The in vitro uptake and incorporation of hemoglobin by adult Haemonchus contortus

The incorporation of radioactivity from [H-3]leucine-labeled hemoglobin (Hb) into adult Haemonchus contortus proteins was investigated. Further, the role of previously described cysteine proteases present in intestinal tissue and excretory/secretory products of H. contortus was assessed in the breakdown of Hb. A cell lysate preparation (predominantly 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 incorporation 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 contrast, PAF at 1.0 mM caused a 78% reduction in incorporated radioactivity. 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)benzenesulfonyl fluoride (AEBSF) at 1.0 mM caused a 40% reduction in incorporation of radioactivity; the aspartic protease inhibitor pepstatin (1 mM) was without effect, Adult H. contortus also incorporated radioactivity from [H-3]leucine-labeled intact reticulocytes. This incorporation 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. contortus degrade Hog and incorporate the radioactivity into their macromolecules. The specific action of the endogenous cysteine protease in the digestion of Hbg could not be demonstrated unequivocally. However, the hypothesis that the secreted cysteine protease functions in extracorporeal digestion was supported.