1141 BRAIN THIAMINE DIPHOSPHATE-ATP PHOSPHOKINASE ACTIVITY

Abstract

That thiamine triphosphate (TTP) has a functional role in the nervous system is indicated by: (1) its deficiency in the brain of patients with Leigh's disease, (2) release of TTP from nerve membrane by pharmacologic or electric stimulation, and (3) the rapid incorporation of 32P into TTP by brain slices or subcellular nerve particles. Although thiamine triphosphatase has been well-characterized, the enzyme which synthesizes TTP in brain has not previously been described. We have been able to demonstrate TTP synthesis by incubating ATP, thiamine diphosphate, MgCl2, TEA (pH 7.4), sucrose and microsomes at 37.5°. Controls (no synthesis) are identical save that the microsomes are heat-denatured (5′ at 100°). TTP is separated by electrophoresis on cellulose acetate, and identified by applying purified TTP on an adjacent strip. Utilizing this method, TTP is synthesized at a linear rate for at least 3 hours. The addition of incremental amounts of microsomal protein is associated with an increased rate of TTP synthesis; i.e. 43.5 nM TTP/mg protein/hr when 0.456 mg of protein is used, and 90 nM/mg protein/hr when 3.08 mg are used. The effect of the inhibitor associated with Leigh's disease on this enzyme(s) will be studied.