Co-immunoprecipitation of the Ly-5 molecule and an endogenous protease: a proteolytic system requiring a reducing agent and Ca2+1.
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 proteins 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.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000502:physiology; D000937:Antigen-Antibody Reactions; D000950:Antigens, Ly / Q000276:immunology / Q000302:isolation & purification*; D000961:Antilymphocyte Serum / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D006868:Hydrolysis; D051379:Mice; D008808:Mice, Inbred CBA; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010447:Peptide Hydrolases / Q000502:physiology*; D011233:Precipitin Tests / Q000379:methods*; D011480:Protease Inhibitors; D014357:Trypsin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000950:Antigens, Ly; D000961:Antilymphocyte Serum; D011480:Protease Inhibitors; D004533:Egtazic Acid; D004492:Edetic Acid; D010447:Peptide Hydrolases; D014357:Trypsin; D002118:Calcium