Grant G A, Bradshaw R A
J Biol Chem. 1978 Apr 25;253(8):2727-31.
The molecular weight of chicken liver D-3-phosphglycerate dehydrogenase, as measured by sedimentation equilibrium analysis, was found to be 165,000, consistent with the measured sedimentation coefficient of 8.13 S. Sedimentation equilibrium studies in 6 M guanidine hydrochloride and gel electrophoresis in sodium dodecyl sulfate gave molecular weights of 40,000 to 43,000, indicating that the protein is composed of 4 subunits of equal molecular weight. Fractionation of the soluble tryptic peptides of the S-[14C]carboxymethylated enzyme by ion exchange chromatography and unidirectional paper electrophoresis indicated a maximum of 40 major peptides, which is approximately one-fourth of the number of tryptic peptides expected from the lysine plus arginine content of 157 of the tetramer. This observation suggests that the subunits possess identical or very similar amino acid sequences. The purified enzyme is a basic protein with an isoelectric point of 8.95. Amino acid analysis and titration with p-hydroxymercuribenzoate indicated that the 12 half-cystinyl residues present/subunit exist in the free sulfhydryl form. NH2-terminal analysis in an automatic protein sequenator indicated that the polypeptide chains of the protein are blocked.
通过沉降平衡分析测得鸡肝D-3-磷酸甘油酸脱氢酶的分子量为165,000，与测得的沉降系数8.13 S相符。在6 M盐酸胍中进行的沉降平衡研究以及在十二烷基硫酸钠中进行的凝胶电泳给出的分子量为40,000至43,000，表明该蛋白质由4个分子量相等的亚基组成。通过离子交换色谱法和单向纸电泳对S-[14C]羧甲基化酶的可溶性胰蛋白酶肽进行分级分离，结果显示最多有40种主要肽段，这大约是从四聚体中157个赖氨酸加精氨酸含量预期的胰蛋白酶肽段数量的四分之一。这一观察结果表明亚基具有相同或非常相似的氨基酸序列。纯化后的酶是一种碱性蛋白质，其等电点为8.95。氨基酸分析以及用对羟基汞苯甲酸滴定表明，每个亚基中存在的12个半胱氨酰残基以游离巯基形式存在。在自动蛋白质测序仪中进行的NH2末端分析表明该蛋白质的多肽链被封闭。
