Isolation, Purification, and Crystallization of Aspartate Aminotransferase from Wheat Grain

A procedure for isolation and purification of aspartate aminotransferase from wheat grain includes chromatography on DEAE cellulose, acidification-alkalization, precipitation with protamine sulfate, fractionation with ammonium sulfate, and chromatography on hydroxyapatite. The yield of protein was 27% with 95% purity. Crystals of the enzyme (0.05 x 0.025 x 0.015 mm3) were obtained from ammonium sulfate solution.     

Evidence of altered histone interactions, as investigated by removal of histones, in chromatin isolated from rat liver nuclei by a conventional method.

It is shown that the release of the slightly lysine-rich histones f2a2 and f2b by 0.4 M ammonium sulfate from conventionally isolated chromatin is diminished in comparison to the lysed nuclei. The change in extractability is further demonstrated by the application of ethidium bromide. At a molar input ratio of 0.09 (moles ethidium bromide/moles nucleotide) and 0.4 M ammonium sulfate the slightly lysine-rich histones are released from the chromatin to 70 - 80% if the lysed nuclei are used. At 0.1 M ammonium sulfate ethidium bromide effected also a release of 50 % of histone f1. Comparable effects could not be observed with chromatin prepared in a conventional way but instead a tendency towards loss of histone f3 in the presence of ethidium bromide was observed.     

Two pathways in the biosynthesis of cadystins (gamma EC)nG in the cell-free system of the fission yeast

Small metal-binding peptides, cadystins, with the general structure of (gamma-Glu-Cys)n-Gly ((gamma EC)nG), were synthesized in a cell-free system of fission yeast to examine the in vivo synthetic pathway. The crude enzyme for cadystin synthesis was prepared by ammonium sulfate precipitation (75% saturation) from the 120,000 x g supernatant of the cell extract, and the excess salt in the enzyme fraction was removed by Sephadex gel filtration. Using this crude enzyme fraction, it was shown that there were two pathways for cadystin biosynthesis. One pathway is gamma-Glu-Cys (gamma EC) dipeptidyl transfer from both glutathione (gamma ECG) and cadystins to glutathione and cadystins. The other one is gamma EC polymerization from (gamma EC)n and glutathione to (gamma EC)n + i, followed by glycine addition with glutathione synthetase.     

Purification and properties of Streptococcus lactis beta-galactosidase

beta-Galactosidase of Streptococcus lactis 7962 was partially purified, and its properties were studied. Enzyme from only this strain of numerous lactic streptococci tested was stable in cell exudates prepared by various means. Cell-free extracts of the 7962 strain were prepared by sonic treatment of washed cells previously grown in presence of lactose to fully induce enzyme synthesis. Protamine sulfate precipitation of the nucleic acids and ammonium sulfate precipitation of protein were used for partial purification of the enzyme. The resulting enzyme, when resuspended in cold (5 C) phosphate buffer, was extremely labile. However, ammonium sulfate in high concentrations (0.85 m) stabilized and stimulated beta-galactosidase activity. Sephadex G-200 gel filtration was used to achieve further purification and to monitor homogeneity of the enzyme. Separation of the beta-galactosidase in buffer at 5 C yielded an enzyme elution pattern showing two peaks of activity. However, addition of the enzyme solution in 0.85 m ammonium sulfate to the column equilibrated with the same salt concentration yielded only one peak of enzyme activity. The data suggested that the native enzyme was dissociating into active subunits which were stabilized in the presence of the ammonium sulfate.     

Inactivation of kanamycin, neomycin, and streptomycin by enzymes obtained in cells of Pseudomonas aeruginoa

Ten strains of Pseudomonas aeruginosa were disrupted and centrifuged. The supernatant fluids from centrifugation at 105,000 x g contained enzymes inactivating kanamycin, neomycin, and streptomycin in the presence of adenosine triphosphate. Kanamycin-inactivating enzyme was precipitated with ammonium sulfate at 66% of saturated concentration, and the inactivated kanamycin was shown to be kanamycin-3'-phosphate in which the C-3 hydroxyl group of 6-amino-6-deoxy-d-glucose moiety was phosphorylated. This is identical with kanamycin inactivated by Escherichia coli carrying R factor. Streptomycin-inactivating enzyme was precipitated with ammonium sulfate at 33% of saturated concentration.     

