Studies on the energy metabolism of Leishmania mexicana mexicana

Mottram, Jeremy C. (1984) Studies on the energy metabolism of Leishmania mexicana mexicana. PhD thesis, University of Glasgow.

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The metabolism of the mammalian form of Leishmania mexicana mexicana has been investigated by comparing the amastigote activities of a number of enzymes involved in the glycolytic sequence, tricarboxylic acid cycle and associated pathways with those of the promastigote. Initial studies suggested that there are only quantitive differences between the two forms with the greatest of the differences found in the area of CO2-fixation. Phosphoenolpyruvate carboxykinase (PEP carboxykinase) was detected at much higher activity in amastigotes than promastigotes. This enzyme was found to be ADP specific and to have an absolute requirement for Mn2+. There appeared to be no regulation of the amastigote enzyme in crude homogenates by ATP, GTP, ITP or the end products malate and succinate. Pyruvate carboxylase was undetectable in either parasite form and malic enzyme (carboxylating) was detected only at low activity in promastigotes. Amastigotes had low pyruvate kinase but high malate dehydrogenase activities in comparison to promastigotes. This suggests that in amastigotes the main catabolic route for PEP is to succinate, a major end-product of metabolism, by way of carboxylation and reduction involving malate dehydrogenase and fumarate reductase. In contrast, the high activity of pyruvate kinase in promastigotes suggests that, in this parasite form, PEP is more likely to be converted to pyruvate which either enters the TCA cycle or is transaminated to alanine. Promastigotes had a high NADH-linked glutamate dehydrogenase activity in comparison to amastigotes and may reflect the higher glutamate utilization by the insect form of the parasite. A low NADPH-linked glutamate dehydrogenase activity was found in amastigotes, whereas none was detected in promastigotes. Isocitrate lyase could not be detected in either form, suggesting that the glyoxylate cycle plays no part in L. m. mexicana metabolism. The theory that the glycolytic kinases, and in particular phosphofructokinase, are primary targets in the action of pentavalent antimonial drugs against Leishmania was investigated with L. m. mexicana. Pentostam (sodium stibogluconate), active in vivo against Leishmania, had no effect on promastigote growth in vitro even at concentrations of 100μg/ml. In contrast, Triostam, the trivalent analogue of Pentostam, inhibited the growth in vitro of L. m. mexicana promastigotes with an LD50 of 20μg/ml and an MLC of 400μg/ml. It was found to be almost as effective on a weight/weight basis as the trivalent arsenical melarsen oxide (LD50, 20μg/ml; MLC, 100μg/ml). The leishmanicidal effect of Triostam, however, could not be correlated with any enzyme inhibitory activity. Neither Pentostam (110μM) nor Triostam (600μM)inhibited hexokinase, phosphofructokinase, pyruvate kinase, malate dehydrogenase or PEP carboxykinase from either amastigote or promastigote. In contrast, melarsen oxide was a potent inhibitor of all leishmanial enzymes tested except hexokinase. The action of Triostam was antagonised by cysteine indicating that the drug's action may involve thiol groups in some way. Triostam, but not Pentostam, inhibited phosphofructokinase of adult Schistosoma mansoni with an I50 of 200μM, suggesting that antimonials may act in different ways against schistosomes and Leishmania. The subcellular organisation of L. m. mexicana promastigotes was investigated using differential and isopycnic centrifugation. Glycosomes and mitochondrial vesicles from culture promastigotes were separated on linear sucrose gradients. Hexokinase, glucose phosphate isomerase, phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase and PEP carboxykinase were recovered largely in association with glycosomes (density; 1.215 g/ml). Phosphoglycerate kinase and glucose-6-phosphate dehydrogenase had some glycosomal activity but were mostly recovered in the soluble fractions. Pyruvate kinase was totally cytosolic. Malate dehydrogenase showed a broad peak corresponding to that of the mitochondrial marker oligomycinsensitive ATPase (density; 1.190g/ml). Glutamate dehydrogenase and alanine aminotransferase both showed small mitochondrial peaks, but most of the activities were recovered elsewhere on the gradient and in the soluble fractions. Amastigotes of L. m. mexicana were not successfully fractionated using the technique developed for promastigotes, therefore the subcellular location of enzymes in amastigotes was investigated by following the release of soluble enzymes from digitonin-treated amastigotes. This revealed distinct cytosolic, mitochondrial and glycosomal compartments. The findings give an insight into the organisation of L. m. mexicana promastigote and amastigote energy metabolism. The importance of PEP carboxykinase and malate dehydrogenase to the metabolism of the amastigote prompted a more detailed investigation of these two enzymes. Isoelectric focusing studies showed that amastigotes possessed particulate malate dehydrogenase isoenzymes apparently absent from promastigotes. The particulate activities of amastigote malate dehydrogenase and PEP carboxykinase were purified to apparent electrophoretic homogeneity by hydrophobic interaction chromatography using Phenyl-Sepharose CL-4B, affinity chromatography using 5' AMP-Sepharose 4B and gel filtration using Sephadex G-100. Malate dehydrogenase was purified 150-fold overall with a final specific activity of 1230 units/mg protein and a recovery of 63%. PEP carboxykinase was purified 132-fold with a final specific activity of 30.3 units/mg protein and a recovery of 20%. Molecular weights determined by gel filtration and SOS gel electrophoresis were 39,8OO and 33,300 for malate dehydrogenase and 63,100 and 65,100 for PEP carboxykinase, respectively. Kinetic studies with malate dehydrogenase assayed in the direction of oxaloacetic acid reduction showed a Km NADH of 41μM and a Km oxaloacetic acid of 39μM. For malate oxidation there was a Km malate of 3.6mM and a Km NAD+ of 0.79mM. Oxaloacetic acid exhibited substrate inhibition at concentrations greater than O.83mM and malate was found to be a product inhibitor at high concentrations, however there was no modification of enzyme activity by a number of glycolytic intermediates and cofactors suggesting that malate dehydrogenase is not a major regulatory enzyme in L. m. mexicana. The results show that these L. m. mexicana amastigote enzymes are in several ways similar to their mammalian counterparts, nevertheless their apparent importance and unique subcellular organisation in the parasite make them potential targets for chemotherapeutic attack.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Subjects: Q Science > QL Zoology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation > Parasitology
Funder's Name: UNSPECIFIED
Supervisor's Name: Coombs, Dr Graham
Date of Award: 1984
Depositing User: Adam Swann
Unique ID: glathesis:1984-8691
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 23 Jan 2018 11:43
Last Modified: 23 Jan 2018 11:43

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