Thallium activation and inhibition of yeast aldehyde dehydrogenase

Abstract

Volume 59, number 1 FEBS LETTERS November 1975 THALLIUM ACTIVATION AND INHIBITION OF YEAST ALDEHYDE DEHYDROGENASE K. BOSTIAN and G. F. BETTS Department of Plant Biology and Microbiology, Queen Mary College, Mile End Road, London, E. 1. England W. K. MAN and M. N. HUGHES Department of Chemistry, Queen Elizabeth College, Campden Hill Road, London, I¢. 8. England Received 1 September i975 1. Introduction T1 ÷ has been shown to replace the physiological ion K ÷ in the activation of several monovalent cation activated enzymes [1-6]. This provides the opportu- nity to employ T1 ÷ as a probe for K ÷ in n.m.r, and fluorescence studies [6-8]. Preliminary to such studies on yeast aldehyde dehydrogenase (EC 1.2.1.3.) T1 ÷ activation of this enzyme was investigated and compared to the previously demonstrated activation by K ÷ [9]. A general mechanism for this activation has been proposed whereby an activating monovalent cation induces and maintains an enzyme conformation suitable for catalytic activity [ 10]. 2. Materials and methods Partially purified aldehyde dehydrogenase was prepared from N.G. and S.F. yeast (British Fermenta- tion Products Ltd.) obtained from a local baker. The method was essentially that of Black [9] except for the inclusion of 1.2 X 10 -3 M phenylmethylsulphonyl fluoride (PMSF) throughout the procedure. The buffer composition at the final stage contained 0.1 M K2HPO4, pH 7.8, 10 -3 M mercaptoethanol, and 1.2 × 10 -3 M PMSF. Enzyme activity was between 7-10 units/ml when assayed with optimal K ÷ (0.1 M) with a specific activity of 1. Samples were stored frozen at -18 ° C, and thawed out immediately prior to use. Enzyme was equilibrated with 0.1 M Tris-NO3 pH 8.0, 10 -3 M mercaptoethanol and activating salt where indicated, by passage through a Sephadex G-25 column at 4°C. Because of the instability of the enzyme in the absence of an activating monovalent cation, the enzyme was stored on ice following chromatography and all assays in a given run were completed within 5 rain. Enzyme activity was measured by following the rate of reduction of NAD ÷ by observing the increase in absorbance at 340 nm in a Pye Unicam SP1800 spectrophotometer using 1 cm quartz cells. Reaction mixtures contained in a final volume of 2.5 ml: 2 X 10 -3 M acetaldehyde, 5 X 10 -4 M NAD ÷, 0.1 M Tris-NO3 buffer, pH 8.0,