肌酸激酶变性过程中圆二色光谱变化及巯基暴露的速度

本文以 CD光谱及巯基测定两种方法监测了肌酸激酶的变性过程.快速可反应巯基的测定表明,巯基暴露速度与从前报道过的荧光光谱及紫外差光谱的变化速度大体相当.但是,在低浓度胍溶液中,隐蔽的芳香氨基酸尚未暴露时,酶分子的椭圆度值已有了明显可察的变化.变性速度的比较表明,220nm平均残基椭圆度值的变化速度明显地慢于芳香氨基酸、巯基的暴露速度.这可能意味着,肌酸激酶分子内某些肽段具有松散的二级结构,又靠近分子表面,易受到低浓度胍的变性作用.而整个酶分子的二级有序结构的破坏,还是慢于卷曲肽链的伸展速度,也即三级结构的破坏速度。     

DECARBOXYLATION DURING THE FORMATION OF THE FLUORESCENT NAD DERIVATIVE OF D-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

During the formation of the fluorescent NAD derivative of rabbit muscle D-glyceral-dehydc-3-phosphate dehydrogenase modified with ~(14)C-1-iodoacetate by ultraviolet irradiation about 1/2 of the total radioactivity is removed as CO_2, when the fluorescent intensity has reached a maximum, With a weak light source, it can be shown that the formation of the fluorescent derivative precedes the decarboxylation. For the Bacillus stearothermophilus enzyme the formation of the fluorescent NAD derivative is an all-sites reaction and is accompanied by the removal of 4 CO_2 molecules showing conclusively that CO_2 has come from the subunits carrying the fluorophore.It is known that the yeast enzyme can be carboxymethylated either at 4, or under carefully controlled conditions, at 2 of the active site SH groups. Experiments with ~(14)C-labelled NAD~ show that both the tetrakis and his carboxymethylated enzymes form 2 molecules of the fluorescent NAD derivative. However, for the biscarboxymethylated enzyme, only on     

LDS与肌酸激酶结合对酶活性的影响

本文用等速电泳技术测定了25℃时LDS对肌酸激酶分子的饱和紧密结合数为87。Hill作图法和Scatchard作图法分析的结果表明,这种结合呈正协同效应。当LDS对酶的高亲和位点充分饱和时,酶完全丧失活力;但部分饱和时,酶的剩余活力却明显地大于被认为是游离酶所能提供的活力。这表明还存在着一些仅被LDS部分饱和的活性酶分子。ATP的存在会导致LDS对酶分子紧密结合的减少和酶活力的部分恢复。     

含有二硫键的蛋白质在6mol/L盐酸胍中变性后不是完全无序的

本文用紫外差光谱、紫外二阶导数光谱和圆二色光谱,对二硫键完整和经还原并氨酰羧甲基化(以下简称还原)的核糖核酸酶A(RNase A),牛血清白蛋白(BSA)和溶菌酶在6 mol/L盐酸胍中变性后的结构进行了比较。尽管在圆二色光谱中,二硫键完整和还原的变性蛋白都看不到α-螺旋和β-折叠等有序二级结构的存在,但是,与二硫键还原的变性蛋白相比,含有完整天然二硫键的变性蛋白在芳香族氨基酸残基侧链的暴露程度上明显较低。这些结果说明含有完整天然二硫键的蛋白在6mol/L盐酸胍中变性后可能仍然保留有一定程度的有序空间结构。     

NAD~ ANALOGS IN FORMATION OF NEW FLUOROPHORES ON ULTRAVIOLET IRRADIATION WITH D-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

A number of NAD~ analogs have been tested in their ability to form fluorescent deriva-tives when UV irradiated with the active site Cys-149 carboxymethylated GAPDH and this hasbeen compared with their properties of acting as hydrogen acceptors and forming the Rackerband. Among the analogs tested, NHD~ , NGD~ , APAD~ and ∈NAD~ give positive results inall the above-mentioned reactions whereas αNAD~ , NMN~ and CPAD~ are all negative.FPAD~ forms a fluorescent derivative on UV irradiation with the carboxymethylated enzymebut is inactive as a hydrogon aceeptor and does not form the Racker band. This is probablydue to thiohemiacetal formation of the pyridine 3-aldehyde of this derivative with the activesite SH group required for both the latter 2 reactions. TPAD~ , although active as a hydrogenacceptor, does not form either a fluorescent derivative or a Racker band. The fact that forthe great majority of the analogs, the property of forming fluorescent derivatives is is parallelwith their hydrogen acceptor activity seems to show that the formation of the fluorescentderivative is indeed at the active site, and hence can be used as an intrinsic probe for thestudy of the conformation of the active site of this enzyme.     

