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Tryptophan phosphorescence of the Ca2+-ATPase of sarcoplasmic reticulum
Biochimica et Biophysica Acta, 957 (1988) 230-236 Elsevier
230
BBA 33260
Tryptophan phosphorescence of the Ca2+-ATPase of sarcoplasmic reticulum Jane M. Vanderkooi a Sandor Papp b, Slawomir Pikula b and Anthony Martonosi b a Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA and b Department of Biochemistry and Molecular Biology, State University of New York, Health Science Center, Syracuse N Y (U. S. A.)
(Received 30 March 1988)
Key words: Phosphorescence; Calcium ion ATPase; Tryptophan residue; Sarcoplasmic reticulum
Phosphorescence of protein tryptophan was analyzed in sarcoplasmic reticulum vesicles, and in the purified Ca 2+ transport ATPase in deoxygenated aqueous solutions at room temperature. Upon excitation with light of 295 nm wavelength, the emission maxima of fluorescence and phosphorescence were at 330 nm and at 445 nm, respectively. The phosphorescence decay was multiexponential; the lifetime of the long-lived component of phosphorescence was --22 ms. ATP and vandate significantly reduced the phosphorescence in the presence of either Ca 2+ or EGTA; ADP was less effective, while AMP was without effect. The quenching by ATP showed saturation consistent with the idea that the ATP-enzyme complex had a lower phosphotcscetlce yield. Upon exhaustion of ATP, the phosphorescence returned to starting level. Significant quenching of phosphorescence with a decrease in phosphorescence lifetime was also caused by NaNO2, methylvinyl ketone and trichioroacetate, without effect on ATPase activity; this quenching did not show saturation and was therefore probably collisional in nature.
Introduction Protein phosphorescence has received little attention so far, perhaps because oxygen at the usual concentration of --250 /tM, reduces the lifetime and intensity of protein phosphorescence, rendering it difficult to detect [1-3]. The powerful quenching of phosphorescence by 02 is due to its ability to penetrate through the proteins, reaching even masked tryptophans with rate constants near the diffusional limit (Ref. 4 see, however, Ref. 5). For reliable measurement of tryptophan phosphorescence the oxygen concentration of the soluCorrespondence: J.M. Vanderkooi, Department of Biochemistry and Biophysics, University School of Medicine, Philadelphia, PA 19104, U.S.A.
tion must be maintained at submicromolar levels by flushing the system with argon, sealing the solutions from atmospheric oxygen with an overlay of heavy mineral oil, and by removing oxygen traces from the solutions with oxygen scavengers, such as glucose oxidase and dithionite [4,6,7]. The relatively easy detection of tryptophan phosphorescence under these conditions in several water-soluble proteins [6,8,9] opened a new approach for the investigation of protein conformational dynamics on a time-scale much longer than that permitted by tryptophan fluorescence. While the photon emission from the singlet state in tryptophan fluorescence occurs within nanoseconds [10], the lifetime of triplet state involved in phosphorescence emission is of the order of 1-1000
ms [6].
0167-4838/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
231 The Ca2+-ATPase of sarcoplasmic reticulum alternates between two major conformations, designated E 1 and E 2, during Ca 2+ transport [11,12]. The equilibrium between the El and E 2 states is influenced by ligands that preferentially react with the enzyme in either of the two states (for review, see Ref. 13). The E1 conformation is stabilized by Ca 2+ or lanthanides [14], leading to the formation of Ca2+-ATPase crystals, with a two-sided plane group of pl [15]. In the absence of Ca 2+, the Ca2+-ATPase reacts with inorganic phosphate or vanadate (V) in the E 2 conformation, inducing the formation of E 2 type Ca 2+-ATPase crystals [16,17], with a two-sided plane group of p2 [18-20]. The tryptophan fluorescence of the Ca 2+ATPase is greater in the E~ state stabilized by Ca 2+ [21-24] or lanthanides [14] than in the E 2 state stabilized by vanadate or inorganic phosphate [25]. Increasing the Ca 2+ concentration in the presence of vanadate disrupts the vanadate-induced p2 crystals of Ca2+-ATPase [17] and increases the intensity of tryptophan fluorescence [25] by shifting the equilibrium from the E 2 toward the E~ state. Although stabilization of the E 2 conformation by vanadate is observed only at submicromolar Ca 2+ concentrations, r,nd Ca 2+ decreases the affinity of vanadate for the Ca 2+ATPase [26], simultaneous binding of Ca 2+ and vanadate to the Ca2+-ATPase with an apparent dissociation constant of = 7 #M has been observed [27]. In this report observations are presented on the phosphorescence of tryptophan in the Ca 2+ATPase of sarcoplasmic reticulum. The intensity of phosphorescence is influenced by ATP, vanadate and water-soluble quenchers, such as NaNO 2, methylvinyl ketone and trichloroacetate. Materials and Methods
Materials Glucose oxidase, catalase, lactic dehydrogenase (rabbit muscle), pyruvate kinase (rabbit muscle), EGTA, NaNO 2, methylvinyl ketone, adenosine 5'-triphosphate, adenosine 5'-diphosphate, adenosine 5'-monophosphate and phosphoenolpyruvate were obtained from Sigma Chemical Co. (St. Louis, MO), sodium dithionite from Virginia Chemical Co. (Portsmouth, VA) and grade 5 argon
gas from Airco (Murray Hill, N J). Sodium vanadate and trichloroacetic acid were supplied by Fisher Scientific (Fairlawn, N J). A23187 was the product of Behring Diagnostics (Calbiochem), La Jolla, CA.
