Phosphorylation and dephosphorylation of Mg2+-independent
R Sikdar, K Roy, A K Mandal and P C Sen*
Department of Chemistry, Bose Institute, 93/1, Acharya Prafulla Chandra Road, Calcutta 700 009, India
*Corresponding author (Fax, 91-33-3506790; Email, firstname.lastname@example.org).
We reported previously that a Ca2+-ATPase in rat testes and goat spermatozoa could be activated by Ca2+ alone without Mg2+, though it has a lot of similarities with the well known Ca2+, Mg2+-ATPase. Recently, we were successful in isolating the phosphorylated intermediate of the former enzyme under control conditions i.e., in the presence of low concentration of Ca2+ and at low temperature. Increase of the concentration of Ca2+ and/or temperature lead to dephosphorylation. Based on our observations, we proposed a reaction scheme comparable to that of Ca2+, Mg2+-ATPase. The findings strengthened our previous report that Mg2+-independent Ca2+-ATPase is involved in Ca2+ transport and Ca2+ uptake like Ca2+, Mg2+-ATPase.
1. IntroductionThe control of intracellular free calcium concentration is crucial for the maintenance of normal cell functions and is regulated through the operation of several mechanisms including ATP driven calcium pump (Carafoli and Crompton 1978; Lynch and Cheung 1979). Changes in free cytosolic calcium concentration play a vital role in the action of certain hormones and other stimuli on cell metabolism (Carafoli and Crompton 1978; Charest
2. Materials and methods
2.1 Chemicals and radiochemicals
ATP disodium salt, 2-mercaptoethanol (2-ME), EDTA, ethylene glycol-bis (2-amino ethylether) N,N,Ną ,Ną tetraacetic acid (EGTA), imidazole, 1,2-cyclohexanediamine tetraacetic acid (CDTA), phenylmethylsulfonyl fluoride (PMSF) and Na-vanadate were purchased from Sigma Chemicals Co., USA. Sucrose, calcium chloride were from SISCO Research Laboratories, Mumbai. [g -32P]ATP (3000 Ci/mmol) from Bhabha Atomic Research Centre, Mumbai. Membrane filters (0·45 m m) from Millipore Corporation. All other reagents used were of analytical grade obtained from local market. Double distilled water was used throughout the study.
2.2 Collection of goat testes, isolation and purification of ATPase enriched membranes
Goat testes were collected from the local slaughter house just after sacrifice of the animal and brought to the laboratory on ice. The caudal region of the testes were minced in 25 mM imidazole buffer containing 0·25 M sucrose, 1 mM EDTA and 1 mM 2-ME (pH 7·5) (buffer A). Membranes enriched with Ca2+, Mg2+- and Ca2+-ATPase were prepared as described earlier (Sikdar et al 1991). Briefly, the sperms were homogenized in a glass homogenizer and spun for 10 min at 600 g in cold. The pellet was resuspended in the buffer, centrifuged again at 600 g. The process was repeated once more. The pooled supernatant was spun at 12,000 g for 10 min at 4° C. The supernatant was collected and centrifuged at 100,000 g for 1 h. The pellet (microsomes) was resuspended in the above buffer and assayed for protein. It was then treated with 0·05% octaethyl glucoside (C12E8) containing 0·1 mM PMSF and 1 mM ATP to a final concentration of 1 mg protein per ml. After stirring for 10 min at 4° C, the mixture was layered on the top of a gradient consisting 5 ml each of 20, 25, 30, 34 and 37% sucrose and spun at 50,000 g for 4·5 h in a SW27 swinging bucket rotor. The band which appeared between 34 and 30% sucrose was collected, diluted three times with 25 mM imidazole buffer (pH 7·5) containing 1 mM 2-ME and spun at 100,000 g for 1 h. The pellet was suspended in buffer A and assayed for protein and ATPase activities. The purified fraction shows two protein bands of molecular mass 110 and 97 kDa on 7·5% SDS-PAGE (Bhattacharyya and Sen 1998). Proteins were estimated following the method of Lowry et al (1951) using bovine serum albumin as standard. The enzyme activities were assayed as described below. However for calcium uptake study non-detergent treated microsomal membranes were used (Sikdar et al 1991).
