Camp. Biochem. Physiol. Vol. 96A, No. I, pp. 87-89, 1990 Printed in Great Britain

0300-9629/90 $3.00 + 0.00 0 1990 Pergamon Press plc

KINETIC STUDIES OF GLUCOSE AND a-METHYL-D-GLUCOSIDE ABSORPTION BY

HYMENOLEPIS DIMINUTA (CESTODA)

M. M. KHAN and Y. K. IP*

Department of Zoology, National University of Singapore, Kent Ridge, Singapore 0511

(Received 26 October 1989)

Abstract-l. Both isomers, G(-(aMG) and b-methyl-D-glucoside (BMG) are competitive inhibitors of glucose uptake by Hymenolepis diminuta with K, values of 0.53 mM and 0.42 mM respectively.

2. Kinetic studies show that glucose and aMG do not have similar kinetic parameters. The V,,. for aMG transport is half that of glucose though they have comparable K, values.

3. Both isomers can be used as analogues to study the mechanism of glucose transport in H. diminuta.

INTRODUCTION

As suggested by Uglem et al. (1978), in order to elucidate the mechanism of hexose transport in Hymenolepis diminuta further, a non-metabolized hexose that exhibits transport characteristics similar to that of glucose must be identified. They recom- mended using \MG as a glucose analogue to study the mechanism of hexose transport and accumulation in H. diminuta. However, a detailed literature search revealed that tl MG rather than its fi -isomer, was used for such function in other systems on many occasions (Segal et al., 1973; Mulin et al., 1980; Kimmich and Randles, 1981; Brown and Sepulveda, 1985; Planas et al., 1986; Kuramochi, 1986). Furthermore, radio- active BMG is not commercially available any more though Uglem et al. (1978) were able to obtain it from New England Nuclear Product (USA) previously.

Moreover, Uglem et al. (1978) stated that PMG had kinetic parameters for transport (V,,,,X and K,) similar to those reported for glucose. However, it is doubtful that they would have similar V,,,,, values as flMG has a much more bulky group on carbon-l instead of simply a hydrogen atom in a glucose molecule. They further concluded that the presence of such methyl group protected /3MG from being phos- phorylated but has little or no effect on its interaction with the glucose transport system. Such a conclusion, if proven wrong, may lead to erroneous interpretation of data when PMG is used as the analogue to study the mechanism of glucose transport in H. diminuta. Based on such analyses, the authors believe that it is important to re-examine the above-mentioned problems concerning c( MG and fl MG before a choice can be made as to which analogue should be used as a non-metabolizable probe to elucidate the mechanism of glucose transport in H. diminuta further.

MATERIALS AND METHODS

H. diminuta was obtained from Carolina Biological Supply Co. (Burlington, NC) as cysticercoids in adult Tenebrio sp. Thirty cysticercoids were force-fed to each rat.

*Author to whom correspondence should be addressed.

Male albino rats of Sprague-Dawley strain, weighing 12@-150g at the time of infection were used as definitive host in all experiments. Before and after infections, the rats were provided with food and water ad libitum. Worms were flushed from the excised gut 10 days postinfection with the balanced saline solution (BSS) of Webb (1985). The worms were randomized and sorted into groups of five, blotted quickly on moistened filter paper and their wet weight (ww) recorded. Samples were preincubated in a water bath shaking at 100 oscillations/min at 37°C for 30 min in sealed beakers containing 5ml of BSS gassed with 5% carbon dioxide in nitrogen. Worms were then incubated under the same conditions for 2 min in 5 ml of BSS containing radiolabelled ‘H-polyethylene&co1 (PEG) (0.5 tiCi/ml) (Podesta, 1977; Phddsta -et aL,- 1977) and a test’ solute, either “C-elucose or 14C-aMG with or without unlabelled inhibitors. Upon removal from the medium, the worms were blotted on moistened filter paper and digested in 1 ml of Protosol (NEN) at 37°C for 12 hr (Gruner and Mettrick, 1984). After complete digestion, 10 ml of Aquasol- (NEN) was added to the digested tissue and dark adapted for 24 hr prior to scintillation counting by a Kontron Betamatic liquid scintillation counter. Counting efficiency was esti- mated by the external standard ratio method. The amount of test solute absorbed by the worms was determined by subtracting the calculated amount of radioactivity adhering to the surface of the worms (as represented by 3H-PEG) from that of the total amount of radioactivity present. Uptake velocities were expressed as pmol/g ww per 2 min.

