A Surface Enzyme in Hymenolepis diminuta (Cestoda)

Abstract

Hymenolepis diminuta is virtually impermeable to "C-fructose or "C-fructose diphosphate. The intact worm hydrolyzes fructose diphosphate (HDP) and barium-soluble fructose is liberated in the external medium. Rate of hydrolysis increases with increasing substrate concentration up to 2 mM, above which concentration substrate inhibition occurs. HDP hydrolysis is inhibited by glucose-6-phosphate or js-glycerophosphate. The pH optimum for HDP hydrolysis is about 7.4. The digestive-absorptive functions of the tapeworm brush border are briefly discussed. Cytochemical studies at the ultrastructure level have indicated that phosphatases are localized in or on the tegumentary brush border of Hymenolepis diminuta (Rothman, 1966; Lumsden et al., 1968). It has been suggested that these tegumentary enzymes act in a digestive capacity, hydrolyzing phosphate esters to which the cell may be virtually impermeable (Read, 1966; Lumsden et al., 1968). Although hydrolysis of phosphate compounds at the cell surface might produce a spatial kinetic advantage for the subsequent absorption of hydrolysis products, there is no evidence for the direct participation of phosphatases in a phosphorylation-dephosphorylation of the substrate during transport of organic compounds (see discussion by Read, 1966). If the phosphatase of the cestode surface has a digestive function and is chemically independent of transport function, it might act on substrates which, upon hydrolysis, yield products not transported by the worm. Furthermore, if hydrolysis occurs on the outer surface of the limiting membrane and the resulting products cannot enter the worm, these hydrolysis products should appear in the external medium. Since it is known that Hymenolepis does not metabolize fructose (Read, 1956; Laurie, 1957) and that fructose does not interact with the mechanism involved in glucose transport (Phifer, 1960; Read, 1961), fructose esters appeared to be substrates whose hydrolysis products might not be absorbed. MATERIALS AND METHODS Hymenolepis diminuta was reared in young male Holtzman rats, each host receiving 30 cysticercoids. Received for publication 30 January 1970. * This work was supported by grants from the NIH (AI 01384 and 5 T01-AI 00106). t Present address: Department of Zoology, The Queen's University, Belfast, U. K. Worms were harvested 9, 10, or 11 days after infecting the host and washed in Krebs-Ringer solution, buffered with 25 mM tris-maleate at pH 7.4. This saline was also used in all incubations. After randomly mixing the worms from a group of rats, 5-worm samples were used in experimental incubations. Samples were preincubated without substrate for 15 min at 37 C in a shaker bath. Each sample was then placed in 5 or 10 ml of incubation medium containing fructose-1, 6-diphosphate, with or without other additions, and incubated for an appropriate time at 37 C in a shaker bath. After incubation, the worms were removed to tared aluminum cups and dried for 24 hr at 95 C. To precipitate unhydrolyzed hexose diphosphate, 4-ml aliquot of incubation medium was adjusted to pH 8.5 with KOH and 1 ml of 1 M barium acetate added. This was allowed to stand for 20 rmin and the precipitate removed by centrifugation. Excess barium was removed from a 3-ml aliquot of the supernatant fluid by adding an equal volume of 1 M sodium sulfate. The precipitated barium sulfate was removed by centrifugation. Controls for fructose recovery were run with each set of samples and recovery ranged from 94 to 97%. Fructose was determined by the method of Roe (1934). Radiocarbon was determined on dried samples in a thin window flow counter. Hexose diphosphate, glucose-6-phosphate, and p-glycerophosphate were obtained from the California Corporation for Biochemical Research or Sigma. Other chemicals were of reagent grade. RESULTS AND DISCUSSION In order to examine the possibility that the phosphatases associated with the tegument of Hymenolepis diminuta were actually acting at the tegument-medium interface it was first necessary to determine whether or not fructose penetrated the worm to a significant extent. Quadruplicate worm samples of about 45 mg were incubated for 60 min with uniformly labeled 0.5 mmx 14C-fructose or 14C-fructose diphosphate, with a specific activity of 1.0 /tc/,umole in a volume of 5 ml. After incubation, the worms were removed, rapidly rinsed in a large volume of saline, and extracted for