铁氰化钾氧化固氮酶钼铁蛋白与铁、钼、硫化合物重组的研究

棕色固氮菌固氮酶钼铁蛋白经铁氰化钾氧化后变为“漂白蛋白”,其活性损失约75％,同时释放出80％的钼和50％左右的铁。与铁、钼、硫重组溶液重组,金属组成和乙炔还原活性均得到明显恢复。穆斯鲍尔(M／issbauer)谱表明,重组溶液中的Fe、Mo、S已形成FeMc6原于簇的类似物,“重组蛋白”的M6ssbauer谱和天然MoFe蛋白相似。     

棕色固氮菌固氮酶铁蛋白的研究(四)

应用荧光探剂——荧光醋酸汞和荧光胺研究了棕色固氮菌固氮酶的两种蛋白组分与MgATP和MgADP的关系。实验确证该菌钼铁蛋白不能与MgATP和MgADP络合,而铁蛋白则能。每分子铁蛋白络合2个分子MgATP,但至少是络合2个分子MgADP,反应的部位是在铁蛋白的巯基上。MgATP和MgADP与铁蛋白络合后,引起铁蛋白构型变化,使铁蛋白中可与荧光胺反应的氨基量增加。MgATP和MgADP二者与铁蛋白作用后所得结果相近似。在没有MgATP存在条件下,钼铁蛋白仍能与铁蛋白相互作用,并在巯基部位上产生变化。对于氨基也有影响。     

高度缺失金属原子簇的钼铁蛋白的制备及其重组

棕色固氮菌固氮酶钼铁蛋白与邻菲罗啉厌氧保温时,尿素可使钼铁蛋白丢失的Ｆｅ原子数目显著增加,在有过量Ｎａ２Ｓ２Ｏ４存在时,每个蛋白分子失去的Ｆｅ原子数猛然增至２６－２８个,经Ｓｅｐｈａｄｅｘ Ｇ－２５柱层析得到的蛋白已失去８５％的乙炔还原活性,而蛋白回收率仍可达６０％。与含Ｍｏ重组液重组后,这种失活蛋白的紫外－可见圆二色谱、凝胶电泳图谱和乙炔还少在性均有所恢复,结果表明,尿素可使厌氧钼铁蛋白的ＦｅＭ     

产碱菌麦芽四糖淀粉酶的化学修饰

不同蛋白质侧链修饰剂对麦芽四糖淀粉酶进行修饰。在一定条件下,分别用ＩＡＡ、ＮＥＭ、ＥＤＣ和ＮＡＩ处理后,酶活力不受影响,仍为１００％,说明巯基、羧基和酪氨酸残基与酶活力无关。用ＤＥＰ、ＮＢＳ和ＨＮＢＢ修饰后,酶活力大幅度下降,说明组氨酸和色氨酸基为酶活力所必需。     

钼铁蛋白铁钼辅因子的有机组分对其功能的影响

棕色固氮菌(Azotobacter vinelandii)固氮酶的钼铁蛋白经邻菲啰啉在厌氧或有氧环境中处理后,变为 P-cluster 单一缺失或 P-cluster 和 FeMoco 同时缺失的失活钼铁蛋白。含柠檬酸盐或高柠檬酸盐的重组液都使这两种失活蛋白能恢复固氮酶重组的 H~ 和 C_2H_2还原活性,活性恢复程度随反映钼铁蛋白中金属原子簇含量变化的圆二色和磁圆二色谱及金属含量的恢复程度的提高而提高,但它们固 N_2能力的恢复程度则不相同:P-cluster 单一缺失的蛋白用两种重组液重组后均可恢复其固 N_2能力,而 P-cluster 和 FeMoco 同时缺失的蛋白,只有用含高柠檬酸盐的重组液重组才恢复其固 N_2能力,表明含不同有机组分的重组液所组装的 P-cluster 均与天然状态相同,只有含高柠檬酸盐的重组液所组装的 FeMoco 才与天然状态相同,从而证明高柠檬酸盐是 FeMoco 的必需的有机组分。     

