Stabilization of trypsin by association to plasma membranes: implications for tryptic cleavage of membrane-bound Na,K-ATPase.

Tryptic cleavage has been a potential method for studying the structure and mechanism of many membrane transport proteins. Here, we report tight association of trypsin to pig kidney plasma membranes enriched in Na,K-ATPase. Trypsin also associated with protein-free vesicles prepared from plasma membrane lipids. Membrane-associated trypsin was found to be highly resistant to autolysis and insensitive to inhibition by PMSF. Na,K-ATPase substrate ions differentially influenced the level of trypsin membrane association. Thus, NaCl significantly increased trypsin membrane association compared to KCl. The ions seem to exert direct effects on the membrane independent of their effects on protein conformation. Bicarbonate anions, which detach peripheral membrane proteins, efficiently released trypsin from the membrane. Trypsin membrane association was found to enhance the cleavage of the Na,K-ATPase gamma-subunit. Comparison between membranes from shark rectal gland and pig kidney showed that trypsin association was significantly higher in the former. This was found to be partly due to the presence of higher cholesterol levels in the membrane. In conclusion, the differential membrane association of trypsin may affect the outcome of proteolytic cleavage of membrane-bound proteins.