Endogenous proteolysis of the human erythrocyte membrane as studied by two-dimensional gel electrophoresis and electron spin resonance.

1. Endogenous proteolysis in human erythrocyte membranes was studied in human erythrocyte membranes incubated at 37 degrees C by monitoring changes in 2-D electrophoretic pattern of membrane polypeptides and in the spectra of maleimide-spin labeled membranes. 2. A strong effect of exogenous proteases derived from contaminating other blood elements was found, resulting in formation of specific spots on 2-D electropherograms, requiring very careful leukocyte removal in investigations of red cell membrane protein composition and proteolysis. 3. Studies of the effects of protease inhibitors and Ca2+ confirmed a complex pattern of endogenous red cell membrane proteolysis ("self-digestion") involving many substrates and enzymes. 4. A promoting effect of high concentrations (150 mM) of Ca2+ on endogenous red cell membrane proteolysis was found.