Mg2+-dependent ATPase activity in Hymenolepis diminuta mitochondria

veterinary parasitology Veterinary Parasitology 58 (1995) 17-26

E LS EV I E R

Ca2+/Mg 2+-dependent ATPase activity in Hymenolepis diminuta mitochondria J a v a i d H. Wani*, V.M.L. Srivastava

Division of Biochemistry, Central Drug Research Institute, Lucknow, India Accepted 18 May 1994

Abstract Ca 2+ and Mg2+ caused a concentration-dependent activation of ATP hydrolysis by mitochondrial membranes of Hymenolepis diminuta, a rat intestinal cestode. Ca 2+ was the more potent, but Mg2+ the more effective. The Lineweaver-Burk plot yielded Km and Vm~x values of 1.15 mM and 217.4 nmol Pi rain-~ rag-~ protein for Ca2+-dependent activity, and 1.86 mM and 333.3 nmol Pi min-~ rag-~ protein for Mg2+-dependent activity, respectively. Neither Na + nor K +, nor a combination of the two cations, induced the hydrolysis of ATP. Ouabain, a specific inhibitor of Na + / K + ATPase, did not affect the rate of ATP hydrolysis induced by Mgz+ alone or in combination with Na + or K +. The membrane-bound enzyme was not affected by neurarninidase and concanavalin A. C a 2+ and Mg2+ also induced appreciable hydrolysis of other nucleoside triphosphates by the membranes. Some known anthelmintics, e.g. niclosamide, praziquantel and mebendazole, had no effect on ATPase activities. In addition to other compounds including respiratory inhibitors and uncouplers of phosphorylation, ruthenium red, which blocks Ca 2+ influx into the cestode mitochondria, had no influence on the rate of ATP hydrolysis induced by the cations. Triton X-100 was found most suitable for solubilization of both activities. The differences between cestode ATPase and its mammalian counterpart have been discussed.

Keywords: ATPase activity; Hymenolepis diminuta; Mitochondria

1. Introduction N u m e r o u s reports have indicated that ATP hydrolysis by m a m m a l i a n ( A n a n d et al., 1971 ) or p r o t o z o a n (Cataldi de F l o m b a u m a n d Stoppani, 1982) mito* Corresponding author at: University of Pennsylvania, School of Medicine, 313 Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104, USA. Telephone: 215-573-3510. Fax: 215-898-9557. 0304-4017/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0304-4017 ( 94 ) 00700-4

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J.H. WanL V.M.L. Srivastava / Veterinary Parasitology 58 (1995) 17-26

chondria can be stimulated by Ca 2+ or Mg 2+. The Ca 2+- and Mg2÷-dependent ATPase, simply called Ca2+/Mg 2+ ATPase (E.C. 3.6. 1.3 ), has also been purified and characterized from mammals and protozoa. However, no such study seems to have been carried out on cestode mitochondria. After the observation of active transport of calcium (Wani et al., 1992) this work was carried out to ascertain the presence of C a 2 + / M g 2+ ATPase in cestode mitochondria. Here, we report some findings on the role of C a 2 + and M g 2+ in ATP hydrolysis by Hymenolepis diminuta mitochondrial membranes. The data indicate that a single ATPase is responsible for both ATP hydrolytic activities. In some cases these two activities can be due to two different enzymes (Anand-Srivastava and Dhalla, 1987). It should be noted that Ca2+/Mg 2+ ATPase activity examined in this study refers to ATP hydrolysis in presence of C a 2 + o r M g 2+ and should not be confused with the Ca2÷-stimulated MgZ+-dependent ATPase commonly seen in microsomes (Dhalla et al., 1981 ). The latter type of ATPase is activated by micromolar concentrations of C a 2 + and utilizes M g 2 + - A T P as substrate.

