Purification and properties of dolphin muscle aspartate and alanine transaminases and their possible roles in the energy metabolism of diving mammals

Abstract

Mitochondrial and supernatant aspartate transaminases and supernatant alanine transaminase were purified 89, 204 and 240 fold respectively, from dolphin muscle. Starch gel electrophoresis of crude and purified preparations revealed that all 3 enzymes exist as single forms. K(m) values of α oxoglutarate, alanine, pyruvate and glutamate for the alanine transaminase were 0.45, 8.2, 0.87 and 15 mM, respectively. For the aspartate transaminases, the K(m) values of α oxoglutarate, aspartate, oxalacetate and glutamate were 0.76, 0.50, 0.10 and 9.4 mM, respectively, for the mitochondrial form and 0.13, 2.4, 0.06, and 3.2 mM, respectively, for the supernatant form. In all cases, as the assay pH value was decreased from pH 7.3, the K(m) values of the α oxo acids decreased whereas those of the amino acids increased. The apparent equilibrium constants for the aspartate transaminases were independent of pH. These values were 9.2 and 6.8 for the mitochondrial and supernatant forms respectively, where K' equil. = [aspartate][α oxoglutarate]/[glutamate][oxaloacetate]. Studies of the inhibition of the aspartate transaminases by dicarboxylic acids indicated that these enzymes may be controlled by pools of metabolic intermediates. Three key roles are suggested for the transaminases in the energy metabolism of the diving animal. First, it is believed that a combined action of the transaminases could enhance energy production during hypoxia by providing fumarate from aspartate for the adenosine triphosphate producing reversal of succinate dehydrogenase, and α oxoglutaratefo glutamate for the guanosine triphosphate producing succinyl thiokinase reaction. Secondly, diving mammal probably accumulate more nicotinamide adenine than other mammals during hypoxia. The aspartate transaminases seem particularly well suited for restoring and maintaining redox balance via the malate aspartate cycle after aerobic metabolism is resumed. Finally, since the preferred fuel for aerobic work is fat, the combined reactions of the transaminases could be instrumental in providing increased supplies of oxaloacetate for sparking the tricarboxylic acid cycle.