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Presence and properties of cAMP phosphodiesterase from promastigote forms of Leishmania tropica and Leishmania donovani
Comp. Biochem. Physiol. Vol. 93B, No. 4, pp. 789-792, 1989 Printed in Great Britain
0305-0491/89 $3.00+ 0.00 © 1989PergamonPress plc
PRESENCE AND PROPERTIES OF cAMP PHOSPHODIESTERASE FROM PROMASTIGOTE FORMS OF L E I S H M A N I A TROPICA AND L E I S H M A N I A DO NO V A N I KUSAI A. K. AL-CHALABI, LAMIAA. ZIZ and BAIDAAL-KHAYAT Department of Biology, College of Science, University of Mosul, Mosul, Iraq (Received 1 December 1988) Abstract--1. This paper reports the isolation and the partial purification of cAMP phosphodiesterase (EC 3.1.4.17) from the promastigote form of Leishmania tropica and a preliminary result from Leishmania donovani. 2. The activity of the fraction obtained from column chromatography was measured. 3. The effects of pH, temperature, time of incubation and various compounds on its activity in vitro were obtained. 4. Two peaks (I and II) exhibiting cyclic nucleotide phosphodiesterase activity were obtained. 5. Both activities were found to require the addition of Mg 2÷ ions for full effect. 6. The apparent Km values for the first and second peaks were 1.43 x 10-3M and 4.1 × 10-3M respectively. L. donovani shows only one peak of activity.
tropica and a preliminary result from the promastigote form of Leishmania donovani.
INTRODUCTION
3',5' c A M P phosphodiesterase (PDE; EC 3.1.4.17) hydrolyses the Y,5'-cyclic nucleotide specifically to the Y-phosphate product AMP. This enzyme is important in the regulation of the intracellular concentration of cAMP, a key intermediate in the glycogenolytic action of glucagon and epinephrine in liver, this cyclic nucleotide has been implicated as the mediator of a n u m b e r of different polypeptide hormones and biogenic amine actions in a variety of different mammalia tissues (Robinson et al., 1968). In other phyla, cAMP has been shown to be a regulatory factor associated with the control of a variety of important biological events which influence the nutritional, survival, growth, development, and/or differentiation of certain organisms (Robinson et al., 1968; Voichick et al., 1973). In the case of the trypanosomatids some evidence is available that cAMP regulates processes of cell differentiation and transformation (Walter et al., 1978; Mancini and Patton, 1981; Rangel et al., 1987) and two enzymes, adenylate cyclase and cyclic A M P phosphodiesterase, which are involved in the synthesis and degradation, respectively, of cAMP have been demonstrated in Trypanosoma cruzi (Zingales et al., 1979; Gonsalves et al., 1980), Trypanosoma gambiense (Walter et aL, 1974; Walter, 1974) and from Trypanosoma brucei (Walter and Opperdoes, 1982). A correlation of the concentration of cyclic A M P in Leishmania with proliferation of promastigotes and the transformation of amastigotes to promastigotes has been described (Walter et al., 1978; Walter, 1981). The present paper reported the occurrence and partial purification of cyclic nucleotide phosphodiesterase from the promastigote form of Leishmania
MATERIALS AND METHODS
Organisms L. tropica (MHDM/IQ) and L. donovani (MCAN, IQ) used throughout this work were cultured promastigote forms which have been obtained from the Department of Parasitology and Medical Microbioligy, the Regional Training Center for Malaria and Medical Insect Control, Ministry of Health, Baghdad, Iraq. The organisms were cultured on NNN medium base with Locke's solution overlay in 3.5 × 1 in/test tubes. To obtain sufficient quantity of parasites for enzymatic study, promastigotes were grown in two media, modified Tobie's medium (Tobie et al., 1950). Parasites were harvested 5-6 days after subculture (i.e. during the log-phase of growth) by centrifugation at 1000g for 15 rain at room temperature and resuspended to a concentration of 1 × 109/ml in 50 mM Tris-HCl buffer at pH 7.9, and then sonicated for 30 s in a MSE ultrasonic disintegrater at a setting of 10 at 4°C. Isolation and enzymatic assay of cAMP phosphodiesterase The homogenate was centrifuged at 20,000 g for 30 min, and the supernatant fraction was used as the first step purified form of crude homogenate. The second step of purification is the ammonium sulfate fractionation which is based upon the degree of saturation (Butcher, 1974). The supernatant fraction in the previous step was brought to 0-50% saturation with ammonium sulphate. The precipitate was recovered by centrifugation at 16,000g for 20min, resuspended in 50mM Tris-HCl, pH 7.9 and dialysed overnight in large volume of 50 mM Tris-HC1 buffer (pH 7.9) at 4°C with continuous stirring. The dialysed enzyme was applied to a ultragel-34 column (30 x 2.6 cm) previously equilibrated with 50 mM Tris-HCl at pH 7.9. Fractions of 10 ml were collected and the enzyme activity and protein content were estimated after each step of purification. 789
KUSAI A. K. AL-CHALAB!et al.
