Effect of low levels of trypsin on erythrocyte membranes

Abstract

The effects of trypsin at concentrations of 1 μg/ml on red blood cell membranes (ghosts) have been investigated. Tryptic digestion induced a gross morphologic change as observed by phase microscopy with the formation of small close vesicles by a process of exocytosis. These vesicles were more tightly sealed (less leaky) to hexoses than the parent ghosts. Coincident with vesiculation there occurred a decrease in particle density from 1.06 to 1.01 as determined by equilibrium density gradient centrifugation in Dextran. Since the equilibrium density in sucrose gradients was much less affected, the creation of new charge groups on the internal membrane surface was suggested. Continued accessibility of all sialic acid residues to attack by neuraminidase after tryptic digestion indicated that the exocytotic vesicles were in fact “right-side-out”. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed that two of the major membrane peptides (mol. wt of 89000 and 77500) were selectively attacked. Under these conditions, proteolysis did not cause the release of protein from the membrane. We suggest that the vesiculation and alteration in membrane permeability seen in association with the cleavage of these two peptides indicates that one or both are critical to the maintenance of erythrocyte membrane structure, probably through their association with lipid.