刺槐幼苗中L-亮氨酸氨基转移酶的提纯和特性

PURIFICATION AND PROPERTIES OF L-LEUCINE AMINOTRANSFERASE FROM BLACK LOCUST SEEDLINGS

刺槐幼苗中的L-亮氨酸氨基转移酶(EC2.6.1.6,LAT)经热处理、硫酸铵沉淀、Sephadex G-100和DEAE-纤维素柱层析被提纯61倍,聚丙烯酰胺凝胶电泳显示单一的带。这种酶的分子量为7×10~4,最适pH为9.0,最适温度为60℃,它对L-亮氨酸的km为2.5mM,对α-酮戊二酸的km为3.5mM;全酶的吸收光谱在230和280nm显示两个主峰,在320和425nm显示两个小峰,透析后320和425nm的小峰基本消失;这种脱辅基酶蛋白无活力,但与100μM磷酸吡哆醛保温1～4h便可恢复活力80～100％,这表示刺槐LAT的辅基是磷酸吡哆醛;一些羰基试剂和底物类似物(二羧酸)能抑制这种酶的活力,除Co^(2+)外,Mg^(2+)、Mn^(2+)、Cu^(2+)、Hg^(2+)、Fe^(3+)和Al^(3+)对酶活力都无抑制,1×10^(-4)MHg^(2+)和1×10^(-3)M金属螯合剂乙二胺四乙酸(EDTA)对酶无抑制作用暗示,刺槐LAT没有对活力所需要的硫氢基和金属离子。

The L-leucine aminotransferase (LAT) from Black locust (R0binia pseudo-acacia L . ) (?)seedlings was purified by heat treatment , ammonium sulfate fractionation, sephadex G-100 and DEAE-cellulose column chromatqgraphy. The data show that final purification obtained was 61 fold. Photographs of polyacrylamide dise gels stained with Coomassie blue R-250 after electrophores is of this LAT preparation showed single band. The MW of the enzyme was 7x 10. The enzyme gave pH optima of 9.0 and temperature optima of 60℃. The Km values to L-lcucine and a-Ketoglutarate were 2.5mM and 3.5mM respectively. The absorption spectrum of the enzyme is typical Of enzymes that contain pyridoxal phosphate with major peaks at 230 and 280nm and smaller coenzyme-specific peaks at 320 and 425nm. Overnight dialysis of this enzyme against 60mM patassium phosphate buffer (ph7.8) (containing 1mM β-mercaptoethanol 1mM EDTA and 5mM L-leucine) essentially removes the coenzyme-specific peaks. The apoenzymc has no detectable activity without added pyridoxal phosphate. However, 80% of the activity of apoenzyme is restored after lh. preincubation period with pyridoxal phosphate, and if a longer period of preincubation is allowed, 100% of the original activity is regained. These results indicate that the cocnzyme of LAT from Black locust seedlings is pyridoxal phosphate. The aminotransferse is inhibited by substrate analogues (dicarboxylic acid) incapable of undergoing transamination. The LAT of Black locust displays sensitivity to carbonyl binding reagents which are typical of pyridoxal phosphate enzyme. Of a number of cationic species tested, only produces an inhibiting effect on activity (10% inhibition), Other cotions found to fexert no effect on the activity include Mg2+, Mn2+, Cu2 + , Hg2 + , Fe3+ , and Al3+. The lack of an effect by I×l0-4 M Hg2 + , as well as by I×l0-3 M EDTA, implies that no sufhydryl group, as well as no involvement of a metal ion.