M-26碱性木聚糖酶的分离纯化及酶学性质研究

从Bacillus pumilus M-26发酵液中分离纯化碱性木聚糖酶,进行酶学性质研究,同时制备工业用碱性木聚糖酶制剂。首先将M-26发酵液进行硫酸铵盐析,制备工业用碱性木聚糖酶干品;然后进行sephadexG-25层析脱盐和cellulose DE-52层析得以纯化。硫酸铵的饱和度50%,酶制剂的酶活可达9 000 IU/g,收率为85%;分离纯化使酶的比活为126.32 IU/mg蛋白,纯化倍数为19.89,酶的回收率12.83%;分子量约为20 ku;M-26碱性木聚糖酶的最适温度和pH分别是55℃和pH 8.0,具有一定的耐碱性;该酶无纤维素酶活性,Fe2＋对其有激活作用;Mn2＋、Zn2＋、Fe3＋、Cu2＋对其具有抑制作用。短小芽胞杆菌M-26碱性木聚糖酶具有纸浆生物漂白应用前景。     

Initial characterization of sheep T-cell growth factor and its species-restricted activity on human, rat, and mouse cells

Sheep T-cell growth factor (TCGF) was prepared from concanavalin A-activated sheep peripheral blood cells and subsequently characterized by ammonium sulfate precipitation, gel exclusion chromatography, and isoelectric focussing. The TCGF was found in the 60-80% ammonium sulfate fraction and was shown to have an apparent molecular weight of 32,500 and an isoelectric point in the range pI 5.2-5.5. The ability of the sheep TCGF to promote proliferation of activated human, sheep, mouse, and rat cells was compared with that of human TCGF prepared by phytohemagglutinin stimulation of lymphocytes from multiple donors and TCGF prepared from concanavalin A-stimulated rat and mouse spleen cells. Human TCGF was found to act across all species barriers, rat TCGF supported the growth of cells of all species except human, and mouse only promoted the growth of activated mouse and rat cells. Sheep TCGF was unique in being unable to support the growth of any cells except autologous cells.     

Activation of hormone-sensitive lipase in extracts of adipose tissue

Rat adipose tissue was homogenized in 0.154 m KCl, and the supernatant fluid, obtained after centrifugation at 15,000 g, was extracted with benzene to remove triglycerides. Most of the lipase activity in the extracted fluid was precipitated with ammonium sulfate between 15 and 40% saturation. The specific activity of the lipase in this fraction was about three times that in the benzene-extracted supernatant fluid. The specific activity of the monoglyceride esterase was increased to a lesser extent. Lipase activity in the benzene-extracted fluid and in the ammonium sulfate fraction was increased 15-45% by incubation with 0.3 mm ATP, 10 mm MgCl(2), and 0.03 mm cyclic AMP for 10 min before assay. None of these compounds alone or in combinations of two was as effective as all three together. The specific activity of the 15-40% ammonium sulfate fraction prepared from fat cells exposed to epinephrine and glucagon was greater than that from portions of the same cell pool not exposed to hormones. In addition, the already elevated lipase activity in preparations from hormone-treated cells was not enhanced by incubation with ATP, MgCl(2), and cyclic AMP. Thus, it seems probable that the lipase activity in the ammonium sulfate fractions represents, at least in part, hormone-sensitive lipase.     

