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Different thermostabilities of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPases from rabbit and trout muscles
Camp. Eiochem.
Pergamon 0742-8413(95)00017-8
Ph.wio/. Vol. I I IC, No. I, pp. 93-98, 1995 Copyright c: 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0742-8413/95 $9.50 + 0.00
Different thermostabilities of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPases from rabbit and trout muscles F. G. S. de Toledo,* E. N. Chinit
M. C. Albuquerque,*
B. H. Goulart”
and
*Departamento de Bioquimica, Instituto de Ciencias Biomedicas, Universidade Federal do Rio de Janeiro, Cidade Universitaria, Ilha do Fundao, Rio de Janeiro, Brazil; and TNephrology Research Unit, Division of Nephrology and Department of Physiology and Internal Medicine, Mayo Clinic and Foundation, Rochester, Minnesota, U.S.A. Trout and rabbit (Ca*+ + Mg’+)-ATPases from sarcoplasmic reticulum were compared for differences in thermal inactivation and susceptibility to trypsin digestion. The trout ATPase is more heat-sensitive than the rabbit ATPase and is stabilized by Ca2+, Na+, K+ and nucleotides. Solubilization of both ATPases shows that the two ATPases have different protein-intrinsic inactivation kinetics. When digested by trypsin, the two ATPases display different cleavage patterns. The present results indicate that the trout and rabbit ATPases have dissimilarities in protein structure that may explain the differences in thermal inactivation kinetics. Rabbit; Key words: (Ca’+ + Mg2+)-ATPase; Muscle; adaptation; Rainbow trout; Conformational states. Comp. Biochem.
Physiol.
I1 lC, 93-98,
Sarcoplasmic
reticulum;
Temperature
1995.
Introduction Muscle relaxation is dependent on the Ca’+pumping ability of the sarcoplasmic reticulum (Ca’+ + Mg’+)-ATPase. This enzyme actively transports cytosolic Ca2+ into the sarcoplasmic reticulum stores at the expense of ATP hydrolysis (de Meis, 1981; Tanford, 1984). The proposed catalytic cycle of the (Ca’+ + Mg’+)ATPase is based on two conformation states: E, and E?. The E, form binds Ca2+ with a high affinity and can be phosphorylated by ATP, while the Ez form has a low affinity for Ca2+ and is phosphorylated by Pi. The sarcoplasmic reticulum (Ca2+ + Mg’+)ATPase of rabbit muscle has been extensively
studied. In contrast, few studies have been focused on this enzyme from other animals such as poikilothermic vertebrates. Comparative studies like this may lead to a better comprehension about the evolution of the sarcoplasmic reticulum and the structure of this protein (Ohnoki and Martonosi, 1980; Dux ef al., 1989; Vrjbar et al., 1990). It is well established that the rabbit (Ca’+ + Mg2+)-ATPase is temperature-dependent (de Meis, 1981; Tanford, 1984). We have previously shown that the trout and rabbit sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPases differ in temperature dependence (Chini et al., 1993). Below 20°C trout vesicles load Ca2+ faster than rabbit vesicles, a phenomenon that could be of physiological importance, since the trout is a cold-water fish. We have also found that in contrast to the rabbit ATPase, the phosphorylation of the trout enzyme shows little variation when the temperature is decreased from 20°C to 0°C and we proposed that
Cowespondence IO: E. N. Chini, Mayo Clinic-200. 1st Street SW-912 Guggenheim Building, Rochester, MN 55905. U.S.A. Received 12 October 1994; revised 19 December 1994; accepted 20 December 1994. Ahhreciakms: SDS, sodium dodecyl sulfate; EGTA, ethyleneglycol-bis-(P-aminoethylether)N,~,~’,~’-tetmacetic acid. 93
94
F. G. S. de Toledo
the differences in the temperature-dependence might be due to a different equilibrium between the E, and E, forms. In low temperatures, the trout ATPase would present higher levels of phosphorylation by Pi than the rabbit ATPase, because more E, conformational states are available at the equilibrium (Chini et al., 1993). This shifted equilibrium towards the E, conformational state may have consequences in thermal stability for the trout ATPase, since it is known that this form is more heat-sensitive (Palecz et al., 1988). The dissimilarities observed between the trout and rabbit (Ca*+ + Mg*+)-ATPases may be attributed to differences in protein structure, lipid environment or both. It was shown (Volmer and Velto, 1985) that the lipid microenvironment affects the locust (Ca*+ + Mg*+)-ATPase stability and that the carp and lobster ATPases have differences in protein structure when compared with the rabbit ATPase (Ohnoki and Martonosi, 1980; Dux et al., 1989; Hieu et al., 1992). Previously, we have shown that in opposition to the rabbit enzyme, the trout (Ca’+ + Mg*+)ATPase can be phosphorylated by Pi when the vesicle lipids are exchanged for the detergent C,*Es, thus suggesting that the two ATPases have differences in protein structure (Chini et al., 1993). These findings led us to investigate further differences between the two ATPases, concerning temperature stability and protein structure.
Materials and Methods Sarcoplasmic reticulum vesicles derived from rabbit skeletal muscle and from rainbow trout Oncorhynchus mykiss dorsolateral muscle were prepared as described by Eletr and Inesi (1972). The fish used were adapted to the temperature range of lo-18°C. Protein concentrations were determined according to Lowry et al. (1951) using bovine serum albumin as a standard. In both preparations the Ca*+-dependent ATPase activity was not inhibited by 1 mM ouabain, but was totally abolished by 1 mM ammonium vanadate. ATP hydrolysis was determined by the calorimetric method of Fiske and Subbarow (1925) and carried out in a reaction medium containing 50 mM MOPS-Tris buffer (pH 7.0) 10mM MgCl?, 0.1 mM CaCl,, 100 mM KCl, 3 mM potassium oxalate and 1 mM ATP. The reaction was quenched after 8 min with ice-cold 10% (w/v) trichloracetic acid. Blanks received acid before the start of the reaction. Tryptophan fluorescence was measured by a Hitachi F-3010 spectrofluorimeter. The excitation and emission wavelengths were set at 295 and 330 nm with 1.5 and 3 nm band widths, respectively. Sarcoplasmic reticulum vesicles
cl al.
