Haemonchus contortus: Enzymes : I. Isoelectric focusing of malate dehydrogenase in sucrose and in polyacrylamide gels

Abstract

Soluble extracts were prepared from adult male and female Haemonchus contortus and fractionated by DEAE-cellulose chromatography. The initial unadsorbed fraction contained 73% of the total malate dehydrogenase activity present in the crude soluble extract. This was fractionated by isoelectric focusing using LKB Ampholine carrier ampholytes in a sucrose-density gradient column. Seven peaks of malate dehydrogenase were obtained with isoelectric points ranging from 5.9 to 9.0. In addition, six hemoglobin peaks were separated. Composites of fractions from each peak of malate dehydrogenase were then fractionated by gel filtration on Sephadex G-200. Aldolase, malate dehydrogenase, and hemoglobin (when present) were separated from one another. Variations in the pH optimum and the pH optimum curves were noted for the various malate dehydrogenase fractions. In addition, twofold differences in the Michaelis constants were found. The maximum purification of malate dehydrogenase was 22 times compared with that of the crude soluble extract. Maximum purification of the other enzymes investigated was 11 times for aldolase, 3 times for inorganic pyrophosphatase, and 123 times for asparate aminotransferase. Isoelectric focusing experiments performed in polyacrylamide gels resolved the malate dehydrogenase into multiple bands. Polyacrylamide or starch gel electrophoretic analyses failed to give discrete bands of MDH activity.