水稻土模拟土柱中肥料氮素的迁移转化特征

为了明确肥料氮素在模拟土柱中的迁移转化特征,通过布置室内模拟土柱试验,研究了3倍常规施肥水平下（360 mg·kg^-1）水稻土中矿质氮的变化.结果表明： 不同处理、不同土层间NH₄⁺-N和NO₃^--N含量差异显著.不施肥对照在整个培养期间养分含量变化不大,不同土层间亦没有显著性差异.施用尿素和硫铵后,土壤NH₄⁺-N和NO₃^--N含量显著提高,尤其是0～50 mm土层内,分别达到186.0～2882.1 mg·kg^-1和268.7～351.5 mg·kg^-1,分别相当于对照的4.8～242倍和5.7～316倍,50 mm以下各土层与对照处理相似,表明肥料氮素的迁移转化主要发生在0～50 mm土层内,并且在培养的前14 d变化最大.整个培养期间不同土层内,硫铵处理不同矿质态氮含量是尿素处理的0.7～2.0倍,硝化率是尿素处理的0.9～1.4倍,表明硫铵在水稻土中的转化效率略高于尿素.     

Preparation of cross-linked tyrosinase aggregates

Tyrosinase from mushroom was immobilized as a cross-linked enzyme aggregate (CLEA) via precipitation with ammonium sulfate and cross-linking with glutaraldehyde. The effects of precipitation and cross-linking on CLEA activity were investigated and the immobilized tyrosinase was characterized. Sixty percent ammonium sulfate saturation and 2% glutaraldehyde were used; a 3-h cross-linking reaction at room temperature, at pH 7.0 was performed; particle sizes of the aggregates were reduced; consequently, 100% activity recovery was achieved in CLEAs with enhanced thermal and storage stabilities. Slight changes in optimum pH and temperature values of the enzyme were recorded after immobilization. Although immobilization did not affect V_max, substrate affinity of the enzyme increased. Highly stable CLEAs were also prepared from crude mushroom tyrosinase with 100% activity recovery.     

Crystallization of water-soluble chlorophyll-proteins from Lepidium virginicum

Water-soluble chlorophyll-proteins were prepared from leaves of Lepidium virginicum, by means of ammonium sulfate fractionation followed by column chromatography on DEAE-cellulose and Sephacryl S-200. After intensive purification the chlorophyll-proteins were crystallized by dialysis against an ammonium sulfate solution.     

Isolation and characterization of the testosterone-estradiol-binding globulin from human plasma. Use of a novel affinity column.

This report concerns the purification and characterization of the testosterone-estradiol-binding globulin (TeBG) from human plasma. Cohn fraction IV was submitted sequentially to ammonium sulfate preciptation, affinity chromatography, gel filtration, and isoelectric focusing. The final product was homogeneous in polyacrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. Its activity was demonstrated by the finding of slightly more than one binding site/mole for dihydrotestosterone. Association constants (M-1) at 4 and 37degreesC were ascertained for three steroids: dihydrotestosterone; 2.4 x 10(9) and 0.99 x 10(9); testosterone, 1.1 x 10(9) and 0.35 x 10(9); estradiol, 0.60 x 10(9) and 0.22 x 10(9). TeBG is a glycoprotein having a molecular weight of 94000 and both the amino acid and carbohydrate content are presented along with other physical properties.     

Chloroplast leucyl-tRNA synthetase from Euglena gracilis. Purification, kinetic analysis, and structural characterization

Euglena gracilis chloroplast leucyl-tRNA synthetase was purified to homogeneity by a series of steps including ammonium sulfate precipitation and chromatography on hydroxylapatite, DEAE-cellulose, Sepharose 6B, phosphocellulose, and Blue Dextran-Sepharose. The purified enzyme exhibits a specific activity of 1233 units/mg of protein, which is one of the highest specific activities obtained for an aminoacyl-tRNA synthetase prepared from plant cells. The enzyme has an apparent Km value of 8 x 10(-6) M for L-leucine, 1.3 x 10(-4) M for ATP, and 1.3 x 10(-6) M for tRNALeu. Chloroplast leucyl-tRNA synthetase appears to be a monomeric enzyme with a molecular weight of 100 000. The amino acid composition of chloroplast leucyl-tRNA synthetase has been determined. It is the first reported for a chloroplast aminoacyl-tRNA synthetase, and it reveals a relatively large proportion of apolar residues, as in the case of prokaryotic aminoacyl-tRNA synthetases.     

