PURIFICATION OF NITRATE REDUCTASE FROM SPINACH (SPINACEA-OLERACEA L) BY IMMUNOAFFINITY CHROMATOGRAPHY USING A MONOCLONAL-ANTIBODY

被引：25
作者：
FIDO, RJ
机构：
来源：
PLANT SCIENCE | 1987年 / 50卷 / 02期
关键词：
D O I：
10.1016/0168-9452(87)90146-4
中图分类号：
Q5 [生物化学]; Q7 [分子生物学];
学科分类号：
071010 ; 081704 ;
摘要：
NADH-Nitrate reductase (EC 1.6.6.1) from spinach (Spinacia oleracea L. v. Noorman) has been purified to apparent homogeneity by immunoaffinity chromatography using a monoclonal antibody linked covalently to Sepharose 4B followed by affinity chromatography. A pre-column of covalently linked non-immune rat .gamma. globulin prevented non-specific binding. The enzyme, released with 1 M KNO3, was purified 1550-fold to a specific activity of 24.8 .mu.mol NO2- produced min-1, mg protein-1 with a recovery of 60% of applied NADH-NR activity. Proteolytically ''nicked'' subunits, detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) were removed by 5''-AMP Sepharose chromatography (Fido and Notton, Plant Sci. Lett., 37 (1984) 87).
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页码：111 / 115
页数：5
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