Susan Lakatos

L-alanine dehydrogenase from Thermus thermophilus.

A heat-stable L-alanine dehydrogenase was isolated and purified from the extremely thermophilic microorganism, Thermus thermophilus, by affinity chromatography. The enzyme has a molecular weight of 290 000, as determined by the sedimentation equilibrium method, and is composed of six subunits of identical molecular weight as concluded from sodium dodecyl sulphate gel electrophoresis. The enzyme has been characterized in terms of pH- and substrate concentration-dependence of activity, substrate specificity, inhibition by D-alanine and D-cysteine and amino acid composition. The parameters obtained are very similar to those reported for L-alanine dehydrogenase from the mesophilic microorganism, Bacillus subtilis (Yoshida, A. and Freese, E. (1965) Biochim. Biophys. Acta 96, 248--262). The thermal stability of the T. thermophilus enzyme is much greater than that of the B. subtilis enzyme. Activation free energy (delta G), activation enthalpy (delta H) and activation entropy (delta S) values were determined for both the alanine deamination and for the heat inactivation reactions of the thermophilic and mesophilic enzymes. The values obtained for the catalytic reaction were practically equal. However, the two enzymes differed significantly in these parameters determined for the enzyme inactivation, which indicates that the factors ensuring the thermoresistance of the enzyme from T. thermophilus do not affect enzyme activity.

THE EFFECT OF SUBSTRATES ON THE ASSOCIATION EQUILIBRIUM OF MAMMALIAN D-GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE

Mammalian D-glyceraldehyde 3-phosphate dehydrogenase, mol. wt 145 000 [ 1,2] , is composed of four identical subunits [3] and it crystallizes with 3.5-4 mol of NAD per mol of tetrameric enzyme [4] . The reversible dissociation of the enzyme into dimers at neutral pH in 0.1 M phosphate buffer at 5”C, below 500 pg per ml enzyme concentration has been reported [5]. In a previous paper [6] we presented a detailed analysis of the molecular weight distribution of the enzyme containing its original NAD complement in the 50-500 yg per ml enzyme concentration range at 5°C. A monomer-dimertetramer equilibrium was found and under 50 pg per ml concentration the monomeric form of the enzyme was predominant. D-glyceraldehyde 3-phosphate dehydrogenase catalyzes the reversible conversion of glyceraldehyde 3-phosphate into 1,3-diphosphoglyceric acid in the presence of inorganic phosphate and NAD. The purpose of our investigation was to study the effect of the various substrates on the association equilibrium of the monomer-dimer-tetramer system and to determine the degree of association of the catalitically active enzyme form.

Subunit interactions in the first component of complement, C1

Interactions between C1q and other subunits of C1 were analyzed by sucrose gradient ultracentrifugation. A zone of dilute, radioiodine labelled C1q was sedimented through uniform concentrations of either C1r2C1s2, C1r2, C1r2 or C1s(2). The dissociation constants were found to be 3 x 10(-9) M and 6 x 10(-9) M for C1r2C1s2 and C1r2 binding respectively. Hill coefficients of 1 indicated no cooperativity in these bindings. Positive cooperativity was found in binding of C1s to C1q. Dissociation constants of 2 x 10(-6) M and 5 x 10(-8) M were obtained form computer modelling of a two step binding mechanism. No interaction was detected between C1q and activated C1r2. The data indicate that most of the interactions between C1q and C1r2C1s2 originates from a strong binding to the C1r2 moiety of the zymogen complex. This interaction is lost upon activation of C1r2.