Aqueous two-phase extraction of 2,3-butanediol from fermentation broths using an ethanol/ammonium sulfate system

Separation of 2,3-butanediol from the fermentation broth is a difficult task that has become a bottleneck in industrial production. Aqueous two-phase systems composed of hydrophilic solvents and inorganic salts could be used to extract 2,3-butanediol from fermentation broth. The ethanol/ammonium sulfate system was investigated in detail, including phase diagram, effect of phase composition on partition, removal of cells and biomacromolecules from the broths and recycling of ammonium sulfate. The highest partition coefficient (7.10) and recovery of 2,3-butanediol (91.7%) were obtained by a system composed of 32% (w/w) ethanol and 16% (w/w) ammonium sulfate. The maximum selective coefficient of 2,3-butanediol to glucose was 30.74 in the experimental range. In addition, cells and proteins could be simultaneously removed from the fermentation broth. The removal ratio of cells and proteins reached 99.7% and 91.2%, respectively. The recovery of ammonium sulfate in the bottom phase reached 97.14% when two volumes of methanol were added to the salt-rich phase.     

双水相系统纯化山楂叶中黄酮类物质的研究

应用水、乙醇和硫酸铵三者形成双水相系统,并利用黄酮在乙醇中的溶解度要大于水中的溶解度,以脱除其中一些易溶于水和在高浓度乙醇中溶解度低的物质。尝试了不同盐、不同体积比的乙醇和水、以及盐加入量对分相的影响并测定了其在不同情况下的纯化效果。发现在硫酸铵中的分相情况最好,在加入量到2．5g时既可以达到分相完全。并测定：（1）在5mL乙醇＋5mL水＋5g硫酸铵＋0．15g粗体物（粗体物中黄酮的百分含量为25％）的双水相中,黄酮类物质的回收率为89．2％,而黄酮百分含量提升为47．6％;（2）在4mL乙醇＋5mL水＋5g硫酸铵＋0．15g粗体物（粗体物中黄酮的百分含量为25％）的双水相中,黄酮类物质的回收率为81．9％,而黄酮含量提升为50．9％。水、乙醇和硫酸铵三者形成双水相系统应用于山楂叶中黄酮类物质的纯化在文献中少有报道,而本文应用此法可以将黄酮含量从25％提升到将近47．6％,并且回收率为89％。     

Straightforward isolation of phosphatidyl-ethanolamine-binding protein-1 (PEBP-1) and ubiquitin from bovine testis by hydrophobic-interaction chromatography (HIC)

Isolation of phosphatidyl-ethanolamine-binding protein-1 (PEBP-1) from bovine brain was described almost three decades ago but it required a large number of steps to reach high purity. After the fractionation of bovine testis proteins by ammonium sulfate precipitation we found that PEBP-1, detected by Western blotting, was among the very few proteins still soluble at 80% ammonium sulfate saturation (3.2M). This soluble fraction (S80) was directly loaded onto a phenyl sepharose column equilibrated at the same ammonium sulfate concentration (3.2M). A stepwise elution of the retained material at 1.0, 0.5, 0.2, 0.1M ammonium sulfate in ammonium hydrogen carbonate was performed and then with ammonium hydrogen carbonate alone and finally with 50% ethylene glycol. All fractions were analyzed by SDS-PAGE and Western blotting and the fractions containing PEBP-1 was further fractionated by size exclusion chromatography on a HR75 Superdex column permitting the isolation of ubiquitin in addition to PEBP-1 as demonstrated by Western blotting and mass spectrometry. This study shows the feasibility of hydrophobic interaction chromatography (HIC) on phenyl sepharose at a very high ammonium sulfate concentration (3.2M; 80% saturation) to efficiently purify the proteins that are still soluble in these extreme conditions.     

Enzymic synthesis of useful chito-oligosaccharides utilizing transglycosylation by chitinolytic enzymes in a buffer containing ammonium sulfate

A chitinase purified from culture filtrates of Trichoderma resei KDR-11 efficiently catalyzed a transglycosylation reaction on tetra-N-acetylchitotetraoside in a buffer medium containing ammonium sulfate, converting the tetrasaccharide into hexa-N-acetylchitohexaose (39.6%) and di-N-acetylchitobiose (55.7%) as the major products. Sugar-chain elongation from di-N-acetylchitobiose as the initial substrate to hexa-N-acetyl-chitohexaose and hepta-N-acetylchitoheptaose was also efficiently induced through lysozyme catalysis in the presence of ammonium sulfate at high (30%) concentration. In this case, the addition of ammonium sulfate to the reaction system resulted in a remarkable increase of the hexamer and heptamer productions, which are desirable as biologically active oligosaccharides.     

