The major substrate of protein kinase C (PKC) in platelets is the 40 kDa protein, pleckstrin. Addition of the homobifunctional reagent, bis(sulphosuccinimidyl)suberate (BS3), to platelet lysate, cytosol fraction or to electropermeabilized platelets resulted in cross-linking of pleckstrin to give higher-molecular-mass complexes of 68 kDa, 90 kDa and 100–120 kDa respectively, which were visualized by immunoblotting with an anti-pleckstrin antibody. Higher levels of cross-linking were observed in permeabilized platelets than in platelet lysates. The yields of the cross-linked complexes were much reduced after dilution of platelet lysate or lysis of electropermeabilized platelets and, in the case of the 90 kDa and 100–120 kDa species, after activation of PKC by phorbol 12-myristate 13-acetate. Similar experiments with purified pleckstrin indicated that the 90 kDa and 100–120 kDa species consist, at least in part, of pleckstrin dimers and higher oligomers. After incubation of purified pleckstrin (0.45 mg/ml) for 1 h with 2 mM BS3, about 25% of the protein was present in cross-linked species. The results indicate that pleckstrin undergoes a reversible self-association that can be prevented by phosphorylation of the protein, and also interacts with an unidentified platelet protein of about 28 kDa.
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Research Article|
July 01 1996
Chemical cross-linking of pleckstrin in human platelets: evidence for oligomerization of the protein and its dissociation by protein kinase C
Alison M. McDERMOTT;
Alison M. McDERMOTT
*Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
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Richard J. HASLAM
Richard J. HASLAM
‡
*Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
†Department of Pathology, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
‡To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
December 22 1995
Revision Received:
March 05 1996
Accepted:
March 12 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 317 (1): 119–124.
Article history
Received:
December 22 1995
Revision Received:
March 05 1996
Accepted:
March 12 1996
Citation
Alison M. McDERMOTT, Richard J. HASLAM; Chemical cross-linking of pleckstrin in human platelets: evidence for oligomerization of the protein and its dissociation by protein kinase C. Biochem J 1 July 1996; 317 (1): 119–124. doi: https://doi.org/10.1042/bj3170119
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