Peter W. Pappas

INACTIVATION OF α- AND β-CHYMOTRYPSIN BY INTACT HYMENOLEPIS DIMINUTA (CESTODA)

When specimens of intact Hymenolepis diminuta were incubated in the presence of α- or β-chymotrypsin, assays for proteolytic activity following removal of the worms showed an inactivation of both enzymes. The amount of inactivation in either case was dependent upon the enzyme concentration, total number of worms (total worm weight) present, period of time worms were incubated with the enzymes, and pH of the assay medium. Inactivation of α- and β-chymotrypsin was independent of available surface area and the presence of polyions, was irreversible, and ceased upon removal of the worms from the medium. Intact worms also inactivited the zymogen, α-chymotrypsinogen A. The data suggest that the inactivation of α- and β-chymotrypsin resembles that previously reported for inactivation of trypsin by intact H. diminuta.

Taenia crassiceps: Absorption of hexoses and partial characterization of Na+-dependent glucose absorption by larvae☆

Abstract The uptake of 14 C-fructose by T. crassiceps larvae was linear with respect to concentration. Uptake of 0.05 m M 14 C-fructose was not inhibited by 5.0 m M unlabeled fructose, tagatose, or sorbose. Fructose appears to enter larvae by diffusion only. The uptake of radioglucose and radiogalactose was not linear with respect to concentration at low substrate concentrations; at high substrate concentrations, the uptake of both hexoses was linear with respect to concentration. Inhibitor studies indicated that both glucose and galactose enter larvae by a combination of diffusion and a mediated process, and that these hexoses are mutually competitive inhibitors of one another. The uptake of glucose and galactose was also inhibited by α-and β-methyl glucoside, fucose, and phlorizin, but not by several amino acids, certain sugar analogs, nor ouabain. Glucose transport is Na + sensitive; K + was demonstrated to be a competitive inhibitor of Na + activation of glucose uptake. After a 90-min incubation in 5 m M unlabeled glucose, larvae accumulated glucose against an apparent concentration difference. Although larvae appear freely permeable to ouabain, this compound had no apparent effect on glucose accumulation. The results of this study are compared with previous studies on Hymenolepis diminuta, Calliobothrium verticillatum, Hydatigera (Taenia) taeniaeformis , and mammalian systems.

Sodium and glucose fluxes across the brush border of a flatworm (Calliobothrium verticillatum, Cestoda)

Summary1.In Na+-free media, 2 min glucose influx(JGi) was sharply reduced. Li+ activated JGi to a small extent in the absence of Na+, while K+, choline and tris did not. When Na+ was varied with tris, choline, or Li+ substituted for deleted Na+, JGi was a hyperbolic function of Na+ concentration. When K+ was substituted, JGi was a linear function of Na+ concentration (Fig. 1). When K+ was varied at a low, constant Na+ concentration, K+ behaved as a fully competitive inhibitor of Na+ activation of JGi (Fig. 2).2.At various fixed concentrations of Na+, JGi obeyed Michaelis-Menten kinetics. With decreasing Na+, there was a decrease in maximal glucose influx (JGimax) and no significant change in the apparent Michaelis constant (Kt) (Fig. 3, Table 2). Similar results were obtained with fixed glucose concentrations and varying Na+ (Fig. 4, Table 2).3.Simultaneous determinations of22Na+ and14C-glucose influxes at 15 sec intervals showed that zero order kinetics pertained over a 2 min incubation period (Fig. 5). Influx coupling coefficients (Jnai/JGi) were determined in double labeling experiments with constant Na+ and varying glucose concentrations or with constant glucose and varying Na+. The experimentally determined coefficient was higher than 1.8 indicating a true coefficient of 2 or more. This coefficient was independent of Na+ and glucose concentrations (Fig. 6, Table 3).4.After 30 min preloading with glucose, worms were incubated for 30 min in media with modified cation compositions containing 10.5 mM glucose. In low Na+, low K+ media, accumulation of glucose continued. In low Na+, high K+ media, there was no change in tissue glucose. In Na+-free, high K+ media, there was a net glucose efflux against a concentration difference (Table 4). Efflux of22Na+ from worms previously equilibrated in 250 mM22Na+ was not affected by the presence of glucose in the external medium.5.The data are consistent with Cranes Na+-gradient hypothesis for the transport and accumulation of sugars.Calliobothrium is compared with some other Na+-glucose coupled animal systems and the data are discussed in terms of a model showing first order dependence on concentration and a coupling coefficient of 2 or more.

