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 of 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 labile 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.

Genetic Effects on the Longevity of Cultured Human Fibroblasts. III

“…Duncan et al [4] observed a heat-labile fraction of enzyme in senescent cultures, but they believed that this was due to a change in the polymeric state of the enzyme. They showed that there are two active fractions separable on polyacrylamide gels, which they suggested were the dimeric and tetrameric forms of the enzyme.…”

“…2). For the two major fractions sep arated by Duncan et al [4] using polyacryl amide gels, the slopes of inactivation differ by approximately a factor of 2. A mixture of such fractions could not possibly give the ini tial rapid rate of inactivation which is consis tently observed.…”

“…A and C show the rates of inactivation of the dimeric and tetrameric forms of G6PD at 60 °C according to results of Duncan et al [4], A mixture of 20% of the more heat-labile form, and 80% of the more stable form, gives the inactivation curve shown in B. The shape of this curve is quite distinct from either figure 2 A or from the very similar result in fig ure 1 of Duncan et al [4], extracts from senescent cells. Since G6PD is intrinsically unstable in low concentrations of NADP.…”

An Evaluation of Some Theories of the Mechanism of Aging

“…In fact, Duncan et al (1977) have recently observed that the accumulation of heat labile glucose-6-phosphate dehydrogenase in aged fibroblasts appears to be due to a shift from the dimeric to tire tetrameric form. Thus the accu mulation of the more heat sensitive enzyme molecules represents an equilibrium shift rather than a mutational change.…”