Philip J. Schofield

A comparative study of the tricarboxylic acid cycle enzymes in Fasciola hepatica and rat liver

Abstract 1. 1. The activities and cellular distribution of the tricarboxylic acid cycle enzymes; citrate synthase, aconitase, isocitrate dehydrogenase, α-oxoglutarate dehydrogenase, succinate dehydrogenase, fumarase, malate dehydrogenase, and glutamate dehydrogenase were determined in Fasciola hepatica and rat liver. 2. 2. The coenzyme specificities of isocitrate dehydrogenase and glutamate dehydrogenase were determined for both tissues. 3. 3. Aconitase and fumarase activities could not be detected in the mitochondrial fraction from F. hepatica, while NAD-specific isocitrate dehydrogenase could not be detected in either the mitochondrial or supernatant fraction from this tissue. 4. 4. Succinate dehydrogenase activity was determined in both reaction direction in F. hepatica and rat liver. The oxidation of NADH2 with fumarate was demonstrated in F. hepatica mitochondria under aerobic conditions. 5. 5. The low levels of aconitase and NADP-specific isocitrate dehydrogenase and the absence of NAD-specific isocitrate dehydrogenase suggest that the tricarboxylic acid cycle may be of minor importance in F. hepatica.

The pathway of arginine catabolism in Giardia intestinalis.

In Giardia intestinalis, arginine is catabolised by the arginine dihydrolase pathway. The enzymes of the pathway (arginine deiminase, ornithine transcarbamoylase and carbamate kinase) were investigated and their basic kinetic parameters determined. The specific activity of arginine deiminase was 270 +/- 23 nmol min-1 (mg protein)-1; ornithine transcarbamoylase, in the direction of citrulline utilisation 170 +/- 22 nmol min-1 (mg protein)-1, and in the direction of ornithine utilisation 2100 +/- 100 nmol min-1 (mg protein)-1; and carbamate kinase 2100 +/- 400 nmol min-1 (mg protein)-1. The activities of these enzymes are between 10 and 250 fold greater than those reported for the enzymes in Trichomonas vaginalis, the only other parasite in which the arginine dihydrolase pathway has been reported. The flux through the pathway in G. intestinalis, as determined by the liberation of 14CO2 from 1 mM [14C-guanidino]arginine was 30 nmol min-1 (mg protein)-1. This flux was not affected by valinomycin (0.1 microM), nigericin (3 microM), azide (5 mM) or cyanide (1 mM). The flux was only marginally affected by glucose up to 10 mM concentration. Conversely, the flux through glucose metabolism, as determined by the release of 14CO2 from 1 mM [1-14C]glucose was only 2 nmol min-1 (mg protein)-1, and was unaffected by arginine concentrations up to 10 mM. These observations suggest that there is no direct metabolic interface between arginine and glucose catabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

The glycolytic pathway in adult liver fluke, Fasciola hepatica

1. 1. The activities of Embden-Meyerhof pathway enzymes and related enzymes of carbohydrate metabolism; hexokinase, glucose-6-phosphate dehydrogenase, glucose-6-phosphatase, phosphoglucomutase, phosphoglucoisomerase, phosphomannoisomerase, phosphofructokinase, aldolase, triosephosphate isomerase, α-glycerophosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase and lactic dehydrogenase were determined in adult F. hepatica and rat liver. 2. 2. Glycolysis in F. hepatica proceeds only to the formation of phosphoenolpyruvate; pyruvate kinase activity could not be detected. 3. 3. Lactic dehydrogenase was much less active in fluke than in rat liver. 4. 4. Phosphofructokinase appears to be a rate-limiting step in that part of the Embden-Meyerhof pathway which is present. 5. 5. The possible fate of phosphoenolpyruvate in relation to the apparent absence of pyruvate kinase is discussed.

The metabolism of phosphoenolpyruvate and pyruvate in the adult liver fluke Fasciola hepatica

