The Effect of α-Tocopheryl Phosphate on Diphosphopyridine Nucleotidase

Abstract

Normal guinea pig hearts were homogenized in distilled water, a 10% suspension being used as a source of diphosphopyridine nucleotidase. Sodium-dl- [alpha] -tocopheryl phosphate was prepared. Diphosphopyridine was prepared by the method of Williamson and Green (1940) and was estimated in the reaction mixtures by the method of Levitas (1947). Individual tubes were made to contain 1 ml. of M/5 phosphate buffer, pH 7.2, 0.5 ml. of homogenate, and 0.5 ml. of buffer containing 500 of DPN. CaCl2 and a-TPh were dissolved in water and added. The vols. were made to 3.0 ml. The tubes were incubated at 37[degree] for 0 time, 15, 30, and 45 min., and 1 hr., after which the enzymes were inactivated by 0.2 ml. of 20% trichloracetic acid. After filtration, the filtrates were analyzed for DPN. The rate of breakdown of DPN was markedly inhibited by a-TPh. CaCl2 relieved the inhibition but did not increase the rate of DPN activity when [alpha]-TPh was not added. In most expts., addition of Ca to the control tubes, without [alpha]-TPh, caused no acceleration of DPN breakdown in the time intervals used. It was shown by direct DPN estimation that [alpha]-TPh inhibited DPN.