(0.2 mg/ml) were incubated at 25 or 30°C in a medium containing 0.02 mM EGTA and 50 mM MOPS-Tris buffer (pH 7.0) and at 5 min Ca’+-induced fluorescence changes were observed by the addition of 0.025 mM CaCl,. The limited proteolysis of sarcoplasmic reticulum vesicles was carried out by incubating sarcoplasmic reticulum vesicles (33 mg/ml) with trypsin (1 .ll mg/ml) at 28°C for O-60 min in a medium containing 10mM MOPS-HOH (pH 6.0) and 0.1 M KCl. The digestion was stopped by addition of an equal volume of a solution containing 10% SDS, 20 mM HCl-Tris (pH 8.0) 2% fl-mercaptoethanol, 20% glycerol and 0.1% Bromophenol Blue and then incubation at 1OO’C for 3 min. Electrophoresis of digested samples was performed with 0.04 mg of protein/lane in a 615% gradient SDS-polyacrylamide gel and stained with Coomassie Blue (Laemmli, 1970). Values shown are averages from two to four experiments.
Results Protein
thermostahility
The thermal inactivation of both trout and rabbit sarcoplasmic reticulum (Ca*+ + Mg*+)ATPase was studied. The ATPases were incubated at 25’ or 3O”C, and at determined intervals, samples were withdrawn and assayed for ATPase activity (Fig. 1). The rabbit ATPase retains full activity when incubated in the temperature range of 25-3O’C in the presence of EGTA. In contrast, the trout ATPase is inactivated at 25’ and 30’C exhibiting a T,,? of 15.7 and 5.2 min, respectively. These data show that
I
01
0
5
10
15
20
Incubation time (min.) Fig. 1. Inactivation of the rabbit and trout (Ca” + Me’+)ATPase activity by temperture sarcoplasmic reticulum-vesicles (I mg/ml) were incubated at 25 C (0.n) or 30 C (*,A) in a medium containing 0.5 mM EGTA and 50 mM MOPS-Tris buffer (PH 7.0). Samples were withdrawn and diluted 50-fold in a‘;eaction medium at 30 C for assay of ATPase activity. (O,@) Rabbit sarcoplasmic reticulum vesicles. (a,A) Trout sarcoplasmic reticulum vesicles.
Thermostabilities
of sarcoplasmic
the trout (Ca’+ + Mg2+)-ATPase is inactivated at lower temperatures when compared with the rabbit enzyme. We also investigated the thermal denaturation of the ATPases by the fluorimetric method. Intrinsic tryptophan fluorescence of the rabbit (Ca*+ + Mg’+)-ATPase is increased by 334% upon Ca 2+ binding to the ATPase (Bigelow and Inesi, 1992). We have found that the trout ATPase also contains tryptophans that respond to the addition of Ca*+ at 25°C as described for the rabbit ATPase (Fig. 2). However, the Ca*+induced change in fluorescence of the trout ATPase is impaired at 30°C suggesting that protein unfolding is occurring. An alternative explanation is that, in contrast with the rabbit ATPase, the E, conformational state may be stabilized in the trout enzyme at 30°C. At the same temperatures, no denaturation was found for the rabbit ATPase (data not shown). EfSect of Iigands on the enzymatic
inactivation
It was previously described that ligands of the rabbit (Ca*+ + Mg*+)-ATPase such as Ca2+, potassium, sodium and nucleotides can yield protection against thermal inactivation of ATPase activity presumably by inducing a shift in the equilibrium of the E, and E, conformational state (Palecz et al., 1988; Kalabokis et al., 1993). We investigated whether this is also true for the trout ATPase. The dose-response of the protective effect of potassium, sodium and lithium in the trout ATPase activity is shown in Fig. 3. Sodium was as effective as potassium. Lithium also protects the ATPase, but to a lesser degree, suggesting that the protective effects of potassium and sodium are not related to an osmotic or ionic strength phenomenon, but to cation binding. We also found that K+ protected against the loss of Ca’+-induced
A,
reticulum
95
“L
80
100
120
140
Cation (mM) Fig. 3. Protection of trout (Ca2+ + Mg2+)-ATPase activity by Na+, K+ and Li+. Trout sarcoplasmic reticulum vesicles (I mg/ml) were incubated at 30°C for 15 min in a medium containing 0.5 mM EGTA and 50 mM MOPS-Tris buffer (pH 7.0) and different concentrations of NaCl (0). KCI (0) or LiCl (a). Samples were withdrawn and diluted 50-fold in a reaction medium at 30°C for ATPase activity measurements and plotted as the per cent of activity expressed by the control (non-incubated) samples.
fluorescence response in the trout ATPase (data not shown). As observed for the rabbit (Ca” + Mg2+)-ATPase (Palecz et al., 1988) Ca2+ was also able to protect the trout ATPase in micromolar concentrations (Fig, 4). Palecz et al. (1988) and Moller et al. (1980) have shown that nucleotides capable of binding to the rabbit (Ca’+ + Mg*+)-ATPase also provide protection against thermal inactivation. This is also true for the trout ATPase (Fig. 5). The protective effect was dose-related and proportional to the binding affinity for the
AF = +3 57%
Ca’+
O-4 0.0
I
0.0
I
2.5
I
5.0
I
7.5
2.5
5.0
7.5
10.0
I
10.0
Minutes Fig. 2. Ca *+-induced fluorescence change of trout ATPase. Trout sarcoplasmic reticulum vesicles (0.2 mg/ml) were incubated at 25” or 30 ‘C in a medium containing 0.02 mM EGTA and 50 mM MOPS-Tris buffer (pH 7.0). Ca’+-induced fluorescence changes were observed by addition of 0.025 mM CaCI, to the incubation medium at 5 min.
