The presence of 14 enzymes was investigated using purified spores of the microsporidian Nosema grylli from fat body of the crickets Gryllus bimaculatus. Glucose 6-phosphate dehydrogenase (EC 1.1.1.49), phosphoglucomutase (EC 5.4.2.2), phosphoglucose isomerase (EC 5.3.1.9), fructose 6-phosphate kinase (EC 2.7.1.11), aldolase (EC 4.1.2.13), 3-phosophoglycerate kinase (EC 2.7.2.3), pyruvate kinase (EC 2.7.1.40) and glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) were detected with activities of 15 +/- 1, 7 +/- 1, 1,549 +/- 255, 10 +/- 1, 5 +/- 1, 16 +/- 4, 6 +/- 1 and 16 +/- 2 nmol/min mg protein, respectively. Hexokinase (EC 2.7.1.1), NAD-dependent malate dehydrogenase (EC 1.1.1.37), malic enzyme (EC 1.1.1.40), lactate dehydrogenase (EC 1.1.1.27), alcohol dehydrogenase (EC 1.1.1.1) and succinate dehydrogenase (EC 1.3.99.1) were not detectable. These results suggest the catabolism of carbohydrates in microsporidia occurs via the Embden-Meyerhof pathway. Glycerol 3-phosphate dehydrogenase may reoxidize NADH which is produced by glyceraldehyde 3-phosphate dehydrogenase in glycolysis.

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ABSTRACT. A new method of fractionation and purification of different life cycle stages of microsporidia Nosema grylli, parasitizing the fat body of cricket Gryllus bimaculatus, by centrifugation in Percoll density gradient is elaborated. The whole procedure can be summarized as: 1) infected fat body preparation, 2) homogenization in buffer and filtration through cotton wad and filter paper, 3) first centrifuging, resulting in the separation of the pellet into three layers containing different life cycle stages, 4) second centrifuging of the chosen layer in Percoll density gradient, 5) washing out the Percoll from the fraction under study. After centrifugation in Percoll density gradient, meronts and early sporonts form a band in the area corresponding to density 1.016 g/ml. Mature spores form the pellet at the bottom of centrifuge tube, while immature spores are distributed throughout the layer of 1.016 g/ml up to the bottom of the centrifuge tube, according to their buoyant densities. The offered technique is simple, it takes about one hour and may become a routine procedure for biochemical studies on microsporidia.

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