Human tissue factor contains thioester-linked palmitate and stearate on the cytoplasmic half-cystine.
The state of the five half-cystine residues in human tissue factor (TF) has been characterized. The results indicate that the four half-cystines in the extracellular domain of TF form two disulfide bonds and the half-cystine in the cytoplasmic region is acylated by palmitic acid and stearic acid. The extracellular disulfide cross-links, Cys49-Cys57 and Cys186-Cys209, were deduced from the analysis of tryptic peptides. Acylation of the cytoplasmic half-cystine was demonstrated by purifying and characterizing fibroblast TF from cells labeled with [3H]palmitic acid. Radiolabeled fibroblast TF was observed by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The tritiated material covalently bound to the protein was identified as [3H]palmitate and [3H]stearate by reverse-phase high-pressure liquid chromatography. Deacylation of TF with hydroxylamine resulted in the spontaneous generation of disulfide-linked TF dimers. This result suggests that the disulfide-linked TF dimer, a minor component of most TF preparations, and the recently described heterodimeric form of TF are artifacts produced by deacylation of Cys245 and subsequent interchain disulfide bond formation.
Bach
R
R
Department of Medicine, Mt. Sinai School of Medicine, City University of New York, New York 10029.
Konigsberg
W H
WH
Nemerson
Y
Y
D001345:Autoradiography; D002851:Chromatography, High Pressure Liquid; D003553:Cystine / Q000032:analysis*; D003593:Cytoplasm / Q000032:analysis; D006801:Humans; D007120:Immunochemistry; D010168:Palmitates / Q000032:analysis*; D010169:Palmitic Acids / Q000032:analysis*; D013228:Stearates / Q000032:analysis*; D013229:Stearic Acids / Q000032:analysis*; D013925:Thromboplastin / Q000032:analysis*
D010168:Palmitates; D010169:Palmitic Acids; D013228:Stearates; D013229:Stearic Acids; D003553:Cystine; D013925:Thromboplastin