CaCl,
WI
Fig. 4. Protection of trout ATPase activity by Ca*+. Trout sarcoplasmic reticulum vesicles (I mg/ml) were incubated at 30’C for I5 min in a medium containing 0.5 mM EGTA and 50 mM MOPSSTris buffer (pH 7.0) and different free Ca’+ concentrations. Samples were withdrawn and diluted 50. fold in a reaction medium at 3O’C ATPase for activity measurements and plotted as the per cent of activity expressed by the control (non-incubated) samples.
96
F. G. S. de
‘oledo et al.
age pattern (Dux et al., 1985a,b; Dux and Martonosi, 1983). The trout ATPase, however, was barely digested showing small amounts of 80,000, 50,000 and 40,000 dalton fragments that appear paralleled to the fading of the 100,000 dalton band of the (Ca2+ + Mg’+)-ATPase. These data lead to the conclusion that the trout ATPase has a different disposition of sites for cleavage by trypsin, and thus differences in the primary structure, when compared with the rabbit ATPase.
“0.00
0.25
0.50
0.75
Discussion
1.00
Nucleotide (mM) Fig. 5. Protection of trout (Ca’+ + Mg’+)-ATPase activity by ATP, ADP and ITP. Trout sarcoplasmic reticulum vesicles (1 mg/ml) were incubated at 30°C for 15 min in a medium containing 0.5 mM EGTA and 50 mM MOPSTris buffer (pH 7.0) and different concentrations of ATP (0). ADP (0) or ITP (A). Samples were withdrawn and diluted 50-fold in a reaction medium at 3O’C for ATPase activity measurements and plotted as the per cent of activity expressed by the control (non-incubated) samples.
nucleotide 1980).
(Palecz
et al.,
Eflect of Triton X-100
1988; Merller et al.,
on protein
stability
The membrane lipids are important in maintaining (Ca*+ + Mg’+)-ATPase conformation structure and, therefore, enzymatic activity (Tanford, 1984). Detergents can cause inactivation of ATPase activity by solubilizing the protein (Msller et ul., 1980). We have found that both trout and rabbit ATPases show similar Triton X- 100 concentration-dependence for solubilization (data not shown). However, when incubated at 30°C with 0.316 mM free-Ca2+ (pCa 3.5) and then assayed for ATPase activity, the solubilized trout ATPase shows a remarkable inactivation rate, while the rabbit ATPase remains stable (Fig. 6). These data suggest that the dissimilarities observed in the rabbit and trout ATPase inactivation rates are related to intrinsic differences in the polypeptide chains between the two enzymes, since the lipids of both vesicles were equally exchanged for the detergent. Trypsin digestion of sarcoplusmic icles
reticulum
ces-
Both trout and rabbit vesicles were digested with trypsin for various times and samples were analyzed by polyacrylamide gel electrophoresis (Fig. 7). The rabbit sarcoplasmic reticulum vesicles showed products of digestion with molecular masses around 45,00&55,000 daltons, which is consistent with the previously described cleav-
Some animals, like fish, are not able to maintain a constant body temperature and therefore are affected by changes in environmental temperature. Since the rabbit (Ca” + Mg’+)ATPase shows a remarkable temperature dependence (de Meis, 1981; Chini et al., 1993) studies of ATPases from other species raise an interesting field of investigation, because changes in body temperature might alter the (Ca’+ + Mg2+)-ATPase function in those species. In previous work (Chini et al., 1993) we have shown that the trout (Ca’+ + Mg’+)-ATPase has a different temperature dependence for Ca2+ uptake and phosphorylation by Pi when compared with the rabbit ATPases, and we attributed the findings to a different equilibrium between the E, and E, conformational states of the enzyme. In this paper we show that the trout
Pre-Incubation
time (min.)
Thermostability of the (Ca’+ + Mg’+)-ATPase and rabbit). Sarcoplasmtc rettculum vestcles (trout (I mg/ml) were solubilized in Triton X-100 (10 mgjml) and incubated at 30 C in a medium containing 0.316 mM free Ca’+ (pCa 3.5) and 50 mM MOPS-Tris buffer (pH 7.0). Samples were withdrawn at determined intervals and diluted 50-fold in a reaction medium at 30 ‘C for assay of ATPase activity and plotted as the per cent of activity expressed by the control (non-incubated) samples. (0) Rabbit sarcoplasmic reticulum vesicles. (0) Trout sarcoplasmic reticulum vesicles.
Thennostabilities
of sarcoplasmic
reticulum
97
A
1234567
Fig. 7. Limited-proteolysis of sarcoplasmic reticulum vesicles. (A) Rabbit sarcoplasmic reticulum vesicles. (B) Trout sarcoplasmic reticulum vesicles. Lane 1: undigested control. Lanes 2-7: samples digested for 0.25, 1, 5, 15, 30 and 60min, respectively. Molecular mass standards are indicated in kDa.
