Glycoprotein Ib and glycoprotein IX in human platelets are acylated with palmitic acid through thioester linkages.
The glycoprotein (GP) Ib-IX complex is a major component of the platelet membrane which mediates adhesion of platelets to exposed subendothelium. GP Ib is a heterodimer with a large alpha chain (Mr = 135,000-145,000) and small beta chain (Mr = 22,000-27,000) linked by a disulfide bond(s). GP Ib is bound in a noncovalent 1:1 complex with GP IX (Mr = 17,000-22,000). We labeled isolated human platelets with [3H] palmitate or surface-labeled platelet membrane glycoproteins with sodium periodate-[3H]sodium borohydride and immunoprecipitated the GP Ib-IX complex from radiolabeled platelet lysates using a mouse monoclonal antibody (SZ.1) which recognizes the intact complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitates from [3H]palmitate-labeled platelets revealed two radiolabeled bands under reducing conditions at 24 and 19 kDa and two bands under nonreducing conditions at 170 and 19 kDa. As demonstrated by the parallel analysis of immunoprecipitates from periodate-[3H]sodium borohydride-labeled platelets, the [3H]palmitate-labeled bands obtained under reducing conditions corresponded to GP Ib beta and GP IX and the ones obtained under nonreducing conditions to intact GP Ib and GP IX, respectively. Using alkaline methanolysis followed by high pressure liquid chromatography analysis of the methanolysis products, we demonstrated that the radioactivity associated with the GP Ib-IX complex from [3H]palmitate-labeled platelets was, in fact, covalently bound [3H]palmitate in ester linkage to protein. The protein-fatty acid linkage was also disrupted by hydroxylamine at neutral pH. Thus, this study demonstrates that GP Ib beta and GP IX in human platelets are both fatty acid-acylated with palmitate through thioester linkages.
Muszbek
L
L
Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia 19104.
Laposata
M
M
D000215:Acylation; D001792:Blood Platelets / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D066298:In Vitro Techniques; D008970:Molecular Weight; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000097:blood*; D010980:Platelet Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D014316:Tritium
D010169:Palmitic Acids; D010980:Platelet Membrane Glycoproteins; D014316:Tritium; D019308:Palmitic Acid