Purification and characterization of a tyrosine aminotransferase from Crithidia fasciculata, a comparative biochemical model for African trypanosomes

Abstract

The catabolism of aromatic amino acids by trypanosomes has been of major research interest because of its involvement in the pathogenesis during chronic African trypanosomiasis. The first step in these pathways is an aminotransferase reaction. A lack of information concerning trypanosomal aromatic aminotransferases has prevented the elucidation of the full significance of these enzymes during the disease. In order to obtain such information, a study of tyrosine aminotransferase (TAT; EC 2.6.1.5) from the 'model' trypanosome, Crithidia fasciculata, was undertaken. The following report describes the extensive purification of a crithidial TAT, representing the first such study of an aromatic aminotransferase from eukaryotic unicellular organisms. The enzyme was purified over 2,000 fold using cofactor-affinity chromatography. It migrated as a single band when electrophoresed under nondenaturing conditions and produced two polypeptides in the presence of SDS. The native form of crithidial TAT had a molecular weight of approximately 100,000 daltons, whereas the subunits were at approximately 50,000 and 48,000 daltons, respectively. Absence of a reaction with the periodic acid-Schiff stain indicated that the crithidial TAT was not a glycoprotein. It was relatively stable and remained active over a wide range of pH and temperature. It exhibited a broad substrate specificity and utilized L-tyrosine, L-tryptophan and L-phenylalanine as amino donors. The apparent Michaelis constant of crithidial TAT for L-tyrosine was at 2.0 mM. Antiserum produced against partially purified crithidial TAT failed to neutralize the enzymatic activity. The same antiserum cross-reacted with a crude, soluble extract from the African trypanosome, Trypanosoma brucei gambiense, but not with that from mouse liver. The cross-reactivity was comparable between T. b. gambiense and C. fasciculata, providing evidence for evolutionary conservatism between the two genera. Furthermore, it provided a justification for the practice of using C. fasciculata as a 'model' for biochemical analyses of African trypanosomes. The anti-TAT should be a good immunochemical probe for analyzing the significance of TAT during chronic African trypanosomiasis. It may also be useful in the design of a diagnostic test.Finally, several advantages concerning the use of C. fasciculata as a model for comparative biochemistry of TATs from various sources have been discussed. Particular emphasis has been placed on a comparison between C. fasciculata and mammals.