790
Table 1. Purification of Leishmania tropica promastigote cyclic AMP phosphodiesterase Total Specific Total protein activity Purification Fraction units (mg) (units/mg) fold Crude homogenate 5100 182.00 28.00 1.00 20,000 g supernatant 3200 95.00 33.70 1.20 Ammonium sulfate precipitate 1700 34.00 50.00 1.79 (0-50% saturation) Dialysed fraction 1500 19.48 77.00 2.75 Gel filtration Peak I 100.90 0.97 103.00 3.68 Peak II 100.07 0.30 334.00 11.93 The activity of phosphodiesterase was assayed essentially as described by Butcher and Sutherland (1962). The procedures consisted in two stages of incubation each for 30 min. The phosphodiesterase reaction was followed by the 5'-nucleotidase reaction in the 2nd stage using an excess amount of snake venom plus MgSO4, and released Pi was determined colorimetrically by the method of Fisk and Subbarow (1925). The standard assay for cyclic-AMP phosphodiesterase contained in a final volume of 0.5 ml: 1.2 mM cAMP 12 mM MgSO4, 100 #g snake venom (Sigma) 2 mM imidazole, 50 mM Tris-HC1 (pH 7.9) and enzyme protein. Control tubes containing buffer instead of the substrate cAMP. The test and control tubes were incubated at 37°C for 60 min. The reaction was then stopped by the addition of 0.1 ml of 55% (w/v) trichloroacetic acid. The denatured protein was removed by centrifugation at 6000 g for 15 min. The Pi content of the supernatant was measured. One unit of activity was defined as the amount of enzyme releasing 1 nmol of inorganic phosphate per rain. Protein concentrations were determined by the method of Lowry et al. (1951), using bovine serum albumin as the standard.
RESULTS AND DISCUSSION Following the disruption of promastigote forms of L. tropica and L. donovani, the crude homogenate was centrifuged and the supernatant was subjected to fractional precipitation with solid a m m o n i u m sulfate at 0 - 5 0 % saturation and then dialyzed. Samples of dialyzed preparations were applied to a column packed with ultragel-34. Fractions were then collected and the activity of the enzyme was determined. The results of difference in enzyme activity due to a m m o n i u m sulphate fractionation and dialysis for promastigote forms of L. tropica are shown in Table 1. Table 1 also shows two active peaks of phosphodiesterase obtained when a dialyzed preparation of L. tropica was applied to a column packed with ultragel-34. Peak II was the dominant component and usually had a specific activity higher than the first peak (Table 1). The presence of more than one cyclic nucleotide phosphodiesterase has been suggested by many workers, two enzymes with different properties exist in rat brain (Kakiuchi et al., 1971) four forms in serum of healthy persons (Juergen and Ogilvie, 1987) Thermononospora curvata (Luann et aL, 1987) and two forms in Rhizobium species (Ong and Lim, 1987) and one type phosphodiesterase in Trypanosoma cruzi (Gonsalves et aL, 1980). In the case of L. donovani, a very low nucleotide phosphodiesterase activity was obtained (8 units/mg protein) when compared with L. tropica and only one peak seen.
Comparative studies were carried out on the enzymatic properties of freshly isolated fractions I and II from L. tropica promastigote.
Effect o f pH, temperature and time on enzyme activity The influence of pH, temperature and time upon the enzyme activity in both peaks were investigated. The optimum temperature for the assay of the activity in L. tropica (grown at 26°C) was 38°C and the optimum pH was 7.9, A value of p H 7.5-8.0 has been reported for the enzyme from most tissues and organisms. The rate of the reaction was linear with respect to both time (for at least 60 min) and to the amount of enzyme added (usually 200-250 # g protein).
