erythrocyte membrane-bound enzymes in

Aust. J. Exp. Biot. Med. Sci., 62 (Pt. 2) 137-143 (1984)

©ERYTHROCYTE MEMBRANE-BOUND ENZYMES IN MASTOMYS NATALENSIS DURING PLASMODIUM BERGHEI INFECTION by S. KHARE, J. K. SAXENA, A. B. SEN* AND S. GHATAK (From the Divisions of Biochemistry and *Parasitology, Central Drug Research Institute, Lucknow-226001, India.) (Accepted for publieation October 21, 1983.) Summary, The pattern of activity of certain membrane-associated enzymes was followed in the erythrocytes of Plasmodium berghei-iniected Mastomys natalensis. Parasitized erythrocytes were separated from non-parasitized populations by percoll-density gradient centrifugation. The activity of adenylate eyclase was markedly increased while those of ATPase, acid phosphatase, ;8-glucuronidase and N-acetyl-/3-D-glucosaminidase were considerably decreased in the membrane preparations of parasitized erythrocytes as compared to normal erythrocytes. There was a decrease in the activity of ATPase and an increase of adenylate eyclase in the membrane preparations of non-parasitized erythrocytes. However, other enzymes did not alter to a significant extent in non-parasitized erythrocytes. Chloroquine (in vitro) stimulated adenylate eyclase, Na* K*-ATPase and Ca**Mg+*-ATPase while acetylcholinesterase was significantly inhibited.

INTRODUCTION Numerous scanning and transmission electron microscopic studies of the fine structure of erythrocytes harbouring malarial parasites have revealed various morphological changes suggestive of metabolic alterations induced by the parasite. These include knob-like protrusions at the surface of Plasmodium falciparum and P. coatneyi parasitized erythrocytes (Kilejian, Abati and Trager, 1977) and pit-like depressions in P. berghei parasitized erythrocytes (Kreier et al., 1972). The spherical shape of parasitized erythrocytes could suggest osmotic transformations caused by changes of electrolyte transport (Seed and Kreier, 1972). Severe alterations in erythrocyte membrane proteins, phospholipids and cholesterol (Wcidekamm et al., 1973; Howard and Sawyer, 1980) and changes in erythrocyte membrane fluidity (Howard and Sawyer, 1980) and increased permeability to essential nutrients (Sherman, 1977; Ncame and Homewood, 1975) have been reported during the course of a malarial infection. Non-parasitized red cells from infected animals also Abbreviations u.?ed in this paper: ATPase, adenosine triphosphatase; PBS, Phosphatebuffered saline; Cyclic AMP, Cyclic adenosine monophosphate; rev/min, revolutions per minute.