2. Materials and methods

2.1. Chemicals Ouabain, phospholipase-A2 and -C, neuraminidase, concanavalin A, nucleoside mono-, di- and triphosphates, ethyleneglycol tetraacetic acid (EGTA), ruthenium red, potassium cyanide, salicylhydroxamic acid (SHAM), phenylmethylsulphonyl fluoride (PMSF), cetylpyridinium chloride (CPC), guanidine HC1, rotenone, antimycin A and dithiothreitol (DTT) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were of analytical grade. The numbered compounds, which, in preliminary studies, have shown some anthelmintic properties, were the products of the Medicinal Chemistry Division of the Central Drug Research Institute, whereas other anthelmintics were obtained as gifts: niclosamide from Bayer, A.G. (Wuppertal, Germany), mebendazole from Cadila Laboratories (Ahmedabad, India) and praziquantel from E. Merck (Darmstadt, Germany).

2.2. Parasite and the isolation of mitochondria Experimental infection of rats with H. diminuta and the isolation of organelles from the parasite have been described elsewhere (Wani et al., 1992). The mitochondrial fraction was disrupted by five successive oscillations with 30 s cooling intervals using a sonicator. The purity of the mitochondrial preparation was checked by measuring the positive and the negative marker enzymes. The washed fraction was rich in cytochrome oxidase (McKelvey and Fioravanti, 1986) and fumarate reductase (McKelvey and Fioravanti, 1985) whereas the activities of glucose-6-phosphatase (Mithieux et al., 1990), lactate dehydrogenase (Kornberg, 1955 ) and 5'-nucleotidase (Gomori, 1957 ) were negligible. The mitochon-

J.H. Wani, V.M.L. Srivastava / Veterinary Parasitology 58 (1995) 17-26

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drial membranes were separated by sucrose density gradient centrifugation. Dialysis was carried out in 2 1 of Tris-HC1 buffer (50 mM, pH 7.5) which was changed five times with intervals of 2-3 h, and finally the process was completed overnight. Protein content was measured by Folin-phenol method (Lowry et al., 1951). 2.3. Cae+ /Mg 2+ ATPase activity The ATPase activity was assayed in a mixture of 1 ml containing Tris-HC1 (50 mM, pH 7.5), CaC12 or MgC12 (2 mM) and mitochondrial membrane preparation (1-2 mg). After 5 min of preincubation at 37°C, the reaction was initiated by adding ATP (disodium salt; 3 mM). After 10 min the reaction was terminated by introducing chilled trichloroacetic acid (TCA) to a final concentration of 5% (McNamara et al., 1974; Cataldi de Flombaum and Stoppani, 1982). The contents were centrifuged and orthophosphate liberated from ATP hydrolysis was determined in the supernatant (Sumner, 1944). Appropriate controls were performed to determine initial inorganic phosphate levels by the addition of TCA before the enzyme source. The possible presence of non-specific phosphatases was determined, and corrected for when necessary, by omitting the cation from the assay system. The activities are expressed as nanomoles of inorganic phosphate (Pi) released per minute per milligram of mitochondrial protein or as a percentage of the control value.

3. Results Neither Na + nor K +, nor a combination of the two cations, induced hydrolysis of ATP by the mitochondrial membrane preparation ofH. diminuta (Table 1 ). Mg2÷ followed by Ca 2+, however, appreciably enhanced hydrolysis. The two divalent cations when present together did not show any additive effect, and the addition of K + or Na ÷ to the reaction mixture containing Ca 2+ or Mg2+ as stimulator did not further increase the hydrolytic rate of ATP. Ouabain, a widely accepted specific inhibitor of Na + / K + ATPase, did not affect the rate of ATP hydrolysis induced by Mg2+ alone or in combination with Na ÷ and K ÷. At equimolar concentrations, Mg2÷ caused more hydrolysis than Ca 2+. Stimulation of ATP hydrolysis by Ca 2÷ or Mg2÷ exhibited a concentration-dependent response. Mg2+-dependent activation was, however, greater and reached saturation earlier than that caused by Ca 2÷. Both activities increased in linear fashion over time. In another series of experiments, phosphohydrolase activities were assayed at different substrate (ATP) concentrations, keeping the cation concentration constant at 2 raM. The Lineweaver-Burk plot of 1/ [ S ] vs. 1/v yielded Km and Vmax values of 1.15 mM and 217.4 nmol Pi min-~ nag-l protein for Ca2+-dependent activity, and 1.86 mM and 333.3 nmol Pi rain-t nag-l protein for Mg2+-dependent activity, respectively. Both activities were decreased by treatment of the en-

