类产碱假单胞菌谷氨酸脱氢酶的提纯、鉴定及某些特性的初步研究

从类产碱假单胞菌纯化出电泳纯的谷氨酸脱氢酶,用聚丙烯酰胺梯度凝胶电泳和ＳＤＳ－聚丙烯酰胺凝胶电泳测得分子量为２９０ ｋＤ,亚基分子量为４７ ｋＤ,提示该酶为六聚体．该酶对ＮＡＤＰ（Ｈ）和底物均具有高度专一性,对谷氨酸、α－酮戊二酸及ＮＡＤＰ＋ 的Ｋｍ 值分别为：２８ ｍ ｍ ｏｌ／Ｌ、１．２ｍ ｍ ｏｌ／Ｌ及０．０６３ ｍ ｍ ｏｌ／Ｌ．用Ｈｉｌｌ作图法求得酶对ＮＨ＋４ 和ＮＡＤＰＨ 的［Ｓ］０．５分别为２４ ｍ ｍ ｏｌ／Ｌ和０．０３７ ｍ ｍ ｏｌ／Ｌ．最适反应温度为５０℃,催化氨化反应和脱氨反应的最适ｐＨ 分别为８．０和８．８,在热稳定性方面不及嗜热细菌的谷氨酸脱氢酶稳定．提纯的谷氨酸脱氢酶在低温（４℃）条件下,可在Ｔｒｉｓ－ＨＣｌ缓冲液中贮存半年以上,活力无明显下降,冷冻则可导致纯酶液迅速失活．氮源对菌体谷氨酸脱氢酶水平有显著影响．     

牛肝L-谷氨酸脱氢酶在压力下的解离

运用荧光光谱方法研究了牛肝Ｌ－谷氨酸脱氢酶（ＧＤＨ）在压力下的解离。研究表明,在２ｋｂａｒ时ＧＤＨ由六聚体解离成亚基,标准解离体积变化为－２９３ｍｌ／ｍｏｌ,解离自由能为４８ｋｃａｌ／ｍｏｌ（１０℃）。ＧＤＨ在压力下的解离还显示出异常的浓度依赖性,表明在天然寡聚蛋白的布居中存在着自由能不同的单体聚合。不同温度下的ＧＤＨ解离研究结果表明,由亚基－六聚体的聚合是一熵增驱动过程。ｂｉｓ－ＡＮＳ存在时观察到的现象,暗示谷氨酸脱氢酶的亚基解离过程中发生了构象漂移（ｃｏｎｆｏｒｍａｔｉｏｎａｌｄｒｉｆｔ）。此外还研究了底物结合对解离的影响     

类产碱假单胞菌杀虫物质的分离纯化和鉴定

类产碱假单胞菌是一株昆虫病原菌,该菌对草地蝗虫、竹蝗等具有良好的致死作用,经鉴定,该菌对蝗虫具有毒杀作用的物质为其代谢分泌到胞外的一种蛋白质。类产碱假单胞菌培养物经硫酸按沉淀,SephadexG-100凝胶过滤及DEAE-SephadexA-50阴离子交换柱层析,纯化获得的杀虫蛋白只含一种亚基,分子量25100,等电点5．16,含17种氨基酸,其中谷氨酸含量最高,胱氨酸含量最低,最大吸收峰为278．3nm。     

文摘

文摘基因工程９８０００１用荧光假单胞菌不同突变株接种的土壤中的微生物活性［英］／Ｋｏｚｄｒｏｊ,Ｊ．∥ＡｃｔａＭｉｃｒｏｂｉｏｌ．Ｐｏｌ．－１９９７,４６（１）．－５７～６３［译自ＤＢＡ,１９９７,１６（１８）,９７－０９３０９］研究了荧光假单胞菌...     

小麦谷氨酸脱羧酶的纯化及部分性质研究

谷氨酸脱羧酶（ｇｌｕｔａｍａｔｅｄｅｃａｒｂｏｘｙｌａｓｅ,ＧＡＤ,ＥＣ４．１．１．１５）催化谷氨酸脱羧生成γ－氨基丁酸（γ－ａｍｉｎｏｂｕｔｙｒａｔｅ,ＢＡ）,植物中已从南瓜［１］、马铃薯和林生山黧豆［２］纯化了ＧＡＤ．ＧＡＤ活性在禾本科作物中作为...     