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exhibited dramatic changes in membrane structure and osmotic fragility when compared to normal erythrocytes of uninfected animals (Gupta et al, 1982). Since membrane-bound enzymes are known to regulate the erythrocyte shape, membrane fluidity and transport of ions, such alterations in erythrocyte membrane structure and function point towards the need for the study of membrane-associated enzymes during the course of malarial infection. The present communication reports the activities of various enzymes in membrane preparations of normal, non-parasitized and parasitized erythrocytes of P. berghei-intected Mastomys natalensis, which has been reported to be a better experimental model for most of the biochemical studies in malaria (Saxena et al, 1983). Further, in vitro effect of chloroquine on the activities of these enzymes has also been investigated. MATERIALS AND METHODS Animals and infection: Eight-weeks-old male M. natalensis (GRA Giessen Strain) were infected with the strain of P. berghei berghei as reported earlier (Khare et al, 1982a). Membrane preparations: Blood was drawn from retro-orbital plexuses of normal and P. berghei-iniected mastomys (45-50% parasitaemia) in heparin and centrifuged to remove plasma and buffy coat. Erythrocytes were washed 4-6 times in phosphate-buffered saline (PBS) (5 mM phosphate buffer—015 M NaCl, pH 8 0). Each wash consisted of resuspending the erythrocytes in 20 volumes of the medium and centrifugation with removal of the supernatant fluid. Parasitized and non-parasitized red blood cells were separated using percoll density gradient centrifugation according to Tosta et al. (1980). After separation, parasitized erythrocytes were lysed and membranes were prepared essentially according to Konigk and Mirtsch (1977). The cells (1 ml) were lysed by mixing rapidly in ice-chilled 5 mMphosphate buffer, pH 8 0 (40 ml). After 30 min at 4° the samples were centrifuged at 7,000 g (15 min at 4°) to remove the pellet containing parasites. Finally, the pellet containing membranes was obtained by centrifugation at 30,000 g (30 min at 4°). Preparations were washed 4-6 times until all the haemoglobin was removed. Membranes prepared in this way were completely free of parasites as observed under phase contrast microscopy. The membrane preparations were homogenized using a glass Potter Elvehjem homogeniser at 800 rev/min., 20 strokes, and the protein concentration was adjusted to about 2 mg/ml with the appropriate buffer. Normal and non-parasitized erythrocytes were lysed similarly after separation by percoll density gradient centrifugation. Enzyme assays: Adenylate eyclase (EC 4.6.1.1) and cyclic AMP phosphodiesterase (EC 3.1.4.17) were determined according to Albano et al (1973) and Butcher and Sutherland (1962), respectively. Acid phosphalase (EC 3.1.3.2) and /3-glucuronidase (EC 3.2.1.31) were determined essentially according to Wootan (1964) and Dodgson, Lewis and Spencer (1953), respectively, while /3-gIucosidase (EC 3.2.1.21), /3-galactosidase (EC 3.2.1.23) and N-acetyI-/3-D-glucosaminidase (EC 3.2.1.30) were analysed according to Beck and Tappel (1968). Acetylcholinesterase (EC 3.1.1.7) and ATPase (EC 3.6.1.3) were measured according to Ellman et al. (1961) and Hanahan and Ekholm (1978) respectively. Protein content of the enzyme preparations was estimated according to Lowry et al. (1951). Specific activity was expressed as units/mg protein. Enzyme units are expressed as nmol of product formed/min for acetylcholinesterase, Na*K*-ATPase, Ca**Mg**-ATPase and acid phosphatase and pmol of product formed/min for N-ace(yl-;9-D-glucosaminidase and ,8glucuronidase. Enzyme units of adenylate eyclase and cyclic AMP phosphodiesterase are expressed as pmol cyclic AMP formed/10 min and pmol cyclic AMP hydrolyzed/10 min, respectively.

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In vitro effect of chloroquine on membrane-bound enzymes: The enzyme in the reaction mixture (devoid of substrate) was preincubated for 5 min with varying concentrations of chloroquine and the activity was measured in the usual manner after addition of the specific substrate.

RESULTS AND DISCUSSION ATPase and acetylcholinesterase It would be seen from Table 1 that the activity of Na+K+-ATPase and Ca+-Mg+-'-ATPase decreased considerably in the membrane preparations of parasitized erythrocytes as compared to normal erythrocytes. The activity of acetylcholinesterase was slightly increased in parasitized erythrocytes when compared to normal red cells; however, the increase was not statistically significant. The activity of acetylcholinesterase did not show any alteration in the membrane preparations of non-parasitized erythrocytes, while a significant decrease in the activities of Na+K+-ATPase and Ca+-Mg+'--ATPase was observed in non-parasitized erythrocytes when compared to normal ones. TABLE 1 Effect of P. berghei infection on the specific activities of erythrocyte membrane-bound acetylcholinesterase, ATPase, adenylate eyclase and cAMP-phosphodiesterase. c " Acetylcholinesterase Na*K*-ATPase Ca*-Mg*2.ATPase Adenylate eyclase Cyclic AMP-phosphodiesterase

Normal Non-parasitized erythrocytes erythrocytes 7 2 1 ± 8-7 75-1 ± 6-9 (NC) 10-7 ± 1-5 5 1 ± 0 9 (—52%) 18-6 ± 2 9 12 8 ± 1 3 (—31%) 37-7 ± 4-4 50 3 ± 5-9 (-1-33%)

Parasitized erythrocytes 9 0 1 ± 7-3 (-1-25%) 3 7 ± 0-6 ( - 6 5 % ) 9 0 ± 1 0 (—52%) 87 0 ± 10 0(-|-130%)

60 0 ± 1 0 7 58 3 ± 1 0 1 ( N C )

62 9 ± I 3 7(NC)

Results represent mean ± SD of four determinations in triplicate. Figures in the parenthesis denotes % change when compared to normal. NC = No change; '-f-' = increase; '—' = decrease.