K m values Reciprocal plots of reaction velocity versus c A M P concentrations were linear for the activities in both peaks (Fig. 1). The apparent K~ values derived from these plots were 1.43 x 10 -3 M and 4.1 x 1 0 - 3 M for the first and second peak respectively. The results show kinetic evidence for the existence of two enzymes as in other systems (Kakiuchi et aL, 1971; Ong and Lim, 1987) and in contrast to the results obtained with Trypanosoma cruzi (Gonsalves et al., 1980).
t /.
3.0 2.5 ~_ 2.0
//
+/
io~ ~ O.
PeakI ,
,
I
2
I/S (mMI 0.14
+,
0.12 x.
/
OAO
+ / 4-
0.08 006 / 0,04 -/+/
Peak]I
O,CrZ
/
hI I/.9 (raM) Fig. 1
h
2
791
Leishmania cAMP phosphodiesterase Table 2. Sensitivity of cAMP phosphodiesterase from L. tropica promastigote to various compounds % of Activity Additions (2 mM) Peak I Peak II Control 100 100 Caffein 41 39 Theophylline 32 26 Trifluroperazine 42 55 Phenobarbiton 69 70 EDTA 37 44 Sodium chloride 91 95 Calcium chloride 93 97 MgSO4 omitted 36 38 Imidazole omitted 23 26 The standard assay, described in Materials and Methods was used. Various compounds were added at 2mM concentrations. Figures obtained from three separate experiments. Table 3. Relative rate of hydrolysis of different cyclic nucleotide monophosphates Peak I Peak II Enzyme % of Enzyme % of activity relative rate activity relative rate Substrate (units/ml) of hydrolysis (units/ml) of hydrolysis Cyclic AMP 1.90 100 1.1 100 Cyclic GMP 1.10 58 1.3 118 Cyclic IMP 0.61 32 0.45 41 Cyclic UMP 0.28 15 0.23 21 Cyclic dAMP 0.62 37 0.52 47
Effect o f cations and other compounds A variety of cations and other compounds were found to affect the activity of phosphodiesterase in vitro. Omission of M g 2+ from the assay reduced c A M P phosphodiesterase activity to about 36% and 38% from peaks I and II respectively (Table 2), i.e., the addition of Mg 2÷ to the assay mixture is necessary. Similarly, Ca 2+ at 2 m M does not stimulate the activity in both peaks (Table 2). Chelators of metal ions, specifically E D T A inhibit the activity of the enzyme of about 37 and 44% from peak I and II respectively (Table 2). The results indicate that L. tropica phosphodiesterase is M g 2+ dependent and Ca 2+ independent, in contrast to the mammalian phosphodiesterase which is Ca 2+ dependent and Mg 2÷ gives slight stimulation (Kakiuchi et al., 1971). While E. coli and the related enteric bacterium Serratia marcescens does not need Mg 2+ for phosphodiesterase activity (Nielson and Rickenberg, 1974). Omission of imadazole from the assay reduces c A M P phosphodiesterase activity to about 23-26% from both peaks. Various compounds such as caffein, theophylline, trifluroperazine and phenobarbiton cause various degrees o f inhibition of the enzyme activity from the two peaks (Table 2). Methylxanthines, such as theophylline and caffein, are poor inhibitors of the trypanosomatid enzyme (Gonsalves et al., 1980), inhibiting approximately 20% of the enzyme activity at a final concentration of 8 mM, while our results shows that methylxanthines cause about 60% inhibition of the leishmanial phosphodiesterase at 2 m M concentration, which is similar to the effect of methylxanthines on mammalian enzymes (Sheppard et al., 1972).
Substrate specificity o f phosphodiesterase The specificities of the phosphodiesterase activity o f the two peaks were assessed relative to c A M P as C.B.P. 93/4B---E
substrate. Cyclic G M P was about 58% as effective as substrate for peak I and about 118% for peak II, this indicates that peak II is more dominant to c G M P . Cyclic I M P was about 3 2 - 4 1 % as effective from the two peaks. The specificities of the two phosphodiesterase activities were found to be relatively broad with regard to cyclic nucleotides. The present studies, for the first time, demonstrate direct evidence for the existence of two phosphodiesterases in the L. tropica promastigote and preliminary results showing the presence of one enzyme in L. donovani of low activity.