除草剂阿特拉津生物降解研究进展

阿特拉津（Ａｔｒａｚｉｎｅ）又称氯乙异丙嗪［２－氯－４（乙基）－６－（异丙氨基）－１,３,５－三嗪］,商品名莠去津,是一种广泛使用的三嗪类除草剂,用于阔叶杂草和禾草的防除,如玉米、高梁、甘蔗和库区杂草等。阿特拉津虽然是一种低毒除草剂,但由于它被微生物矿化的过程十分缓慢,在土壤中的半存留期长达４-５７周,所以在施用过这种除草剂的土壤中以及地下水和表面水中,其浓度远远超过３ｐｐｂ的最大允许值,造成对环境的污染［１］。阿特拉津在世界范围内已经使用了近４０年,其在环境中的扩散引起广泛重视,因此研究这种化合物的生物降解机理十分必要。虽然自１９８２年以来先后在诺卡氏菌属（Ｎｏｃａｒｄｉａ）［２,３］、红球菌属（Ｒｈｏｄｏｃｏｃｕｓ）［４,５］、不动杆菌属（Ａｃｉｎｅｔｏｂａｃｔｅｒ）［６］、土壤杆菌属（Ａｇｒｏｂａｃｔｅｒｉｕｍ）［７］和假单胞菌属（Ｐｓｅｕｄｏｍｏｎａｓ）［１,８,９］等多个细菌属中分离到降解阿特拉津的菌株,但直到９０年代中期,对这一生物降解过程所涉及到的基因、酶和中间代谢物仍知之甚少。自１９９５年Ｗａｃｋｅｔ实验室从施用过阿特拉津的土壤中分离到假单胞菌ＡＤＰ菌株以后［１］,阿特拉津的生物降解机理研究获得了迅速发展。此后,又从根瘤菌属（Ｒｈｉｚｏｂｉｕｍ）［１０］以及棍状杆菌属（Ｃｌａｖｉｂａｃｔｅｒ）、产碱杆菌属（Ａｌｃａｌｉｇｅｎｓ）［１１］和Ｒａｌｓｔｏｎｉａ等多个细菌属中分离到降解阿特拉津的细菌。本文主要对假单胞菌ＡＤＰ菌株降解阿特拉津的酶学、遗传学和生物工程研究概况作简要介绍 。     

羧甲基壳聚糖对玉米籽粒氮代谢关键酶和种子贮藏蛋白含量的影响

以羧甲基壳聚糖（ＮＣＭＣ） 处理玉米开花期果穗,发育中籽粒的谷氨酰胺合成酶（ＧＳ） 、谷氨酸脱氢酶（ＧＤＨ） 及谷丙转氨酶（ＧＰＴ） 活性均明显增强,而蛋白水解酶活性下降。羧甲基壳聚糖处理离体玉米籽粒酶提取液,酶液中ＧＤＨ 活性明显提高。羧甲基壳聚糖处理玉米果穗,其发育籽粒中可溶性蛋白和成熟种子中贮藏蛋白含量明显提高。这都表明,羧甲基壳聚糖对玉米氮代谢、蛋白质合成与积累具有明显的生理调节作用。     

羧甲基壳聚糖对玉米籽粒氮代谢关键酶和种子贮藏蛋白含量的影响

以羧甲基壳聚糖（ＮＣＭＣ）处理玉米开花期果穗,发育中籽粒的谷氨酰胺合成酶（ＧＳ）,谷氨酸脱氢酶民（ＧＤＨ）及谷丙转氨酶（ＧＰＴ）活性均明显增强,而蛋白水解酶活性下降。羧甲基壳聚糖处理离体玉米籽粒酶提取液,酶液中ＧＤＨ活性明显提高。羧甲基壳聚糖处理玉米果穗,其发育籽粒中可溶性蛋白和成熟种子中贮藏蛋白含量明显提高,这都表明,羧甲基壳聚糖对玉米氮代谢、蛋白质合成与积累具有明显的生理调节作用。     