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J.H. WanL V.M.L. Srivastava VeterinaryParasitology 58 (1995)17-26

Table 1 Effect of various cations and their combinations on ATP hydrolysis by mitochondrial membranes of Hymenolepis diminuta Cation(s)

Concentration (mM)

ATPase activitya

Ca Mg K Ca+K Ca+Mg K+Mg K+Mg+Ca Na Na+Mg Na+K Na+K+Mg Na+ K + Mg+Ouabain

2 2 10 2,10 2,2 10,2 10,2,2 10 10,2 10,10 10,10,2 10,10,2,1

188.5_+ 8.1a 295.9_+ 9.5a 8.7_+0.6c 192.1 _+9.7a 301.8_+ 12.1a 299.3_+ 11.6a 288.0_+ 11.5a 12.5_+ 1.4c 287.9_+ 12.2a 13.3_+ 1.8c 289.3_+ 10.1a 280.0_+ 10.4a

aActivities are in nmol Pi released min-J mg-t protein and are expressed as means + S.D. of four observations. P value: a, P< 0.005; b, P< 0.05; c, P> 0.05. Table 2 Effect of some bioactive proteins on H. dirninuta mitochondrial membrane Ca2+/Mg 2+ ATPase Compound

Control Phospholipase A2 Phospholipase A2 Phospholipase C Phospholipase C Neuraminidase Concanavalin A

Concentration (m1-1 )

4U 8U 3U 6U 1U 0.2 mg

ATPase activity~

Ca2+-Dependent

Change (%)b

Mg2+-Dependent

Change (%)b

193.4_+ 9.7 135.2_+ 10.Sa 92.3_+4.6a 129.8_+ 8.2a 119.3-+ 9.7a 216.6_+8.9c 172.4_+ I0.9c

-30.1 -52.3 -32.9 -38.3 + 12.0 - 10.8

308.4_+13.3 201.9_+10.9a 163.8_+10.0a 212.4_+ll.4a 179.0-+9.8a 280.7_+12.7c 289.8+ 11.3c

-34.5 -46.9 -31.3 -42.0 -9.0 -6.0

~Activities are in nmol Pi released min - t mg - l protein and are expressed as means_+ S.D. of four observations. Plus and minus signs indicate increase and decrease in activity, respectively. bP value: a, P< 0.005; b, P< 0.05; c, P> 0.05. z y m e p r e p a r a t i o n w i t h p h o s p h o l i p a s e s A2 a n d C ( T a b l e 2 ) . N e u r a m i n i d a s e a n d c o n c a n a v a l i n A h a d n o s i g n i f i c a n t effect o n t h e e n z y m e . C a 2+ a n d M g 2+ i n d u c e d the hydrolysis of other nucleoside mono-, di- and triphosphates. GTP, UTP, and A D P w e r e h y d r o l y s e d t o a s u b s t a n t i a l level, w h e r e a s C T P , C D P a n d G D P w e r e hydrolysed to a lesser extent. Mononucleotides were found least susceptible (Tab l e 3 ). Several anthelmintics, including niclosamide, praziquantel, mebendazole, and C D R I c o m p o u n d s 8 2 / 4 3 7 , 8 7 / 6 3 9 , a n d 9 0 / 5 5 , h a d n o effect o n t h e A T P a s e .

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Table 3 Spectrum of Ca 2+- and Mg2+-induced hydrolysis of substrates by H. diminuta mitochondrial membranes Compound (3raM)

+ Ca 2+ -Dependent Activity~

+ Mg 2+-Dependent Relative

Activitya

Relative activity

335.3 _+10.7 192.8_+ 11.4 172.3 -+ 7.4 92.5 -+ 4.9 135.1 _+ 10.4 60.0_+ 3.6 34.2_+4.1 54.7_+4.9 34.2 _+4.4 7.4 _+0,5

