Relationship of heat labile glucose-6-phosphate dehydrogenase and multiple molecular forms of the enzyme in senescent human fibroblasts

Abstract

Human fibroblasts derived from newborn foreskin and designated CF‐3 were assayed for heat labile glucose‐6‐phosphate dehydrogenase (G6PD) when they had grown to confluency, as well as when they were arrested in an essentially nonmitotic state. Under both culture conditions there was an increase in heat labile G6PD (up to 25% of the total activity) as cells progressed through their in vitro lifespan; however, arrested cells exhibited less heat labile G6PD than did comparable growth controls (5–10% decrease). Acrylamide gel electrophoresis of crude G6PD preparations revealed three distinct bands of enzymatic activity. One of the bands was tentatively identified as the dimeric form of the enzyme and another as the tetrameric form. The tetrameric form was more heat sensitive, and the percent to the total activity of this form increased as the cells became senescent. The percent of total activity of the tetrameric form was comparable to the percent heat liable enzyme assayed at a given population doubling level. These results indicated that the observed increase in heat lability of G6PD with age may be due to a shift in equilibrium to the tetrameric form of the enzyme rather than the synthesis of aberrant enzyme molecules.