神经节苷脂GM_3对人白血病J6－2细胞磷脂酰胆碱代谢的影响

神经节苷脂ＧＭ３诱导人单核样白血病Ｊ６－２细胞沿单核／巨噬细胞途径分化．在ＧＭ３诱导分化同时,Ｊ６－２细胞磷脂代谢发生了显著变化．采用（（３２）Ｐ）Ｐｉ、［ＧＨ３－３Ｈ］胆碱和［ＣＨ３－３Ｈ］ＳＡＭ参入实验对ＧＭ３影响Ｊ６－２细胞ＰＣ代谢的机制进行了初步的探讨．ＧＭ３促进［（３２）Ｐ］Ｐｉ参入Ｊ６－２细胞ＰＣ;抑制［ＣＨ３－３Ｈ］胆碱参入ＰＣ及ＰＣ合成的前体磷酸胆碱及ＣＤＰ－胆碱;ＧＭ３促进［ＣＨ３－３Ｈ］ＳＡＭ参入ＰＣ,但抑制［ＣＨ３－３Ｈ］ＳＡＭ参入ＰＣ合成的前体胆碱、磷酸胆碱和ＣＤＰ－胆碱．上述结果提示,ＧＭ３抑制Ｊ６－２细胞ＰＣ合成的ＣＤＰ－胆碱途径,促进ＰＣ合成的ＰＥ甲基化途径．     

产碱假单胞菌一个新变种

关于假单胞菌的研究,已有许多文献报道。我们于1979—1980年从黑斑泥鳅肠腔内先后分离出假单胞菌83株。经鉴定该菌与产碱假单胞菌(Pseudomonas alcaligenes)很相似,但也有些不同。故认为该菌是产碱假单胞     

Endogenous substrates of protein kinase in rat liver cell sap under different dietary conditions

Liver cell sap from normally fed rats, rats fed with a highly-carbohydrate diet and fasted rats was chromatographed on DEAE-cellulose (pH 7.0). The chromatogram from each diet group was analyzed for pyruvate kinase activity and endogenous substrates of cyclic AMP-stimulated protein kinase. The materials were pooled into five phosphorylatable fractions, in each of which phosphate incorporation at 0.1 mM and 1.0 mM [³²P]ATP in the presence of cyclic AMP and protein kinase was determined.For characterization of the phosphorylatable components, thin-layer gel chromatography on Sephadex G-200 and polyacrylamide gel electrophoresis in detergent were used for determination of native and minimal molecular weights, respectively.Except for pyruvate kinase, eight components which incorporated at least 0.05 nmol of [³²P]phosphate/g of liver were detected. The phosphorylation of four of them was stimulated by cyclic AMP. Their minimal molecular weights were 42 000, 21 000, 52 000 and 49 000. The component with a minimal molecular weight of 42 000 seemed to have a native molecular weight of 160 000. Both the 21 000 and the 52 000 component had a native molecular weight of about 110 000–120 000. The protein with a minimal molecular weight of 49 000 could not be correlated with certainty to a native molecular weight. The proteins whose phosphorylation was not stimulated by cyclic AMP had minimal molecular weights of 54 000, 39 000, 34 000 and 22 000.     

The molecular structure of different polymorphic forms of canine C3 and C4

The subunit composition of different electrophoretic forms of canine C3 and C4 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of reduced immune precipitates. Canine C4 comprises alpha, beta, and gamma chains of approximate molecular weight 90 000–95 000, 72 000, and 33 000, respectively. The molecular weight of the alpha chain of the C4 1 allelic product was approximately 95 000, but 90 000 for the C4 2 and C4 4 allotypes. No differences were observed in the molecular weights of the beta or gamma chains of any canine C4 phenotype tested. Canine C3 appears to be encoded by a single locus. The subunit composition comprises an alpha and beta chain with molecular weights of approximately 106 000 and 71000, respectively. Unlike C4, no differences in the molecular weights of the subunits were observed in different electrophoretic forms of canine C3.     

Detection of a membrane-associated protein on detached membrane ribosomes in Staphylococcus aureus

Membrane ribosomes from Staphylococcus aureus which were detached from the membrane by extraction with the nonionic detergent Triton X-100 retained a protein (MBRP) with a molecular weight of 60 000, which was absent from cytoplasmic ribosomes. MBRP was detected and quantified by immunological methods. When membrane ribosomes were dissociated into 50S and 30S subunits, MBRP remained associated with the 50S particle. MBRP was found both on membrane ribosomes and in the cytoplasm in roughly equal amounts. When added to Triton X-100-solubilized protoplasts, antibodies to MBRP produced immunoprecipitates which contained a complex of MBRP and three other proteins with molecular weights of 71 000, 46 000 and 41 000. Four proteins with the same molecular weights as those of the MBRP complex were found associated with membrane ribosomes. The proteins of molecular weight 71 000, 60 000, 46 000 and 41 000 seemed to be present in stoichiometrically equivalent amounts in the complex.     