100 57.5 51.4 27.6 40.3 17.9 10.2 16.3 10.2 2.2

activity ATP GTP UTP CTP ADP GDP CDP AMP GMP cAMP

183.9 ± 7.5 154.5+_ 10,8 138.5 + 6.6 28.3 ± 1.8 93.1 -+7.2 59.0±2.6 50.1 _+4.0 13.6_+0.5 2.2_+ 0.3 4.6 _+0.4

100 84.0 75.3 15.4 50.6 32.1 27.2 7.4 1.2 2.5

aActivities are in nmol Pi released m i n - ' mg-~ protein and are expressed as means_+ S.D. of four observations. Table 4 Effect of some compounds on Ca 2+/Mg 2+ ATPase ofH. dirninuta mitochondria a Compound added (raM)

Control EGTA (5) EDTA (5) Ruthenium red (0.1) KCN (0.5) K3Fe(CN)6 (0.5) SHAM (1) Ouabain ( 1 )

Ca 2+ -Dependent

Mg 2+-Dependent

Activity

Change

Activity

Change

196.6+9.1 38.5 + 1.8a 62.4+4.1a 227.2 + 9.8c 174.4+ 10.5c 336.6+ 11.1a 170.6_+ 10.8c 176.9 + 12.4c

- 80.4 -68.3 + 15.6 - 11.3 +71.2 -13.2 - 10.0

315.5+ 10.8 345.1 + 10.9c 97.1 + 5.2a 344.6+ 1 l.lc 335.4+ 10.7c 477.4+ 11.6a 291.5_+ 10.7c 287.4 _+9.2c

+ 9.4 -69.2 +9.2 +6.3 + 51.3 -7.6 - 8.9

(%)b

(%)b

aActivities are in nmol Pi released min-~ mg-1 protein and are expressed as means + S.D. of four observations. Plus and minus signs indicate increase and decrease in the activity, respectively. bp values; a, P < 0.005; b, P < 0.05; c, P > 0.05.

EGTA specifically blocked Ca 2 +-stimulated hydrolysis, whereas EDTA inhibited both the activities (Table 4). Potassium ferricyanide stimulated both the activities, whereas other compounds, namely ouabain, DTT, and ruthenium red, had no significant effect. Similarly, antimycin A, sodium azide, rotenone, potassium cyanide, potassium thiocyanide, and SHAM had no significant effect on the enzyme activities. The detergents CPC, deoxycholate, digitonin, guanidine HCI, saponin, sodium dodecyl sulphate (SDS), Triton X-100 and urea had inhibitory effects of varying degrees on both the activities (Table 5 ). The inhibition of Ca2+-

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J.H. WanL V.M.L. Srivastava / Veterinary Parasitology 58 (1995) 17-26

Table 5 Effect of some detergents on Ca 2÷ / M g 2+ ATPase o f H. diminuta mitochondriaa Detergent

Concentration

None CPC Deoxycholate Digitonin Guanidine HCI Saponin SDS Triton X-100 Urea

0.1% 0.1% 0.1% 3M 0.1% 0.1% 0.2% 8M

Ca 2+-Dependent

Mg2÷-Dependent

Activity

Inhibition

Activity

Inhibition

194.3_+9.3 81.8_+4.5a 90.0 _+7.6a 125.2 + 9.4a 47.7 -+ 3.8a 158.9-+ 7.3c 78.0_+4.9a 165.1 -+ 10.8c 44.5 +4.8a

57.9 53.7 35.6 75.5 18.2 59.9 15.0 77.1

310.1_+10.7 132.8_+ 6.6a 142.2 _+7.8a 214.5-+ 12.2a 50.9 -+ 6.8a 257.2_+ 12.7c 105.4_+ 7.1a 38.5-+ 4.9a 54.3-+ 3.8a

57.2 54.1 30.8 83.6 17.1 66.0 87.6 82.5

(%)

(%)

"The activities are in nmol Pi released min-~ m g - l protein and are expressed as _+S.D. of three observations. P values: a, P < 0.005; b, P < 0.05; c, P > 0.05. Table 6 Effect of detergent treatment on the submitochondrial distribution of Ca 2÷/Mg 2÷ ATPase of H. diminuta Detergent

Concentration (%)