ATPase is more temperature-sensitive for inactivation than the rabbit enzyme. The temperature dependence of the rabbit ATPase is explained by changes in the equilibrium between the conformational states of the enzyme (Pick and Karlish, 1982). In the rabbit ATPase the E, form of the enzyme is stabilized by low temperatures (below 20°C); in contrast the E, conformational state is the stable form at temperatures above 25530°C (Loomis et al., 1982; Pick and Karlish, 1982). This equilibrium probably does not vary significantly for the trout enzyme, since no temperature dependence is observed for the phosphorylation of ATPase by Pi (Chini et al., 1993). Since the E, isoform is more heatsensitive, the higher sensitivity to thermal inactivation of the trout ATPase might be explained by the fact that more E, isoforms are present in
the trout ATPase than in the rabbit ATPase at lower temperatures (Chini et al., 1993). It is interesting to note that the winter flounder and carp (Ca’+ + Mg’+)-ATPases are also more easily inactivated by temperature when compared with the rabbit ATPase (Dux et al., 1989; Vrjbar et al., 1990). We have also investigated the effect of ligands of the ATPase against thermal denaturation in the trout ATPase. The E2 conformational state is the stable form of the rabbit enzyme in the absence of ligands at temperatures above 25-C (Loomis et al., 1982). We have found that ligands capable of changing the equilibrium between E, and ET in favor of the E, conformational state, such as Ca2+ and nucleotides, were able to protect the trout enzyme against thermal inactivation. These data are consistent
98
F. G. S. de Toledo
with the view that the E, form, the stable conformational state in the absence of ligands, is more heat-sensitive, leading to an increased heat-sensitivity of the trout ATPase. The differences found here in heat-induced inactivation between rabbit and trout cannot be explained solely by distinct lipid compositions in the vesicle membranes, because both solubilized (Ca’+ + Mg*+)-ATPases still display differences in rates of inactivation, suggesting that the two ATPases must have dissimilarities in protein structure. The trypsin-digested ATPases displayed different cleavage patterns, and this finding reinforces the hypothesis that the two proteins have differences in structure. This might explain the differences in enzymatic inactivation rates. It is also interesting to note that the lobster and carp (Ca*+ + Mg*+)-ATPases also show 80,000 and 51,000 dalton fragments (Ohnoki and Martonosi, 1980; Dux et ul., 1989) hence suggesting that these isoforms are very closely related. Further research comparing differences in primary and secondary structure may lead to answers about how these ATPases have evolved among species and also how the amino acid sequence influences the equilibrium between the E, and E2 conformational state. For this purpose, it should be interesting to determine the primary structure of the trout ATPase. AcknoM,led~ements-This work was partially supported by Financiadora de Estudos e Projetos (FINEP) and by the Conselho National de Desenvolvimento Cientifico e Technologico (CNPq). We would like to thank Felipe 0. de Faria for the helpful discussion of the data and Kelly Beers for the discussion of the manuscript.
References Bigelow D. _I. and Inesi G. (1992) Contributions of chemical derivatization and spectroscopic studies to the characterization of the Ca” transport ATPase of sarcoplasmic reticulum. Biochim. biophps. Acra 1113, 323-338. Chini E. N.. de Toledo F. G. S.. Albuquerque M. C. and de Meis L. (1993) The Ca”-transporting ATPases of rabbit and trout exhibit different pH- and temperature dependences. Biochem. J. 293, 469473. de Meis L. (1981) In The Sarcoplasmic Reticulum: Transport and Energy Transduction: Transport in the L@ Sciences, Vol. 2 (Edited by Bittar, E. E.). John Wiley, New York. Dux L. and Martonosi A. (1983) Ca’+-ATPase membrane crystals in sarcoplasmic reticulum. J. biol. Chem. 258, 10111~10115. Dux L., Lelkes G., Hieu L. H. and Nemcsok J. (1989) Structural differences between the Cal+ -ATPase enzymes
et ul.
of sarcoplasmic reticulum membrane from rabbit and carp muscles. Comp. Biochem. Physiol. 92B, 263-270. Dux L., Papp S. and Martonosi A. (1985a) Conformational responses of the tryptic cleavage products of the CaLiATPase of sarcoplasmic reticulum. J. biol. Chem. 260, 13454-13458. Dux L.. Taylor K. A., Ping Ting-Beall H. and Martonosi A. (1985b) Crystallization of the Ca’+-ATPase of sarcoplasmic reticulum by calcium and lanthanide ions. J. biol. Chem. 260, 11730-I 1743. Eletr S. and Inesi G. (1972) Phospholipid orientation in sarcoplasmic membranes: spin-label ESR and proton MNR studies. Biochim. biophys. Acta 282, 246 269. Fiske C. H. and Subbarow Y. (1925) The calorimetric determination of phosphorus. J. biol. Chem. 66, 375400. Hieu L. H., Nemcsok J.. Molnar E. and Dux L. (1992) Different sensitivity of the sarcoplasmic reticulum Ca’+-ATPase enzyme to fluorescein-isothiocyanate in rabbit and carp muscles. Comp. Biochem. Physiol. 102B, 19923. Kalabokis V. N., Santoro M. M. and Hardwicke P. M. D. (1993) Effect of Na+ and nucleotide on the stability of solubilized Ca2+-free Ca-ATPase from scallop sarcoplasmic reticulum. Biochemistry 32, 4389-4396. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage - T4. Nat& (Lund.) 227,. 680~ 685. Loomis C. R., Martin D. W.. McCaslin D. R. and Tanford C. (1982) Phosphorylation of calcium adenosinetriphosphate by inorganic phosphate: Reversible inhibition at high magnesium ion concentration. Biochemurr~~ 21, 151-156. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurements with the Folin phenol reagent. J. biol. Chem. 193, 2655275. Msller J. V., Lind K. E. and Andersen J. P. (1980) Enzyme kinetics and substrate stabilization of detergent-solubilized and membranous (Ca’+ + Me” )-activated ATPase from sarcoplasmic reticulum. 7. biol. Chem. 255, 1912~1920. Ohnoki S. and Martonosi A. (1980) Structural diff‘erences between Ca’+ transport ATPases isolated from sarcoplasmic reticulum of rabbit. chicken and lobster muscle. Comp. Biochem. PI~~srol. 65B, 18 I-1 89. Palecz D.. Grzelinska E, Bartosz G., Leyko W. and Moller J. (1988) Ligand and lipid domain stabilization of a membranous Ca’ ’ -ATPase during hyperthermia. Biochim. bioDhv.v. Acre 931, 23-30. Pick U. and Karlish S. J. D. (1982) Regulation of the conformational transition in the Ca-ATPase from sarcoplasmic reticulum by pH, temperature. and calcium ions. J. biol. Chem. 257, 6120-6126. Tanford C. (1984) Twenty questions concerning the reaction cycle of the sarcoplasmic reticulum calcium pump. Crir. Ret,. Biochem. 17, 123-15 I. Volmer H. and Velter D. (1985) The effect of phospholipids on the thermostability of the ATPase from locust and crayfish sarcoplasmic reticulum. Camp. Biochem. Physio/. SlA, 761m 768. Vrbjar N., Simatos G. A. and Keough K. M. W. (1990) Temperature dependence of kinetic parameters of (Ca’+ + Mg’+)-ATPase in rabbit and winter flounder sarcoplasmic reticUlUm. Biochim. bioph~x. Acru 1030, 944100.
