Enzymatic Characterization of a Human Acyltransferase Activity 英文参考文献.docVIP

EnzymaticCharacterizationofaHumanAcyltransferase Activity AkihikoOzawa,RichardB.SpeakerIII,IrisLindberg* DepartmentofAnatomyandNeurobiology,UniversityofMaryland—Baltimore,Baltimore,Maryland,UnitedStatesofAmerica Abstract Background:Non-histoneproteinacylationisincreasinglyrecognizedasanimportantposttranslationalmodification,but littleisknownastothebiochemicalpropertiesofproteinserineacylatingenzymes. Methodology/Principal Findings: We here report that we have identified a metal-stimulated serine octanoyltransferase activityinmicrosomesfromhumanerythroleukemic(HEL)cells.TheHELacylatingenzymewaslinearwithrespecttotime and protein, exhibited a neutral pH optimum (stimulated by cobalt and zinc), and inhibited by chelating reagents. Hydroxylaminetreatmentremovedmost,butnotall,oftheattachedradioactivity.Asaltextractofmicrosomalmembranes containedthemajorportionofenzymeactivity,indicatingthatthisacyltransferaseisnotanintegralmembraneprotein. Sucrose density fractionation showed that the acyltransferase activity is concentrated in the endoplasmic reticulum. In competitionexperiments,theacyltransferasewaswellinhibitedbyactivatedformsoffattyacidscontainingatleasteightto fourteen carbons, but not by acetyl CoA. The zinc-stimulated HEL acyltransferase did not octanoylate proenkephalin, proopiomelanocortin, His-tagged proghrelin, or proghrelin lacking the amino-terminal His-tag stub of Gly-Ala-Met. The peptidesdes-acylghrelinandACTHwerealsonotacylated;however,des-acylghrelincontainingtheN-terminaltripeptide Gly-Ala-Metwasacylated.Mutagenesisstudiesindicatedarequirementforserinefiveresiduesfromtheaminoterminus, reminiscent of myristoyl transferase, but not of ghrelin acylation. However, recombinant myristoyl transferase could not recapitulate the hydroxylamine sensitivity, zinc-stimulation, nor EDTA inhibition obtained with HEL acyltransferase, propertiespreservedintheHELcellenzymepurifiedthroughfoursequentialchromatographicsteps. Conclusions/Significance: In conclusion, our da