Trypsin Inactivation by Intact Hymenolepis diminuta

Abstract

Using azoalbumin as a substrate, it was demonstrated that intact Hymenolepis diminuta inactivate trypsin when incubated with this enzyme. At a given enzyme concentration ([E]), the per cent inactivation (%I) was relatively constant as the substrate concentration ([S]) was varied. However, at a given [S], the %I increased as the [E] decreased. It was also demonstrated that inactivation was dependent upon the time worms were exposed to the enzyme, the total worm weight present in the assay, and the pH of the assay medium. The %I was not related to worm surface area, and was unaffected by polyions and calcium ions in the incubation medium. These results are discussed in relation to the possible mechanism and physiological significance of this phenomenon. The mucosal surface of the mammalian intestine and the outer surface of the syncytial epidermis of the cestode, Hymenolepis diminuta, contain intrinsic membrane-bound enzymes (Crane, 1962; Newey et al., 1963; Arme and Read, 1970; Dike and Read, 1971a). Both surfaces are reported to adsorb enzymes of pancreatic origin (Ugolev, 1965; DeLaey, 1966a; Goldberg et al., 1968, 1969; Read, 1972). According to DeLaey (1966b), adsorption of amylase involves interaction with the mucosal glycocalyx and is readily reversible (Jesuitova et al., 1964). Amylase adsorption to the surface of H. diminuta is also reversible and is partially blocked by polyions (Read, 1972). However, the adsorption of proteolytic enzymes to isolated mucosal cells or subcellular particles is not readily reversible (Goldberg et al., 1968, 1969). According to Ugolev (1965), the adsorption of amylase on the mammalian mucosa results in an increase in enzyme activity. However, doubt has been cast on Ugolev's interpretation with the demonstration that the mammalian mucosa has intrinsic membrane-bound amylases (McMichael and Dahlqvist, 1968; Alpers and Solin, 1970). On the other hand, while Hymenolepis does not exhibit intrinsic amylase or disaccharidase activity, adsorption of pancreatic amylase to the worm surface results in an increase in amylolytic activity (Taylor and Thomas, 1968; Read, 1972). Adsorption of trypsin and chymotrypsin to isolated human Received for publication 24 February 1972. * This study was supported by a grant (AI-01384) from the NIH, U. S. Public Health Service. t U. S. Public Health Service Postdoctoral Fellow, 1-F02-AI-45108-01. mucosal cells or subcellular particles does not ter the kinetic parameters of these enzymes (Goldberg et al., 1969). However, in the presence of intact mammalian mucosa, proteolytic enzymes of pancreatic origin are inctivated (Borgstrom et al., 1957; Goldberg et al., 1968, 1969; Pappas and Gallogly, unpublished). Reichenbach-Klinke and Reichenbach-Klinke (1970) reported that trypsin is inactivated in the presence of the tapeworm, Proteocephalus longicollis. The present investigation was concerned with the effects of the tapeworm, Hymenolepis diminuta, on pancreatic trypsin. MATERIALS AND METHODS Hymenolepis diminuta were reared in the beetle, Tenebrio molitor. Male Sprague-Dawley rats (Holtzman Co.) weighing 80 to 100 g were infected with 30 H. diminuta cysticercoids and the worms removed 11 days postinfection. Worms were rinsed in 3 changes of Krebs-Ringer solution containing 25 mM Tris(hydroxymethyl) aminomethane-maleate buffer (pH 7.2) (KRT of Read, Rothman, and Simmons, 1963), randomized into groups (usually 10 worms/group), and incubated in fresh KRT at 37 C for 15 min prior to their addition to the assay medium. The assay medium consisted of 4 ml of KRT and 1 ml of an enzyme solution (Trypsin-type XI: DCC treated. 8,000 BAEE units/mg. Sigma Chemical Co., St. Louis) maintained at 37 C in a shaking water bath. When groups of worms were to be used during assays they were removed from the previous incubation medium (KRT) and allowed to incubate in the KRT-enzyme solution for 15 min prior to the addition of substrate (this period in the KRT-enzyme solution is hereafter referred to as the preincubation period); after preincubation the worms were removed. The reaction was initiated by the addition of 1 ml of an azoalbumin (bovine origin, Sigma) solution prewarmed to 37 C, and allowed to continue for a prede-