Pergamon 0742-8413(95)00017-8
Ph.wio/. Vol. I I IC, No. I, pp. 93-98, 1995 Copyright c: 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0742-8413/95 $9.50 + 0.00
Different thermostabilities of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPases from rabbit and trout muscles F. G. S. de Toledo,* E. N. Chinit
M. C. Albuquerque,*
B. H. Goulart”
and
*Departamento de Bioquimica, Instituto de Ciencias Biomedicas, Universidade Federal do Rio de Janeiro, Cidade Universitaria, Ilha do Fundao, Rio de Janeiro, Brazil; and TNephrology Research Unit, Division of Nephrology and Department of Physiology and Internal Medicine, Mayo Clinic and Foundation, Rochester, Minnesota, U.S.A. Trout and rabbit (Ca*+ + Mg’+)-ATPases from sarcoplasmic reticulum were compared for differences in thermal inactivation and susceptibility to trypsin digestion. The trout ATPase is more heat-sensitive than the rabbit ATPase and is stabilized by Ca2+, Na+, K+ and nucleotides. Solubilization of both ATPases shows that the two ATPases have different protein-intrinsic inactivation kinetics. When digested by trypsin, the two ATPases display different cleavage patterns. The present results indicate that the trout and rabbit ATPases have dissimilarities in protein structure that may explain the differences in thermal inactivation kinetics. Rabbit; Key words: (Ca’+ + Mg2+)-ATPase; Muscle; adaptation; Rainbow trout; Conformational states. Comp. Biochem.
Physiol.
I1 lC, 93-98,
Sarcoplasmic
reticulum;
Temperature
1995.
Introduction Muscle relaxation is dependent on the Ca’+pumping ability of the sarcoplasmic reticulum (Ca’+ + Mg’+)-ATPase. This enzyme actively transports cytosolic Ca2+ into the sarcoplasmic reticulum stores at the expense of ATP hydrolysis (de Meis, 1981; Tanford, 1984). The proposed catalytic cycle of the (Ca’+ + Mg’+)ATPase is based on two conformation states: E, and E?. The E, form binds Ca2+ with a high affinity and can be phosphorylated by ATP, while the Ez form has a low affinity for Ca2+ and is phosphorylated by Pi. The sarcoplasmic reticulum (Ca2+ + Mg’+)ATPase of rabbit muscle has been extensively
studied. In contrast, few studies have been focused on this enzyme from other animals such as poikilothermic vertebrates. Comparative studies like this may lead to a better comprehension about the evolution of the sarcoplasmic reticulum and the structure of this protein (Ohnoki and Martonosi, 1980; Dux ef al., 1989; Vrjbar et al., 1990). It is well established that the rabbit (Ca’+ + Mg2+)-ATPase is temperature-dependent (de Meis, 1981; Tanford, 1984). We have previously shown that the trout and rabbit sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPases differ in temperature dependence (Chini et al., 1993). Below 20°C trout vesicles load Ca2+ faster than rabbit vesicles, a phenomenon that could be of physiological importance, since the trout is a cold-water fish. We have also found that in contrast to the rabbit ATPase, the phosphorylation of the trout enzyme shows little variation when the temperature is decreased from 20°C to 0°C and we proposed that
Cowespondence IO: E. N. Chini, Mayo Clinic-200. 1st Street SW-912 Guggenheim Building, Rochester, MN 55905. U.S.A. Received 12 October 1994; revised 19 December 1994; accepted 20 December 1994. Ahhreciakms: SDS, sodium dodecyl sulfate; EGTA, ethyleneglycol-bis-(P-aminoethylether)N,~,~’,~’-tetmacetic acid. 93
94
F. G. S. de Toledo
the differences in the temperature-dependence might be due to a different equilibrium between the E, and E, forms. In low temperatures, the trout ATPase would present higher levels of phosphorylation by Pi than the rabbit ATPase, because more E, conformational states are available at the equilibrium (Chini et al., 1993). This shifted equilibrium towards the E, conformational state may have consequences in thermal stability for the trout ATPase, since it is known that this form is more heat-sensitive (Palecz et al., 1988). The dissimilarities observed between the trout and rabbit (Ca*+ + Mg*+)-ATPases may be attributed to differences in protein structure, lipid environment or both. It was shown (Volmer and Velto, 1985) that the lipid microenvironment affects the locust (Ca*+ + Mg*+)-ATPase stability and that the carp and lobster ATPases have differences in protein structure when compared with the rabbit ATPase (Ohnoki and Martonosi, 1980; Dux et al., 1989; Hieu et al., 1992). Previously, we have shown that in opposition to the rabbit enzyme, the trout (Ca’+ + Mg*+)ATPase can be phosphorylated by Pi when the vesicle lipids are exchanged for the detergent C,*Es, thus suggesting that the two ATPases have differences in protein structure (Chini et al., 1993). These findings led us to investigate further differences between the two ATPases, concerning temperature stability and protein structure.
Materials and Methods Sarcoplasmic reticulum vesicles derived from rabbit skeletal muscle and from rainbow trout Oncorhynchus mykiss dorsolateral muscle were prepared as described by Eletr and Inesi (1972). The fish used were adapted to the temperature range of lo-18°C. Protein concentrations were determined according to Lowry et al. (1951) using bovine serum albumin as a standard. In both preparations the Ca*+-dependent ATPase activity was not inhibited by 1 mM ouabain, but was totally abolished by 1 mM ammonium vanadate. ATP hydrolysis was determined by the calorimetric method of Fiske and Subbarow (1925) and carried out in a reaction medium containing 50 mM MOPS-Tris buffer (pH 7.0) 10mM MgCl?, 0.1 mM CaCl,, 100 mM KCl, 3 mM potassium oxalate and 1 mM ATP. The reaction was quenched after 8 min with ice-cold 10% (w/v) trichloracetic acid. Blanks received acid before the start of the reaction. Tryptophan fluorescence was measured by a Hitachi F-3010 spectrofluorimeter. The excitation and emission wavelengths were set at 295 and 330 nm with 1.5 and 3 nm band widths, respectively. Sarcoplasmic reticulum vesicles
cl al.
