Pathways of glucose catabolism and the fate of reducing equivalents in the in vitro-propagated

Abstract

Theileria parva schizonts were cultured in bovine peripheral lymphocytes. After schizogony, when the majority of the daughter cells were infected, selective lysis of the lymphoblasts was effected with aerolysin. The optimum concentration of aerolysin was determined using rabbit erythrocytes and was found to be approximately 540 ug/ml for 2 x 108 erythrocytes. This concentration of aerolysin was used throughout the study to lyse the infected bovine lymphocytes in order to release the schizonts for biochemical studies. To carry out enzyme assays the enzymes were released from the parasites using 0.02 % (w/v) Triton-Xl 00 which disrupts the cell membranes. Some glycolytic enzymes were assayed and all the following were found to have activities greater than 20.0 nmoles/min/mg protein at 25°C: hexokinase, glucosephosphate isomerase, phosphofructokinase, fructose 1,6- diphosphate aldolase, phosphoglycerate kinase, enolase, pyruvate kinase and lactate dehydrogenase. The glycerol kinase and a-glycerophosphate dehydrogenase which are required during glycerol metabolism were found to have very low activities which were less than 3.0 nmoles/rnin/mg protein. It was proposed that the catabolism of glucose by the Theileria parva schizont involves the Embden-Meyerhofpathway and that glycerol is hardly utilized as substrate for energy production. From the mean activity of glucose 6-phosphate dehydrogenase which was found to be 3.8 nmoles/min/rng protein, it was proposed that the pentose phosphate pathway exists in T parva schizonts. Pyruvate carboxylase and glutamate pyruvate transaminase activities were greater than 15.0 nmoles/minlmg protein. It was suggested that there could be significant carboxylation of pyruvate to oxaloacetate and that transamination of glutamate with pyruvate also occurs. a-Ketoglutarate is formed via this transamination. It was also proposed that pyruvate carboxylase plays an important role in further metabolism of pyruvate. To determine how the a-ketoglutarate formed via the glutamate pyruvate transaminase-catalysed reaction is used, the activity of glutamate oxaloacetate transaminase was also determined and found to be 18.0 nmoles/minlmg protein. It was suggested that a-ketoglutarate in T .parva is also transaminated with aspartate to form oxaloacetate. The following Krebs cycle enzyme activities were less than 2.0 nmoles/minlmg protein and were considered insignificant: citrate synthase, aconitase, NAD+-isocitrate dehydrogenase, NADP+-isocitrate dehydrogenase and a-ketoglutarate dehydrogenase. It was proposed that the Krebs cycle is incomplete in T parva schizont and that it does not contribute significantly towards energy production. The Krebs cycle enzymes found to have significant activities were: malate dehydrogenase, succinate dehydrogenase and fumarase. Their activities were more than 6.0 nmoles/minlmg protein. It was proposed that these enzymes catalyse reactions involved in other pathways leading to formation of oxaloacetate.