Effect on activity (%)" Mg2+-Dependent

Ca 2+-Dependent

Sediment Undialysed Dialysed Sediment Undialysed Dialysed supernate superuate supernate supernate CPC Deoxycholate Saponin SDS Triton X-100

0.1 0.1 0.1 0.1 0.2

-99.5a - 1.9c + 10.7b -88.9a +7.4c

-73.9a + 121.Ta -8.7c -82.6a + 169.6a

+68.4a + 100.3a - 105.0a +415.8a +484.2a

-92.9a - 1.9c -7.2c -82.8a -19.1b

- 35.5b + 232.2a -21.6b -23.2b +983.9a

+ ll2.4a + 565.6a - 85.2a + 1207.3a + 1525.9a

Disrupted mitochondrial preparation ( 1 ml) was incubated with detergent solution (0.2 ml ) for 5 h at 37 ° C. After incubation, the volume was made to 5.1 ml with Tris-HC1 (50 raM, pH 7.5 ) and centrifuged for I h at 105 000 g. Sediment obtained was washed, and the supernatant (5.0 ml ) was divided into two parts: one assayed as such and the other assayed after dialysis. ~The percentage data have been derived from mean activities _+S.D. of four observations (data not shown). P value: a, P < 0.005; b, P < 0.05; c, P > 0.05.

dependent hydrolysis by Triton X-100 and of both the activities by saponin was negligible. However, when detergent treatment was followed by separation into sediment and soluble fractions (sediment was washed whereas some portion of soluble fraction was dialysed to remove the detergent), it was observed that Triton X-100 solubilized both activities most effectively, resulting in a 5-15-fold increase in activities in the soluble fraction (Table 6). It should be noted that the presence of a detergent greatly changed ATPase

J.H. Wahl, V.M.L. Srivastava / Veterinary Parasitology 58 (1995) 17-26

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activities. For example, CPC and SDS strongly inhibited the activities. Saponin failed to solubilize the enzyme, and its removal from the supernatant by dialysis resulted in a tremendous decrease in both the activities (Table 6). Removal of deoxycholate by dialysis did not change C a 2 + - d e p e n d e n t activity in the supernatant whereas Mg 2+-dependent activity was increased. Nonetheless, Triton X- 100 effectively solubilized the enzyme, and its removal by dialysis increased the above activities by approximately 2.9- and 1.6-fold, respectively.

4. Discussion

Like inorganic pyrophosphatase, the ATPase activities are also universally distributed in helminth parasites (Barrett, 1981 ). ATP hydrolysis by the mitochondrial membranes of H. d i m i n u t a is markedly induced by both C a 2+ and Mg 2+. Surprisingly, stimulation caused by the combination of both the cations is nearly equal to that by Mg 2+ alone, indicating the presence of one enzyme with two kinds of ATPase activities: one dependent o n C a 2+ and the other on Mg 2+. The former activity is strongly inhibited by the specific C a 2+ chelator EGTA, whereas both the activities are blocked by EDTA, a general chelator of divalent cations. The results indicate that the dependence of the ATPase o n Ca 2+ or Mg 2+ is exclusive, i.e. not interdependent. C a 2+- and Mg2+-dependent activities are kinetically different; the former has lower Km and Vmaxvalues. Thus, the extent of stimulation of ATP hydrolysis by the cations and the maximum rates at which they induce ATP hydrolysis are consistent. Although M g 2+ is the more potent stimulator, the enzyme has a lower affinity for this cation than for Ca 2+. Saponin appears to be the detergent of choice for the solubilization of both the activities whereas Triton X-100 is suitable for Ca2+-dcpendent activity only. However, when the detergent treatment is followed by the separation of the soluble fraction and its dialysis, Triton X- 100 is most suitable for the purpose of solubilization of both the activities (Table 6). This detergent seems to be membrane specific with regard to its solubilizing capacity. It inactivates both C a 2 +- and Mg 2+-ATPases of rat heart plasma membrane (Zhao and Dhalla, 1991 ), whereas it has successfully been employed for the purification of C a 2 + / M g 2+ ATPase of rat heart sarcolemmal membranes (Tuana and Dhalla, 1988 ). C a 2 + / M g 2+ ATPase of the cestode mitochondria differs from that of its mammalian counterpart in its response to certain inhibitors. For example, sodium azide does not affect either of the two activities of H. d i m inuta mitochondria but it significantly inhibits the enzyme in rat heart mitochondria. Similarly, ruthenium red, which has no effect on the cestode ATPase, inhibits the mammalian enzyme (Anand et al., 1971 ). That C a 2 + / M g 2+ ATPase ofH. d i m i n u t a mitochondria is different from microsomal Ca2+-stimulated Mg2+-dependent ATPase is suggested by the observed stimulation of hydrolysis by the cations individually and in combination (Table 1 ). If the latter type of activity had been present in the cestode mitochondria, the addition of C a 2 + to a M g 2 +-containing reaction mixture would have effected an elevation in ATP hydrolysis compared