(0.2 mg/ml) were incubated at 25 or 30°C in a medium containing 0.02 mM EGTA and 50 mM MOPS-Tris buffer (pH 7.0) and at 5 min Ca’+-induced fluorescence changes were observed by the addition of 0.025 mM CaCl,. The limited proteolysis of sarcoplasmic reticulum vesicles was carried out by incubating sarcoplasmic reticulum vesicles (33 mg/ml) with trypsin (1 .ll mg/ml) at 28°C for O-60 min in a medium containing 10mM MOPS-HOH (pH 6.0) and 0.1 M KCl. The digestion was stopped by addition of an equal volume of a solution containing 10% SDS, 20 mM HCl-Tris (pH 8.0) 2% fl-mercaptoethanol, 20% glycerol and 0.1% Bromophenol Blue and then incubation at 1OO’C for 3 min. Electrophoresis of digested samples was performed with 0.04 mg of protein/lane in a 615% gradient SDS-polyacrylamide gel and stained with Coomassie Blue (Laemmli, 1970). Values shown are averages from two to four experiments.
Results Protein
thermostahility
The thermal inactivation of both trout and rabbit sarcoplasmic reticulum (Ca*+ + Mg*+)ATPase was studied. The ATPases were incubated at 25’ or 3O”C, and at determined intervals, samples were withdrawn and assayed for ATPase activity (Fig. 1). The rabbit ATPase retains full activity when incubated in the temperature range of 25-3O’C in the presence of EGTA. In contrast, the trout ATPase is inactivated at 25’ and 30’C exhibiting a T,,? of 15.7 and 5.2 min, respectively. These data show that
I
01
0
5
10
15
20
Incubation time (min.) Fig. 1. Inactivation of the rabbit and trout (Ca” + Me’+)ATPase activity by temperture sarcoplasmic reticulum-vesicles (I mg/ml) were incubated at 25 C (0.n) or 30 C (*,A) in a medium containing 0.5 mM EGTA and 50 mM MOPS-Tris buffer (PH 7.0). Samples were withdrawn and diluted 50-fold in a‘;eaction medium at 30 C for assay of ATPase activity. (O,@) Rabbit sarcoplasmic reticulum vesicles. (a,A) Trout sarcoplasmic reticulum vesicles.
Thermostabilities
of sarcoplasmic
the trout (Ca’+ + Mg2+)-ATPase is inactivated at lower temperatures when compared with the rabbit enzyme. We also investigated the thermal denaturation of the ATPases by the fluorimetric method. Intrinsic tryptophan fluorescence of the rabbit (Ca*+ + Mg’+)-ATPase is increased by 334% upon Ca 2+ binding to the ATPase (Bigelow and Inesi, 1992). We have found that the trout ATPase also contains tryptophans that respond to the addition of Ca*+ at 25°C as described for the rabbit ATPase (Fig. 2). However, the Ca*+induced change in fluorescence of the trout ATPase is impaired at 30°C suggesting that protein unfolding is occurring. An alternative explanation is that, in contrast with the rabbit ATPase, the E, conformational state may be stabilized in the trout enzyme at 30°C. At the same temperatures, no denaturation was found for the rabbit ATPase (data not shown). EfSect of Iigands on the enzymatic
inactivation
It was previously described that ligands of the rabbit (Ca*+ + Mg*+)-ATPase such as Ca2+, potassium, sodium and nucleotides can yield protection against thermal inactivation of ATPase activity presumably by inducing a shift in the equilibrium of the E, and E, conformational state (Palecz et al., 1988; Kalabokis et al., 1993). We investigated whether this is also true for the trout ATPase. The dose-response of the protective effect of potassium, sodium and lithium in the trout ATPase activity is shown in Fig. 3. Sodium was as effective as potassium. Lithium also protects the ATPase, but to a lesser degree, suggesting that the protective effects of potassium and sodium are not related to an osmotic or ionic strength phenomenon, but to cation binding. We also found that K+ protected against the loss of Ca’+-induced
A,
reticulum
95
“L
80
100
120
140
Cation (mM) Fig. 3. Protection of trout (Ca2+ + Mg2+)-ATPase activity by Na+, K+ and Li+. Trout sarcoplasmic reticulum vesicles (I mg/ml) were incubated at 30°C for 15 min in a medium containing 0.5 mM EGTA and 50 mM MOPS-Tris buffer (pH 7.0) and different concentrations of NaCl (0). KCI (0) or LiCl (a). Samples were withdrawn and diluted 50-fold in a reaction medium at 30°C for ATPase activity measurements and plotted as the per cent of activity expressed by the control (non-incubated) samples.
fluorescence response in the trout ATPase (data not shown). As observed for the rabbit (Ca” + Mg2+)-ATPase (Palecz et al., 1988) Ca2+ was also able to protect the trout ATPase in micromolar concentrations (Fig, 4). Palecz et al. (1988) and Moller et al. (1980) have shown that nucleotides capable of binding to the rabbit (Ca’+ + Mg*+)-ATPase also provide protection against thermal inactivation. This is also true for the trout ATPase (Fig. 5). The protective effect was dose-related and proportional to the binding affinity for the
AF = +3 57%
Ca’+
O-4 0.0
I
0.0
I
2.5
I
5.0
I
7.5
2.5
5.0
7.5
10.0
I
10.0
Minutes Fig. 2. Ca *+-induced fluorescence change of trout ATPase. Trout sarcoplasmic reticulum vesicles (0.2 mg/ml) were incubated at 25” or 30 ‘C in a medium containing 0.02 mM EGTA and 50 mM MOPS-Tris buffer (pH 7.0). Ca’+-induced fluorescence changes were observed by addition of 0.025 mM CaCI, to the incubation medium at 5 min.
