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     

KINETICS OF IRREVERSIBLE MODIFICATION OF ENZYME ACTIVITY IN A SYSTEM OF COUPLED ENZYME ASSAY (Ⅱ)——BOTH PRIMARY AND AUXILIARY ENZYMES MODIFIED BY REAGENT Y

Following the previous paperr, we examined the kinetics of the irreversible modification of enzyme activity in coupled assay when the modifier reacted with both primary and auxiliary enzymes. It was shown that Tsou's kinetic method can also be used in the present situation in some conditions.     

偶联酶测活系统中酶活性不可逆改变的动力学——Ⅰ.修饰剂仅与待测酶反应的情况

本文对偶联酶测活系统中酶活性不可逆改变的动力学进行了系统的理论研究,导出了不同情况下的动力学方程.结果表明,邹承鲁提出的确定不可逆修饰动力学常数的方法以及区分不同抑制类型的判据可以很方便地推广到偶联酶测活体系.     

甘油醛-3-磷酸脱氢酶活性部位巯基与配体结合的关系

本文用荧光滴定、透析平衡和分子筛柱层析等方法对甘油醛-3-磷酸脱氢酶(以下简称为GAPDH)与配体的结合进行了研究. 荧光滴定的实验结果,GAPDH酶肮与εNAD⁺结合为负协同性,如果酶的Cys-149巯基经碘代乙酸或四硫硫酸钠修饰后,则不仅酶与εNAD⁺结合减弱,并且其结合的负协同性也明显减弱.与此相应,εNAD⁺与酶蛋白结合后εA部分的荧光增强也不如未修饰酶那样明显.分子筛柱层析的结果指出,当酶的Cys-149巯基经化学修饰后,酶与ATP的结合能力也大大下降.这些结果都说明,酶活性部位Cys-149巯基不仅和酶与NAD⁺的菸酰胺部分的结合有关,它的修饰还直接影响到酶的腺嘌呤结合部位,从而影响到酶与配体结合的协同性质.荧光滴定和透析平衡的结果还表明,温度升高酶与εNAD⁺结合的负协同性增强,酶与ATP的结合则由非协同性变为负协同性.     

甘油醛-3-磷酸脱氢酶形成荧光NAD衍生物过程中的脱羧

本文证明兔肌甘油醛-3-磷酸脱氢酶,经紫外光照射,形成荧光NAD衍生物的过程中发生脱羧。用[1-~(14)C]碘乙酸示踪此反应当荧光强度达最大值时,大约有一半的总放射性以二氧化碳的形式放出。用弱光源照射,可以观察到荧光衍生物的生成先于脱羧。嗜热脂肪芽孢杆菌酶,荧光NAD衍生物的生成为全位反应,并伴随有四分子二氧化碳放出。表明二氧化碳只能从形成荧光团的亚基上放出。控制实验条件,酵母酶四个活性部位的巯基可以全被羧甲基化,也可以仅有两个被羰甲基化。这两种酵母酶均形成两分子的荧光NAD衍生物,但二羰甲基酵母酶却只能脱去一分子二氧化碳。这一现象可能表示亚基间存在着构象的不对称性。     

羧甲基D-甘油醛-3-磷酸脱氢酶光照产生新荧光物质的研究——十二烷基硫酸钠的影响

对于甘油醛-3-磷酸脱氢酶,无论是酶朊、羧甲基化酶朊,或经光照产生新荧光物的光照酶,在加入十二烷基硫酸钠(SDS)并扫描差吸收光谱时,均产生295nm及288nm两个负峰以及267nm的正峰,同样SDS也引起酶朊和羰甲基化酶朊的蛋白荧光淬灭,这些无疑都是由于原来位于酶蛋白内部非极性环境的色氨酸及酪氨酸残基转移到分子外部极性环境所致。新荧光物的形成对SDS非常敏感,在光照前加入SDS使其与酶的重量比为0.5时新峰的形成已经大为减弱,但与此同时其蛋白荧光却比上述对照样品为高,SDS对已经形成的荧光新峰的强度也有影响,但需要更多的SDS才能使新峰荧光强度有较明显的下降,在SDS与酶的比值约为0.5时,新峰荧光强度下降还不明显,但蛋白荧光强度却反而略有增加,这些结果表明,在酶分子内部的色氨酸侧链、NAD⁺以及活性部位巯基上的羰甲基之间的空间关系受到SDS破坏时,形成新荧光物的光化学反应就不能进行,在新荧光物形成之后,它与色氨酸之间的能量转移关系,也同样是对SDS敏感的。     

KINETICS OF THE IRREVERSIBLE MODIFICATION OF ENZYME ACTIVITY IN A SYSTEM OF COUPLED ENZYME ASSAY (Ⅰ)——ONLY PRIMARY ENZYME MODIFIED BY REAGENT Y

Kinetic equations are derived for the substrate reaction during simultaneous irreversible modification of enzyme activity for systems using coupled enzyme assay. It has been shown that the method proposed previously by Tsou for determining the irreversible inhibition constants can also be used in the present situation. Moreover, the criteria proposed to distinguish between different inhibition types are also applicable.     

QUANTITATIVE RELATION BETWEEN MODIFICATION OF FUNCTIONAL GROUPS OF PROTEINS AND THEIR BIOLOGICAL ACTIVITY——A COMPUTER PROGRAM FOR ASCERTAINING THE NUMBER OF ESSENTIAL RESIDUES

Based on a statistical treatment of the quantitative relation between the extents of mod-ification of protein functional groups and the decrease of their biological activity, a meth-od was proposed (Tsou, Sci. Sin., 1962, 11:1535) and widely used for the determination ofthe number of residues essential for the biological activity ot the protein modified. How-ever, the original method depends on a trial and error approach and manual calculation tofind the best fit for the data obtained. A computer program is described in this paper forthe treatment of experimental data to produce the number of essential and non-essentialgroups modified and the ratio of rates for their modification which best fit the data ob-tained. Applications of this method show that in some cases wrong conclusions were reachedin literature and in other cases quantitative conclusions can sometimes be drawn when orig-inally either no quantitative data treatment was presented or attempts at manual curve fit-ting were unsuccessful.     

偶联酶测活系统中酶活性不可逆改变的动力学——Ⅱ.修饰剂同时作用于待测酶和辅助酶

继文献[1],我们进一步研究了修饰剂同时也作用于辅助酶的情况下,偶联法测活体系中酶活性不可逆改变动力学。结果表明,在一定条件下,邹承鲁动力学方法仍可应用于偶联法测活体系。