The Effect of α-Ketoglutarate and Other α-Keto Acids on the Recovery of Tyrosine Aminotransferase Activity from Chick Embryo Liver

Abstract

Abstract The presence of α-ketoglutarate or other α-keto acids during homogenization of chick embryo liver largely prevented the decrease in tyrosine aminotransferase activity which otherwise occurred. This decrease in activity generally occurred when the specific activity of the enzyme in a 20,000 x g supernatant fraction prepared in the absence of α-keto acids was relatively low (2 to 6 milliunits per mg). Pyridoxal phosphate alone only partially stabilized the enzyme under these conditions but increased recovery when added together with α-ketoglutarate. In some instances, activity was the same in the presence or absence of α-ketoglutarate, and in such cases the specific activity of the enzyme was rather high (13 to 22 milliunits per mg). Passage of the 20,000 x g supernatant fraction through a Bio-Gel P-10 column converted tyrosine aminotransferase to the apoenzyme form to the extent of 90 to 95%. The enzyme then became heat-labile, losing approximately 40% of its activity during heating at 45° for 3.5 min. Under these conditions, the addition of the monocarboxylic and dicarboxylic α-keto acids not only completely protected the enzyme activity but in addition activated the enzyme to varying degrees. Activation did not occur at 0°. The amino acid substrates, l-tyrosine and l-glutamate, were completely ineffective in protecting the enzyme. Similar results were obtained upon heating the enzyme at 60° for 5 min. The extent of activation appeared to depend on the specific activity of the enzyme: the lower the activity, the greater the degree of activation. The effect of pyridoxal phosphate was variable and appeared to be independent of the action of α-ketoglutarate. The fact that α-ketoglutarate almost completely protected and also activated apoenzyme suggests that the keto acid is not acting by converting phosphopyridoxamine enzyme to phosphopyridoxal enzyme but by some other mechanism. The concentrations of α-keto acids required to give half-maximal stabilization during homogenization or half-maximal protection during heating were 1 order of magnitude lower than the apparent Km values for these α-keto acids. These results suggest that α-keto acids stabilize tyrosine aminotransferase during homogenization and protect it during heating by a similar mechanism and also that the binding site involved in these processes may be different from the catalytic binding site. On the basis of observations made during these studies, it is suggested that chick embryo tyrosine aminotransferase may exist in vivo in two forms differing in activity and stability. The relationship between these postulated forms and the forms that we have observed in vitro is discussed.