Purification and characterisation of alpha-L-fucosidase from human placenta. pH-dependent changes in molecular size.

alpha-L-Fucosidase has been purified 12 000 fold from human placenta. The enzyme is a glycoprotein containing, by weight: 0.9% galactose; 1.9% mannose, 1.9% N-acetylglucosamine and 1.9% N-acetylneuraminic acid. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate separated proteins with molecular weights ot 55 000, 51 400 and 25 000. Resolution of the two larger protein bands varied with the gel system and these proteins may differ only in carbohydrate content. Gel filtration of te purified enzyme failed to separate the three proteins. Treatments with the cross-linking reagent dimethyl suberimidate prior to electrophoresis, resulted in a diminution of the original protein bands and the formation of oligomers with molecular weights of 80 000, 100 000, 130 000, and 144 000. These results suggest that the heavy (55 000 and 51 400) and light (25 000) proteins are structurally associated. The molecular weight of the native enzyme, measured by gel filtration, was dependent on the pH of the eluting buffer. At pH 5.0 or 6.0 a catalytically active peak was observed, with a molecular weight of 305 000. At pH 7.5 this peak was completely absent and the enzyme eluted as an asymmetrical peak with an apparent molecular weight of about 60 000. The reduction in apparent molecular weight at pH 7.5 was reversible by dialysis of isolated fractions at pH 6.0. In agreement with these findings the sedimentation coefficient was 8.5 S at pH 5.0 but only 3.6 S at pH 7.5. The results can be accounted for by the existence of a pH-dependent equilibrium between aggregated and dissociated forms of the enzyme or by pH-depedent conformational changes.     

Studies on structural proteins of the rat liver ribosomes. I. Molecular wights of the proteins of large and small subunits.

Structural proteins of active 60-S and 40-S subunits of rat liver ribosomes were analysed by two-dimensional polyacrylamide gel electrophoresis. 35 and 29 spots were shown on two-dimensional gel electrophoresis of proteins from large and small subunits, respectively. It was noted that the migration distances of stained proteins with Amido black 10B remained unchanged in the following sodium dodecyl sulfate-acrylamide gel electrophoresis, although some minor degradation and/or aggregation products were observed in the case of several ribosomal proteins, especially of those with high molecular weights. This finding made it possible to measure the molecular weight of each ribosomal protein in the spot on two-dimensional gel electrophoresis by following sodium dodecyl sulfate-acrylamide gel electrophoresis. The molecular weights of the protein components of two liver ribosomal subunits were determined by this 'three-dimensional' polyacrylamide gel electrophoresis. The molecular weights of proteins of 40-S subunits ranged from 10 000 to 38 000 and the number average molecular weight was 23 000. The molecular weights of proteins of 60-S subunits ranged from 10 000 to 60 000 and the number average molecular weight was 23 900.     

An analysis of the protein, glycoprotein and monosaccharide composition of Dictyostelium discoideum plasma membranes during development.

Qualitative and quantitative changes in the protein and glycoprotein components of the plasma membrane of the cellular slime mould Dictyostelium discoideum have been detected by analysis of sodium dodecyl sulphate-polyacrylamide gel electrophoretic patterns. The amounts of proteins of subunit molecular weight 220 000, 91 000, 63 000, 59 000, 56 000 increased during the acquisition of aggregation competence, while proteins of subunit molecular weight 82 000 and 22 000 decreased. The amounts of glycoproteins with apparent subunit molecular weights 285 000, 150 000, 137 000, 100 000, 53 000, 50 500 and 30 500 increased during differentiation while a 125 000 dalton component decreased dramatically in amount. The neutral and amino sugar composition of the plasma membrane was also analyzed and found to remain essentially unchanged during the first 12 h of differentiation. The major sugars were mannose, fucose, and glucosamine; galactose and galactosamine were also present, but in lower amounts.     