Ammonium sulfate precipitation of the glucocorticoid receptor from rat liver

The transformed glucocorticoid receptor (GR) from rat liver precipitated at 30% saturation of ammonium sulfate and sedimented at 4.3 S on glycerol gradient centrifugation, whereas the nontransformed GR precipitated at higher concentrations of ammonium sulfate (40-50% saturation) and sedimented at 8.6 S on a gradient. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that heat shock protein 90 (hsp 90) precipitated at 40-50% saturation of ammonium sulfate. Moreover, hsp 90 and the nontransformed GR were eluted from DEAE high performance ion-exchange chromatography at similar salt concentrations (0.22-0.23 M NaCl), whereas the transformed GR was eluted at 0.1 M NaCl. Therefore, hsp 90 seems to be responsible for the surface charge characteristics of the nontransformed GR.     

Purification of lysosomal membrane-bound glucocerebrosidase from human cultured fibroblasts using high-performance liquid chromatography

Glucocerebrosidase from human skin fibroblasts was purified more than 2300-fold to apparent homogeneity with an overall yield of 39% using taurocholate extraction, ammonium sulfate fractionation, and high-performance hydrophobic interaction and gel permeation column chromatography. This relatively high yield is attributed to two modifications from previously published procedures: (i) the elimination of a butanol delipidation step that resulted in substantial loss of enzyme activity; and (ii) the use of 2% (w/v) sodium taurocholate instead of 1-2% sodium cholate that resulted in more than 90% solubilization of total membrane-bound enzyme activity. Confluent monolayers of human cultured skin fibroblasts (approximately 3.6 x 10(8) cells) were harvested from 10 roller bottles. Glucocerebrosidase in the cell pellet was solubilized with 2% (w/v) sodium taurocholate, fractionated in 14% ammonium sulfate, and applied to a high-performance hydrophobic interaction phenyl-5PW column. After an ammonium sulfate descending linear gradient step, glucocerebrosidase was eluted from the column at 4% cholate concentration using a 0-5% linear cholate gradient. There was a 36-fold purification and 80% recovery. In the subsequent step, concentrated glucocerebrosidase fractions from the phenyl column were injected into two Bio-Sil TSK-250 gel permeation columns joined in series. Glucocerebrosidase peak activity was eluted at 263 ml corresponding to Mr 76,000. There was an 18-fold purification and 78% recovery. The enzyme preparation was then recycled through the phenyl-5PW column in order to remove a remaining contaminant.(ABSTRACT TRUNCATED AT 250 WORDS)     

A simplified methylcoenzyme M methylreductase assay with artificial electron donors and different preparations of component C from Methanobacterium thermoautotrophicum delta H.

Different preparations of the methylreductase were tested in a simplified methylcoenzyme M methylreductase assay with artificial electron donors under a nitrogen atmosphere. ATP and Mg2+ stimulated the reaction. Tris(2,2'-bipyridine)ruthenium (II), chromous chloride, chromous acetate, titanium III citrate, 2,8-diaminoacridine, formamidinesulfinic acid, cob(I)alamin (B12s), and dithiothreitol were tested as electron donors; the most effective donor was titanium III citrate. Methylreductase (component C) was prepared by 80% ammonium sulfate precipitation, 70% ammonium sulfate precipitation, phenyl-Sepharose chromatography, Mono Q column chromatography, DEAE-cellulose column chromatography, or tetrahydromethanopterin affinity column chromatography. Methylreductase preparations which were able to catalyze methanogenesis in the simplified reaction mixture contained contaminating proteins. Homogeneous component C obtained from a tetrahydromethanopterin affinity column was not active in the simplified assay but was active in a methylreductase assay that contained additional protein components.