蓝藻固氮酶钼铁蛋白的研究

改进了蓝藻固氮酶的分离、提纯方法。首次用小型厌氧聚丙烯酰胺凝胶制备电泳法,代替常用的层析法,获取了电泳纯的蓝藻固氮酶钼铁蛋白,简化了程序,缩短了实验周期。SDS凝胶电泳和分子筛凝胶过滤测定分子量结果表明,钼铁蛋白分子量为360,000,由4个分子量为90,000的同一类型亚单位构成。每个钼铁蛋白分子含1个钼,18个铁和3290个氨基酸残基。其中酸性氨基酸占优势。研究了柱孢鱼腥藻(Anabaena cylindrica)固氮酶粗提物和钼铁蛋白的某些特性,其结果是:米氏常数为3.33×10~(-3)大气压乙炔,等电点为5—5.5。紫外、可见光谱与其它固氮生物的类似。盐对蓝藻固氮酶较之对其它固氮生物的固氮酶有更大的抑制作用。     

铁钼辅因子激活氧钝化钼铁蛋白的初步研究

棕色固氮菌(Azotobacter vinelandii)固氮酶钼铁蛋白氧钝化后,未断裂出任何含钼、铁原子的断片。用有机溶剂从钼铁蛋白中提取得到的铁钼辅因子(FeMoCo)可以激活被氧钝化了的钼铁蛋白,使其还原乙炔能力得到部分或完全恢复。这种激活作用的效率随着钼铁蛋白氧钝化程度的加深而降低。初步结果表明,氧钝化的钼铁蛋白中最先受到损伤的可能是 FeMoCo。如果氧钝化程度进一步加剧,其它部分也可能受到损伤。     

有催化活性的固氮酶二聚态钼铁蛋白

利用离子交换层析和凝胶过滤,第一次成功地从棕色固氮菌提取液中分离出接近电泳纯的能重组固氮酶活性的二聚态钼铁蛋白。它的分子量约为150,000,每分子含钼约0.5原子。当与铁蛋白重组固氮酶时,表现出相当高的活性。这个结果对固氮酶底物络合中心的结构提供了重要的启示,指出与目前较为流行的双钼多核中心相比,含单钼的多核中心至少同样是不可忽视的可能结构。     

棕色固氮菌固氮酶铁蛋白的提纯及其某些特性的研究

用DEAE-纤维素和凝胶过滤法,可将棕色固氮菌固氮酶铁蛋白组分提纯43倍,比活性述512毫微克分子NH3／毫克蛋白·分。用聚丙烯酰胺凝胶电泳检定,呈均一状态,不含色氨酸、钼。每分子铁蛋白含3．89个原子Fe,分子量约64,000。氨基酸组分分析结果表明,铁蛋白中酸性氨基酸含量为碱性氨基酸含量的一倍。在380—650毫微米区内有宽的吸收带,但无明显吸收峰。     

棕色固氮菌固氮链中的能量转化

应用Fld_(SR)天然荧光和1,5—Br—AEDANS及5—IAF荧光探针偶标记铁蛋白和钼铁蛋白作荧光测定。Fld在与铁蛋白络合时所损失能量为铁蛋白所接受。同样,铁蛋白与钼铁蛋白络合时也有能量转移现象。铁蛋白与MgATP形成复合物后仍能将能量转移给钼铁蛋白,铁蛋白与钼铁蛋白络合物间荧光标记基团距离为35～58A,铁蛋白·MgATP与钼铁蛋白间荧光标记基因距离为38～63A。     

N2和NH4^＋培养下粪产碱菌固氮酶钼铁蛋白的合成及其特性

应用柱层析和制备电泳分别将N_2和 NH_4~ 培养的粪产碱菌固氮酶钼铁蛋白(Af 1和Af 1)分离并纯化,两者理化性质十分相似。分子量分别为226 kD和 222 kD;α亚基和β亚基分子量分别为57 kD和60 kD;氨基酸种类相同,总残基数分别为1790和1750;每分子Af 1和Af 1均含有2个原子Mo和32个原子 Fe;金属原子簇的氧化还原当量数为6。Af 1的比活性为 1477 nmolC_2H_4 mg~(-1)protein min~(-1),Af 1无活性;分子结构两者有差异。     