REFERENCES
Butcher R. W. (1974) Cyclic 3',5'-nucleotide phosphodiesterase from bovine heart. Meths Enzymol. 38, 218-223. Butcher R. W. and Sutherland E. W. (1962) Adenosine 3",5'-phosphate in biological material. 1. Purification and properties of cAMP phosphodiesterase and use of this enzyme to characterize adenosine 3",5'-phosphate in human urine. J. biol. Chem. 237, 1244-1250. Fisk C. H. and Subbarow Y. (1925) The colorimetric method of estimation of inorganic phosphate. J. biol. Chem. 66, 375. Gonsalves M. F., Zingales B. and Colli W. (1980) cAMP phosphodiesterase and the activator protein of mammalian cAMP phosphodiesterase from Trypanosoma cruzi. Molec. Biochem. Parasitol. 1, 107-118. Juergen L. and Ogilvie A. (1987) 5-nucleotide phosphodiesterase isoenzymes in human serum: Quantitative measurement and some biochemical properties. Clin. chim. Acta 164, 275-284. Kakiuchi S., Yamazaki R. and Teshima Y. (1971) Cyclic 3',5'-nucleotide phosphodiesterase. IV. Two enzymes with different properties from brain. Biochem. Biophys. Res. Comm. 42, 968-974. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275.
792
KUSAI A. K. AL-CHALAB1 et al.
Luann G., Neubauer G. D. and Stutzenberger J. (1987) Cyclic AMP phosphodiesterase in Thermomonospora curvata. J. Baeteriol. 169, 2267 2271. Mancini P. E. and Patton C. L. (1981) Cyclic 3':5' monophosphate levels during the developmental cycle of Trypanosoma brucei brucei in the rat. Molee. Biochem. Parasitol. 3, 19 31. Nielsen L. D. and Rickenberg H. V. (1974) Cyclic AMP phosphodiesterase of Eseherichia coli. In Methods in Enzymology (Edited by Hardman J. G. and O'Malley B. V.), Vol. 38, pp. 249-256. Ong K. K. and Lim S. T. (1987) Two forms of cyclic 3,5-nucleotide phosphodiesterase in Rhizobium spp. Biochem. Int. 14, 561-568. Rangel-Aldao R., Allende D., Triana F., Piras R., Henriquez D. and Piras M. (1987) Possible role of cAMP in the differentiation of Trypanosoma cruzi. Molec. Biochem. Parasitol. 22, 39 43. Robinson G. A., Butcher R. W. and Sutherland E. W. (1968) Cyclie AMP. Ann. Rev. Biochem. 37, 149 174. Sheppard H., Wiggan G. and Tsien W. H. (1972) Structure activity relationships for inhibitors of phosphodiesterase from erythrocytes and other tissues. Adv. cyclic nueleotide Res. 1, 103-112. Tobie E. J., Von Brand M. and Mehlman B. (1950) Cultural and physiological observations on Trypanosoma rhodesiense and Trypanosoma gambiense. J. Parasitol. 36, 48 54.
Voichick J., Elson C., Granner D. and Shrago E (1973) Relationship of adenosine 3',5'-monophosphate to growth and metabolism of Tetrahymena pyr(/brmis. J. Bacteriol. 115, 68-72. Walter R. D. (1974) 3':5'-cyclic-AMP phosphodieslerase from Trypanosoma gambiense. Hoppe-Seyler's Z. Physiol. Chem. 355, 1443-1450. Walter R. D. (1981) Regulation of cyclic-AMP metabolism in Leishmania promastigotes and amastigotes. In The Biochemistry of Parasites (Edited by Slutzky G. M.), pp. 151 167. Pergamon Press, Oxford, New York. Walter R. D. and Opperdoes F. R. (1982) Subcellular distribution of adenylate cyclase, cAMP phosphodiesterase, protein kinases and phosphoprotein phosphatase in Trypanosoma hrucei. Mole~ . Biochem. Parasitol. 6, 287-295. Walter R. D., Nodmeyer J. P. and Konigk E. ([974) Adenylate cyclase from Trypanosoma ganbiense. HoppeSeyler's Z. Physiol. Chem. 355, 427 430. Walter R. D., Buse E. and Ebert F. (1978) Effect of cAMP on transformation and proliferation of Leishmania cells. Tropenmed. Parasitol. 29, 439-442. Zingales B., Carniol C., Abrahamsohn P. A. and Colli W. (1979) Purification of an adenyl cyclase-containing plasma membrane fraction from Trypanosoma cruzi. Bioehem. biophys. Acta 550, 233-244.
