Department of Chemistry, Bose Institute, 93/1, Acharya Prafulla Chandra Road, Calcutta 700 009, India

*Corresponding author (Fax, 91-33-3506790; Email, parimal@boseinst.ernet.in).

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.

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 10–15 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 with the
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 nM–250 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.

MS received 24 November 1998; accepted 3 May 1999