Abstract 1. 1. The role of carboxylating reactions in the formation of dicarboxylic acids in adult Fasciola hepatica was investigated. The mechanism of pyruvate formation from phosphoenolpyruvate, without the involvement of a pyruvate kinase (EC 2.7.1.40) step, was determined. 2. 2. In the production of organic acids from carbohydrate, phosphoenolpyruvate carboxylase (EC 4.1.1.32) acts as the key carboxylating enzyme, while under the same conditions malate dehydrogenase (decarboxylating) (NAD/NADP) (EC 1.1.1.39/1.1.1.40) acts primarily as a decarboxylating enzyme in the production of pyruvate. No pyruvate carboxylase (EC 6.4.1.1) activity could be detected in F. hepatica . 3. 3. CO 2 fixation by phosphoenolpyruvate results in the rapid formation of a large pool of succinate. The formation of succinate from carbohydrate appears to involve the production of phosphoenolpyruvate by a glycolytic series of reactions, carboxylation to oxaloacetate, reduction to malate, fumarate formation and finally the reduction of fumarate to succinate. Fixation of labelled CO 2 by phosphoenolpyruvate also yields large amounts of labelled citrate. 4. 4. Phosphoenolpyruvate carboxylase was found to be readily reversible and thus this enzyme may function either in the degradation or synthesis of carbohydrate. 5. 5. Malate dehydrogenase (decarboxylating) activity was greater in the presence of NAD + than in the presence of NADP + . 6. 6. The activity of pyruvate dehydrogenase (EC 1.2.4.1) was determined in the mitochondrial fraction from F. hepatica . 7. 7. The low level of pyruvate kinase activity in F. hepatica precludes the formation of significant amounts of pyruvate directly from phosphoenolpyruvate. However, it is suggested that pyruvate can be formed from phosphoenolpyruvate by a pathway involving the action of phosphoenolpyruvate carboxylase, malate dehydrogenase (EC 1.1.1.37) and malate dehydrogenase (decarboxylating). The significance of such a pathway is discussed.

Glycolysis in Haemonchus contortus larvae and rat liver.

Abstract 1. 1. The glycogen content of third-stage inefective Haemonchus contortus larvae was found to average 9·2 per cent on a dry weight basis 2. 2. The enzymes of the Embden-Meyerhof pathway of glycolysis were investigated in H. contortus and their activities compared with those of the corresponding enzymes in rat liver. 3. 3. Following enzymes were found to be present in H. contortus and possessed reaction rates very similar to the respective enzymes in rat liver; hexokinase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, glucose-phsophate isomerase, mannosephosphate isomerase, phosphofructokinase, aldolase, triosephosphate isomerase, glyceraldehydephsophate dehydrogenase, phophoglycerate kinase, phosphoglyceromutse and phosphophyruvate hydratase. 4. 4. Under the assay conditions employed no pyruvate kinase could be detected in H. contortus . 5. 5. Lactate dehydrogenase requiring NAD as cofactor was present in H. contortus but its activity was extremely low compared to that of rat liver. 6. 6. Negligible glucose-6-phosphatase activity was detected in H. contortus in contrast with the considerable activity in rat liver. 7. 7. It appears that it is only in the steps involving the metabolism of phosphoenolpyruvate that glycolysis in the nematode differs markedly from that in vertebrate tissue.

Comparative activity and intracellular distribution of tricarboxylic acid cycle enzymes in Haemonchus contortus larvae and rat liver.

1. 1. The enzymes involved in the tricarboxylic acid cycle were investigated in Haemonchus contortus and the results compared with those obtained for the corresponding enzymes in rat liver. 2. 2. Succinic dehydrogenase and the oxoglutarate dehydrogenase system were detected in a particulate preparation from H. contortus, their specific activities being about one-third those of the corresponding enzymes in rat liver. The succinic dehydrogenase of H. contortus resembled that of facultative anaerobes in its relative rates of succinate oxidation and fumarate reduction. 3. 3. The level of citrate synthase activity in H. contortus crude homogenates was much greater than that in rat liver, the bulk of this activity being retained by the supernatant fraction. In rat liver the specific activities of the supernantant and particulate fractions were almost equal. 4. 4. The level of NADP specific isocitrate dehydrogenase in H. contortus was about one-sixth that in rat liver, although the intracellular distribution of this enzyme was similar in both tissues, the supernatant containing most of the activity. 5. 5. In contrast to the exclusive localization of NAD specific isocitrate dehydrogenase in the mitochondrial fraction of rat liver homogenates, considerable activity was detected in the supernatant fraction of H. contortus homogenates as well as in the particulate fraction. Like that of vertebrate tissue, the NAD specific isocitrate dehydrogenase of H. contortus was activated by ADP. 6. 6. Aconitate hydratase, fumarate hydratase and malate dehydrogenase were present in H. contortus and their intracellular distribution and specific activities were very similar to those of the corresponding rat liver enzymes. 7. 7. The particulate fraction from H. contortus was found to be a poor source of some of the enzymes of the tricarboxylic acid cycle, especially of aconitate hydratase and NADP specific isocitrate dehydrogenase, much higher levels of activity being retained by the supernatant. Similar results were obtained with rat liver homogenates and the implications of these findings in relation to previous reports on the existence of this cycle in other parasites is discussed. 8. 8. It appears that a full tricarboxylic acid cycle can operate in H. contortus under aerobic conditions and the possible role of these enzymes under anaerobic conditions is discussed.

