| dc.description.abstract | Flight metabolism in tsetse flies of the genus
Glossina is largely supported by the partial oxidation
of proline to alanine. Reconstitution of proline from
alanine supposedly occurs in the fat body of Glossina,
using alanine, lipid and bicarbonate carbons.
Comparison of enzyme activities in the fat body
of the proline metabolising Glossina morsitans and carbohydrate
metabolising fleshfly Sarcophaga tibialis
showed that the activixYes of alanine aminotransferase
and 3-hydroxyacyl-CoA de~ydrogenase were much higher
in G. morsitans. Also, enzymes of the oxaloacetatecitrate-
rr-oxoglutarate section of the tricarboxylic
acid cycle, namely citrate synthase, aconitase and
in particular NADP-dependent isocitrate dehydrogenase
were more active in G. morsitans. Conversely, certain
enzymes of the rr-oxoglutarate-succinate-fumarate-malateoxaloaGetate
section of the cycle, rr-oxoglutarate dehydrogenase,
fumarase and malate dehydrogenase were
more active in S. tibialis. NAD-dependent isocitrate
dehydrogenase was also more active in S. tibialis.
Some enzymes of carbohydrate metabolism investigated,
namely hexokinase, phosphofructokinase, aldolase and
pyruvate kinase were all much more active in S. tibialis.
The above observations conform with the idea
that proline synthesis in the fat body of G. morsitans
would involve B oxidation of fatty acids and the
section of the tricarboxylic acid cycle from oxaloacetate
to -oxoglutarate, and that carbohydrate metabolism
1S not as important in the fat body of G. morsitans
as in S. tibialis.
Whereas NADP-dependent isocitrate dehydrogenase
and alanine aminotransferase were exclusively cytosolic
in S. tibialis, these two enzymes were largely
mitochondrial in G. morsitans. This observation, and
the particularly low activity of NAD-dependent isocitrate
dehydrogenase in G. morsitans strongly suggested
that the mitochondrial oxidation of isocitrate to -
oxoglutarate is catalysed mainly by NADP-dependent
isocitrate dehydrogenase.
In G. morsitans fat body, NADP-malic enzyme was
exclusively cytosolic and appeared to be better geared
to working in the direction of pyruvate formation than
pyruvate carboxylase. On the other hand, pyruvate
carboxylase was largely mitochondrial, and its Km
values for pyruvate and bicarbonate were lower and
nearer the likely physiological concentrations of
these substrates than the Kms of NADP-malic enzyme.
Pyruvate carboxylase activity was also totally dependent
on acetyl-CoA. These observations indicated that carboxylation
of pyruvate in G. morsitans is probably
catalysed physiologically by pyruvate carboxylase.
NADP-malic enzyme would probably be implicated in
cytosolic lipogenesis. Dependenceof pyruvate carboxylase
activity on acetyl-CoA concentration could also offer
a mechanism whereby proline synthesis is increased by
S oxidation of fatty acids .
Kinetic studies of .alanine aminotransferase
from the flight muscle and the fat body of G. morsisitans
showed that the fat b6dy enzyme had lower Km
values for alanine and -oxoglutarate than the flight
muscle enzyme, while Km values for pyruvate and glutamate
were slightly lower for the flight muscle
enzyme. This indicated that the enzymes from the
two tissues are different proteins.
In vitro studies showed that the fat body cells
of G. morsitans are capable of synthesizing proline
from glutamate, and to a lesser extent from alanine.
The rates of synthesis were increased by addition of
NADH, NADPH or ATP. Addition of all three coenzymes
together more than doubled the rate obtained in their
absence. Isocitrate in the presence of coenzymes produces
a further stimulation of synthesis. Citrate had
a similar but lesser stimulatory effect, but cr-OXOglutarate
had none. Isocitrate could substitute and
augment the effects of added NADPH. Stimulation of
proline synthesis by isocitrate was probably due to
the generation of NADPH via the NADP-dependent isocitrate
dehydrogenase.
The in vitro formation of cr-oxoglutarate from
isocitrate by fat boqy·cells in G. morsitans was much
less in the absence of ~lutamate (the substrate for
proline synthetase) thgn in its presence. This suggested
that isocitrate oxidation via NADP-dependent
isocitrate dehydrogenase is coupled to proline synthesis,
so that reoxidation of mitochondrial NADPH
by the proline synthetase reaction provides the coenzyme
that allows the oxidation to continue efficiently. | en |
