Inactivation of Muscle Triosephosphate Dehydrogenase by Reduced Diphosphopyridine Nucleotide at Physiological Concentrations

Recent studies (l-8) have shown that enzymes can become less stable to physical agents in the presence of substrates and cofactors, and that variations in concentration and combination of necessary components profoundly affect enzyme stability.On the other hand, it was shown with malic dehydrogenase that ionic strength and other environmental conditions must be rigidly controlled to evaluate enzyme stability properly (9).However, the fairly common (l-8) increased instability of enzymes in the presence of substrates and cofactors under conditions in vitro, led to the proposal that the phenomenon might have important consequences in biology (4, 6).It has therefore become of primary importance to determine whether this type of effect occurs under physiological conditions.The enzyme triosephosphate dehydrogenase has been thoroughly studied in the past and many of its properties are known.As briefly reported previously, this enzyme is very susceptible to inactivation in the presence of reduced diphosphopyridine nucleotide (5, 10).Thus, it was of interest to extend these studies, particularly since it has been suggested that this enzyme may be rate-limiting (11).As illustrated in this paper, there was enzyme inactivation by reduced diphosphopyridine nucleotide at physiological concentrations with formation of its hydrated product (12) and disappearance of -SH groups.However, no changes were noted in a number of physical parameters.EXPERIMENTAL PROCEDURE Materials and Methods-Crystalline muscle triosephosphate dehydrogenase, DPN, DPNH (enzymatically reduced), TPNH, TPN, and nL-glyceraldehyde phosphate were obtained from Sigma Chemical Company or from California Foundation for Biochemical Research (Boehringer).Yeast triosephosphate dehydrogenase was purified by the method of Krebs (13).DPNH-X1 was prepared and measured as described by Chaykin et al. (12).A sample of wDPN was a gift from Drs. Kaplan and Ciotti.In all experiments, the enzyme (kept in approximately 0.68 saturated ammonium sulfate at pH 7.0 to 7.1 and at 0') was centrifuged for 10 minutes at 20,000 g before use, and the protein precipitate was taken up in water.