The Cytosolic and Mitochondrial Aspartate Aminotransferases from Pig Heart. A Comparison of their Primary Structures, Predicted Secondary Structures and Some Physical Properties

Abstract

1.A primary structure is presented for mitochondrial aspartate aminotransferase from pig heart based on previously published results [Barra et al. (1977) FEBS Lett. 83, 241 -244; ( 1 979 3. Hydrophobic chromatography of the isozymes using alkyl agaroses suggests that the cytosolic but not the mitochondrial isozyme has a hydrophobic patch or pocket capable of binding to the alkyl side chains of the affinity medium.4. Evidence is presented to show that the cytosolic isozyme contains some basic amino acids whose ionizations are not expressed under native conditions. The mitochondrial isozyme does not show this effect.5. Predictions of the secondary structures of the isozymes are presented based on four predictive models. Minimum predictions are derived on the basis of agreement of three out of four of the individual predictions. These agregate predictions underestimate the amount of ordered secondary structure in both isozymes and suggest a relatively low degree of similarity between the isozymes. Individual predictive models, on the other hand, reveal greater similarities in secondary structures between the two isozymes.Aspartate aminotransferase, in common with a small number of other enzymes, exists in two distinct molecular forms one associated with the cell cytosol and the other with the mitochondria [l-31. These two isozymes differ in chemical and physical properties (for a review, see [4]) and indeed it is now known that, in humans, they are coded by genes on different chromosomes (I. Craig, quoted in [5]). Some time ago we [6] and another group [7] reported the complete primary structure of the cytosolic isozyme from pig heart and it seemed logical to extend these investigations to the corresponding mitochondrial form in order to provide a basis for understanding the comparative enzymology, evolutionary history and compartmen-

Structure of the genes of two homologous intracellularly heterotopic isoenzymes

“…The length of the entire gene, as estimated from the genomic Southern blot, is approximately 10 kb, less than half of the approximately 25 kb of the mouse mAspAT gene [19]. The first exon of the sequenced mAspAT clone begins within the codon of Ser3, the first amino acid residue of mature mAspAT (the numbering of amino acid residues corresponds to that in porcine cAspAT [2,4]). In almost all genes of imported mitochondrial proteins the first intron tends to separate the exon of the presequence from the rest of the gene and is larger than all other introns.…”

Mitochondrial aldehyde dehydrogenase from human liver

“…Structures of a few other such pairs are known, e.g. for aspartate aminotransferases (47% identity between pig heart cytosolic and mitochondrial forms [52]); adenylate kinases (30 -42% identity between the beef heart cytosolic form and known parts of the mitochondrial matrix 1351 and outer compartment [53] enzymes); and malate dehydrogenases (24% N-terminal identity between the pig heart cytosolic and mitochondrial forms [54]). As with aldehyde dehydrogenase, hoinotopic forms of malate dehydrogenase and asparatate aminotransferase from different species appear to be more highly conserved than the heterotopic forms from the same species [54, 551.…”

Aspartate aminotransferase isoenzymes in Leotosphaeria michotii

“…Since most of the well-analyzed aspartate aminotransferases, in Neurospora [35] and in several animal tissues [2,36,371, are isologous a2-dimers, M I 90000 -95000, the most probable structure for L. michotii holoenzymes could be an isologous a2-dimer, M , 92000. The last purification step allowed separation of the holoenzymes to give a protein of M I 25 000 for form A and 40 000 -44000 for form B.…”