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.