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:
xiao-ying, CHEN
;
jing, XU
;
jun-wei, YANG
;
yi-xuan, ZHANG
- Publication Type:Journal Article
- Keywords:
platelet-activating factor acetylhydrolase isoformⅠ;
plasma platelet-activating factor acetylhydrolase;
recombinant protein;
purification;
enzyme activity;
substrate
- From:Journal of Shanghai Jiaotong University(Medical Science)
2006;0(10):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct and purify the recombinant protein of platelet-activating factor acetylhydrolase(PAF-AH) isoformⅠ,and study the enzyme activity by different substrates. Methods The ? subunit of PAF-AH isoformⅠwas 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,l-O-hexadecyl-2-deoxy-2-thioacetyl-sn-glycero-3-phosphocholine(2-Thio PAF) and 1-myristoyl-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 ? subunit of PAF-AH isoformⅠwas 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 1-myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine.Conclusion Recombinant protein of ? subunit of PAF-AH isoformⅠcan be successfully constructed.There are differences in the substrate specification to the two commercial PAF substrates for PAF-AH isoformⅠand plasma type PAF-AH,which provides a quick method to differentiate PAF-AH isoformⅠfrom plasma type PAF-AH.