CaCl,
WI
Fig. 4. Protection of trout ATPase activity by Ca*+. Trout sarcoplasmic reticulum vesicles (I mg/ml) were incubated at 30’C for I5 min in a medium containing 0.5 mM EGTA and 50 mM MOPSSTris buffer (pH 7.0) and different free Ca’+ concentrations. Samples were withdrawn and diluted 50. fold in a reaction medium at 3O’C ATPase for activity measurements and plotted as the per cent of activity expressed by the control (non-incubated) samples.
96
F. G. S. de
‘oledo et al.
age pattern (Dux et al., 1985a,b; Dux and Martonosi, 1983). The trout ATPase, however, was barely digested showing small amounts of 80,000, 50,000 and 40,000 dalton fragments that appear paralleled to the fading of the 100,000 dalton band of the (Ca2+ + Mg’+)-ATPase. These data lead to the conclusion that the trout ATPase has a different disposition of sites for cleavage by trypsin, and thus differences in the primary structure, when compared with the rabbit ATPase.
“0.00
0.25
0.50
0.75
Discussion
1.00
Nucleotide (mM) Fig. 5. Protection of trout (Ca’+ + Mg’+)-ATPase activity by ATP, ADP and ITP. Trout sarcoplasmic reticulum vesicles (1 mg/ml) were incubated at 30°C for 15 min in a medium containing 0.5 mM EGTA and 50 mM MOPSTris buffer (pH 7.0) and different concentrations of ATP (0). ADP (0) or ITP (A). Samples were withdrawn and diluted 50-fold in a reaction medium at 3O’C for ATPase activity measurements and plotted as the per cent of activity expressed by the control (non-incubated) samples.
nucleotide 1980).
(Palecz
et al.,
Eflect of Triton X-100
1988; Merller et al.,
on protein
stability
The membrane lipids are important in maintaining (Ca*+ + Mg’+)-ATPase conformation structure and, therefore, enzymatic activity (Tanford, 1984). Detergents can cause inactivation of ATPase activity by solubilizing the protein (Msller et ul., 1980). We have found that both trout and rabbit ATPases show similar Triton X- 100 concentration-dependence for solubilization (data not shown). However, when incubated at 30°C with 0.316 mM free-Ca2+ (pCa 3.5) and then assayed for ATPase activity, the solubilized trout ATPase shows a remarkable inactivation rate, while the rabbit ATPase remains stable (Fig. 6). These data suggest that the dissimilarities observed in the rabbit and trout ATPase inactivation rates are related to intrinsic differences in the polypeptide chains between the two enzymes, since the lipids of both vesicles were equally exchanged for the detergent. Trypsin digestion of sarcoplusmic icles
reticulum
ces-
Both trout and rabbit vesicles were digested with trypsin for various times and samples were analyzed by polyacrylamide gel electrophoresis (Fig. 7). The rabbit sarcoplasmic reticulum vesicles showed products of digestion with molecular masses around 45,00&55,000 daltons, which is consistent with the previously described cleav-
Some animals, like fish, are not able to maintain a constant body temperature and therefore are affected by changes in environmental temperature. Since the rabbit (Ca” + Mg’+)ATPase shows a remarkable temperature dependence (de Meis, 1981; Chini et al., 1993) studies of ATPases from other species raise an interesting field of investigation, because changes in body temperature might alter the (Ca’+ + Mg2+)-ATPase function in those species. In previous work (Chini et al., 1993) we have shown that the trout (Ca’+ + Mg’+)-ATPase has a different temperature dependence for Ca2+ uptake and phosphorylation by Pi when compared with the rabbit ATPases, and we attributed the findings to a different equilibrium between the E, and E, conformational states of the enzyme. In this paper we show that the trout
Pre-Incubation
time (min.)
Thermostability of the (Ca’+ + Mg’+)-ATPase and rabbit). Sarcoplasmtc rettculum vestcles (trout (I mg/ml) were solubilized in Triton X-100 (10 mgjml) and incubated at 30 C in a medium containing 0.316 mM free Ca’+ (pCa 3.5) and 50 mM MOPS-Tris buffer (pH 7.0). Samples were withdrawn at determined intervals and diluted 50-fold in a reaction medium at 30 ‘C for assay of ATPase activity and plotted as the per cent of activity expressed by the control (non-incubated) samples. (0) Rabbit sarcoplasmic reticulum vesicles. (0) Trout sarcoplasmic reticulum vesicles.
Thennostabilities
of sarcoplasmic
reticulum
97
A
1234567
Fig. 7. Limited-proteolysis of sarcoplasmic reticulum vesicles. (A) Rabbit sarcoplasmic reticulum vesicles. (B) Trout sarcoplasmic reticulum vesicles. Lane 1: undigested control. Lanes 2-7: samples digested for 0.25, 1, 5, 15, 30 and 60min, respectively. Molecular mass standards are indicated in kDa.
