COMPARATIVE ANALYSIS OF PKCa AND PKCZ ACTIVITIES IN RAT AND LAMPREY ERYTHROCYTES OF DIFFERENT AGES
The present study aims to investigate the link between ageing of rat and lamprey erythrocytes and activity of two isoforms of protein kinase C (PKC), PKCa and PKC^. The entire cell population was separated into fractions of different ages in Percoll density gradient. The reliability of separation was confirmed by the immature erythrocyte (reticulocyte) count. PKC activity was analyzed in cytosolic and membrane cell fractions. Rat erythrocytes express both PKC isoforms, PKCa and PKCZ, whereas lamprey erythrocytes express only PKC^. PKCa is identified as a major band at ~ 80 kDa and minor bands at ~ 55-65 kDa; PKCC, is presented by a single band at ~ 80 kDa. In young rat erythrocytes, PKCa is detected mainly in cytosolic fractions, while in membrane fractions its content is by far lower. As cells age, PKCa is translocated from the cytosol to membranes and undergoes proteolytic degradation due to repeated cycles of activation. As a result, in aged erythrocytes, relative total PKCa expression (as a sum of expressions in the cytosol and membranes per total protein content) diminishes, indicating a depletion of the PKCa pool and a decrease in its functional activity. In both animal species studied, a highest PKCC, level is observed in the cytosol of young erythrocytes. Erythrocyte ageing is accompanied by a gradual decline in expression of free cytosolic PKCC, and concurrent increase in the level of its membrane-bound forms. However, in contrast to PKCa, PKCC, is not proteolyzed; its total content in cells and perhaps functional activity do not change throughout the erythrocyte lifespan.