Passive Movements of CA and K in Human Red Blood Cells

Plishker, Gordon

Abstract

Specialized functions of a wide variety of cells are thought to involve the passive movement of calcium. Little is known about the biochemical mechanisms that mediate the passive calcium transport processes across the plasma membrane. The overal objective of this proposal is to test the hypothesis that a glutathione S-transferase inhibits passive Ca uptake by enzymatically reacting with the Ca channels in the membrane. The work proposed is primarily a biochemical analysis of the GST reaction and a determination of its relationship to Ca uptake. By establishing the substrates and products of this reaction, we hope to identify the proteins and lipids which form CA channels in the red cell. We will study the glutathione S-transferase reaction by assessing the incorporation of radioactive glutathione into the cell membrane. The proteins will be examined by SDS polyacrylamide gel electrophoresis. The lipids will be examined by thin layer chromatography. The small molecular weight components will be examined by high pressure liquid chromatography. Changes in labelling will be quantitatively compared to passive Ca uptake and Ca-dependent K efflux. Atomic absorption spectrophotometry will be used to measure intracellular Ca and K. The long-term objective of this research is to apply the information obtained in red cells to understanding the passive movements of Ca and K in other cells and in disease processes. There are a number of diseases in which defects in membrane permeability to Ca has been implicated (sickle cell anemia, hereditary spherocytosis, epilepsy, and myotonic muscular dystrophy). It is possible that definition of the biochemical basis for passive Ca will be relevant to understanding such disease processes.