固氮酶内部结构揭秘

固氮酶由钼铁蛋白和铁蛋白组成 ,钼铁蛋白包含ＦｅＭｏｃｏ和Ｐ Ｃｌｕｓｔｅｒ。为解释固氮机理 ,Ｋｉｍ等 ( 1 992 )、Ｐｅｔｅｒｓ等 ( 1 997)和Ｍａｙｅｒ等 ( 1 999)分别解析出分辨率为 2 8 、2 .0 和1 .6 的钼铁蛋白晶体结构。Ｅｉｎｓｌｅ等 ( 2 0 0 2 )又获得了 1 .1 6 的钼铁蛋白结晶 ,在对其进行结构解析后发现 :ＦｅＭｏｃｏ的内部有一个轻的原子与六个铁原子键连接。此轻原子不可能是硫 ,最可能是氮 ,但不排除是碳或氧的可能。已有极好的证据表明ＦｅＭｏｃｏ是固氮酶的底物结合位点和还原中心 ,但底物是如何结合到ＦｅＭｏｃ…     

电子传递促进的ATP水解

铁蛋白在结合MgATP时,MgATP基本上不水解,只有在与钼铁蛋白结合并传递电子给钼铁蛋白时,MgATP才酶促水解为MgADP和Pi(磷酸根),电子传递和ATP的水解是两个快速的偶联过程。[Fe_4S_4(SPh)_4]~(-2)     

棕色固氮菌固氮酶钼铁蛋白两种聚合体的研究

 棕色固氮菌固氮酶钼铁蛋白八聚体相当于两个钼铁蛋白四聚体的聚合体。在细胞生长过程中,胞内钼铁蛋白两种聚合体的相对含量出现规律性变化:在对数期,细胞固氮酶比活力成上升趋势,而钼铁蛋白主要以高活力的四聚体形式存在;在对数期结束至稳定期,细胞固氮酶比活力下降至一个低水平的稳定值,此时的钼铁蛋白基本上为八聚体形态。在细胞固氮生长时,向培养基中加入过量氨可明显地导致钼铁蛋白由四聚体向八聚体的转化。我们推断,生长过程中胞内钼铁蛋白聚合态的变化可能是调节固氮酶活力的一种方式。胞外,钼铁蛋白的两种聚合态可以相互转化。     

纯化柱胞鱼腥藻(Anabaena cylindrca)固氮酶组份(铁蛋白)的厌氧制备电泳法

各种固氮生物的固氮酶对氧都很敏感,无论是制备固氮酶的组份Ⅰ(钼铁蛋白)或组份Ⅱ(铁蛋白),也无论采取什么方法(如DEAE-纤维素层析法、硫酸鱼精朊沉淀法、胶滤、制备凝胶电泳等等)都必需在严格厌氧条件下进行,铁蛋白对氧更加敏感,因此要获得较纯而又具活力的铁蛋白,其分离、纯化过程既要严格厌氧又要迅速。到目前为止,仅红螺菌(Rhodospirillum rubrum)和棕色     

The molecular weight of, and evidence for two types of subunits in, the molybdenum-iron protein of Azotobacter vinelandii nitrogenase.

The weight-average molecular weight of the Mo-Fe protein isolated from Azotobacter vinelandii has been determined by sedimentation-equilibrium techniques. In buffer, the value is 245000+/-5000; in 8M-urea, the value is 61000+/-1000. The protein was separated into two components by chromatography on CM-cellulose in 7M-urea, pH 4.5. These components have similar molecular weights but were shown to differ in charge, amino acid content and arginine-containing peptides. It is proposed that the tetramer has the subunit composition (nalpha2nbeta2).     

ISOLATION OF AN ACID-SOLUBLE PROTEIN WITH LOW MOLECULAR WEIGHT FROM MONKEY BRAIN

Abstract— The isolation of a perchloric acid-soluble low molecular weight protein from brain of Macaca irus is reported. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel isoelectric focusing indicate that the protein is free of impurities. The molecular weight, as determined by gel filtration and sodium dodecyl sulphate gel electrophoresis, is shown to be 10,400 and 9900, respectively. This is in agreement with the value of 10,700 obtained from amino acid analysis. The protein contains 27 per cent acid amino acids and 15 per cent basic amino acids. However, the relatively high amide content gives the protein a neutral nature as shown by isoelectric point determination using gel isoelectric focusing.     

