|
|
 Previous Article | Table of Contents | Next Article 
Thiol-disulfide isomerization in thrombospondin: effects of conformation
and protein disulfide isomerase
EM Huang, TC Detwiler, Y Milev and DW Essex
Department of Biochemistry, State University of New York, Health Science
Center at Brooklyn, 11203, USA.
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.
Volume 89,
Issue 9,
pp. 3205-3212,
05/01/1997
Copyright © 1997 by The American Society of Hematology
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
P. Lagadec, O. Dejoux, M. Ticchioni, F. Cottrez, M. Johansen, E. J. Brown, and A. Bernard
Involvement of a CD47-dependent pathway in platelet adhesion on inflamed vascular endothelium under flow
Blood,
June 15, 2003;
101(12):
4836 - 4843.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. A. Bennett, B. S. Edwards, L. A. Sklar, and S. Rogelj
Sulfhydryl Regulation of L-Selectin Shedding: Phenylarsine Oxide Promotes Activation-Independent L-Selectin Shedding from Leukocytes
J. Immunol.,
April 15, 2000;
164(8):
4120 - 4129.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. O'Neill, A. Robinson, A. Deering, M. Ryan, D. J. Fitzgerald, and N. Moran
The Platelet Integrin alpha IIbbeta 3 Has an Endogenous Thiol Isomerase Activity
J. Biol. Chem.,
November 17, 2000;
275(47):
36984 - 36990.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. G. Rodrigues, N.-h. Guo, L. Zhou, J. M. Sipes, S. B. Williams, N. S. Templeton, H. R. Gralnick, and D. D. Roberts
Conformational Regulation of the Fibronectin Binding and alpha 3beta 1 Integrin-mediated Adhesive Activities of Thrombospondin-1
J. Biol. Chem.,
July 20, 2001;
276(30):
27913 - 27922.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