Relative molecular mass determination of a major, highest relative molecular mass extracellular amelogenin of developing bovine enamel

Proteins of developing bovine enamel were fractionated by molecular sieving and ion-exchange chromatography. The major fraction corresponding to the highest Mr amelogenin of Mr approximately 26 000-30 000 was isolated and its Mr determined by SDS-PAGE, molecular sieving on G-100 resin and high performance liquid chromatography and by sedimentation-equilibrium ultracentrifugation, the latter three procedures in guanidine hydrochloride. SDS-PAGE and HPLC molecular sieving, employing commonly used Mr standards, gave Mr values of approximately 22 000-26 000. SDS-PAGE and HPLC molecular sieving, using proline-rich CNBr peptides of collagen as standards, and sedimentation-equilibrium ultracentrifugation, gave Mr values of approximately 15 000-18 000 and approximately 17 385, respectively. These latter values correspond well with those reported earlier and with the Mr of the major amelogenin computed from recent amino acid sequence data (approximately 19 000). It is concluded that the recently described, highest Mr amelogenin of Mr = 26 000-30 000 is not a new component but is identical to the proline-rich components having relative molecular masses ranging from 15 000 to 18 000 described much earlier by several groups of workers.     

Studies on mitochondrial proteins I. Separation and characterization by polyacrylamide gel electrophoresis

Rat liver mitochondria were fractionated into inner and outer membranes and soluble intermembrane space and matrix. The protein components of these fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mitochondria contained at least 20 components ranging in molecular weights from 10 000 to 140 000. Inner membranes differed markedly from outer membranes both in number of components and size distribution. The intermembrane space contained a few polypeptide species. These were of low molecular weight. The matrix was characterized by a high molecular weight component (130 000) which comprised 30% of this fraction. A major carbohydrate-containing polypeptide with an approximate molecular weight of 93 000 was detected in outer membrane preparations.     

Developmental changes in proteins of purified membranes of chicken lenses and evidence for contamination by cytoplasmic delta-crystallin.

Urea-washed membranes from embryonic chick lenses (15 days old) and from the cortical and nuclear regions of adult chicken lenses (1 year) have been prepared by repeated centrifugation through discontinuous density gradients. The protein components of the isolated membranes have been examined by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate and urea. Proteins with molecular weights of 75 000, 56 000, 54 000, 48 000, 34 000, 32 000, 25 000, and 22 000 were present in all the membrane preparations, although their proportions changed during development. One additional protein, molecular weight 70 000, was seen only in the embryonic lens membranes. The greatest developmental change was the increase in 25 000 molecular weight protein from 12% in the embryonic lens to about 45% in the adult lens. Since it has been suggested that this protein is associated with gap junctions, its increase during development may reflect a corresponding increase in the number of gap junctions in the lens. The 50 000 molecular weight protein of embryonic lens membranes and membranes of adult nuclear lens fibers consisted at least partly of delta-crystallin, since delta-crystallin peptides could be identified in tryptic peptide maps of the isolated protein after in vitro radioiodination. Peptide maps of the 50 000 molecular weight protein of cortical lens fiber membranes contained no identifiable delta-crystallin peptides, although it is possible that modified delta-crystallin peptides may be present. The level of cytoplasmic contamination of the membrane fraction was estimated by preparing lens membranes in the presence of added delta-[35S]crystallin. The results indicated that cytoplasmic contamination contributes significantly to the presence of delta-crystallin in lens membrane preparations.     

The influence of humic substances on the molecular weight distributions of phosphate and iron in epilimnetic lake waters

1. The influence of humic substances on the association of free inorganic iron and phosphate with material of larger molecular weight was investigated in epilimnetic samples from two humus‐rich lakes of contrasting ionic strength. After modification of the molecular weight distribution of the humic substances in the samples using dialysis and ultrafiltration, the molecular weight distribution of added radioisotopes ( Fe3+ and PO43?) was assessed using gel filtration chromatography.
2. The association of Fe3+ and PO43? with larger molecular weight fractions (>50 000 and 10 000–50 000 Da) was not in general related to the quantity of humic substances of the same molecular weight in the samples. However, the proportions of Fe3+ and PO43? observed in higher molecular weight peaks were strongly correlated to the quantity of humic substances of the same molecular weight in (a) the 10 000–50 000 Da peak in the sample of low ionic strength at pH 5.5 and pH 7.0, and (b) the> 50 000 Da peak in the sample of higher ionic strength at pH 4.0.
3. It was concluded that humic substances promote the association of Fe3+ and PO43? with higher molecular weight fractions primarily by acting as peptizing agents for inorganic colloids containing Fe and P. Association of Fe3+ and PO43? with material of higher molecular weight via the formation of humic substance‐Fe3+–PO43? complexes was identified but only at specific pH and within specific molecular weight ranges for each of the epilimnetic lake water samples studied.