锰对部分缺失金属原子族的固氮酶钼铁蛋白的重组作用

棕色固氮菌固氮酶钼铁蛋白经邻菲罗啉的Ｏ２处理后,变为部分缺失Ｐ－ｃｌｕｓｔｅｒ和ＦｅＭｏｃｏ的失活蛋白、经与由ＫＭｎＯ４、高柠檬酸铁、Ｎａ２Ｓ和二硫苏糖醇组成的重组液保温后,重组蛋白的吸收光谱和对Ｃ２Ｈ２、Ｈ＋和Ｎ２的还原活性都恢复至与还原钼铁蛋白相似的状态,而它的α－螺旋度和在３８０－５５０ｎｍ,６２０－６７０ｎｍ的ＣＤ谱虽有明显的恢复,但仍与还原钼铁蛋白有所不同。表明：（１）重组蛋白液既含有在     

棕色固氮菌不固氮变种UW45与固氮酶钼铁蛋白铁钼辅因子重组的某些特性

以UW45抽提液的DEAE-纤维素柱层析的0.15M NaCl或0.25M NaCl洗出液与Smith法或Shah法制备的铁铝辅因子重组,则0.25 M NaCl洗出液的重组对乙炔还原的活性远较0.15 M NaGl的为高。0.15 M NaGl的洗出液较0.25 M NaCl洗出液的组分明显不纯。不全钼铁蛋白与铁钼辅因子和铁蛋白重组后能还原基质氰化钾,但对分子氮或迭氮化钠的还原却较微弱甚至不还原。铁钼辅因子按Smith和Shah法制备,其分子量范围分别为低于1000D和接近1500D。由于Smith和Shah法两种铁钼辅因子还原乙炔和氰化钾的比活不同,电泳图谱有差异、分子量又有大小,因此这两种铁钼辅因子的分子结构可能不尽相同。     

水稻疏基蛋白酶抑制剂的分子修饰与其对稻瘟病菌的抑制作用

水稻巯基蛋白酶抑制剂经用二硫苏糖醇,对氯汞苯甲酸和碘乙酸修饰后,对木瓜蛋白酶的抑制活性并无改变;用Ｎ－乙基顺丁烯二酰亚胺与ＣＰＩ反应,可以测出ＣＰＩ分子内有１９个巯基被修饰,被修饰后,抑制活性仍无改变,表明水稻ＣＰＩ的抑制活性不需要巯基参与;应用Ｎ－溴代丁二酰亚胺与ＣＰＩ反应,可测出ＣＰＩ分子内有２个Ｔｒｐ被修饰,修饰后,抑制活性全部丧失,表明Ｔｒｐ是保持抑制活性所必需的基因,水稻巯基蛋白酶抑制剂     

锰,铬和钼重组液及其与部分缺金属原子簇钼铁蛋白重组的比较研究

棕色固氮菌（Ａｚｏｔｏｂａｃｔｅｒｖｉｎｅｌａｎｄｉ）钼铁蛋白经邻菲口罗啉和Ｏ２处理后,缺失一部分ＦｅＭｏｃｏ和Ｐｃｌｕｓｔｅｒ,并失去大部分活性。高柠檬酸铁、Ｎａ２Ｓ和二硫苏糖醇（ＤＴＴ）分别与Ｋ２ＣｒＯ４、ＫＭｎＯ４和Ｎａ２ＭｏＯ４按一定比例混合的具有不同颜色的重组液,均可显著激活失活的钼铁蛋白;然而,除ＤＴＴ可略提高失活蛋白的活性外,其它化合物或缺一、二种化合物的混合液均无激活能力。含９９Ｍｏ重组液与失活蛋白重组后的微分扰动角关联测定显示,该蛋白中的４１％的９９Ｍｏ的状态与克氏肺炎杆菌９９ＭｏＦｅ蛋白中的９９Ｍｏ相似,表明这种激活主要是以金属原子簇含量恢复为基础的。由此可推论出,含Ｍｎ或Ｃｒ的重组液,通过与失活钼铁蛋白重组得到含Ｍｎ或Ｃｒ的固氮酶是可能的。     

胎盘膜胰岛素受体结合活性与巯基和二硫键的关系

部分纯化的人胎盘膜经DTT还原,NEM,DTNB修饰蛋白巯基后,改变了胰岛素受体的结合活性。Scatchard分析表明,当DTT浓度较低时,亲和常数基本不变;高亲和位点数略微升高,较高浓度的DTT处理时,结合位点数和亲和常数均有所下降。DTT还原膜蛋白二硫键后再用NEM,DTNB修饰巯基,胰岛素结合活性进一步下降。NEM或DTNB单独处理结合活性下降较少。胰岛素与受体结合后,用DTT洗后剩余的结合胰岛素比缓冲液洗低,表明有一部分胰岛素以二硫键与受体共价结合。在0—5mmol/L浓度范围内,随着DTT处理浓度的升高,这种以二硫键共价结合胰岛素增加。     

alpha-Bromo-4-amino-3-nitroacetophenone, a new reagent for protein modification. Modification of the methionine-290 residue of porcine pepsin.

