Effect of pH and Temperature on Lipase and Phospholipase of Adult Haemonchus contortus (Nematoda: Trichostrongylidae)

Abstract

Haemonchus contortus (Rud., 1803), one of the most pathogenic parasitic species found in the abomasum of ruminants, has attracted the attention of investigators from different parts of the world. Lipid biosynthesis of H. contortus has been studied extensively (Kapur and Sood, 1984, Zentralblatt fur Veterinarmedizin 31: 225-230); the worm is capable of lipid utilization during starvation, in vitro (Kapur, 1983, Ph.D. Thesis, Punjab Agricultural University, Ludhiana, India; Kapur and Sood, 1984, loc. cit.). However, no attempt has been made to detect the enzymes involved in lipid metabolism. The present study examines lipase and phospholipase activity. In addition, the effect of pH and temperature on enzyme activity was also observed. Adults of H. contortus were collected from the abomasa of goats (Capra hircus) procured from local abattoirs. For assaying the enzymes of the worms only, these were repeatedly washed in physiological saline in order to remove adhering host material. Worms were then homogenized in 0.25 M sucrose for 5 min and centrifuged at 1,250 g for 10 min at 4 C. The supernatant thus obtained was assayed for the enzyme activity. The entire operation was performed under cold conditions. Lipase (EC 3.1.1.3) and phospholipase (EC 3.1.1.32 [A1] and EC 3.1.1.4 [A2]) were estimated by the method of Mahadevan et al. (1969, Analytical Biochemistry 27: 387-396). For estimation of lipase, 0.1 ml tris-HCl buffer (1 M), 0.2 ml aqueous sodium taurocholate (15 mg/ ml), 0.3 ml water, 0.2 ml NaCl (3 M), 0.1 ml CaCl2 2H20 (75 mM), 0.5 ml substrate, and 0.1 ml enzyme preparation were incubated for 30 min. Substrate was prepared by mixing 41.3 ml gum arabic (10% w/v), 3.25 g crushed ice, and 5 ml olive oil. Reaction was terminated with 2 ml 95% ethanol and 0.2 ml 1 N HC1. Free fatty acids thus liberated were extracted in petroleum ether. Controls, one without substrate and the enzyme, and the other with the boiled enzyme preparation but with the substrate, were similarly incubated. Fatty acids were estimated by the method of Lowry and Tinsley (1976, Journal of American Oil Chemists Society 53: 470-472). Aliquots of fatty acids extracted above were taken and dried at 50 C. To each sample was added 5 ml slightly warmed benzene. Subsequently, 1 ml of 5% aqueous cupric acetate pyridine reagent (pH 66.2) was added and mixed on a cyclomixer for 2-3 min. The upper layer was read at 715 nm against a blank with no fatty acid. For phospholipase, 0.5 ml substrate (10 mg lecithin/ml water), 0.5 ml acetate buffer (0.2 M), and 0.5 ml enzyme preparation were incubated for 90 min. Reaction was terminated in the same manner as in the case of lipase, and controls were similarly incubated. All the assays were repeated 3 times. For determining optimum pH, buffers with a pH range 6.6-9.4 in the case of lipase, and pH 3.4-6.2 in the case of phospholipase, were used. Incubations were carried out at 37 C. The pH at which maximum activity was obtained was selected for studying the effect of temperature. The range of temperatures used was 22-52 C. At each pH and temperature, controls were similarly incubated. Protein content was determined by the method of Lowry et al. (1951, Journal of Biological Chemistry 193: 265-275). Results of the effect of pH and temperature on the activity of lipase and phospholipase are given in Figures 1 and 2. For lipase, optimum activity was at pH 7.8. Practically no change was observed from pH 9 to 9.4. Also, there was a progressive increase in enzyme activity with increasing temperature, with optimum activity at 42 C. Thereafter, there was a decrease followed again by an increase at 52 C. High phospholipase activity at pH 3.4 was recorded. Optimum activity was at pH 5.0; at pH 6.2 no activity could be recorded. As in lipase, optimum activity was attained at 37 C. Thereafter, there was a slight decrease at 42 C, followed by a significant decrease at 47 C. Surprisingly, with a further increase in temperature, phospholipase activity increased dramatically. This was not an artifact, since blanks were also incubated at this high temperature.