The results were presented as means k SD. Difference between means were examined by Student’s f-test for statistical significance. The percentage data was subjected to arcsine transformation prior to the application of t-test. Data presented graphically were plotted as least square regression lines. Linear transformation of the Michaelis- Menten kinetics was performed according to the Lineweaver-Burk plot

( 1934, _:+.‘+L mm s VIII,, >

(

the Woolf-Augustinsson-Hofstee plot

1959, v = -KS; + V,.,’ V >

and the Direct linear plot

( Eisenthal and Cornish-Bowden, 1974, % - $ = 1

> .

The method of Dixon (1953) was used to determine inhibition constants.

87

88 M. M. tiN and Y. K. IP

RESULTS AND DISCUSSION

In order to determine the inhibitor constant (K,) and to verify the nature of aMG and /.?MG inhibition on glucose uptake in H. diminuta, the data were plotted (Figs 1 and 2) according to the method of Dixon (1953). Both isomers are competitive inhibitors of glucose uptake by H. diminuta with K, values of 0.53 mM and 0.42 mM respectively indi- cating that /IMG is indeed a more effective inhibitor of glucose transport in H. diminuta.

However, the difference in their efficacy as inhibitors to glucose uptake is not as great as that claimed by Uglem et al. (1978). When tested as inhibitors, the authors found that a MG and /I MG at the concentration of 10 mM inhibited 0.1 mM 14C glucose uptake by 90.1% and 94.6% (Table 1) respec- tively (P < 0.01); different from the report of 80 and 100% by Uglem et al. (1978). No significant differ- ence (P > 0.05) between their effect on glucose uptake was observed at the concentrations of 1,2 and 5 mM of both isomers. Hence, it would appear that both aMG and /?MG can be used as analogues to study the glucose transport mechanism in H. diminuta.

However our data indicate that glucose and aMG do not have similar kinetic parameters (Table 2). The K, value (0.73 mM) for aMG obtained by the Woolf-Augustinsson-Hofstee transformation in the present studies is identical to that of BMG uptake (0.73 mM) reported by Uglem et aI. (1978) using the Lineweaver-Burk plot. The K, values for glucose and aMG absorption (Table 2) in the present studies were found to be similar though H. diminuta may have slightly higher affinity to the latter. However, the V,,, value for a MG transport was approximately half that of glucose. This may be explained by the presence of a more bulky methyl group in aMG at the carbon-l position instead of hydrogen as in glucose. The I’,,,,, value of 13.2 pmol/g of ethanol extracted dry weight per 2 min for /IMG reported by Uglem et al. (1978) does fall within the range of I’,,,,, values (10.3-26.6) for glucose as stated in the review article by Pappas and Read (1975). However it is well known that such kinetic parameters are highly variable as they can be

Substrate concentration

1 mM a-methyl-D-glucoside (n = 3) I mM p-methyl-D-glucoside (n = 3)

% Inhibition

61.9 f 12.4 61.0 k 5.1

Fig. I. Dixon plots of stable a-methyl-o-glucoside (I) inhibition of 14C-glucose absorption by H. diminutu. “C-Glucose concentrations were 0.2 mM (0) 0.3 mM (a), 0.5 mM (0) and I.0 mM (B). V = pmol/g ww per 2 min; I = inhibitor concentration in mM. r = 0.92, 0.92, 0.92 and 0.74 for 0.2mM. 0.3mM, OSmM and l.OmM of “C-glucose respectively. Each point represents the mean of four determinations; vertical bars around the points show

2 mM a-methyl-o-glucoside (n = 3) 2 mM B-methyl-o-glucoside (n = 3)

5 mM a-methyl-o-glucoside (n = 3) 5 mM /l-methyl-o-glucoside (n = 3)

IO mM ol-methyl-D-glucoside (n = 3)

70.6 + 4.5 72.4 f 6.4

81.4 f 2.2 86.8 f 2.8

90. I _+ 0.8*

10 mM ,9-methyl-D-glucoside (n = 3) 94.6 f 0.3*

standard deviations. l P < 0.01, significantly different at the concentration of IOmM.

Fig. 2. Dixon plots of stable /I-methyl-o-glucoside (I) inhibition of “C-glucose absorption by H. diminuta. “C-Glucose concentrations were 0.2 mM (O), 0.3 mM (e), 0.5 mM (0) and 1.0 mM (m). V = pmol/g ww per 2 mitt; I = inhibitor concentration in mM. r = 0.90, 0.90, 0.95 and 0.89 for 0.2mM, 0.3mM, 0.5mM and l.OmM of “C-ghtcose respectively. Each point represents the mean of four determinations; vertical bars around the points show

standard deviations.

affected by the strain of the specimen, the age of the worms, their nutritional status and experimental con- ditions. Since Uglem et al. (1978) did not obtain any kinetic parameters for glucose uptake by H. diminuta simultaneously with /IMG for their batch of worms under exactly similar experimental conditions, such statement concerning their similar V,,, values may not be justifiable. Hence, allowance must be made for such differences in kinetic parameters in analysing data obtained by using methylglucosides as probes to study the glucose transport mechanism in H. diminuta in the future.

