A novel ATP-Adpase from Mimosa pulvinus

June 8, 2017 | Autor: Takahide Tsuchiya | Categoria: ATPase
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Comp. Biochem. Physiol. Vol. 78B, No. 1, pp. 59-61, 1984

0305-0491/84 $3.00+0.00 © 1984 Pergamon Press Ltd

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A NOVEL ATP-ADPASE FROM MIMOSA PULVINUS HIROSHI ISHIKAWA,TORU TAMIYA,TAKAHIDETSUCHIYA, and JUICHIRO J. MATSUMOTO Department of Chemistry, Faculty of Science and Technology, Sophia University, 7-1, Kioi-cho Chiyoda-ku, Tokyo 102, Japan (Received 9 September 1983) Abstract--1. The ATPase of Mimosa pudica pulvinus hydrolyzed various nucleoside triphosphates, but the presence of divalent cations was required for obtaining positive activity. 2. The enzyme also hydrolyzed the nucleosidediphosphates but it did not hydrolyze AMP, PPi or PNPP. 3. On the basis of its activity with both triphosphates and diphosphates, quantitative relationships between ATP, ADP and AMP were traced by HPLC. 4. The present enzyme did not hydrolyze ATP and ADP at the same time, but it first hydrolyzed the ADP rapidly and then the ATP rather slowly. 5. The ATPase isolated from Mimosa pudica pulvinus is an ATP-ADPase of a type which differs from the known enzymes such as apyrase. INTRODUCTION

Mimosa demonstrates a motile property on touch which is remarkably rare in the kingdom of plants. It was reported that the ATP content in the motor organ 'pulvinus' is reduced to 1/4-1/3 the value (Lyubimova et aL, 1964) before the movement. The main pulvinus of Mimosa contains ATPase [EC 3.6.1.3] (Toriyama, 1963) which is activated by divalent cations. The ATPase must be playing a key role in the mechanisms of the movement of Mimosa leaves. Several studies (Biswas and Bose, 1972; Lyubimova et al., 1978; Mukherjee and Biswas, 1980) have been done on the properties of ATPase namely, pH optimum, effects of cations and substrate specificity. But the results do not always agree with each other and no definite picture of the properties has been obtained so far. We succeeded in isolating and purifying the ATPase from Mimosa main pulvinus. Our studies on its activity profiles allowed us to conclude that the Mimosa enzyme is a novel type of the ATP hydrolase. MATERIALS AND METHODS

The main pulvinus of Mimosa pudica L. was cut off, immersed in 50~ glycerol, and stored in deep freezer until use. The pulvinus was submitted to the extraction after less than 2 months of the storage. The stored pulvinus was homogenized in a Waring blender for a few minutes and the water soluble fraction was removed by centrifugation. By suspending the fraction in 0.6 M KC1 (pH 8.5) solution, the enzyme was extracted and purified by ion exchange chromatography with DEAE Toyopearl 650M (Try6 Soda Co. Ltd.), followed by gel filtration with Toyopearl HW55F (Try6 Soda Co. Ltd.). The enzymatic activity was assayed by two methods. In one, the liberated inorganic phosphate was determined by Fiske-SubbaRow's method; in the other, each of the reactants and reaction products namely, ATP, ADP and AMP, was separated and determined by means of HPLC. RESULTS AND DISCUSSION

To investigate the substrate specificity of the Mimosa pulvinus enzyme, the overall phosphate

