Kinetic Studies of Mouse Brain Transketolase

Abstract

The activity of transketolase in mouse brain was 5.7 nmol/min/mg protein measured by an enzyme-coupled spectrophotometric assay. The apparent Km for ribose-5-phosphate was 330 microM, for D-xylulose-5-phosphate was 120 microM, and for thiamine pyrophosphate was 7 microM. However, thiamine pyrophosphate remained tightly bound to transketolase in homogenates in which it dissociated completely from another thiamine pyrophosphate-dependent enzyme, the pyruvate dehydrogenase complex. These data suggest that loss of transketolase activity is likely to be a later consequence of thiamine deficiency in mammalian brain than is decreased activity of pyruvate dehydrogenase complex.

Properties and functions of the thiamin diphosphate dependent enzyme transketolase

“…The enzyme has been at least partially puri®ed from a variety of sources including baker's yeast [25,65,154], Candida utilis [77,79], spinach [165], pig liver [120,147], rat liver [116], rabbit liver [98], mouse brain [8], human leukocytes [105] and human erythrocytes [1,10,56,58,135,158,170]. Recently, TK has been puri®ed from E. coli [37,153].…”

Effect of a Deficiency of Thiamine on Brain Pyruvate Dehydrogenase: Enzyme Assay by Three Different Methods

“…The whole frozen brain was suspended in ice-cold "sample buffer" (9 ml/g of brain) containing 50 mM potassium phosphate, 1 mM 2-mercaptoethanol, 1 mM potassium EDTA, and 0.1% (wt/vol) Triton X-100 at pH 7.8 (Hinman and Blass, 1981;Blass et al, 1982;Ksiezak-Reding et al, 1982) and homogenised with an Ultra-Turrax electric blender (Janke und Kunkel K.G., Staufen-in-Breisgau, F.R.G.). The homogenates were kept frozen (-20°C) for 2 1 h before use.…”

“…(a) In some studies, PDH activity was measured in the absence, or in the presence of suboptimal concentrations, of exogenous TPP. In brain homogenates, PDH, unlike transketolase, readily loses TPP (Heinrich et al, 1973;Blass et al, 1982). It is probable that TPP, once released from the binding site, is hydrolysed to thiamine monophosphate by phosphatase in brain homogenates and thereby loses its cofactor activity.…”

Myelin

“…In the thiamine-deficient rat, severe symptoms and pathology became apparent after the brain thiamine levels had decreased to less than 20% and transketolase activity to 50% of normal, and assays of this enzyme in tissue from various regions of the human CNS gave the highest activity in the mammillary body, a region particularly vulnerable to pathological changes in Wernicke's disease (Dreyfus, 1965). However, the results of kinetic studies of transketolase and pyruvate dehydrogenase suggest that the lauer may be the more vulnerable enzyme activity under conditions of thiamine deficiency (Blass et al, 1982). In animal studies it was not, in fact, possible to correlate lesion formation with inactivation of any of three thiamine-dependent enzymes, pyruvate dehydrogenase, a-ketoglutarate dehydrogenase, and transketolase (Pincus and Wells, 1972), and over 60% of the transketolase activity could be depressed without decreasing the nux through the pentose phosphate pathway (McCandless et al, 1976).…”