A Study of Transaminases in Bloodstream Trypanosoma Brucei Brucei

Abstract

With glutamate pyruvate transaminase as a cytosolic marker and a -glycerol phosphate dehydrogenase as a glycosomal marker the intracellular location of transaminases utilising leucine, isoleucine, valine, phenylalanine, tyrosine and tryptophan as substrates was investigated. The transaminase activities were released from the trypanosomes by increasing the concentrations of Triton X-IOO ranging from 0 to 0.02% v /v. To release maximal activities of GPT, leucine: a -ketoglutarate a-ketoglutarate transaminase, transaminase, phenylalanine: utransaminase, isoleucine: valine: a-ketoglutarate ketoglutarate transaminase, tyrosine: a -ketoglutarate transaminase and tryptophan: a-ke t ogl.u t.a rate transaminase, 0.01% (v/v) Triton X-IOO was required. The concentration of Triton X-IOO required to release maximum activity of a-GPDH was 0.015% (v/v). It was concluded that these transaminases are localized within the cytosol since their pattern of release corresponded to that of GPT and was different from that ofa-GPDH. The specificity of the transaminases for the acceptor of the amino group from L-valine, L-Ieucine L-isoleucine, L-Glutamine, L-methionine, L-aspartate, L-phenylalanine, Ltyrosine and L-tryptophan was investigated using a-ketoglutarate and pyruvate. The rate of transamination with pyruvate as the acceptor of a-amino group was lower than when (Lt ) ~ketoglutarate was the amino group acceptor. Branched chain amino acids were transaminated at approximately the same rate around 1.10 ± 0.030 vmoles glutamate/hr/mg protein with a-ketoglu tara te and 0.30 ± 0.01 umo Les alanine /hr /mg protein with pyruvate as a-amino group acceptor. Methionine gave the highest rate of transamination with pyruvate as the uamino group acceptor with 0.414 ± 0.012 V moles L-alanine formed/hr/mg protein. It was concluded that both a - ketoglutarate and pyruvate could be acceptors of the -amino group during transamination although a-ketoglutarate is the preferred substrate. The effect of pH on the enzymes catalysing the transamination of L-leucine, L-isoleucine, L-valine, Lphenylalanine, L-tyrosine and L":'tryptophan with a - ketoglutarate as a -amino group acceptor was investigated within pH values ranging from 6.5 to 9.0. The highest transaminating activity was observed at the pH ranging between 7.8-8.5, beyond which the activity of the enzymes decreased. It was therefore concluded that the optimum pH for the transaminases present in bloodstream T.b. brucei lie between 7.8 to 8.5 pH range. Results obtained on the stability of these transaminases when stored at 40C and 2SoC over 48 hours period indicated a gradual decrease in activity with time. About 50-80% of the original transaminase activity was lost at 40C within 48 hours. Little or no activity remained at 2SoC over the same period. It was observed that the rate at (d I L) which transaminase activity for the branched chain amino acids; leucine, isoleucine and val ine was Los tat 40C and 250C was approximately the same. However, transaminase activity for the aromatic amino acids was lost at differing rates at 40C and 2SoC. There was no significant loss of transaminase activity for both branched chain and aromatic amino acids in the presence of dithiothreito1. Results from this study suggest that, branched chain amino acid are cata1ysed by either very closely similar or common t ransaminases whereas the aromatic amino acids are transaminated by different enzymes.