2.3 Enzyme activity assay
The Mg2+-dependent Ca2+-ATPase activity was assayed according to previously established procedures (Sikdar et al 1991; Nandi et al 1981). The values were expressed as the difference in activities between Mg2+, Ca2+ and Mg2+ alone. The Mg2+-independent Ca2+-ATPase was assayed as described previously (Sikdar et al 1991). The assay medium (1 ml) contained 25 mM imidazole, 25 mM sucrose, 0·5 mM EDTA, 1 mM 2-ME (pH 8·5), 4 mM CaCl2 and 3 mM ATP. The mixture was preincubated for 5 min at 37° C and the reaction was initiated with the addition of 1015 m g detergent treated membrane protein and incubated for 30 min. It was terminated with the addition of 0·2 ml of 30% ice-cold TCA. The liberated inorganic phosphate was estimated colorimetrically following the method of Sen et al (1981). A tube containing all the ingredients except membrane protein was run simultaneously as blank. The free concentration of calcium in the reaction mixture was adjusted by the addition of EGTA (Sillen and Martell 1971). After chelating endogenous Mg2+ (which was found to be very low when measured by atomic absorption spectrophotometer) with CDTA, the ATPase activity obtained was solely due to free Ca2+ alone.
2.4 45Ca uptake study with microsomal membrane vesicles
Calcium uptake by the Mg2+-independent Ca2+-ATPase was measured in a 1 ml reaction mixture containing 25 mM imidazole buffer (pH 8·5), 10 mM KCl, 0·5 mM CaCl2 containing 45Ca (sp. activity 2,000 cpm/pmol) and 4 mM ATP. The reaction was started by the addition of non-detergent treated microsomal vesicles (100 m g protein) and the mixture incubated at 37° C. At a different time an aliquot of 20 m l was removed and diluted to 1 ml with ice-cold buffer containing 0·5 mM CaCl2 and 2 mM EGTA. The suspension was rapidly filtered through a 0·45 m m Millipore filter, which was then washed with 20 ml cold CaCl2-EGTA (1 mM). The filters were taken in scintillation fluid and the radioactivity counted in a liquid scintillation counter.
To examine if the uptake is energy dependent, we have carried out the uptake experiment in the absence of ATP. To study the effect of A23187 on Ca2+ uptake by the ATPase, we added the ionophore at a final concentration of 1 m M after 10 min; at a different time after addition, an aliquot of 20 m l was withdrawn and radioactivity measured as described above. The ionophore was prepared in dimethyl sulphoxide. The solvent alone had no effect on Ca2+ uptake at the concentration used.
2.5 Phosphorylated intermediate study
Mg2+-independent Ca2+-ATPase enriched membranes
This has been done under different conditions to standardize the optimum conditions for the formation of phosphorylated intermediate (E ~ P).
2.5a Time course of phosphorylation: Rate of phosphorylation was measured at a different time of incubation at 4° C in the presence of 1·7 m M free Ca2+ under above optimum conditions with 50 m g membrane protein. The level of E ~ P formed was measured as described above.
2.6 Effect of different concentrations of Ca2+ on phosphorylated intermediate (E ~ P) formation
ATPase enriched membranes were phosphorylated with
[g -32P]ATP in different concentrations of free Ca2+
(50 nM250 m M) at 4° C for 3 s in a buffer containing 50 mM
Tris-HCl, 1 mM EDTA, 80 mM KCl, 0·5 mM
2-ME (pH 6·8) in a total reaction volume of 1 ml. The free calcium concentration was calculated according to the method of Sillen and Martell (1971). The reaction was stopped with 0·2 ml of ice-cold 30% TCA and filtered through 0·45 m m membrane filter and washed thoroughly with 10 ml cold 10 mM Na2 HPO4 containing 1 mM cold ATP. The radioactivity on the membrane filters was counted in a scintillation counter.
2.6a Incorporation of 32P to membrane ATPase at different concentrations of ATP: The incorporation was carried out at different concentrations of [g -32P]ATP under above conditions at 1·7 m M free Ca2+.
2.6b Dephosphorylation of phosphorylated intermediates: After the intermediate was allowed to form for 3 s in the presence of 1·7 m M free Ca2+, protein and 5 m M [32P]ATP, calcium concentration in the reaction mixture was increased and incubated for 30 s more. The reaction was terminated with TCA and the radioactivity was measured as described before.
2.7 Effect of vanadate on phosphorylation and dephosphorylation
To examine which reaction step phosphorylation or dephosphorylation-is affected by vanadate (an inhibitor of Ca2+-ATPase), we have performed the phosphorylation and dephosphorylation experiments in the presence of 50 m M vanadate under optimum conditions. After termination of the reaction with ice-cold TCA, the radioactive counts were determined in each case as described above.
3. Results and discussion
Mg2+-dependent and independent Ca2+-ATPase activities are shown in table 1. Though the activities of both Mg2+-dependent and independent enzymes are predominant in this fraction, the activity of the latter is higher than the former. The high level of Mg2+-independent Ca2+-ATPase activity found in goat spermatozoa is comparable with that of rat testicular membranes (Nagdas et al 1988). The treatment with octyl glucoside enhances the enzyme activity under control conditions.