The enzyme Na+K+-ATPase is responsible for the maintenance of low Na+ and high K+ concentration within the cell by an active transport of sodium from inside to outside the cell while Ca+-Mg+-'-ATPase maintains low Ca+- concentration inside the cell by transporting calcium from inside to outside the cell concomitantly with the hydrolysis of ATP (Hinds, Raess and Vincenzi, 1981). Earlier reports have shown that during the course of P. falciparum, P. knowlesi and P. berghei infections the concentrations of Na+ and Ca+- were elevated while that of K+ was depressed considerably in both parasitized and non-parasitized erythrocytes (Boehm and Dunn, 1970; Gupta e? al, 1982). According to Seed and Kreier (1972), this imbalance in ionic concentration during malarial infection was responsible for the increase in volume and osmotic fragility of erythrocytes. In the present study the observed decline in the activities of Na+K+-ATPase and Ca+-Mg+-ATPase could account for increased concentrations of Na+ and Ca++ during malarial infection. However, the cause of inhibition of ATPase during P.

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berghei infection was not clearly understood. Alterations in lipid fluidity of the membrane have also been found to affect the activities of ATPase and acetylcholinesterase, e.g., an increase in lipid fluidity was accompanied by a parallel decrease in the activity of ATPase and increase in the acetylcholinesterase (Bloj et al, 1973). During the course of malarial infection increased erythrocyte membrane lipid fluidity (Howard and Sawyer, 1980) probably could be responsible for tiie depressed activities of Na+K+-ATPase and Ca+Mg+-'-ATPase and enhanced activity of acetylcholinesterase as observed in the present study. Adenylate eyclase and cyclic AMP phosphodiesterase The activity of adenylate eyclase, an enzyme responsible for the synthesis of cyclic AMP, was increased considerably in the membrane preparations of parasitized and non-parasitized erythrocytes as compared to normal red cells (Table 1). However, infection did not produce any significant change in the activity of cyclic AMP phosphodiesterase, an enzyme responsible for the degradation of cyclic AMP (Table 1). The observed increase in the activity of adenylate eyclase could account for elevated levels of cyclic AMP in parasitized and non-parasitized erythrocytes during a malarial infection (Khare et al, 1982b; Khare et al, unpublished data). The mechanisms responsible for increasing cyclic AMP levels remain to be elucidated and the function of cyclic AMP in the erythrocyte-parasite relationship is still not clear. According to Hertelendy, Toth and Fitch (1979), increased production of cyclic AMP could represent the communication mechanism and regulatory interactions that exist between the parasite and host erythrocyte. However, the cause of increased activity of adenylate eyclase was not clearly understood. It might be due to an activating factor that contributes to host enzyme activity as observed in the case of cholera and other infections (Vine and Cuatrecasas, 1981; Gill, Evans and Evans, 1976; Field, 1974). P-Glucosidase, p-galactosidase, P-glucuronidase, N-acetyl-P-Dglucosaminidase and acid phosphatase It is clear from Table 2 that during P. berghei infection the activities of acid phosphatase, /J-glucuronidase and N-acetyl-;8-D-glucosaminidase were TABLE 2 Effect of P. berghei infection on the specific activities of erythrocyte membrane-hound acid phosphatase, N-acetyl-fi-D-glucosaminidase and ji-glucuroiiidase.

