| dc.description.abstract | The main purpose of this study was to establish the pathway(s) by which
PEP from glucose catabolism is catabolized, the end products formed in the
presence and absence of SHAM, the sul.ceinuar localisation of some key
enzymes involved in PEP catabolism and to partially characterise PEPCK in
bloodstream T congolense.
When the trypanosomes were incubated with glucose as the substrate in
the absence of SHAM, the main end products observed were, acetate, glycerol
and pyruvate. The amounts observed were 292.9 ± 56.3; 308 ±54 and 154 ± 19
nmoles/30 rnin/mg protein respectively. Addition of SHAM reduced the
production of glycerol, acetate and pyruvate to 165 ± 36; 44.6 ± 30 and 26 ± 2
nmoles/30 min/rng protein respectively. Succinate which was not detectable in
the absence of SHAM was found to be 1.32 ± 0.29 nmoles/30 min/mg protein.
Lactate was not produced as an end product. It was therefore concluded that,
under aerobic conditions, bloodstream T congolense produce glycerol, acetate
and pyruvate as the main end products-of glucose catabolism. Under anaerobic
conditions simulated by addition of SHM, glycerol, acetate and pyruvate were
still produced with succinate as a minor end product.
The rate of respiration was also measured in the presence and absence of
SHAM and cyanide, It was observed that SHAM totally inhibited the rate of
respiration whereas cyanide had no effect. It was therefore proposed that
molecular oxygen is the terminal electron acceptor which reoxidises the
reducing equivalents in (NADH) via the trypanosome alternate oxidase (TAO).
The bloodstream T congolense appears to have no cytochrome systems which
could be inhibited by cyanide but has an alternative oxidase which was
inhibited by SHAM.
The amount of pyruvate production was determined in the presence of
SHAM, cyanide or in their absence for 30 minutes. It was observed that in the
absence of both SHAM and cyanide, 155± 12 nmoles/30 min/mg protein vias
produced while in the presence of both SHAM and cyanide, only 28 ± 4
nmoles/30 min/mg protein was produced. When SHAM alone was present, 76
± 8 nmoles/30 min/mg protein was produced while in the presence of cyanide
alone 156 ± 12 nmoles/30 min/mg protein was produced. When the production
of pyruvate was measured with time under aerobic conditions , there was an
increase from 0 to 280 nmoles/mg protein after 3 hrs of incubation. The
maximum quantity achieved in the presence of SHAM was 24 nmoles/mg
protein. This was achieved after 30 minutes and remained constant for 3 hrs.
This was attributed to death and lysis of the trypanosomes after 30 minutes in
the presence of SHAM. It was concluded that bloodstream T congolense only
possesses trypanosome alternate oxidase (TAO) as a means of oxidising the
reducing equivalents.
The activities of the enzymes likely to be involved in the catabolism of
PEP derived from glucose oxidation to pyruvate were assayed. The enzymes
which had specific activities greater than 36 nmoles/min/mg protein were PEP
carboxykinase, NADP-linked malic enzyme and malate dehydrogenase. Those
that had- specific activities less than 6 nmoles/min/rng protein were pyruvate
kinase and NAD-linked malic enzyme: From these observations it was
suggested that bloodstream T congolense catabolise PEP to pyruvate via
another pathway not involving pyruvate kinase. It was also concluded that it
was unlikely that pyruvate could be converted to lactate due to the low specific
activity of lactate dehydrogenase of <0.43 nmoles/min/mg protein.
The activities of the enzymes likely to catabolise pyruvate further to
either acetate or TeA cycle intermediates were assayed. The enzymes which
had specific activities greater than 18 nmoles/min/rng protein were fumarase,
pyruvate dehydrogenase, phosphotransacetylase, acetate kinase and malate
dehydrogenase. Those that had specific activities less than 2 nmoles/min/mg
protein were aconitase, citrate synthase, α-ketoglutarate dehydrogenase and
NADP-linked isocitrate dehydrogenase. From these observations, it was
suggested that bloodstream T congolense was unlikely to have a fully
functional TCA cycle and that pyruvate could be converted to acetate.
The subcellular localization of some enzymes that could be involved in
the catabolism of PEP to pyruvate were determined using various methods. It
was observed that PEPCK, MDH and α-GPDH activities were more latent than
enolase and NADP-linked malic enzyme. From the pattern of release of
PEPCK and MDH which was similar to that of α-GPDH, it was suggested that
PEPCK and MDH could be glycosomal while NADP-linked malic enzyme
could be cytosolic.
When the activity of PEPCK from the bloodstream T congolense lysate
was assayed in the presence of either magnesium or manganese ions, it was
observed that the specific activity in the presence of both metal ions was 48 ± 6
nmoles/minlmg protein. Manganese ions only gave activity of 48 ± 6
nmoles/minlmg protein while magnesium ions alone gave activity of 9.0 ± 1.5
nmoles/mg protein. When both metal ions were excluded from the assay
mixture, the specific activity was <0.43 nmoles/minlmg protein. It was
suggested that bloodstrearr(T congolense PEPCK requires manganese ions for
activity- The specific activity of the enzyme was determined in imidazole
buffer with pH ranging from 5.6 - 7.6.· The activity increased from 2 to a
maximum of 48 nmollminlmg protein at pH 6.6 before gradually falling. From
these observations it was concluded that the activity of PEPCK is dependent on
the pH. | en |
