Ascaridia galli: lactic acid production, glycogen content, glycolytic enzymes and properties of purified aldolase, enolase and glucose-6-phosphate dehydrogenase

Abstract

SUMMARYGlycogen reserves of whole worms and their body wall, intestines, ovarian and testicular tubules of the avian intestinal nematode, Ascaridia galli, were assayed and on dry-weight basis found to be 14–20 %, except in the testicular tissue which contained only 7 % glycogen.Segments of whole male worms or testicular tubules were found to produce more lactic acid than segments of whole female worms or ovarian tubules. The body wall and intestine of the worms had also appreciable glycolytic activity. Whereas in the segments of whole worms, male and female alike, glycolysis was more active under anaerobic conditions, no differences in glycolytic rates were seen between aerobiosis and anaerobiosis when isolated tissues were used. Exogenously added glucose did not stimulate glycolysis either aerobically or anaerobically to any extent greater than observed in the absence of glucose.Homogenates of whole worms or their different anatomical parts were assayed for enzymic activities associated with the Embden-Meyerhof pathway of glucose assimilation. The crude extracts prior to differential centrifugation, were found to contain glycogen phosphorylase, aldokinases, pyruvate kinase, phosphatases acting on glucose 6-phosphate and hexose diphosphate and 6-phospho-gluconate dehydrogenase. The 105000 g particulate-free supernate was found to have significant activities of the following enzymes: fructokinase, phosphoglucomutase, phosphoglucoseisomerase, phosphofructokinase, aldolase, glyceraldehyde 3-phosphate dehydrogenase, lactic dehydrogenase and glucose-6-phosphate dehydrogenase. This fraction was devoid of NADH or NADPH oxidase activities in the absence of added substrates.Although there was some indication of a negative correlation between low glycogen reserves and high glycolytic activity in the testicular tubules, in general, there was no relationship between glycogen reserve and glycolytic activity on the one hand or between the rate of glycolysis and the specific activities of some of the key glycolytic enzymes in either whole worm or in tissues other than the testicular tubules.The 105000 g supernate was fractionated with ammonium sulphate. The fraction precipitating between 25 and 80 % saturation of the salt was recovered, dialysed and chromatographed on DEAE cellulose column. By a step-wise elution schedule using increasing molarity of NaCl in tris-HCl buffer, pH 7·4, three main protein fractions were obtained representing respectively enolase, aldolase and glucose-6-phosphate dehydrogenase.The recovery of enzyme activity after chromatography on DEAE cellulose was higher than the amount applied to the column suggesting that during the fractionation some naturally occurring inhibitors were removed. About 10- to 20-fold purification of the enzymes was achieved by anion-exchange chromatography.Some properties of the purified enzymes were studied with respect to the affects of enzyme and substrate concentrations, temperature of preincubation and action of divalent cations, some anions, metal chelating agents and SH reagents. The Km values of enolase, aldolase and glucose-6-phosphate dehydrogenase of A. galli were 5·9 × 10−4M, 4·5 × 10−3M and 2·4 × 10−3M respectively. Glucose-6-phosphate dehydrogenase was found to be very sensitive to both heat and cold losing activity rapidly even at 43 °C or by freezing and thawing.The SH groups of aldolase were readily blocked by pCMB and presumably by o–phenanthroline. No requirement of any divalent cations was shown by this enzyme which was, however, inhibited by borate ions.Enolase of A. galli showed a requirement of Mg2+ for full activation. Phosphate, fluoride, EDTA, o–phenanthroline, αα-bipyridyl inhibited the enzyme. Veronal was found to inactivate the enzyme.Glucose-6-phosphate dehydrogenase of A. galli was also found to be sensitive to SH reagents and metal chelating agents. The enzyme was activated by Co2+, Mn2+ and Mg2+.The evidence presented indicates that segments of whole worms of A. galli or its anatomical parts are equipped with the enzymatic machinery required to mediate anaerobic breakdown of glucose and to derive energy by this mechanism.The authors are grateful to Dr R. K. Kaushik for help in identifying the worms, to Mr P. A. George for help in the statistical analysis of the data and to Messrs A. C. Kol and S. K. Bose for skilful technical assistance.