Phosphoenolpyruvate carboxykinase in the adult liver fluke, Fasciola hepatica

Abstract 1. 1. Fasciola hepatica incorporates C14O2 via an active phosphoenolpyruvate carboxykinase. 2. 2. Phosphoenolpyruvate carboxykinase activity is associated with both the soluble and mitochondrial fractions. 3. 3. Maximum activity occurred in the mitochondrial fraction at pH 5·9. 4. 4. The activity of the phosphoenolpyruvate carboxykinase was dependent on the presence of a nulceotide and divalent cation. IDP was the most effective nucleotide, but high activity was demonstrated with either ADP or GDP. Mg2+ was much less effective than Mn2+. 5. 5. The role of phosphoenolpyruvate carboxykinase as possibly the key enzyme linking glycolysis with the production of C4-dicarboxylic acids in F. hepatica is discussed.

Arginine metabolism during culture of Giardia intestinalis

The effect of arginine on the growth and metabolism of Giardia intestinalis trophozoites was determined. Supplementation of the normal growth medium (Diamonds TYI-S-33) with 5 or 10 mM arginine accelerated trophozoite growth over the first 2 days. There was a corresponding rapid utilisation of arginine, with none being detectable after this time. The decrease was associated with the appearance in the growth medium of 1 mol of ornithine and 2 mol of ammonia per mol of arginine utilised, the stoichiometry being consistent with the operation of the arginine dihydrolase pathway. Subsequently, there was a decrease in the ammonia concentration in the medium. Removal of arginine from the medium by pretreatment with arginase substantially decreased cell growth. In TYI-S-33 medium containing no added glucose, instead of the normal 50 mM glucose concentration, arginine supplementation also increased cell growth over the first 2 days, with concurrent stoichiometric production of ornithine and ammonia. However, in these conditions, the ammonia concentration remained elevated. This suggests that under normal conditions there is re-uptake of ammonia, which is glucose dependent. The observations confirm the operation of a functional arginine dihydrolase pathway in G. intestinalis. The concordance of cessation of rapid growth with the depletion of arginine, and the beneficial effect on growth of arginine supplementation suggests that arginine availability is a limiting factor during the initial stages of rapid growth. It would appear that arginine is a major potential energy source during the initial stages of giardial growth, and that supplementation of Diamonds TYI-S-33 medium with additional arginine may provide an improved in vitro culture medium.

Glucose metabolism in Giardia intestinalis

The effect of glucose and other monosaccharides on Giardia intestinalis was investigated by growing G. intestinalis trophozoites in Diamonds TYI-S-33 medium modified by changes in the monosaccharide component, and observing changes in the trophozoite growth and product formation (alanine, ethanol and acetate). Reducing the glucose concentration from 50 mM to 10 mM had little effect on trophozoite growth and product formation. Below 10 mM glucose, ethanol production was markedly reduced, there was a lesser effect on alanine, but acetate production was unaffected. In medium in which no glucose had been added, trophozoites grew at about half the rate of controls (50 mM glucose) and continued to form the same products. Growth in medium containing 10 mM ribose or 10 mM fructose substituted for glucose produced a metabolic profile similar to that of the no glucose added condition. The activity of a number of glycolytic and related enzymes was also determined, but the enzymic profile was not affected by the monosaccharide status of the medium. Ethanol production by trophozoites was specifically depressed by the aldehyde reductase inhibitor, valproate; 3 mM valproate reduced ethanol production by 90%. The alcohol dehydrogenase inhibitor pyrazole had no effect on ethanol production or any other parameter. This differential inhibition suggests that ethanol is produced by an aldehyde reductase or related enzyme. The observations that G. intestinalis trophozoites can continue to grow, replicate and produce the same metabolites in medium containing little or no glucose suggest that G. intestinalis is not solely dependent on glucose as a metabolic fuel.(ABSTRACT TRUNCATED AT 250 WORDS)

The arginine dihydrolase pathway is present in Giardia intestinalis.

Growth of Giardia intestinalis in Diamonds TYI-S-33 medium is characterized by a rapid depletion of the arginine in the medium, and concurrent production of ornithine and ammonia. [Guanidino-14C] arginine was converted to 14CO2 by extracts of G. intestinalis suggesting the presence of the arginine dihydrolase pathway. This was confirmed by the detection of arginine deiminase, catabolic ornithine transcarbamylase, carbamate kinase and ornithine decarboxylase in giardial extracts. The findings demonstrate for the first time the existence of the arginine dihydrolase pathway in Giardia, and suggest that arginine metabolism via this pathway plays a significant role in energy metabolism by providing a site for anaerobic substrate level phosphorylation.