ISOLATION OF AN ACID-SOLUBLE BASIC PROTEIN FROM MONKEY BRAIN

—A basic protein, soluble in 0·1 m -perchloric acid, has been purified from brain of Macaca irus. The protein is homogeneous as indicated by ultracentrifugation, gel filtration, gel isoelectric focusing and gel electrophoresis at pH 2·9, 4·3 and 7·5. The molecular weight is estimated to be 16,000 by electrophoresis in sodium dodecyl sulphate–polyacrylamide gels. This result is in agreement with the value of 16,728 obtained from the amino acid analysis. The protein dimerizes under alkaline conditions. The predominant amino acid is glycine (15%) and the protein also contains 4% cysteine. The ratio of acidic to basic amino acids is 1·6, but a high amide content gives the protein a basic character. An isoelectric point of 9·5 is observed in gel isoelectric focusing.     

Regulatory Properties of Sucrose Synthetase in Sweet Potato Roots

The molecular weight of the yeast tannase [E.C. 3.1.1.20, tannin acyl-hydrolase] of Candida sp. was determined to be 250,000 by gel filtration on Sephadex G–200. The enzyme was dissociable into two identical subunits with molecular weight of 120,000 on SDS-polyacrylamide gel electrophoresis. The amino acid analysis revealed that the enzyme consisted of 786 amino acid residues per protein molecule. The polypeptide moiety of the enzyme was 38 % by the Lowry-Folin reaction and 35% by the amino acid analysis. The enzyme contained 62% neutral sugars, which were identified as mannose and galactose on cellulose thin-layer chromatogram and 2.2 % hexosamines.     

The molybdenum--iron protein of Klebsiella pneumoniae nitrogenase. Evidence for non-identical subunits from peptide ''mapping''.

The molybdenum- and iron-containing protein components of nitrogenase purified from Klebsiella pneumoniae, Azotobacter vinelandii, Azotobacter chroococcum and Rhizobium japonicum bacteroids all gave either one or two protein-staining bands after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, depending on the commercial brand of sodium dodecyl sulphate used. The single band obtained with K. pneumoniae Mo-Fe protein when some commercial brands of sodium dodecyl sulphate were used in the preparation of the electrode buffer was resolved into two bands by the addition of 0.01% (v/v) dodecanol to the buffer. Protein extracted from the two bands obtained after electrophoresis of K. pneumoniae Mo-Fe protein gave unique and distinct peptide 'maps' after tryptic digestion. Undissociated Mo-Fe protein contained both sets of tryptic peptides. These data are consistent with Mo-Fe protein from K. pneumoniae being composed of non-identical subunits. Amino acid analyses of the subunit proteins revealed some clear differences in amino acid content, but the two subunits showed close compositional relatedness, with a different index [Metzer, H., Shapiro, M.B., Mosiman, J.E. & Vinton, J.G. (1968) Nature (London) 219, 1166-1168] of 4.7.     

Characterization of the apolipoproteins of rat plasma lipoproteins.

Purified fractions of three major rat high-density lipoproteins (HDL) and one rat very low-density lipoprotein (VLDL) were isolated by Sephadex gel chromatography or preparative sodium dodecyl sulfate gel electrophoresis. These proteins were characterized by amino acid analysis, end-group analysis, molecular-weight determination, polyacrylamide gel electrophoresis, and circular dichroism. One of these rat proteins, of molecular weight 27 000, appears to be homologous with the human A-I protein. However, rat HDL possesses two additional major components not reported in human HDL - an arginine-rich protein of molecular weight 35 000 and a protein of molecular weight 46 000. The arginine-rich protein of the rat is similar in size and amino acid analysis to the arginine-rich protein reported in human VLDL. A major component of rat VLDL of 35 000 molecular weight appears similar or identical to the arginine-rich protein in rat HDL by every criterion employed for their characterization.     

