EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF THE CGMP-DEPENDENT PROTEIN-KINASE-I-BETA AND PROTEIN-KINASE-II USING THE BACULOVIRUS SYSTEM

被引:52
作者
POHLER, D [1 ]
BUTT, E [1 ]
MEISSNER, J [1 ]
MULLER, S [1 ]
LOHSE, M [1 ]
WALTER, U [1 ]
LOHMANN, SM [1 ]
JARCHAU, T [1 ]
机构
[1] MAX PLANCK INST BIOCHEM, GENZENTRUM, D-82152 MARTINSRIED, GERMANY
来源
FEBS LETTERS | 1995年 / 374卷 / 03期
关键词
CGMP-DEPENDENT PROTEIN KINASE; BACULOVIRUS EXPRESSION; AFFINITY PURIFICATION; KINASE ACTIVITY;
D O I
10.1016/0014-5793(95)01168-E
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
Detailed studies of differences in distinct cGMP kinase isoforms are highly dependent on expression of large amounts of these enzyme isoforms that are not easily purified by conventional methods, Here cGMP-dependent protein kinases, the type I beta soluble form from human placenta, and the type II membrane-associated form from rat intestine, were each expressed in a baculovirus/Sf9 cell system and purified in milligram amounts by affinity chromatography. The expressed recombinant proteins displayed characteristics like those of their native counterparts, cGK I beta was expressed as a 76 kDa protein predominantly found in the cytosol fraction, whereas cGK II was expressed as an 86 kDa protein predominantly associated with the membrane fraction. The apparent K-a and V-max of cGMP for activation of cGK I beta were 0.5 mu M and 3.4 mu mol/min/mg, and for cGK II were 0.04 mu M and 1.8 mu mol/min/mg.
引用
收藏
页码:419 / 425
页数:7
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