In:
The Journal of Immunology, The American Association of Immunologists, Vol. 134, No. 4 ( 1985-04-01), p. 2513-2519
Abstract:
Sodium [3H]borohydride- and [35S] methionine-labeled Ly-5 molecules from mouse thymocytes and T lymphoma cells were isolated with specific antibody and Staphylococcus aureus Cowan I (SaCI) strain; after extensive washing of the complexes, elution with Laemmli's reducing buffer (0.05 M Tris [pH 6.8 or 6.0], 4% sodium dodecyl sulfate [SDS] , and 2% 2-mercaptoethanol [2-ME]) resulted in partial breakdown of the isolated Ly-5 molecules from a Mr = 175,000 to 150,000. Other pr oteins present during the elution step showed no evidence of proteolysis. 2-ME and SDS were required for proteolysis; although addition of exogenous Ca2+ during elution was not necessary, both EDTA and EGTA inhibited breakdown of the molecule that could be overcome by excess Ca2+. Of a variety of protease inhibitors and thiol-reactive agents tested, only TAME and oxidized glutathione blocked proteolysis almost completely. SaCI, serum, and contaminating antibodies were ruled out as the source of the proteolytic activity. More stringent preclearing and washing conditions did not eliminate endogenous proteolysis of the Ly-5 molecule. The endogenous proteolytic fragment had a Mr distinct from the tryptic fragment of the Ly-5 molecule. We conclude that the Ly-5 molecule may be autolytic or tightly associated with a distinct cellular protease.
Type of Medium:
Online Resource
ISSN:
0022-1767
,
1550-6606
DOI:
10.4049/jimmunol.134.4.2513
Language:
English
Publisher:
The American Association of Immunologists
Publication Date:
1985
detail.hit.zdb_id:
1475085-5
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