Effect of proteolysis on the activity of the sodium channel in isolated lobster nerve membrane

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Abstract
The effect of emymatic proteolysis on structural and functional properties of the isolated lobster nerve membrane was investigated. The membranes were treated with diffcrcnt amounts of either trypsin or unspecific protease. Sodium channel activity was determined by measuring the veratridine‐tetrodotoxin‐sensitive sodium influx in proteoliposomes prepared with nerve membrane and soybean lipids. The changes in the membrane proteins were followed by electrophoresis in polyacrylamide gradient gels. From the densitometric scan of the gels the relative area for each protein was obtained, and the ratio of enzyme‐treated to control areas was evaluated. Under a similar degree of proteolysis catalyzed either by trypsin or by unspecific protease, the sensitive sodium influx is not affected by trypsin, whereas it is about 60% diminshed by the unspecific protease. In this condition thc zoncs corresponding to molecular weights of 240,000 and 166,000 daltons appear modified in the electrophoretic gels by both enzymes. The 117,000‐dalton range is modified only by the unspecific proteasc. Increasing trypsin concentration diminishes sodium influx about 60%; and the 240,000‐, 166,000‐, and 117,000‐dalton zoncs appear modified. A further increase of the protease concentration totally abolishes the sensitive sodium influx and modifies practically all of the membrane proteins. The present results indicate the rathcr high sensitivity of thc mcmbranc sodium channel activity to proteolytic action, and show that the membrane sites that respond to veratridine appear to be highly affected by proteolysis. In contrast, the tetrodotoxin receptor retains its binding capacity even after treatment of the membrane with proteasc conccntrations 1,000 times higher than those affecting the sensitive sodium influx [Benzcr and Raftery, 1972; Villegas et al, 1973].