Localization and Role of Calcium in the Erythrocyte Coat: Effects of Enzymes and Storage

S ummary . The effects of various treatments on erythrocyte shape, surface, cell coat and calcium binding sites have been investigated by means of high voltage electron microscopy (HVM), scanning electron microscopy (SEM) and conventional electron microscopy (TEM). Papain caused the formation of small blisters within the cellular surface as well as crenation and'budding'of the erythrocytes. Afer neuraminidase treatment, long filaments were observed to radiate from the surface of the erythrocyte. The other enzymes investigated, RNAse, DNAse, phospholipase, protease and trypsin, produced no demonstrable effect on the cellular structure, nor (with the possible exceptionof trypsin) on the cell coat as seen by subsequent staining with ruthenium red. Putative calcium binding sites on and in the erythrocyte membrane were demonstrated. Following incubation with radioactive calcium, activity was found in the erythrocyte membranes. Calcium binding could be reduced by prior treatment of the erythrocyte with EDTA, neuraminidase, and to a lesser extent, by papain and trypsin. Other enzymes had no demonstrable effect. Stored erythrocytes showed a progressive diminution in calcium binding over a period of up to 4 weeks.