A new coloured reagent for protein modification, alpha-bromo-4-amino-3-nitroacetophenone (NH2BrNphAc), was synthesized. The reagent was found to alkylate specifically the methionine-290 residue of porcine pepsin below pH 3 at 37 degrees C, which lead to a 45% decrease of enzyme's activity towards haemoglobin. The effect of this reagent as well as that of other phenacyl bromides on the activity of pepsin appeared to be a result of steric hindrance caused by the attachment of bulky reagent residue to the edge of the cleft harbouring the enzyme active site. Only marginal reaction with the co-carboxy group of aspartic acid-315 was found under the above conditions. More pronounced esterification of carboxy groups (up to one residue per enzyme molecule) occurred when the pH was shifted to 5.2. The latter modification had no noticeable effect on enzyme activity, thus disproving a previously held assumption that pepsin inactivation by phenacyl bromide is due to the carboxy-group esterification. alpha-Bromo-4-amino-3-nitroacetophenone forms derivatives with characteristic u.v. spectra when it reacts with methionine, histidine, aspartic and glutamic acid residues, and may be recommended as a reagent for protein modification.     

Chemical modification of D-amino acid oxidase. Amino acid sequence of the tryptic peptides containing tyrosine and lysine residues modified by fluorodinitrobenzene

D-Amino acid oxidase can be inactivated by covalent modification of predominantly tyrosine residue(s) at pH 7.4 by a low molar excess of fluorodinitrobenzene, which appears to act as an active site-directed reagent (Nishino, T., Massey, V., and Williams, C. H., Jr. (1980) J. Biol. Chem. 255, 3610-3616). Peptide mapping by high performance liquid chromatography of tryptic digests of protein modified with radiolabeled reagent revealed two major radioactive fractions with substantially different retention times which were not observed in protein modified in the presence of benzoate, a potent competitive inhibitor. Isolation and sequence analysis of the major radiolabeled peptides, as well as other direct chemical analyses, are presented which unambiguously demonstrate that these fractions represent modification of two different regions of the protein. The majority of the radiolabel was found within a 61-amino acid residue peptide containing an O-DNP-tyrosine residue exclusively at position 17. The substantial sequence surrounding this tyrosine residue indicates that it is different from that shown to react with N-chloro-D-leucine (Ronchi, S., Galliano, M., Minchiotti, L., Curti, B., Rudie, N. G., Porter, D. J. T., and Bright, H. J. (1980) J. Biol. Chem. 255, 6044-6046). The second fraction consisted of a 12-residue peptide containing an epsilon-DNP-lysine residue at position 5. Together, these two modified amino acids represented 0.89 mol of DNP incorporated/protein monomer. Both modifications must contribute to inactivation to account for the 90% decrease in enzymatic activity. Evidence is presented which suggests that both groups are within the active center of the enzyme and are modified in a mutually exclusive manner.     

Chemical modification of spinach ferredoxin: evidence for the involvement of a complex between ferredoxin and ferredoxin:NADP oxidoreductase in NADP photoreduction.

Spinach ferredoxin was modified chemically with trinitrobenzene sulfonic acid (TNBS), a reagent which reacts specifically with amino groups. The trinitrophenylated ferredoxin (TNP-Fd) can accept electrons from Photosystem I as indicated by its full activity in the photoreduction of cytochrome $\text{c}$. The modified protein is inactive, however, in the photoreduction of NADP and cannot form a complex with the flavoprotein, ferredoxin: NADP oxidoreductase. The data presented indicate that the inactivity of the modified protein is the result of modification of a single amino group.     

Effect of diethyl pyrocarbonate modification on spectral and steady-state kinetic properties of bovine heart cytochrome oxidase.