SUMMARY

Both aMG and BMG are competitive inhibitors of glucose uptake by H. diminuta with Ki values of 0.53 mM and 0.42 mM respectively, indicating that the latter isomer is indeed a more effective inhibitor of glucose transport in H. diminuta. However, glucose and aMG do not have similar kinetic parameters as suggested elsewhere. The I’,, value for aMG transport is approximately half that of glucose. This may be explained by the presence of a more bulky group in aMG at the carbon-l position instead of hydrogen as glucose.

Table I. Effects of different concentrations of OL- and p-methyl-o- glucoside on the absorption of 0.1 mM “C-glucose by H. diminura.

Da-& represent means *SD

Membrane transport in H. diminuta 89

Table 2. The kinetic parameters, obtained by different types of plots, for glucose and a-methyl-o- glucoside uptake by H. diminuru. K, and V,,,,, were calculated from lines obtained from the mean

values of 4+5 determinations at each of the substrate concentrations (0.1-5 mM) tested

Type of plots

Lineweaver-Burk plot Woolf-Augustinsson-Hofstee plot Direct linear plot

Glucose a-Methyl-o-glucoside

V pmol/Mww

V pmolrww

(mKh) per 2 min (mKh) per 2 min

0.66 5.81 0.68 2.50 0.77 6.25 0.73 3.23 0.70 5.65 0.72 2.48

REFERENCES

Brown P. D. and Sepulveda F. V. (1985) A rabbit jejunal isolated enterocyte preparation suitable for transport studies. J. Physiol. 363, 257-270.

Dixon M. (1953) The determination of enzyme inhibitor constant. Biochem. J. 55, 170-171.

Eisenthal R. and Cornish-Bowden A. (1974) The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters. Biochem. J. 139, 715-720.

Gruner S. and Mettrick D. F. (1984) The effect of 5-hydroxy- tryptamine on glucose absorption by Hymenolepis diminuta (Cestoda) and by the mucosa of the rat small intestine. Can. J. Zool. 62, 798-803.

Hofstee B. H. J., Dixon M. and Webb E. C. (1959) Non-inverted versus inverted plots in enzyme kinetics. Nature 184, 12961298.

Kimmich G. A. and Randles J. (1981) a-Methylglucoside satisfies only Na+-dependent transport system of intesti- nal epithelium. Am. J. Physiol. 241, C227-C232.

Kuramochi G. (1986) Effect of n-glucose on oxygen consumption of renal proximal tubules of the Triturus. Jpn. J. Physiol. 36, 287-293.

Lineweaver H. and Burk D. (1934) The determination of enzyme dissociation constants. J. Am. Chem. Sot. 56, 658-666.

Mulin J. M., Weibel J., Diamond L. and Kleinzeller A. (1980) Sugar transport in the LLC-PK, renal epithelial

cell line: Similarity to mammalian kidney and the influence of cell density. J. Cell Physiol. 104, 375-389.

Pappas P. W. and Read C. P. (1975) Parasitological Review: Membrane transport in helminth parasites: A review. Exp. Parasitol. 37, 469-530.

Planas J. M., Villa M. C., Ferrer R. and Moreto M. (1986) Hexose transport by chicken cecum during development. PJlugers Arch. 407, 216-220.

Podesta R. B. (1977) Hymenolepis diminuta: Marker distri- bution volumes of tissues and mucosal extracellular spaces. Exp. Parasitol. 42, 289-299.

Podesta R. B., Stallard H. E., Evans W. S., Lussier P. E., Jackobson D. J. and Mettrick D. F. (1977) Hymenolepis diminuta: Determination of unidirectional uptake rates for nonelectrolytes across the surface ‘epithelial’ mem- brane. Exp. Parasitol. 42, 300-317.

Segal S., Rosenhagen M. and Rea C. (1973) Developmental and other characteristics of a-methyl-n-ghtcoside trans- port by rat kidney cortex slices. Biochim. Biophys. Acfa. 291, 519-530.

Uglem G. L., Love R. D. and Eubank J. H. (1978) Hymenolepis diminuta: Membrane transport of glucose and /?-methyl-glucoside. Exp. Parasirol. 45, 88-92.

Webb R. A. (1985) The uptake and metabolism of S-hydroxy- tryptamine by tissue slices of the cestode Hymenolepis diminuta. Comp. Biochem. Physiol. 8OC, 305-312.