Comparative biochemistry of entomocidal parasporal crystals of selected Bacillus thuringiensis strains.

Parasporal crystals of Bacillus thuringiensis subspp. kurstaki, tolworthi, alesti, berliner, and israelensis were compared by electron microscopy, polyacrylamide gel electrophoresis, amino acid analysis, tryptic peptide mapping, immunological analysis, and insecticidal activity. Spore coats also were compared by polyacrylamide gel electrophoresis. B. thuringiensis subsp. israelensis crystals were lethally toxic to mosquito larvae and nontoxic to tobacco hornworm larvae. Conversely, crystals from the other subspecies killed tobacco hornworm larvae but were ineffective against mosquitoes. Crystalline inclusion bodies of all subspecies contained a protoxic subunit that had an apparent molecular weight of approximately 1.34 X 10(5). However, polyacrylamide gel electrophoretic patterns of solubilized crystals revealed a small-molecular-weight component (apparent molecular weight, 26,000) in B. thuringiensis subsp. israelensis that was absent in the other subspecies. Also, differences were noted in amino acid composition and tryptic peptide fingerprints. Crystal proteins were found in spore coats of all subspecies. The results suggest that insecticidal specificity is due to unique polypeptide toxins.     

Pumpkin (Cucurbita sp.) seed globulin I. Purification, characterization, and subunit structure

A heat stable globulin present in the cotyledons of pumpkinseeds was prepared as crystals which were soluble in a dilutesaline solution below pH 4.5 or in a solution with a high ionicstrength at neutral pHs. The protein was nearly homogeneousby ultracentrifuge analysis, and had a molecular weight of about112,000 daltons. Sodium dodecyl sulfate-polyacrylamide gel electrophoresisseparated the globulin into two subunits, and ß,corresponding to molecular weights of about 63,000 and 56,000daltons, respectively. By reduction of disulfide bonds, thetwo subunits were each separated into two polypeptide chainswith molecular weights of around 36,000 and 22,000 daltons,judged by gel electrophoresis. The amino acid composition ofwhole globulin indicated high contents of arginine, glutamicacid and aspartic acid. The total number of half-cystine residuewas nine and only one residue was shown to be free. The subunitstructure of the globulin is discussed. The protein has beenshown to have oxaloacetate decarboxylase activity, and thisfact was confirmed. However, the activity decreased markedlyat pH 4.5 in a fairly short period. It did not require Mn⁺⁺,and the K_m for oxaloacetate was determined to be 4.1 mM. (Received April 9, 1976; )     

A clottable protein (coagulogen) from amoebocyte lysate of Japanese horseshoe crab (Tachypleus tridentatus). Its isolation and biochemical properties.

A clottable protein, named coagulogen, was highly purified from the amoebocyte lysate of Japanese horseshoe crab (Tachypleus tridentatus) by a method similar to that used for the lysate of Limulus polyphemus amoebocytes. The isolated material gave a single protein band on analytical gel electrophoresis at pH 3.2, gel electrofocusing, and sodium dodecyl sulfate (SDS) gel electrophoresis with or without 2-mercaptoethanol. It was 90 percent coagulable, and the total yield from 10 ml of the amoebocyte lysate was about 40 mg. The sedimentation coefficient of purified coagulogen was 2.6 S and its molecular weight was estimated to be about 15,300 by sedimentation equilibrium analysis. The molecular weight estimated by SDS-gel electrophoretic analysis was 19,500 +/- 1,000. This discrepancy was apparently due to abnormal mobility arising from the basic nature of this protein on electrophoresis. The protein had a high isoelectric point of pH 10.0 +/- 0.2, as measured by the isoelectric focusing technique. It consisted of a total of 132 to 135 amino acid residues and contained high levels of basic amino acids, which accounted for more than 16 per cent of the total amino acid residues. No methionine was detected. High contents of valine, half-cystine, glutamic acid (glutamine), and phenylalanine were found. The N-terminal sequence of the first three residues of the coagulogen was Ala-Asx-Thr, and its C-terminal residues was identified as phenylalanine, indicating that it consists of a single polypeptide chain. It is of interest that the first three N-terminal residues are homologous with those of the Aalpha-chain of non-human primate fibrinogen.     

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.