Thiamine Uptake by Hymenolepis diminuta

Over the concentration range from 0.005 to 100 mM the uptake of thiamine (vitamin B1) by Hymenolepis diminuta was linear with respect to concentration (product-moment correlation coefficient > 0.99). Radiothiamine uptake was inhibited by thiamine, thiamine monophosphate (TMP), oxythiamine, and neopyrithiamine. At 0.1 mM substrate, maximum inhibition was 57% at 800:1 inhibitor/substrate ratio. Iodoacetate (1 mM) inhibited thiamine uptake only if worms were preincubated in the inhibitor for 5 min. Thiamine uptake was not inhibited by thiamine pyrophosphate (TPP), 2,4-dinitrophenol, alanine, arginine, glutamic acid, histidine, methionine, glucose, galactose, glycerol, uracil, cytosine, thymine, riboflavin, folic acid, or nicotinamide. After a 30-sec incubation in labeled thiamine, 93% of the radioactivity (extractable as thiamine, TMP, and TPP) was present as thiamine; 3% was present as TMP, and 4% at TPP. After 30 min incubation, the relative amounts of labeled thiamine, TMP, and TPP were 61, 7, and 32%, respectively. Thiamine uptake appeared to take place by combination of both mediated and nonmediated mechanisms, and the mediated transport system is relatively specific. Hymenolepis diminuta absorbs numerous compounds when incubated in vitro. These include amino acids (Read, Rothman, and Simmons, 1963), monosaccharides (Laurie, 1957; Phifer, 1960), purines and pyrimidines (MacInnis et al., 1965; MacInnis and Ridley, 1969), and fatty acids (Arme and Read, 1968; Chappell et al., 1969). Although it appears that some vitamins (of either host or exogenous origin) are important in the developmental physiology of cestodes (Hager, 1941; Addis and Chandler, 1944, 1946; Platzer and Roberts, 1969, 1970), and that H. diminuta absorbs thiamine (vitamine B1) from the digestive tract of its host (Chandler et al., 1950), no information is available concerning the mechanism(s) of vitamin absorption in this parasite. This paper reports the results of experiments in which thiamine uptake by H. diminuta was studied. MATERIALS AND METHODS Young, male Sprague-Dawley rats (Holtzman Co.) were infected with 30 H. diminuta cysticercoids and the worms removed 11 days postinfection. After removal, worms were rinsed in 3 changes of Krebs-Ringer solution containing 25 mM Tris-(hydroxymethyl)-aminomethane-maleate buffer (pH 7.4) (KRT of Read et al., 1963), randomized into groups of 5 worms, and preincubated Received for publication 30 September 1971. * This work was supported in part by a grant from the NIH (AI-01384). t U. S. Public Health Service Postdoctoral Fellow, 1-FO2-AI-45108-01. at 37 C for 15 min prior to incubations. Worms were then transferred to an incubation medium (in a shaking water bath at 37 C) consisting of 5 ml of KRT to which 4C-thiamine (thiamine [thiazole-2-14C]) or 5S-thiamine (14 mCi/mM and 65 mCi/mM, respectively; Amersham Serale Corp., Arlington Heights, Illinois) had been added. The concentration of thiamine in the incubation medium was adjusted by the addition of unlabeled thiamineHC1 (Sigma Chemical Co., St. Louis). All incubations were of 2-min duration and consisted of 5 replicates, unless otherwise noted. After incubation, worms were removed and rinsed in 2 changes of fresh KRT, blotted on filter paper, and placed in 5 ml of 70% ethanol for 24 hr (ethanol extracted 98%o+ of the absorbed radioactivity). Radioactivity in the ethanol extracts was determined with a Nuclear-Chicago Gas-Flow Counter and the worms dried for 24 hr at 95 C before weighing. Data were converted to ,umoles thiamine absorbed/g alcohol extracted dry wt/hr unless otherwise noted. Inhibitor studies were conducted as described above, except that single inhibitors were added to the incubation media. The following inhibitors were used: Thiamine HCl, thiamine monophosphoric acid chloride HCl (thiamine monophosphate, TMP), thiamine pyrophosphate chloride (TPP, cocarboxylase), oxythiamine (5-[2-hydroxyethyl ] 3[ ( 4-hydroxy -2-methyl5-pyrimidinyl) methyl]-4-methylthiazolium chloride), neopyrithiamine (1-[(4-amino-2-methyl)-5-pyrimidylmethyl]2-methyl-3-[P,-hydroxyethyl] pyridinium bromide hydrobromide), histidine, alanine, methionine, glutamic acid, arginine, glucose, galactose, glycerol, cytosine, uracil, thymine, nicotinamide, riboflavin, folic acid, 2,4-dinitrophenol (DNP), and iodoacetate (all obtained from Sigma Chemical or CalBiochem, Los Angeles). The concentration of all inhibitors was 5 mM, with the following exceptions: iodoacetate and DNP were 1 mM, and

