牛蛙乳酸脱氢酶同工酶的分离纯化及其性质研究

Purification and Characterizations of Lactate Dehydrogenase Isozyme of Bullfrog

牛蛙心脏中乳酸脱氢酶在聚丙烯酰胺凝胶电泳上显示3种同工酶区带,分别命名为LDH1、LDH2、LDH3,其中LDH1的活力占绝对优势。采用HiTrapTMBlue HP亲和层析和DEAE-Sephadex A离子交换层析对牛蛙骨骼肌中的LDH3进行了分离纯化。纯化的LDH3比活力为295U/mg,Km NADH=0·028,Km丙酮酸=1·242,在SDS-PAGE上显示两条带,提示该同工酶是由两种亚基组成的,亚基的分子量分别为35·3kD和37·6kD。

Lactate dehydrogenase of bullfrog heart showed three isozymes on polyacrylamide gel electrophoresis, LDH1, LDH2, and LDH3. LDH1 exhibited the highest activity. LDH3 was separated and purified from bullfrog muscle by HiTrap^TM Blue HP affinity chromatography and DEAE-Sephadex A ion exchange chromatography. The relative activity of purified LDH3 was 295 U/rag. Protein kinetic analysis showed that the Michaelis constants （Kin） value for NADH was0.028, and Km value for pyruvate was 1.242. The purified LDH3 exhibited two bands on SDS-PAGE, with molecular weight of 35.3 kD and 37.6 kD respectively, suggesting that the LDH3 isozyme be composed of two subunits.