0305-0491/89 $3.00+ 0.00 © 1989PergamonPress plc
PRESENCE AND PROPERTIES OF cAMP PHOSPHODIESTERASE FROM PROMASTIGOTE FORMS OF L E I S H M A N I A TROPICA AND L E I S H M A N I A DO NO V A N I KUSAI A. K. AL-CHALABI, LAMIAA. ZIZ and BAIDAAL-KHAYAT Department of Biology, College of Science, University of Mosul, Mosul, Iraq (Received 1 December 1988) Abstract--1. This paper reports the isolation and the partial purification of cAMP phosphodiesterase (EC 3.1.4.17) from the promastigote form of Leishmania tropica and a preliminary result from Leishmania donovani. 2. The activity of the fraction obtained from column chromatography was measured. 3. The effects of pH, temperature, time of incubation and various compounds on its activity in vitro were obtained. 4. Two peaks (I and II) exhibiting cyclic nucleotide phosphodiesterase activity were obtained. 5. Both activities were found to require the addition of Mg 2÷ ions for full effect. 6. The apparent Km values for the first and second peaks were 1.43 x 10-3M and 4.1 × 10-3M respectively. L. donovani shows only one peak of activity.
tropica and a preliminary result from the promastigote form of Leishmania donovani.
INTRODUCTION
3',5' c A M P phosphodiesterase (PDE; EC 3.1.4.17) hydrolyses the Y,5'-cyclic nucleotide specifically to the Y-phosphate product AMP. This enzyme is important in the regulation of the intracellular concentration of cAMP, a key intermediate in the glycogenolytic action of glucagon and epinephrine in liver, this cyclic nucleotide has been implicated as the mediator of a n u m b e r of different polypeptide hormones and biogenic amine actions in a variety of different mammalia tissues (Robinson et al., 1968). In other phyla, cAMP has been shown to be a regulatory factor associated with the control of a variety of important biological events which influence the nutritional, survival, growth, development, and/or differentiation of certain organisms (Robinson et al., 1968; Voichick et al., 1973). In the case of the trypanosomatids some evidence is available that cAMP regulates processes of cell differentiation and transformation (Walter et al., 1978; Mancini and Patton, 1981; Rangel et al., 1987) and two enzymes, adenylate cyclase and cyclic A M P phosphodiesterase, which are involved in the synthesis and degradation, respectively, of cAMP have been demonstrated in Trypanosoma cruzi (Zingales et al., 1979; Gonsalves et al., 1980), Trypanosoma gambiense (Walter et aL, 1974; Walter, 1974) and from Trypanosoma brucei (Walter and Opperdoes, 1982). A correlation of the concentration of cyclic A M P in Leishmania with proliferation of promastigotes and the transformation of amastigotes to promastigotes has been described (Walter et al., 1978; Walter, 1981). The present paper reported the occurrence and partial purification of cyclic nucleotide phosphodiesterase from the promastigote form of Leishmania
MATERIALS AND METHODS
Organisms L. tropica (MHDM/IQ) and L. donovani (MCAN, IQ) used throughout this work were cultured promastigote forms which have been obtained from the Department of Parasitology and Medical Microbioligy, the Regional Training Center for Malaria and Medical Insect Control, Ministry of Health, Baghdad, Iraq. The organisms were cultured on NNN medium base with Locke's solution overlay in 3.5 × 1 in/test tubes. To obtain sufficient quantity of parasites for enzymatic study, promastigotes were grown in two media, modified Tobie's medium (Tobie et al., 1950). Parasites were harvested 5-6 days after subculture (i.e. during the log-phase of growth) by centrifugation at 1000g for 15 rain at room temperature and resuspended to a concentration of 1 × 109/ml in 50 mM Tris-HCl buffer at pH 7.9, and then sonicated for 30 s in a MSE ultrasonic disintegrater at a setting of 10 at 4°C. Isolation and enzymatic assay of cAMP phosphodiesterase The homogenate was centrifuged at 20,000 g for 30 min, and the supernatant fraction was used as the first step purified form of crude homogenate. The second step of purification is the ammonium sulfate fractionation which is based upon the degree of saturation (Butcher, 1974). The supernatant fraction in the previous step was brought to 0-50% saturation with ammonium sulphate. The precipitate was recovered by centrifugation at 16,000g for 20min, resuspended in 50mM Tris-HCl, pH 7.9 and dialysed overnight in large volume of 50 mM Tris-HC1 buffer (pH 7.9) at 4°C with continuous stirring. The dialysed enzyme was applied to a ultragel-34 column (30 x 2.6 cm) previously equilibrated with 50 mM Tris-HCl at pH 7.9. Fractions of 10 ml were collected and the enzyme activity and protein content were estimated after each step of purification. 789
KUSAI A. K. AL-CHALAB!et al.
