Enterobacter cloacae toxin able to form oligomers and binding to proteins

A leukotoxic and hemolytic toxin was purified from cultures of Enterobacter cloacae. Stimulation of oxidative stress was observed and the production of reactive oxidant species was measured in leukocytes treated with toxin by means of nitroblue tetrazolium and chemiluminescence assays. Molecular weight of toxin was estimated by chromatography and SDS-PAGE. Two protean peaks with toxic activity were found in Sephadex G-100 (P1, 42.0 kDa; and P2, 13.3 kDa). The relative amounts between the peaks (P1/P2 = 0.36) changed when 2-mercaptoethanol was employed (P1/P2 = 0.59). When Sephadex G-200 chromatography was performed, a protean peak of Ve = 113 mL (100 kDa) was found; its was dissociated with 3 M urea in toxic proteins of lower mass: 42, 27, and 13.3 kDa. SDS-PAGE (15%) showed a single toxin band of purified monomer (13.3 kDa), but electrophoresis of a 42-kDa toxin with urea presented three bands of trimer, dimer, and monomer. An increase of casein hydrolysate and albumin molecular weight was observed by chromatography after incubation with toxin due to the binding of both proteins with toxin.     

Isolation and characterization of fourteen ribosomal proteins from small subunits of yeast.

A method for preparation of a large amount of ribosomal subunits from Saccharomyces cerevisiae by a Ti-15 zonal rotor is described. The proteins of the small subunits (ca. 50 000 A260 units) were separated into 22 fractions by chromatography on carboxymethylcellulose columns. Fourteen proteins were then purified from the ten chromatographic fractions by filtration through Sephadex G-100 or Sephacryl S--200. The isolated proteins are YP 6, YP 7, YP 9, YP 12, YP 14', YP 14', YP 28, YP 38, YP 45, YP 50, YP 52, YP 58, YP 63, and YP 70. The molecular weight and amino acid compositions of these proteins are presented.