Construction, purification and substrate specificity identification of recombinant human platelet-activating factor acetylhydrolase isoformⅠ
- VernacularTitle:人细胞内Ⅰ型血小板激活因子乙酰水解酶重组蛋白的构建、纯化及酶活性研究
- Author:
Xiaoying CHEN
;
Jing XU
;
Junwei YANG
;
Yixuan ZHANG
- Publication Type:Journal Article
- Keywords:
platelet-activating factor acetylhydrolase isoform I;
plasma platelet-activating factor acetylhydrolase;
recombinant protein;
purification;
enzyme activity;
substrate
- From:
Journal of Shanghai Jiaotong University(Medical Science)
2009;29(10):1152-1156
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct and purify the recombinant protein of platelet-activating factor acetylhydrolase (PAF-AH) isoform I , and study the enzyme activity by different substrates. Methods The (3 subunit of PAF-AH isoform I was cloned and expressed in E. coli. Exogenously expressed recombinant protein was purified to SDS-PAGE homogeneity, and its activity was identified by arylesterase detection. Phenylacetate, 1-O-hexadecyl-2-deoxy-2-thioacetyl-sn-glycero-3-phosphocholine ( 2-Thio PAF) and l-myristoy1-2-( 4-nitrophenylsuccinyl) phosphatidylcholine (the latter two were commercial plasma PAF-AH substrates) were used for the substrate identification. The plasma type PAF-AH was served as positive control. Results Recombinant protein of β subunit of PAF-AH isoform I was successfully constructed and expressed in E. coli after purification. Compared with positive control, the recombinant protein could hydrolyze phenylacetate and 2-Thio PAF, but could not hydrolyze l-myristoyl-2-( 4-nitrophenylsuccinyl) phosphatidylcholine. Conclusion Recombinant protein of β subunit of PAF-AH isoform I can be successfully constructed. There are differences in the substrate specification to the two commercial PAF substrates for PAF-AH isoform I and plasma type PAF-AH, which provides a quick method to differentiate PAF-AH isoform I from plasma type PAF-AH.
