Abstract
Abstract dl-Glyceraldehyde 3-phosphate in low concentrations has been found to be a time-dependent inhibitor of aspartate aminotransferase (EC 2.6.1.1) isozymes. The cationic isozyme is more susceptible than the anionic isozyme to this inhibition. The l-isomer of glyceraldehyde 3-phosphate was as effective as the d-isomer for each isozyme. Study of various glycolytic intermediates indicated that the conjoint presence of the free aldehyde group and the phosphoryl residue of glyceraldehyde 3-phosphate was necessary for inhibition. The extent of inhibition was dependent on the duration of preliminary incubation, the concentration of glyceraldehyde-3-P, and the pH of the preliminary incubation mixture, but was independent of the concentration of enzyme. Half-maximum inhibition of each isozyme was obtained between pH 6.5 and 6.7. Maximum inhibition of the anionic isozyme was obtained at pH values of 8.4 and above, while the cationic isozyme was optimally inhibited at pH 7.4. The presence of α-ketoglutarate in the preliminary incubation mixture decreased inhibition while aspartate accentuated it. After 30 min of preliminary incubation with glyceraldehyde-3-P, dialysis of the anionic isozyme against buffer, aspartate, α-ketoglutarate, or pyridoxal 5-phosphate resulted in a substantial release of inhibition. In the case of the cationic isozyme, substantial release of inhibition was obtained only by dialysis against α-ketoglutarate. The addition of α-ketoglutarate to either the anionic or cationic isozyme-inhibitor preliminary incubation mixtures released inhibition, but only in the case of the anionic isozyme was the extent of release dependent on the duration of preliminary incubation. Prolonged incubation of this isozyme with glyceraldehyde-3-P resulted in the appearance of a second phase of inhibition. The mechanism of the inhibition of the isozymes by glyceraldehyde-3-P is discussed.
Highlights
Enzyme Assay-The activity of aspartate aminotransferase was measured by a modification of the coupled reaction described by Karmen [15]
Aminotransferase Isozymes-Table I shows the effect of preliminary incubation of aspartate aminotransferase isozymes with various glycolytic intermediates for 30 min at 37”
The cationic isozyme was more sensitive than the anionic isozyme to this inhibition
Summary
Purijication of Aspartate Arninotransferase Isozymes-The isozymes of aspartate aminotransferase were purified 150- to 200fold from rat liver essentially as described by Nisselbaum and Bodansky [5]. The final preparation of each isozyme was dissolved in 5 mM Tris-acetate buffer, pH 7.4. The specific activity of the anionic isozyme was 150 units per mg and that of the cationic isozyme was 210 units per mg. Cross-contamination of the isozyme preparations was less than 0.1% as determined by starch gel electrophoresis [14]. Enzyme Assay-The activity of aspartate aminotransferase was measured by a modification of the coupled reaction described by Karmen [15]. One unit is equal to the utilization of 1 pmole of substrate per min at 37”. Reaction mixtures were buffered with sodium barbital-HCl for the following reasons. Tris has Glyceraldehyde-S-P and Aspartate Aminotransferase Isoxymes
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