Mechanisms and specificity of amino acid transport in Taenia crassiceps larvae (Cestoda)

Abstract The absorption of lysine, arginine, phenylalanine and methionine by Taenia crassiceps larvae is linear with respect to time for at least 2 min. Arginine uptake occurs by a mediated system and diffusion, and arginine, lysine and ornithine (in order of decreasing affinity) are completely competitive inhibitors of arginine uptake. The basic amino acid transport system has a higher affinity for l -amino acids than d -amino acids, and blocking the α-amino group of an amino acid destroys its inhibitory action. Phenylalanine uptake by T. crassiceps larvae is inhibited in a completely competitive fashion by serine, leucine, alanine, methionine, histidine, phenylalanine, tyrosine and tryptophan (in order of increasing affinity). Methionine apparently binds non-productively to the phenylalanine (aromatic amino acid-preferring) transport system. l -methionine uptake by larvae is inhibited more by d -alanine and d -valine than by their respective l -isomers, while d - and l -methionine inhibit l -methionine uptake equally well. The presence of an unsubstituted α-amino group is essential for an inhibitor to have a high affinity for the methionine transport system. Uptake of arginine, phenylalanine and methionine is Na + -insensitive, and both phenylalanine and methionine are accumulated by larvae against a concentration difference in the presence or absence of Na + . Arginine accumulation is precluded by its rapid metabolism to proline, ornithine and an unidentified compound.

Na+-dependent and Na+-independent glycerol fluxes inHymenolepis diminuta (Cestoda)

Summary1.Labeled glycerol influx (JGi) in the cestode,Hymenolepis diminuta, occurs by diffusion and two mediated systems which were distinguished by their sensitivity to Na+ and inhibitors. The Na+-activated system had a maximal glycerol influx (JGi max) of 91.5 μmoles/g ethanol extracted dry wt/hr and an apparent transport constant (Kt) of 0.24 mM; fluxes of glycerol and Na+ did not appear to be coupled. The second transport system, which operated in the absence of external Na+, had aJGi max of 83.8 μmoles/g ethanol extracted dry wt/hr and aKt of 0.69 mM (Figs. 1 and 2).2.DL-β-glycerophosphate, glyceraldehyde, monoacetin, and phloretin inhibited both transport systems, while 1, 2-propanediol inhibited only the Na+-dependent system (Fig. 3).3.The inhibition ofJGi by DL-β-glycerophosphate was relieved by ammonium molybdate, adenosine triphosphate, adenosine monophosphate and glucose-6-phosphate (Fig. 4, Table 1). These compounds had little or no effect onJGi indicating that inhibition was produced by glycerol liberated from the hydrolysis of the phosphorylated compound by surface phosphohydrolases of the worm.4.Li+, K+, Tris and choline did not replace Na+ as an activator ofJGi (Fig. 7). Glycerol diffusion rates in media where Li+ or K+ were used to replace Na+ were identical; however, replacement of Na+ with Tris lowered the diffusion rate (Table 2). Diffusion-corrected rates forJGi were the same in Li+-, K+-, and Tris-substituted media, but were significantly lower in choline-substituted media; choline appeared to act as a competitive inhibitor of mediatedJGi (Fig. 8, Table 2).5.Phloretin competitively inhibitedJGi while phlorizin had no effect. The effects of pH on the inhibition by phloretin indicated that the ketonic form, and not the enolic form, of phloretin was responsible for inhibition (Fig. 5).

Hymenolepis diminuta: Absorption of nicotinamide

Abstract The uptake of 14C-nicotinamide was linear over the concentration range of 0.025 to 20 mM (Y = 58.1[x] + 0.8; r > 0.99), and the uptake of 0.05 m M 14 C-nicotinamide was not inhibited by unlabeled nicotinamide at concentrations as high as 10 mM (200:1 inhibitor/substrate ratio). Absorption appeared to occur by diffusion only. The uptake of 0.1 m M 14 C-nicotinamide was not linear with respect to time, again indicating a nonmediated mechanism of absorption. Analyses of worm extracts and incubation media after 30 min incubations demonstrated that all the radioactivity remained as labeled nicotinamide. Worms removed from rats which had received intraperitoneal injections of 14C-nicotinamide were found to contain radioactivity. On the basis of comparison of chromatograms of worm extracts with chromatograms of standard nicotinamide derivatives, this radioactivity was present as nicotinamide, nicotinic acid, and two unidentified compounds which did not correspond to nicotinuric acid, 1-methyl nicotinamide, or the reduced forms of nicotinamide adenine dinucleotide or trinucleotide.