790
Table 1. Purification of Leishmania tropica promastigote cyclic AMP phosphodiesterase Total Specific Total protein activity Purification Fraction units (mg) (units/mg) fold Crude homogenate 5100 182.00 28.00 1.00 20,000 g supernatant 3200 95.00 33.70 1.20 Ammonium sulfate precipitate 1700 34.00 50.00 1.79 (0-50% saturation) Dialysed fraction 1500 19.48 77.00 2.75 Gel filtration Peak I 100.90 0.97 103.00 3.68 Peak II 100.07 0.30 334.00 11.93 The activity of phosphodiesterase was assayed essentially as described by Butcher and Sutherland (1962). The procedures consisted in two stages of incubation each for 30 min. The phosphodiesterase reaction was followed by the 5'-nucleotidase reaction in the 2nd stage using an excess amount of snake venom plus MgSO4, and released Pi was determined colorimetrically by the method of Fisk and Subbarow (1925). The standard assay for cyclic-AMP phosphodiesterase contained in a final volume of 0.5 ml: 1.2 mM cAMP 12 mM MgSO4, 100 #g snake venom (Sigma) 2 mM imidazole, 50 mM Tris-HC1 (pH 7.9) and enzyme protein. Control tubes containing buffer instead of the substrate cAMP. The test and control tubes were incubated at 37°C for 60 min. The reaction was then stopped by the addition of 0.1 ml of 55% (w/v) trichloroacetic acid. The denatured protein was removed by centrifugation at 6000 g for 15 min. The Pi content of the supernatant was measured. One unit of activity was defined as the amount of enzyme releasing 1 nmol of inorganic phosphate per rain. Protein concentrations were determined by the method of Lowry et al. (1951), using bovine serum albumin as the standard.
RESULTS AND DISCUSSION Following the disruption of promastigote forms of L. tropica and L. donovani, the crude homogenate was centrifuged and the supernatant was subjected to fractional precipitation with solid a m m o n i u m sulfate at 0 - 5 0 % saturation and then dialyzed. Samples of dialyzed preparations were applied to a column packed with ultragel-34. Fractions were then collected and the activity of the enzyme was determined. The results of difference in enzyme activity due to a m m o n i u m sulphate fractionation and dialysis for promastigote forms of L. tropica are shown in Table 1. Table 1 also shows two active peaks of phosphodiesterase obtained when a dialyzed preparation of L. tropica was applied to a column packed with ultragel-34. Peak II was the dominant component and usually had a specific activity higher than the first peak (Table 1). The presence of more than one cyclic nucleotide phosphodiesterase has been suggested by many workers, two enzymes with different properties exist in rat brain (Kakiuchi et al., 1971) four forms in serum of healthy persons (Juergen and Ogilvie, 1987) Thermononospora curvata (Luann et aL, 1987) and two forms in Rhizobium species (Ong and Lim, 1987) and one type phosphodiesterase in Trypanosoma cruzi (Gonsalves et aL, 1980). In the case of L. donovani, a very low nucleotide phosphodiesterase activity was obtained (8 units/mg protein) when compared with L. tropica and only one peak seen.
Comparative studies were carried out on the enzymatic properties of freshly isolated fractions I and II from L. tropica promastigote.
Effect o f pH, temperature and time on enzyme activity The influence of pH, temperature and time upon the enzyme activity in both peaks were investigated. The optimum temperature for the assay of the activity in L. tropica (grown at 26°C) was 38°C and the optimum pH was 7.9, A value of p H 7.5-8.0 has been reported for the enzyme from most tissues and organisms. The rate of the reaction was linear with respect to both time (for at least 60 min) and to the amount of enzyme added (usually 200-250 # g protein).