liberating activity was determined of 14 phosphate compounds (Table 1). The enzyme preparation hydrolyzed various nucleoside triphosphates, but the presence of divalent cations was essential for obtaining a positive activity. The effect was outstanding with Mn 2+, except for the case with GTP where the effect with Ca 2+ was the largest. The activity appeared higher for the purine nucleosides (GTP, ITP and ATP) than for the pyrimidine nucleosides (UTP and CTP). On the other hand, the Mimosa enzyme preparation also hydrolyzed the nucleoside diphosphates. The presence of the divalent cations was essential too, but the effect was most marked with Ca 2+, except for the case with GTP. In general, the relative activity level for each diphosphate was higher than for the corresponding triphosphate. The activity value for ADP was the highest among the tested diphosphates and 3-10 times as high as that for ATP. The enzyme preparation did not hydrolyze AMP, PPi or PNPP. This indicated that the present enzyme is by no means a non-specific phosphohydrolase. In view of its positive activity on both the triphosphates and diphosphates, it was suggested that the enzyme belongs to none of the known ATPases but is of a novel category. For elucidating the details of the above point, quantitative relationships between the substrate ATP and the enzymic products namely, ADP and AMP, were traced by HPLC, as shown in Fig. 1. With the decrease of ATP, AMP and inorganic phosphate increased to match the ATP decrease stoichiometrically, but the increase of ADP was not appreciable. There was no PPi formation detected. After 24 hr incubation, AMP was the only product nucleotide, while adenosine was not detected. The relative affinity of the enzyme for ATP and for ADP was sought by incubating the enzyme preparation with a mixture of ATP and ADP (Fig. 2). With each added divalent cation, ADP decreased first and AMP increased in a speed corresponding to the former. However, ATP did commence to decrease after ADP had fallen down to a low level. The 59

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HIROSHI [SHIKAWA et al.

Table 1. Substrate specificity of Mimosa ATPase Relative activity (°/~,) Sabstrate (3 mM) Mg2+ Ca2+ Mn:+ EDTA GTP 179 235 189 3 ITP 176 162 244 3 ATP 100 80 196 3 UTP 83 77 230 2 CTP 47 47 141 0 GDP 299 488 557 nd IDP 522 769 488 nd ADP 457 782 572 3 UDP 541 772 672 nd CDP 427 751 724 nd AMP 2 3 2 2 PPi 3 3 4 1 PPPi 1 1 3 nd PNPP 1 1 1 2 Assay conditions: Tris-maleate buffer pH 7.0 40 raM; protein concn 0.05,ttgcm-3; KCI 60mM; substrate 3mM; MgCI2, CaCI2, MnCI2 or EDTA 6 mM, 25°C, 30 min. The liberated inorganic phosphate was determined by FiskeSubbaRow's method on the supernatants after addition of TCA to the reaction mixture. decrease of A T P was fairly slow a n d the p r o d u c t arising from A T P decomposition was A M P . These results suggest the following: The present enzyme did not hydrolyze b o t h A T P a n d A D P at the same time, but it first hydrolyzed A D P rapidly and then A T P r a t h e r slowly. T h e difference in the speed is in accordance with the results o f Table 1. Apyrase ( A D P a s e ) [EC 3.6.1.5] from insects (Gil-

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Fig. 2. Time course of hydrolysis of mixture of ATP (1.8raM) and ADP (l.2mM) by Mimosa ATPase. Both ATP and ADP were incubated with the M i m o s a ATPase preparation and the concentrations of ATP, ADP and AMP were determined by HPLC. (A) 6 mM Mg :+ present; (B) 6 mM Ca :+ present; (C) 6 mM Mn 2+ present. - - 0 - - ATP: - - I I - - ADP; - - A - - AMP. The reaction conditions are as described in Table 1.

A

0

A

3o0

Time/rain

Fig. 1. Time course of ATP hydrolysis by M i m o s a ATPase. ATP was incubated with the Mimosa ATPase preparation and the concentration of ATP (3 mM) and the reaction products were determined by HPLC. (A) 6 m M Mg 2÷ present; (B) 6 m M Ca 2+ present; (C) 6 mM Mn 2+ present. - - 0 - - ATP; - - m - - ADP; - - A - - AMP; ---O--- Pi.

