Thiol-disulfide isomerization in thrombospondin: effects of conformation and protein disulfide isomerase.
Thiol-disulfide isomerization in thrombospondin may affect the function of this adhesive protein. Two assays were developed to analyze the determinants of thiol-disulfide exchange and to correlate this exchange with thrombospondin conformation. (1) A competitive immunoassay for the EDTA-conformation of thrombospondin was developed with monoclonal antibody D4.6. (2) The free thiol(s) in thrombospondin was labeled with [3H]N-ethylmaleimide (NEM) under various conditions (the presence or absence of calcium, temperature, and pH), and thrombin digests of the labeled protein were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Consistent with previous reports, thrombin digest fragments of 150, 120, 20, and 14 kD were observed, each with radioactivity under some condition, plus a 25-kD peptide that was not labeled. Sequence data for these fragments and comparisons of SDS-PAGE analyses under reducing and nonreducing conditions indicated that Cys974 was the free thiol. The appearance of thiol label in the 120-kD fragment was previously shown to be a consequence of thiol-disulfide exchange (J Biol Chem 265:17859,1990) and label was recovered in this peptide only under conditions (absence of calcium, 37 degrees C and pH 8.4) that led to the appearance of the EDTA-conformation of thrombospondin. Additional evidence for the correlation of EDTA-conformation and thiol-disulfide exchange was the enhanced conversion of thrombospondin to its EDTA-conformation in the presence of protein disulfide isomerase and the inability of thrombospondin pretreated with NEM to attain the EDTA-conformation. Flow cytometry with antibody D4.6 revealed platelet-associated thrombospondin in the EDTA-conformation in the presence of calcium, suggesting that the EDTA-conformation is a physiological conformation that does not necessarily require EDTA.
Huang
E M
EM
Department of Biochemistry, State University of New York, Health Science Center at Brooklyn, 11203, USA.
Detwiler
T C
TC
Milev
Y
Y
Essex
D W
DW
D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides; D004492:Edetic Acid; D005033:Ethylmaleimide / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007118:Immunoassay; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D015539:Platelet Activation; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases; D013438:Sulfhydryl Compounds; D013816:Thermodynamics; D019699:Thrombospondins
D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D004220:Disulfides; D008562:Membrane Glycoproteins; D013438:Sulfhydryl Compounds; D019699:Thrombospondins; D000001:Calcimycin; D004492:Edetic Acid; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005033:Ethylmaleimide; D002118:Calcium