ATPase is more temperature-sensitive for inactivation than the rabbit enzyme. The temperature dependence of the rabbit ATPase is explained by changes in the equilibrium between the conformational states of the enzyme (Pick and Karlish, 1982). In the rabbit ATPase the E, form of the enzyme is stabilized by low temperatures (below 20°C); in contrast the E, conformational state is the stable form at temperatures above 25530°C (Loomis et al., 1982; Pick and Karlish, 1982). This equilibrium probably does not vary significantly for the trout enzyme, since no temperature dependence is observed for the phosphorylation of ATPase by Pi (Chini et al., 1993). Since the E, isoform is more heatsensitive, the higher sensitivity to thermal inactivation of the trout ATPase might be explained by the fact that more E, isoforms are present in
the trout ATPase than in the rabbit ATPase at lower temperatures (Chini et al., 1993). It is interesting to note that the winter flounder and carp (Ca’+ + Mg’+)-ATPases are also more easily inactivated by temperature when compared with the rabbit ATPase (Dux et al., 1989; Vrjbar et al., 1990). We have also investigated the effect of ligands of the ATPase against thermal denaturation in the trout ATPase. The E2 conformational state is the stable form of the rabbit enzyme in the absence of ligands at temperatures above 25-C (Loomis et al., 1982). We have found that ligands capable of changing the equilibrium between E, and ET in favor of the E, conformational state, such as Ca2+ and nucleotides, were able to protect the trout enzyme against thermal inactivation. These data are consistent
98
F. G. S. de Toledo
with the view that the E, form, the stable conformational state in the absence of ligands, is more heat-sensitive, leading to an increased heat-sensitivity of the trout ATPase. The differences found here in heat-induced inactivation between rabbit and trout cannot be explained solely by distinct lipid compositions in the vesicle membranes, because both solubilized (Ca’+ + Mg*+)-ATPases still display differences in rates of inactivation, suggesting that the two ATPases must have dissimilarities in protein structure. The trypsin-digested ATPases displayed different cleavage patterns, and this finding reinforces the hypothesis that the two proteins have differences in structure. This might explain the differences in enzymatic inactivation rates. It is also interesting to note that the lobster and carp (Ca*+ + Mg*+)-ATPases also show 80,000 and 51,000 dalton fragments (Ohnoki and Martonosi, 1980; Dux et ul., 1989) hence suggesting that these isoforms are very closely related. Further research comparing differences in primary and secondary structure may lead to answers about how these ATPases have evolved among species and also how the amino acid sequence influences the equilibrium between the E, and E2 conformational state. For this purpose, it should be interesting to determine the primary structure of the trout ATPase. AcknoM,led~ements-This work was partially supported by Financiadora de Estudos e Projetos (FINEP) and by the Conselho National de Desenvolvimento Cientifico e Technologico (CNPq). We would like to thank Felipe 0. de Faria for the helpful discussion of the data and Kelly Beers for the discussion of the manuscript.
References Bigelow D. _I. and Inesi G. (1992) Contributions of chemical derivatization and spectroscopic studies to the characterization of the Ca” transport ATPase of sarcoplasmic reticulum. Biochim. biophps. Acra 1113, 323-338. Chini E. N.. de Toledo F. G. S.. Albuquerque M. C. and de Meis L. (1993) The Ca”-transporting ATPases of rabbit and trout exhibit different pH- and temperature dependences. Biochem. J. 293, 469473. de Meis L. (1981) In The Sarcoplasmic Reticulum: Transport and Energy Transduction: Transport in the L@ Sciences, Vol. 2 (Edited by Bittar, E. E.). John Wiley, New York. Dux L. and Martonosi A. (1983) Ca’+-ATPase membrane crystals in sarcoplasmic reticulum. J. biol. Chem. 258, 10111~10115. Dux L., Lelkes G., Hieu L. H. and Nemcsok J. (1989) Structural differences between the Cal+ -ATPase enzymes
et ul.
of sarcoplasmic reticulum membrane from rabbit and carp muscles. Comp. Biochem. Physiol. 92B, 263-270. Dux L., Papp S. and Martonosi A. (1985a) Conformational responses of the tryptic cleavage products of the CaLiATPase of sarcoplasmic reticulum. J. biol. Chem. 260, 13454-13458. Dux L.. Taylor K. A., Ping Ting-Beall H. and Martonosi A. (1985b) Crystallization of the Ca’+-ATPase of sarcoplasmic reticulum by calcium and lanthanide ions. J. biol. Chem. 260, 11730-I 1743. Eletr S. and Inesi G. (1972) Phospholipid orientation in sarcoplasmic membranes: spin-label ESR and proton MNR studies. Biochim. biophys. Acta 282, 246 269. Fiske C. H. and Subbarow Y. (1925) The calorimetric determination of phosphorus. J. biol. Chem. 66, 375400. Hieu L. H., Nemcsok J.. Molnar E. and Dux L. (1992) Different sensitivity of the sarcoplasmic reticulum Ca’+-ATPase enzyme to fluorescein-isothiocyanate in rabbit and carp muscles. Comp. Biochem. Physiol. 102B, 19923. Kalabokis V. N., Santoro M. M. and Hardwicke P. M. D. (1993) Effect of Na+ and nucleotide on the stability of solubilized Ca2+-free Ca-ATPase from scallop sarcoplasmic reticulum. Biochemistry 32, 4389-4396. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage - T4. Nat& (Lund.) 227,. 680~ 685. Loomis C. R., Martin D. W.. McCaslin D. R. and Tanford C. (1982) Phosphorylation of calcium adenosinetriphosphate by inorganic phosphate: Reversible inhibition at high magnesium ion concentration. Biochemurr~~ 21, 151-156. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurements with the Folin phenol reagent. J. biol. Chem. 193, 2655275. Msller J. V., Lind K. E. and Andersen J. P. (1980) Enzyme kinetics and substrate stabilization of detergent-solubilized and membranous (Ca’+ + Me” )-activated ATPase from sarcoplasmic reticulum. 7. biol. Chem. 255, 1912~1920. Ohnoki S. and Martonosi A. (1980) Structural diff‘erences between Ca’+ transport ATPases isolated from sarcoplasmic reticulum of rabbit. chicken and lobster muscle. Comp. Biochem. PI~~srol. 65B, 18 I-1 89. Palecz D.. Grzelinska E, Bartosz G., Leyko W. and Moller J. (1988) Ligand and lipid domain stabilization of a membranous Ca’ ’ -ATPase during hyperthermia. Biochim. bioDhv.v. Acre 931, 23-30. Pick U. and Karlish S. J. D. (1982) Regulation of the conformational transition in the Ca-ATPase from sarcoplasmic reticulum by pH, temperature. and calcium ions. J. biol. Chem. 257, 6120-6126. Tanford C. (1984) Twenty questions concerning the reaction cycle of the sarcoplasmic reticulum calcium pump. Crir. Ret,. Biochem. 17, 123-15 I. Volmer H. and Velter D. (1985) The effect of phospholipids on the thermostability of the ATPase from locust and crayfish sarcoplasmic reticulum. Camp. Biochem. Physio/. SlA, 761m 768. Vrbjar N., Simatos G. A. and Keough K. M. W. (1990) Temperature dependence of kinetic parameters of (Ca’+ + Mg’+)-ATPase in rabbit and winter flounder sarcoplasmic reticUlUm. Biochim. bioph~x. Acru 1030, 944100.