The histidine-specific reagent diethyl pyrocarbonate has been used to chemically modify bovine heart cytochrome oxidase. Thirty-two of sixty-seven histidine residues of cytochrome oxidase are accessible to modification by diethyl pyrocarbonate. Effects on the Soret and alpha bands of the heme spectrum indicate disturbance in the environment of one or both of the heme groups. However, diethyl pyrocarbonate modification does not alter the 830-nm absorbance band, suggesting that the environment of CuA is unchanged. Maximal modification of cytochrome oxidase by diethyl pyrocarbonate results in loss of 85-90% of the steay-state electron transfer activity, which can be reversed by hydroxylamine treatment. However, modification of the first 20 histidines does not alter either activity or the heme spectrum, but only when 32 residues have been modified are the activity and heme spectral changes complete. The steady-state kinetic profile of fully modified oxidase is monophasic; the phase corresponding to tight cytochrome c binding and low turnover is retained, whereas the high turnover phase is abolished. Proteoliposomes incorporated with modified oxidase have a 65% lower respiratory control ratio and 40% lower proton pumping stoichiometry than liposomes containing unmodified oxidase. These results are discussed in terms of a redox-linked proton pumping model for energy coupling via cytochrome oxidase.     

Biochemical aspects of the visual process. XXVIII. Classification of sulfhydryl groups in phodopsin and other photoreceptor membrane proteins.

Reaction of isolated bovine rod outer segment membrane with radioactive N-ethylmaleimide, both in the presence and absence of 1% dodecyl sulfate followed by dodecyl sulfate-polyacrylamide gel electrophoresis, shows that six sulfhydryl groups (96% of total sulfhydryl in this membrane) are located on the rhodopsin molecule. On the basis of their reactivity towards rho-chloromercuribenzoate and rho-chloromercuribenzene sulfonate in suspensions of outer segment membranes, the sulfhydryl groups of rhodopsin can be divided into three pairs. One pair is rapidly modified, both in light and darkness. This modification does not impair the recombination capacity of opsin with 11-cis retinaldehyde under regeneration of rhodopsin. A second pair is modified upon prolonged interaction with the rho-chloromercuriderivatives in darkness. Modification of this pair leaves the typical rhodopsin absorbance at 500 nm intact, but a proportional loss of recombination capacity does occur. The third pair is only modified after illumination and isprobably located in the vicinity of the chromophoric center. The differences between these results and those obtained by modification with dithiobis-(2-nitrobenzoic acid) or N-ethylmaleimide in suspension, where even upon prolonged exposure to light as well as in darkness only two sulfhydryl groups of rhodopsin are modified, is explained by the detergent-like character of the rho-chloromercuri-derivatives.     

The modification of tryptophan in bovine thrombin.

2-Hydroxy-5-nitrobenzyl bromide, at a 100-fold molar excess, was observed to react withthrombin at pH 4.0 to give a modified enzyme which possessed 20% of the fibrinogen clotting activity and 80% of the esterase activity compared to a control preparation. Spectrophotometric analysis of the modified protein indicated that this effect on catalytic activity was associated with the incorporation of 1 mol of reagent per mol of thrombin. Amino acid analysis showed no loss of amino acids other than tryptophan. The reaction of N-bromosuccinimide with thrombin at 2-fold molar excess resulted in the modification of one tryptophan per mol of enzyme with the loss of 80% of the fibrinogen clotting activity with, as above, a considerably smaller loss of esterase activity. Oxidation of thrombin with N-bromosuccinimide decreased the extent of subsequent tryptophan modification with 2-hydroxy-5-nitrobenzyl bromide. Thrombin modified with 2-hydroxy-5-nitrobenzyl bromide showed a 3-4 fold increase in Km and a decrease in V for the ester substrate. The reaction of thrombin with 2-acetoxy-5-nitrobenzyl bromide, a substrate analogue, also resulted in the inactivation of the enzyme. The data are interpreted to show the presence of a tryptophan residue at or near the enzyme's substrate binding site.     

The use of p-fluorobenzenesulfonyl chloride as a reagent for studies of proteins by fluorine nuclear magnetic resonance

