Abstract
We have cloned and sequenced the gene for the
glycerol kinase of Trypanosoma brucei (TbGLK1), obtained
by RT-PCR. The corresponding mRNA is 2.3 kb
in size and contains an ORF encoding a protein with
high homology to known glycerol kinases of other organisms.
It is 512 amino acids in length with a PTS1-like targeting sequence (AKL) at its C-terminus, suggesting
glycosomal compartmentalization of this enzyme.
Although Northern blot analysis revealed higher
mRNA levels in slender bloodstream forms than in the
procyclic insect forms, specific glycerol kinase activities
were found to be virtually identical in both life
stages. Southern blot analysis suggested a single
copy gene, but we were able to clone two alleles utmost
similar to each other. Heterologous expression
of the trypanosomal glycerol kinase in E. coli enabled
us to perform a kinetic analysis of this enzyme. In particular,
we have been able to monitor ATP production
from glycerol-3-phosphate and ADP, a reaction which,
although thermodynamically very unfavorable, is regarded
essential for the survival of Trypanosoma brucei
under anoxic conditions. Since the unique spatial
separation of glycolysis in the kinetoplastida imposes
important consequences for the regulation of the
energy metabolism in these organisms, we discuss
the observed differences between TbGLK1 and
glycerol kinases from other organisms in view of its
physiological relevance.
Characterization of Saccharomyces cerevisiae thymidylate kinase, the CDC8 gene product. General properties, kinetic analysis, and subcellular localization.
