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.
Publisher
University of Nairobi
Rights
Attribution-NonCommercial-NoDerivs 3.0 United StatesUsage Rights
http://creativecommons.org/licenses/by-nc-nd/3.0/us/Collections
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