mour, 1955) or potatoes (Valenzuela et al., 1973) is k n o w n for its hydrolytic activity on b o t h A T P a n d A D P . However, the apyrase hydrolyzed A T P to A D P to the first step a n d then A D P a n d A M P to the second step. Usually, their affinity to A T P is comparable with or higher t h a n t h a t to A D P . In view of these results, the M i m o s a enzyme, which is preferably active on A D P , is distinct from any of the apyrase a n d other k n o w n enzymes a n d m i g h t m a k e a novel type. Recently, an enzyme which is slightly more active to A D P t h a n to A T P was f o u n d in chick peas (Vara a n d Serrano, 1981) a n d peas (Tognoli a n d Marre, 1981). A n enzyme from the porcine pancreas (LeBel et al., 1980) was reported as equally active to A T P a n d A D P . A n u c l e o s i d e d i p h o s p h a t a s e (IDPase) (EC 3.6.1.6.] which has been f o u n d to exist latent in Golgi a p p a r a t u s (Ishibashi et al., 1978) is also different from the present enzyme, because the substrate specificity a n d cation dependence are different. In spite o f the a b o v e views, some d o u b t s remain to be settled before confirming the presence o f a n ATPase of a unique type. One d o u b t is t h a t there m i g h t have been b o t h an A T P a s e a n d a n A D P a s e in the present enzyme preparation. In t h a t case, each of A T P a n d A D P m u s t have decreased simultaneously in the experiment of Fig. 2, b u t the fact was t h a t A T P decreased after A D P h a d decreased.

A novel ATP-ADPase from Mimosa pulvinus Another doubt is that the present enzyme preparation might have contained both ATPase and adenylate kinase. If the ATPase was more active than the adenylate kinase, the decrease of ATP must have preceded that of ADP, which is not the case with Fig. 2. If the ATPase is less active than the adenylate kinase, the ATP concentration curve of Fig. 2 must have demonstrated a rise at the early stage, but no such rise was found in the results illustrated. To confirm this, the present enzyme preparation was incubated with ADP. The ADP was converted to AMP but no ATP was detected for analyses every 20 min throughout 5 hr incubation. Hence the presence of the adenylate kinase was definitely rejected. At any rate, in several stages of the separation procedures namely, ion exchange chromatography and gel filtration, the enzymic activities on ATP and ADP were never separated but appeared always together in the same fraction. Therefore, the above doubts about the concomitant existence of two kinds of enzymes have been settled. It is proposed now that the ATPase isolated from the Mimosa main pulvinus is an ATPase of a novel type. It catalyzed the hydrolysis of both ATP and ADP with preference of the latter to the former. Its hydrolysis products are AMP and inorganic phosphate, regardless of the substrate species.

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Gilmore D. (1955) Insect ATPase. In Methods in Enzymology (Edited by Colowick S. P. and Kaplan N. O.), Vol. 2., pp. 595-598. Academic Press, New York. Ishibashi T., Gasa S., Ohkubo I. and Makita, A. (1978) Affinity purification and some properties of nucleoside diphosphatase from rat liver cytosol. Biochim. biophys. Acta 525, 265-274. LeBel D., Poirier G. G., Phaneuf S., St-Jean P., Laliberte J. F. and Beaudoin A. R. (1980) Characterization and purification of a calcium-sensitive ATP diphosphohydrolase from pig pancreas. J. biol, Chem. 255, 1227-1233. Lyubimova M. N., Burnasheva S. A., Fain F. S., Mitani N. A. and Poprykina Ya. M. (1978) Study of adenosine triphosphatase from Mimosa pudica. Biokhimiya 43, 748-760. Lyubimova M. N., Demyanovskaya N. S., Fedorovich I. B. and Itomlenskite I. V. (1964) Participation of ATP in the motor function of the Mimosa pudica leaf. Biokhimiya 29, 774-779. Mukherjee J. and Biswas S. (1980) Purification and characterization of a nucleoside triphosphatase from Mimosa pudica. Indian J. Biochem. Biophys. 17, 452-456. Tognoli L. and Marre E. (1981) Purification and characterization of a divalent cation-activated ATP-ADPase from stem mierosomes. Biochim. biophys. Acta. 642, 1-14. Toriyama H. (1963) Histochemical detection of ATPase activity in motor tissue of Mimosa pudica. Bot. Mag., Tokyo. 76, 79-80. Valenzuela M. A., Campo G. D., Marin E. and TraversoCori (1973) Effects of protein-modifying reagents on an isozyme of potato apyrase. Biochem. J. 133, 755-763. REFERENCES Vara F. and Serrano R. (1981) Purification and characterBiswas S. and Bose D. M. (1972) An ATPase in sensitive ization of a membrane-bound ATP diphosphohydrolase plant Mimosa pudica I. Purification and characterization. from Cicer arictinum (chick-pea) roots. Biochem. J. 197, Archs Biochem. Biophys. 148, 199-207. 637-643.

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