K m values Reciprocal plots of reaction velocity versus c A M P concentrations were linear for the activities in both peaks (Fig. 1). The apparent K~ values derived from these plots were 1.43 x 10 -3 M and 4.1 x 1 0 - 3 M for the first and second peak respectively. The results show kinetic evidence for the existence of two enzymes as in other systems (Kakiuchi et aL, 1971; Ong and Lim, 1987) and in contrast to the results obtained with Trypanosoma cruzi (Gonsalves et al., 1980).
t /.
3.0 2.5 ~_ 2.0
//
+/
io~ ~ O.
PeakI ,
,
I
2
I/S (mMI 0.14
+,
0.12 x.
/
OAO
+ / 4-
0.08 006 / 0,04 -/+/
Peak]I
O,CrZ
/
hI I/.9 (raM) Fig. 1
h
2
791
Leishmania cAMP phosphodiesterase Table 2. Sensitivity of cAMP phosphodiesterase from L. tropica promastigote to various compounds % of Activity Additions (2 mM) Peak I Peak II Control 100 100 Caffein 41 39 Theophylline 32 26 Trifluroperazine 42 55 Phenobarbiton 69 70 EDTA 37 44 Sodium chloride 91 95 Calcium chloride 93 97 MgSO4 omitted 36 38 Imidazole omitted 23 26 The standard assay, described in Materials and Methods was used. Various compounds were added at 2mM concentrations. Figures obtained from three separate experiments. Table 3. Relative rate of hydrolysis of different cyclic nucleotide monophosphates Peak I Peak II Enzyme % of Enzyme % of activity relative rate activity relative rate Substrate (units/ml) of hydrolysis (units/ml) of hydrolysis Cyclic AMP 1.90 100 1.1 100 Cyclic GMP 1.10 58 1.3 118 Cyclic IMP 0.61 32 0.45 41 Cyclic UMP 0.28 15 0.23 21 Cyclic dAMP 0.62 37 0.52 47
Effect o f cations and other compounds A variety of cations and other compounds were found to affect the activity of phosphodiesterase in vitro. Omission of M g 2+ from the assay reduced c A M P phosphodiesterase activity to about 36% and 38% from peaks I and II respectively (Table 2), i.e., the addition of Mg 2÷ to the assay mixture is necessary. Similarly, Ca 2+ at 2 m M does not stimulate the activity in both peaks (Table 2). Chelators of metal ions, specifically E D T A inhibit the activity of the enzyme of about 37 and 44% from peak I and II respectively (Table 2). The results indicate that L. tropica phosphodiesterase is M g 2+ dependent and Ca 2+ independent, in contrast to the mammalian phosphodiesterase which is Ca 2+ dependent and Mg 2÷ gives slight stimulation (Kakiuchi et al., 1971). While E. coli and the related enteric bacterium Serratia marcescens does not need Mg 2+ for phosphodiesterase activity (Nielson and Rickenberg, 1974). Omission of imadazole from the assay reduces c A M P phosphodiesterase activity to about 23-26% from both peaks. Various compounds such as caffein, theophylline, trifluroperazine and phenobarbiton cause various degrees o f inhibition of the enzyme activity from the two peaks (Table 2). Methylxanthines, such as theophylline and caffein, are poor inhibitors of the trypanosomatid enzyme (Gonsalves et al., 1980), inhibiting approximately 20% of the enzyme activity at a final concentration of 8 mM, while our results shows that methylxanthines cause about 60% inhibition of the leishmanial phosphodiesterase at 2 m M concentration, which is similar to the effect of methylxanthines on mammalian enzymes (Sheppard et al., 1972).
Substrate specificity o f phosphodiesterase The specificities of the phosphodiesterase activity o f the two peaks were assessed relative to c A M P as C.B.P. 93/4B---E
substrate. Cyclic G M P was about 58% as effective as substrate for peak I and about 118% for peak II, this indicates that peak II is more dominant to c G M P . Cyclic I M P was about 3 2 - 4 1 % as effective from the two peaks. The specificities of the two phosphodiesterase activities were found to be relatively broad with regard to cyclic nucleotides. The present studies, for the first time, demonstrate direct evidence for the existence of two phosphodiesterases in the L. tropica promastigote and preliminary results showing the presence of one enzyme in L. donovani of low activity.