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J.H. Wani, V.M.L. Srivastava / VeterinaryParasitology58 (1995) 17-26

with that by Mg 2+ alone. However, no such elevation was observed in the present study. Intestinal epithelial cells of Ascaris s u u m possess a number of phosphatases, including Mg2+-ATPase (Borgers and Van den Bossche, 1972 ). In the case of rat heart sarcolemma, Ca 2+ ATPase activity has been reported to be higher than M g 2+ ATPase, whereas stimulation by both cations was identical in mitochondria and microsomes (Anand et al., 1971 ). As neuraminidase had no effect, it is evident that sialic acid moieties either are not present in the ATPase molecule or, if present, they are not required for the maintenance and/or the regulation of the enzyme activities. DTT, which has no effect on cestode Ca 2+/Mg 2÷ ATPase, has been found to reverse inhibition of soluble beef heart mitochondrial ATPase (Mg 2+ ATPase ) by 7-chloro-4-nitrobenzo-2-oxa- 1,3-diazole (NBD-C1), which is believed to cause the inactivation by modifying certain amino acid residues at the active site (Ferguson et al., 1974). Ca 2 + / M g 2+ ATPase is known to be involved in a number of functions in various organelles. The ATPase in the transverse tubules of the rabbit skeletal muscle is considered to be responsible for ATP-dependent calcium transport (Hidalgo et al., 1983). In contrast, in rat heart sarcolemma, the enzyme may be involved in the stimulus-induced C a 2+ influx into heart cells (Dhalla et al., 1977) and the regulation of extracellular ATP concentration (Tuana and Dhalla, 1988 ). Some ATPases are involved in the energy generation process in mammalian system (Hatefi et al., 1975; Yang and Lacuara, 1978; Frisell, 1982). An earlier notion that mitochondrial Ca 2 + transport is meant for the buffering of cytosolic Ca 2+ levels is less accepted, and it is now believed the cation transport in the organelle is primarily meant for maintaining its own ion homeostasis and the regulation of mitochondrial enzymes such as dehydrogenases (Denton et al., 1978; McCormack and Denton, 1979). In general, helminth ATPase activities are lower than mammalian ones. For example, ATPase activity of Ascaris lumbricoides myosin is three to four times lower than that of rabbit myosin, despite the fact that myosin in rabbit and in the parasite have identical function, chain composition, molecular weight, and shape (Nakamura et al., 1975). Because, in actomyosin preparation, A. lumbricoides myosin contracts at lower ATP levels than mammalian myosin (Yamaguchi et al., 1973), it has been suggested that this is an adaptation to the relatively low ATP production in parasitic helminths. Thus A. lumbricoides eggs, which are aerobic in their mode of energy generation, have an ATPase similar to that of mammalian mitochondria (Barrett, 1981 ). Thus, it is concluded that H. d i m i n u t a mitochondria, like their mammalian counterparts, possess an ATPase with Ca 2+- and Mg2+-dependent activities. Comparison between the observations from this study and those reported earlier for the corresponding mammalian system indicates that there are similarities as well as dissimilarities between the enzymes of the two sources.

Acknowledgements J.H.W. is grateful to the University Grants Commission, New Delhi, for financial assistance. The authors thank Miriam Shapiro for typing the manuscript.

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