Acid phosphatase N-acetyl-/3-Dglucosaminidase /3-glucuronidase

Normal erythrocytes 577± 58

Non-parasitized erythrocytes 55 9 ± 6 7 (NC)

Parasitized erythrocytes 28- 9 ± 4 3 (—50%)

8 9 4 ± 11 73 55 ± 10 9

8 0 ± 1 9 (NC) 76 9 ± 9 3 (NC)

3- 2 ± 0 5 ( - 6 4 % ) 50- 7 ± 5 9 ( - 3 1 % )

Results represent mean ± SD of four determinations in triplicate. Figures in the parenthesis denotes % change when compared to normal. NC = No change; ' - ' = decrease.

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considerably decreased in the membrane preparations of parasitized erythrocytes when compared to normal red cells. However, the level of these enzymes did not alter to a significant extent in the membrane preparations of nonparasitized erythroeytes. In spite of repeated attempts, the activity of j8-glucosidase and /8-galactosidase could not be detected. Hence, it is not possible to eomment on the later two membrane-bound enzymes during parasitization with P. berghei. Recently, Schrier (1977) has reported the presence of various hydrolytic enzymes in erythrocyte membrane preparations. Hydrolytic enzymes play an important role in the digestion and elimination of infective organisms and also in the processing of antigens. The observed decline in the activities of these enzymes during P. berghei infection could possibly be due to increased consumption in response to the multiplying organisms or to their inactivation by the parasite itself. In vitro effect of chloroquine on the activities of membrane-bound enzymes of parasitized erythrocytes In vitro incubation of the membrane preparations of parasitized erythroeytes with chloroquine significantly affected the activities of adenylate eyclase, Na+K+-ATPase, Ca+-Mg+--ATPase and acetylcholinesterase. Adenylate eyclase, Na+K+-ATPase, Ca+-Mg+-ATPase were stimulated 96%, 100% and 8 3 % , respectively, by as low as 0 1 mM chloroquine. Acetyleholinesterase activity was, however, inhibited by the drug exhibiting 78% deerease at 0 1 mM chloroquine (Table 3). . Activation of adenylate eyclase by chloroquine would support our earlier findings where chloroquine treatment Jias been shown to enhance significantly the intra-erythrocytic concentration of cyclic AMP (Khare et al, 1982b; Khare et al, unpublished data). The results of the present study clearly TABLE 3 In vitro effect of chloroquine on the activities of various membrane-bound enzymes of parasitized erythrocytes. Chloroquine concentration % Effect Enzymes a^:hSs* (mM) 87.0 180.1 170.5 37 Na*K*-ATPase 95 7.4 90 Ca*2Mg-'-ATPase 18.0 16.4 90.1 Acetylcholinesterase 19.8 45.0 47.7 * Specific activities—as described in Materials and Methods. ' + ' = activation '—' = inhibition

Adenylate eyclase

0 1 0.1 0 1 01 0 1 01 0 01 005 0025

+ 107 + 157 + 100 + 100 + 83 —78 —50 -47

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demonstrate that enhanced activity of adenylate eyclase in the presence of chloroquine was responsible for the increased production of cyclic AMP. Sherman and Tanigoshi (1972) have shown that incubation of erythrocytes with chloroquine significantly depressed the level of A T P inside erythrocytes. The observed elevation in the activities of adenylate eyclase, Na+K"'ATPase and Ca+-Mg+--ATPase in the membrane preparations of parasitized erythrocytes in the presence of chloroquine may indicate active utilization of ATP, thus providing an explanation for the decreased concentration of ATP in the presence of chloroquine. These studies in general demonstrate the changes in the activities of various erythrocyte membrane-bound enzymes during the course of malarial infection. Some of these alterations undoubtedly reflect damage to the membrane, but some of them may be helpful in facilitating the survival of the intracellular parasites. Further, proper elucidation and characterization of membrane-bound enzymes may provide knowledge of the intaetness and integrity of the membrane during parasitization. Acknowledgements. Authors are grateful to Dr. Nitya Nand, F.N.A., Director, CDRI for his keen interest and encouragement. The award of a senior research fellowship to S. Khare by the Ministry of Health, Gover.nment of India, is gratefully acknowledged.

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