PRELIMINARY CRYSTALLOGRAPHIC STUDIES ON THE COENZYME BINDING TO GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM Palinurus versicolor

The three forms of glyceraldehyde-3-phosphate dehydrogenase from Palinurus versicolor muscle, namelyapo native enzyme, apo calboxymethylated enzyme and the enzyme carrying two fluorescent NAD deriva-tives per tetramer have been crystallized. The space groups and unit cell dimensions of these crystals areisomorphous to each other and to those of three corresponding holo-enzymes saturated with NAD~+ reportedin a previous paper suggesting that binding of coenzyme to the apo enzymes does not lead to significantconformational changes involving domain movement as demonstrated in the case of glyceraldehyde-3-phos-phate dehydrogenase from Bacillus stearothermophilus.     

肌酸激酶在胍溶液中的变性与失活速度的比较

本文以紫外差光谱、荧光光谱为监测手段测定肌酸激酶的变性与失活过程均为一级反应。在3M胍溶液中,30℃测得的失活速度常数为5.9秒~(-1),变性速度常数为1.9秒~(-1)。1M胍变性时,失活速度常数为4.3秒~(-1),而交性速度常数明显降低为0.04秒~(-1)。0.5M胍变性时,失活为两个一级过程,其速度常数分别为3.6秒~(-1)与0.003秒~(-1),此外酶还保持有3.4％的剩余活力;变性速度常数为0.004秒~(-1)。在0.3M胍中,酶的紫外差吸收及荧光变化都很小,但是60％的活力仍然迅速丧失,最后还保持有30％剩余活力。上述结果表明,酶的活性部位可能比较脆弱。酶分子双体的解聚或盐酸胍的抑制作用也可能引起酶的快速失活。低浓度胍变性时,失活过程的多相性意味着可能存在有部分活力的中间态。     

A CIRCULAR DICHROIC STUDY OF THE INTERACTION OF INSULIN A AND B CHAINS

It has been shown previously that the S-thiomethyl forms of the insulin A and B chains interact in solution leading to a partial transfer of Tyr side chains from hydrophilic to hydrophobic environments. In the present work, a circular dichroic study has indicated that the α-helix contents of the chains show a gradual increase upon mixing of the chains reaching completion in about 2h. The separated S-thiomethyl protected chains contain some ordered structure (A: α=15%, β=27%: B: α=22%, β=23%). Contrary to reports in the literature, the reduced chains also show some ordered structure and upon mixing of the reduced chains, the α-helix content also shows some increase. The ordered structure of the reduced chains decrease with increasing concentrations of dithiothreitel and in presence of a large excess of DTT both the reduced chains have very little, if any, α-helix structure. These latter results are in accord with those of Wu and Yang. In agreement with the results obtained previously with ultravio     

羧甲基甘油醛-3-磷酸脱氢酶光照产生新萤光物质的研究——生成条件与性质

本文报道兔肌和猪肌GAPDH经碘代乙酸修饰活性部位半胱氨酸巯基后,在有NAD~+存在下,经紫外光照射可以产生一萤光新峰,其发射波长为410nm,激发光谱为双峰形,峰值分别为293nm和325nm。形成新峰的最适光照波长为280—290 nm,说明新萤光物的形成与芳香族氨基酸的激发有关.在光照过程中,随410nm萤光的增强,由295nm紫外光激发的340 nm蛋白萤光则明显降低,这说明在色氨酸与新生成的萤光发色团之间有能量转移.生成萤光新峰的光化学反应具有高度特异性,用碘代乙酰胺和四硫硫酸钠代替碘代乙酸对Cys-149进行修饰不能产生新峰.在NAD⁺类似物中,ADPR,AMP,ATP和NADH不能生成新峰.而β-NMN⁺和NADP⁺在加大浓度的情况下,可代替NAD⁺与CM-GAPDH生成新的萤光斗句质.文中对新峰发色团与酶蛋白的关系也进行了初步探讨。