THE IN-VITRO UPTAKE AND INCORPORATION OF HEMOGLOBIN BY ADULT HAEMONCHUS-CONTORTUS

The incorporation of radioactivity from [H-3]leucine-labeled hemoglobi n (Hb) into adult Haemonchus contortus proteins was investigated. Furt her, the role of previously described cysteine proteases present in in testinal tissue and excretory/secretory products of H. contortus was a ssessed in the breakdown of Hb. A cell lysate preparation (predominant ly Hb) was obtained from reticulocytes metabolically labeled, in vitro , with [H-3]leucine. Following 24-h incubation in the presence of [3H] Hb, adult H. contortus incorporated radioactivity. The presence of the protein synthesis inhibitor puromycin (200 mu g ml(-1)) reduced incor poration by 72%, indicating that this process was dependent on protein synthesis. The specific cysteine protease inhibitor Z-phe-alaFMK (PAF ) at 0.1 mM had no effect on incorporation of radioactivity; however, the breakdown of Hbg in the culture medium was reduced by 50%. In cont rast, PAF at 1.0 mM caused a 78% reduction in incorporated radioactivi ty. Parasite viability was also decreased by 1.0 mM PAF, and thus the reduction of incorporation of radioactivity may not be due to specific enzyme inhibition. The serine protease inhibitor 4-(2-aminoethyl)benz enesulfonyl fluoride (AEBSF) at 1.0 mM caused a 40% reduction in incor poration of radioactivity; the aspartic protease inhibitor pepstatin ( 1 mM) was without effect, Adult H. contortus also incorporated radioac tivity from [H-3]leucine-labeled intact reticulocytes. This incorporat ion was inhibited by 1.0 mM PAF and AEBSF in a manner similar to that for the cell lysate preparation. These data indicate that adult H. con tortus degrade Hog and incorporate the radioactivity into their macrom olecules. The specific action of the endogenous cysteine protease in t he digestion of Hbg could not be demonstrated unequivocally. However, the hypothesis that the secreted cysteine protease functions in extrac orporeal digestion was supported.