Figure 1 shows the rate of Ca2+ uptake at points of different time under various conditions. It is evident from the figure that uptake reaches a maximum level at 20 min. The lowering of uptake beyond 20 min may be due to the leakiness of the membranes thereby loosing the vesicular structure. It can also be seen that in the absence of ATP, no Ca2+-uptake takes place suggesting that Ca2+-uptake by Ca2+-ATPase is energy dependent. Accumulated Ca2+ is rapidly and completely released by the calcium ionophore A 23187. The fact that the uptake is absolutely ATP dependent suggests that a significant proportion of the prepared vesicles are oriented inside out (Enyedi et al 1988; Sumida et al 1988).
Figure 2 shows time course of phosphorylation of Mg2+-independent Ca2+-ATPase. It is seen from the figure that maximum phosphorylation of the ATPase is obtained at 3 s, beyond which it decreases. It could so happen that the phosphorylated intermediate (E1 ~ P) which forms at 3 s may undergo dephosphorylation at longer time of incubation in the presence even of low concentration of Ca2+.
Figure 3 shows the rate of formation of phosphorylated intermediate of ATPase at different concentrations of Ca2+. It is evident from the figure that phosphorylation increases up to 1·7 m M Ca2+ concentration above which dephosphorylation takes place. The finding suggests that higher concentration of Ca2+ is responsible for dephosphorylation of the intermediate. This step of the reaction is comparable to the dephosphorylation of the Ca2+, Mg2+-ATPase in the presence of Ca2+ (Schurmans-Stekhoven and Bonting 1981). The concentration of free Ca2+ required for optimum phosphorylation is relatively high but interesting, since the finding is reproducible (Nagdas et al 1988; Sikdar et al 1991; Bhattacharyya and Sen 1998). Possibly the transport of calcium takes place through the binding of low affinity site. It has been shown by Quist and Roufogalis (1975) that Ca2+-ATPase with high affinity for Ca2+ can easily be removed from membranes and this suggests that low affinity site plays basic role in active calcium transport.
The study of the phosphorylation of ATPase at different concentrations of ATP shows that optimum concentration of substrate (ATP) to obtain maximum phosphorylation is 5 m M (data not shown) which is comparable to Ca2+, Mg2+-ATPase reported in other sources (Miessner 1973).
Vanadate is an inhibitor of Mg2+-independent Ca2+-ATPase (Sikdar et al 1991) and also an inhibitor of P-type ATPases (Stryer 1988). To examine which step of the overall reaction sequence (phosphorylation or dephosphorylation) is inhibited, we have performed an experiment in the presence of vanadate and the finding is shown in table 2. From the table it may be suggested that vanadate inhibits the phosphorylation step of the overall reaction sequence. The inhibition by vanadate suggests that Mg2+-independent Ca2+-ATPase is a P-type ATPase. Since in all P-type ATPases phosphorylation takes place on Asp residue (Stryer 1988), it is therefore logical to conclude that in Mg2+-independent Ca2+-ATPase in the present study, phosphorylation takes place on Asp residue.
It is pertinent to mention here that recently we have reported a Ca2+, Mg2+- and a Ca2+-ATPase from goat testes microsomal membranes which belong to two isoforms of SERCA family having different sensitivity to Mg2+ (Bhattacharyya and Sen 1998). From the ongoing findings we propose a scheme for the overall reaction of this Ca2+-ATPase comparable with the Mg2+, Ca2+-ATPase (Ikemoto 1975; Schatzmann 1975) except that phosphorylation and dephosphorylation is regulated by different concentrations of calcium ion i.e., phosphorylation of the enzyme takes place at lower concentration of calcium and dephosphorylation at higher concentration:
Bhattacharyya D and Sen P C 1998 Purification and functional characterization of a low molecular mass Ca2+, Mg2+- and Ca2+-ATPase inhibitor protein from rat brain cytosol; Biochem. J. 330 95101
Carafoli E and Crompton M 1978 The regulation of intracellular calcium; Curr. Top. Memb. Trans. 10 151216
Charest R, Blackmole P F, Berthon B and Exton J H 1983 Changes in free cytosolic Ca2+ in hepatocytes following a -adrenergic stimulation: studies on quin-2 loaded hepatocytes; J. Biol. Chem. 258 87698773
Enyedi A., Jminami J, Caride A J and Penniston J T 1988 Characteristics of the Ca2+-pump and Ca2+-ATPase in the plasma membrane of rat myometrium; Biochem. J. 252 215220
Garbers D L and Kopf G S 1980 The regulation of spermatozoa by calcium and cyclic nucleotides; Adv. Cyclic. Nucl. Res. 13 251306
Gupta R P and Venktitasubramanian T A 1983 Ca2+, Mg2+-ATPase in lung lamellar bodies; Indian J. Biochem. Biophys. 20 381385
Ikemoto N 1975 Transport and inhibitory Ca2+ binding sites on the ATPase enzyme isolated from the sarcoplasmic reticulum; J. Biol. Chem. 250 72197224
Jackowski S, Petro K and Shaaf R J 1979 A Ca2+-stimulated ATPase activity in rabbit neutrophil membrane; Biochim. Biophys. Acta 558 348372
Joseph S K and Williams J R 1983 The origin, quantitation and kinetics of intracellular calcium mobilisation by vasopresin and phenylephrine in hepatocytes; J. Biol. Chem. 258 1042510432
Lowry O H, Rosebrough N J, Farr A L and Randall R J 1951 Protein measurement with folin phenol reagent; J. Biol. Chem. 193 265275.