The reagent p-fluorobenzenesulfonyl chloride modifies the protein side chains of tyrosine, lysine, and histidine and the alpha-NH2 group. The p-fluorobenzenesulfonyl (Fbs-) group, identified by the 19F nuclear magnetic resonance signal, exhibits a different 19F chemical shift for each functional group modified. The Fourier-transformed spectra of the Fbs- group displayed the expected nine-line multiplet in Fbs- amino acids and simple Fbs- peptides but not in the Fbs- proteins, where the resolution was less. Lysozyme, RNase, DNase, and chymotrypsin react with this reagent and each Fbs- protein exhibits a distinctive pattern of 19F NMR signals due to the label, suggesting that the reaction of the reagent varies with the reactivity of the side chains in a protein. The three major 19F signals of the unfolded Fbs-RNase in 8 M urea are due to the Fbs- label on the imidazolium, alpha-NH2, and epsilon-NH2 groups. Based upon results from amino acid and 19F NMR analyses of the tryptic-chymotryptic peptides of Fbs-RNase, portions of the imidazolium and epsilon-NH2 resonances were assigned to the Fbs- label on His-105 and Lys-41, respectively, while the alpha-NH2 resonance was entirely due to the Fbs- label on the alpha-NH2 of Lys-1. Because Fbs-RNase has an unchanged, near-ultraviolet circular dichroism spectrum and because it retains approximately 80% of the RNase activity, the conformation of Fbs-RNase is probably not altered from the folded conformation of the native enzyme. Upon unfolding in 8 M urea or heating at 70 degrees C, Fbs-RNase gave a 19F NMR spectrum differing from that of the folded Fbs-RNase. In the presence of uridylic acid, Lys-41 was the only residue protected from modification by the reagent with a concomitant reduction of the epsilon-NH2 resonance, and the RNase thus modified was fully active. Hence, 19F NMR analysis of protein, via the reaction with p-fluorobenzenesulfonyl chloride, provided not only information about the protein conformation but also direct measurements of the modification status.     

Modification of maize phosphoenolpyruvate carboxylase by Woodward's reagentK

Maize leaf phosphoenolpyruvate carboxylase was completely and irreversibly inactivated by treatment with micromolar concentrations of Woodward's reagentK (WRK) for about 1 min. The inactivation followed pseudo-first-order reaction kinetics. The order of reaction with respect to WRK showed that the reagent causes formation of reversible enzyme inhibitor complex before resulting in irreversible inactivation. The loss of activity was correlated to the modification of a single carboxyl group per subunit, even though the reagent reacted with 2 carboxyl groups per protomer. Substrate PEP and PEP + Mg²⁺ offered substantial protection against inactivation by WRK. The modified enzyme showed a characteristic absorbance at 346 nm due to carboxyl group modification. The modified enzyme exhibited altered surface charge as seen from the elution profile on FPLC Mono Q anion exchange column. The modified enzyme was desensitized to positive and negative effectors like glucose-6-phosphate and malate. Pretreatment of PEP carboxylase with diethylpyrocarbonate prevented WRK incorporation into the enzyme, suggesting that both histidine and carboxyl groups may be closely physically related. The carboxyl groups might be involved in metal binding during catalysis by the enzyme.     

Modification of maize phosphoenolpyruvate carboxylase by Woodward's reagentK

Maize leaf phosphoenolpyruvate carboxylase was completely and irreversibly inactivated by treatment with micromolar concentrations of Woodward's reagentK (WRK) for about 1 min. The inactivation followed pseudo-first-order reaction kinetics. The order of reaction with respect to WRK showed that the reagent causes formation of reversible enzyme inhibitor complex before resulting in irreversible inactivation. The loss of activity was correlated to the modification of a single carboxyl group per subunit, even though the reagent reacted with 2 carboxyl groups per protomer. Substrate PEP and PEP + Mg²⁺ offered substantial protection against inactivation by WRK. The modified enzyme showed a characteristic absorbance at 346 nm due to carboxyl group modification. The modified enzyme exhibited altered surface charge as seen from the elution profile on FPLC Mono Q anion exchange column. The modified enzyme was desensitized to positive and negative effectors like glucose-6-phosphate and malate. Pretreatment of PEP carboxylase with diethylpyrocarbonate prevented WRK incorporation into the enzyme, suggesting that both histidine and carboxyl groups may be closely physically related. The carboxyl groups might be involved in metal binding during catalysis by the enzyme.     

A simplified method of quantitating protein using the biuret and phenol reagents.

A new modification of the Lowry method of quantitating protein is introduced, whereby the protein sample is mixed first with a diluted biuret reagent and later with 2 n phenol reagent (undiluted) for color development. The method is superior to the original in (i) extremely stable color development (0.3% change from 20 min to 2 hr), (ii) good reproducibility (±2% for 50–600 μg/ml of protein), (iii) elimination of the need to mix reagents for each assay, (iv) good storability (the diluted biuret reagent is storable for months), (v) simplicity (both reagents are available commercially), and (vi) the biuret method can be immediately converted to the Lowry method if the former does not yield a sufficient absorbance. It was found that the relationship between absorbance and protein concentration is expressed by a straight line with a slope of 1 in the Hill plot.