REFERENCES
Butcher R. W. (1974) Cyclic 3',5'-nucleotide phosphodiesterase from bovine heart. Meths Enzymol. 38, 218-223. Butcher R. W. and Sutherland E. W. (1962) Adenosine 3",5'-phosphate in biological material. 1. Purification and properties of cAMP phosphodiesterase and use of this enzyme to characterize adenosine 3",5'-phosphate in human urine. J. biol. Chem. 237, 1244-1250. Fisk C. H. and Subbarow Y. (1925) The colorimetric method of estimation of inorganic phosphate. J. biol. Chem. 66, 375. Gonsalves M. F., Zingales B. and Colli W. (1980) cAMP phosphodiesterase and the activator protein of mammalian cAMP phosphodiesterase from Trypanosoma cruzi. Molec. Biochem. Parasitol. 1, 107-118. Juergen L. and Ogilvie A. (1987) 5-nucleotide phosphodiesterase isoenzymes in human serum: Quantitative measurement and some biochemical properties. Clin. chim. Acta 164, 275-284. Kakiuchi S., Yamazaki R. and Teshima Y. (1971) Cyclic 3',5'-nucleotide phosphodiesterase. IV. Two enzymes with different properties from brain. Biochem. Biophys. Res. Comm. 42, 968-974. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275.
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KUSAI A. K. AL-CHALAB1 et al.
Luann G., Neubauer G. D. and Stutzenberger J. (1987) Cyclic AMP phosphodiesterase in Thermomonospora curvata. J. Baeteriol. 169, 2267 2271. Mancini P. E. and Patton C. L. (1981) Cyclic 3':5' monophosphate levels during the developmental cycle of Trypanosoma brucei brucei in the rat. Molee. Biochem. Parasitol. 3, 19 31. Nielsen L. D. and Rickenberg H. V. (1974) Cyclic AMP phosphodiesterase of Eseherichia coli. In Methods in Enzymology (Edited by Hardman J. G. and O'Malley B. V.), Vol. 38, pp. 249-256. Ong K. K. and Lim S. T. (1987) Two forms of cyclic 3,5-nucleotide phosphodiesterase in Rhizobium spp. Biochem. Int. 14, 561-568. Rangel-Aldao R., Allende D., Triana F., Piras R., Henriquez D. and Piras M. (1987) Possible role of cAMP in the differentiation of Trypanosoma cruzi. Molec. Biochem. Parasitol. 22, 39 43. Robinson G. A., Butcher R. W. and Sutherland E. W. (1968) Cyclie AMP. Ann. Rev. Biochem. 37, 149 174. Sheppard H., Wiggan G. and Tsien W. H. (1972) Structure activity relationships for inhibitors of phosphodiesterase from erythrocytes and other tissues. Adv. cyclic nueleotide Res. 1, 103-112. Tobie E. J., Von Brand M. and Mehlman B. (1950) Cultural and physiological observations on Trypanosoma rhodesiense and Trypanosoma gambiense. J. Parasitol. 36, 48 54.
Voichick J., Elson C., Granner D. and Shrago E (1973) Relationship of adenosine 3',5'-monophosphate to growth and metabolism of Tetrahymena pyr(/brmis. J. Bacteriol. 115, 68-72. Walter R. D. (1974) 3':5'-cyclic-AMP phosphodieslerase from Trypanosoma gambiense. Hoppe-Seyler's Z. Physiol. Chem. 355, 1443-1450. Walter R. D. (1981) Regulation of cyclic-AMP metabolism in Leishmania promastigotes and amastigotes. In The Biochemistry of Parasites (Edited by Slutzky G. M.), pp. 151 167. Pergamon Press, Oxford, New York. Walter R. D. and Opperdoes F. R. (1982) Subcellular distribution of adenylate cyclase, cAMP phosphodiesterase, protein kinases and phosphoprotein phosphatase in Trypanosoma hrucei. Mole~ . Biochem. Parasitol. 6, 287-295. Walter R. D., Nodmeyer J. P. and Konigk E. ([974) Adenylate cyclase from Trypanosoma ganbiense. HoppeSeyler's Z. Physiol. Chem. 355, 427 430. Walter R. D., Buse E. and Ebert F. (1978) Effect of cAMP on transformation and proliferation of Leishmania cells. Tropenmed. Parasitol. 29, 439-442. Zingales B., Carniol C., Abrahamsohn P. A. and Colli W. (1979) Purification of an adenyl cyclase-containing plasma membrane fraction from Trypanosoma cruzi. Bioehem. biophys. Acta 550, 233-244.