Aspartate aminotransferase in Leptasphaeria michotii has previously been shown to have an activity rhythm in constant conditions. The enzyme is present as two isoforms whose levels were quantified along the activity rhythm by ELISA, using specific polyclonal antibodies raised against polypeptides purified to a state of apparent homogeneity. The time-course of the level of the cytosolic isoform remained unchanged along the experiment. On the contrary, the cyclic variations in amount and transaminase activity (using cysteine sulfinate as substrate) of the mitochondrial isoform gave rise to the aspartate aminotransferase activity rhythm of the fungus. The mRNA levels of the two isoforms, as determined by in vitro heterologous translation, remained monotonous along the daily cycle. These results and the sensitivity of the rhythm towards protein synthesis inhibitors are consistent with the hypothesis that the aspartate aminotransferase activity rhythm in this species is caused by some mechanism controlling the efficiency of translation of mitochondrial isoform mRNA.

Two forms of aspartate aminotransferase were obtained from the fungus Leptosphaeria michotii and purified to a state of apparent homogeneity by a five-step purification procedure ending with blue Ultrogel chromatography. Holoenzyme specific activities were 13430 and 91 10 nkat oxalacetate/mg protein-' and isoelectric points were 7.1 and 7.0 for forms A and B, respectively. Both isoenzymes were isologous dimers of M , 92000. They differed mainly in their K,,, for 2-oxoglutarate and aspartate, their ability to use cysteine sulfinate as a substrate and their ability in vitro to be specifically tightly associated as follows: form A with a malate dehydrogenase monomer of M , 25000; form B with an unidentified protein of M , 40000-44000. Rabbit antiserum raised against the form A holoenzyme was not reactive against the form B holoenzyme and vice versa. Association of the holoenzyme with the complex essentially provoked a shift of the isoelectric point to 5.8 for form A and to 5.2 for form B, without affecting kinetic parameters. In order to localize in situ the two transaminase forms, ultrastructural detection was carried out by immunogold staining of thin sections of Lowicryl-K4M-embedded colonies. Antiserum against form A essentially labelled cytoplasm and cell wall and, to some extent, mitochondria, while antiserum against form B heavily labelled mitochondria and cell wall and to a lesser extent cytoplasm. Moreover, mitochondria were isolated and purified by Percoll-density-gradient centrifugation. Only form A was identified in this subcellular fraction using ELISA.Aspartate aminotransferase (~-aspartate-2-oxoglutarate aminotransferase) exists as two distinct dimeric isoenzymes in eukaryotes. One is located in the cytosol and the other in mitochondria [l], their structural and catalytic properties have been extensively studied [2 -41. Furthermore, there is evidence that these transaminases can be associated with other enzymes in weak binary or ternary complexes [5-71. Several results point to a possible role in vivo for these complexes. Complexes between pig heart aspartate aminotransferase and malate dehydrogenase are specific in that the cytoplasmic forms of the enzymes interact with each other, as do the mitochondrial forms. No complex between a cytoplasmic enzyme and a mitochondrial one could be detected [S]. Leucine, carbamyl phosphate synthase-I, and its substrate and co-factor, ATP and Mg2 +

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