Lynch T J and Cheung W Y 1979 Human erythrocyt Ca2+, Mg2+-ATPase: mechanism of stimulation by Ca2+; Arch. Biochem. Biophys. 194 165170
Mazumder B, Sikdar R and Sen P C 1991 Inhibition of Ca2+-ATPase by gossypol and chlorpromazine in the microsomal membranes of rat testes; in Biomembranes in health and diseases (eds) A M Kidwai, R K Upreti and P K Ray (New Delhi: Today and Tomorrows Printers and Publishers) pp 407412
Meissner G 1973 ATP and Ca2+ binding by the Ca2+ pump protein of sarcoplasmic reticulm; Biochim. Biophys. Acta 298 906926
Moolenaar W H, Defize L H K and De Laat S W 1986 Calcium and the cell (Chichester: Wiley) (Ciba Foundation Symposium 122) pp 212231
Moore L, Chen T, Knapp H R and London E J 1975 Energy dependent calcium sequestration activity in rat liver microsomes; J. Biol. Chem. 250 45624568
Nagdas S K, Mukherjee S, Mazumder B and Sen P C 1988 Identification and characterization of a Mg2+-dependent and an independent Ca2+-ATPase in microsomal membranes of rat testis; Mol. Cell. Biochem. 79 161169
Nandi J, Ray T K and Sen P C 1981 Studies of gastric Ca2+- stimulated ATPase: characterization and general properties; Biochim. Biophys. Acta 646 457464
Niggli V, Ronner P, Carafoli E and Penniston J T 1979 Effects of calmodulin on the Ca2+, Mg2+-ATPase partially purified from erythrocyte membranes; Arch. Biochem. Biophys. 198 124130
Nishizuka Y 1992 Intracellular signalling by hydrolysis of phospholipids and activation of protein kinase C; Science 258 607614
Potter J D and Johnson J D 1982 Troponin; in Calcium and cell function (ed.) Cheung Wy (New York: Academic Press) vol. 2, pp. 145173
Quist E E and Roufogalis B D 1975 Calcium transport in human erythrocytes. Separation and reconstitution of high and low calcium affinity (Mg, Ca)-ATPase activities in membranes prepared at low ionic strength; Arch. Biochem. Biophys. 168 240251
Robinson J D 1976 Ca2+, Mg2+-ATPase activity of rat brain microsomal preparation; Arch. Biochem. Biophys. 176 366374
Schatzmann H J 1975 Active calcium transport and calcium ion-activated ATPase in human red cells; Curr. Top. Memb. Trans. 6 125168
Schurmans-Stekhoven F and Bonting S L 1981 Transport adenosine triphosphatase: properties and functions; Physiol. Rev. 61 176
Sen P C, Kapakos J G and Steinberg M M 1981 Modification of Na+, K+-ATPase by fluorescein isothiocyanate: Evidence for the involvement of different amino groups at different pH values; Arch. Biochem. Biophys. 211 652662
Shami Y and Radde J C 1971 Calcium stimulated ATPase
of guinea pig placenta; Biochim. Biophys. Acta 249 345
Sikdar R, Ganguly U, Pal P, Mazumder B and Sen P C 1991 Biochemical characterization of a calcium stimulated ATPase from goat spermatozoa; Mol. cell. Biochem. 103 121130
Sillen L G and Martell A E 1971 Stability constant of metal ion complexes, Special publications 17 and 25 (London: The Chemical Society)
Stryer L 1988 Biochemistry (New York: W.H. Fremann) pp 957958
Sumida M, Hamada M, Shimowaka A, Morimoto C and Okuda H 1988 Ca2+ uptake in bovine adrenocortical microsomes: formation of phosphorylated intermediate of Ca2+-dependent ATPase; J. Biochem. (Tokyo) 104 687692
Vijaysarathai S, Shivaji S and Balaram P 1980 Plasma membrane bound Ca2+ in bull sperm; FEBS Lett. 114 4548
MS received 24 November 1998; accepted 3 May 1999
Corresponding editor: Samir Bhattacharya
BACK TO CONTENTS