Methods Sarcoplasmic reticulum vesicles were isolated from predominantly white muscles of rabbits, as described earlier [28]. The purified Ca2+-ATPase was prepared according to Meissner et al. [29]. The sarcoplasmic reticulum lipids were extracted with chloroform/methanol (2:1, v/v) according to Folch et al. [30]. The organic solvent phase was evaporated to dryness under a nitrogen stream at 35-40°C. The phospholipid phosphorus was determined according to Bartlett [31]. The Lowry method [32] or the biuret procedure [33] were used for the determination of proteins. For phosphorescence measurements, the samples were prepared under the conditions described by Englander et al. [4] to exclude oxygen. Sarcoplasmic reticulum or purified ATPase preparations (1.4-1.6 mg protein/ml) were dispersed in a medium of argon-saturated 0.1 M KCI, 0.01 M Tris-HCl (pH 7.4), 5 mM MgCI 2 containing the deoxygenating system of glucose oxidase (80 nM), catalase (16 riM) and glucose (3 mg/ml). Other additions are indicated in the figure legends. After incubation at room temperature for 10 min, sodium dithionite was added to a final concentration of 80 /zM, the samples were overlaid with argon-saturated mineral oil, and the cuvette was closed with a glass stopper. The phosphorescence spectra were measured at 350-650 nm in a Perkin Elmer model LS-5 phosphorescence spectrometer using light of 295 nm wavelength for excitation with a time delay of 0.5 ms and a gate time of 2 ms. For measurement of phosphorescence lifetimes a Perkin Elmer MPF-2A fluorometer was used, interfaced with an A T & T 6300 computer. The CW lamp of the fluorometer was replaced by a xenon flash lamp [7]. Data were analyzed using the Asystant software package (MacMillan Software Company, New York, NY). The phosphorescence lifetime was measured at an emission wavelength of 440 nm, using light of 295 nm for e~citation. The lifetimes were calculated from the average of 200-500 decay curves from data col-
232 lected between 1-150 ms after the light flash of --5 /ts duration. Since emissions that occurred within 1 ms after the exciting light flash were not included in the evaluation, the estimated lifetimes reflect the long-lived components of the phosphorescence.
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Fluorescence and phosphorescence spectra of sarcoplasmic reticulum Excitation of sarcoplasmic reticulum vesicles with light of 295 nm wavelength in a medium of 0.1 M KCI, 10 mM Tris-HCl buffer (pH 7.4), and 5 mM MgCI ~, causes the emission of fluorescence and phosphorescence (Fig. 1). The emission maximum of fluorescence is near 340 nm (Fig. 1A), and that of the much weaker phosphorescence at
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Fig. 1. Fluorescence and phosphorescence spectra of sarcoplasmic reficulum vesicles, purified Ca2+-ATPase, and extracted lipids. The sarcoplasmic reticulum (sample 1), purified ATPase (sample 2) and the iiposomesprepared from extracted lipids (sample 3) weredispersed in 0.1 M KCi, 10 mM Tris-HC! (pH 7.4), 5 mM MgCI, and prepared for deoxygenation, as described under Methods. The final protein concentrations in A and B were 1.6 mg protein/ml; the concentration of lipids in C was 1.5 mg/ml. Excitation wavelength: 295 nm. Temperature: 24°C. The fluorescence (A) and phosphorescence (B) spectra were measured as described under Methods.
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Fig. 2. The effect of ATP on the phosphorescence of sarcoplasmic reticulum vesicles. The phosphorescence of sarcoplasmic reticulum vesicles (protein concentration 1.46 mg/ml) was measured at 20°C in a medium of 0.1 M KCI, 10 mM Tris-HCI (pH 7.4), 5 mM MgCI2, and =10 -5 M CaC!2, without further addition (line 1), and after the addition of 3 mM ATP (line 2) or 6 mM ATP (line 3).
445 nm (Fig. 1B). The intensity ratio of fluorescence/phosphorescence emission is greater than 100. The sharp band in Fig. 1B near 590 nm is due to second-order scattering of the exciting light by the turbid vesicle suspension. The phosphorescence emission of purified Ca 2+-ATPase is weaker than that of the sarcoplasmic reticulum vesicles (Fig. 1B). Thi~ difference is probably due to the presence of highly phosphorescent accessory proteins in the sarcoplasmic reticulum, that were removed during the purification of the Ca 2+ATPase. The extracted sarcoplasmic reticulum lipids have only slight phosphorescence in the wavelength range of 350-550 nm (Fig. 1B). Solubilization of sarcoplasmic reticulum with deoxycholate (1 mg per mg protein) did not alter significantly the wavelength distribution or intensity of phosphorescence. Therefore the lower phosphorescence emission of purified ATPase, as compared with sarcoplasmic reticulum (Fig. 1), is not attributable to the use of deoxycholate for the purification of the Ca2+-ATPase. Deoxycholate (1 m g / m g phospholipid) also had no influence on the phosphorescence of extracted sarcoplasmic reticulum lipids.
The effect of.4 TP on phosphore.~cence The phosphorescence was significantly reduced by 3 - 6 mM ATP in the presence of ---10 -5 M Ca 2+ (Fig. 2) or 1 m M EGTA (not shown). Since
233
lowering the Ca 2+ concentration by EGTA inhibits the phosphorylation of Ca2+-ATPase by ATP, these observations suggest that the decrease in phosphorescence caused by ATP is due to the formation of enzyme-ATP complex. Under similar conditions ADP (3-6 mM) had only slight effect on the phosphorescence, while AMP was ineffective. Therefore the phosphorescence quenching by ATP cannot be due to absorption of the exciting light or to quenching by free nucleotides in solution. The phosphorescence returned to starting level after the added ATP was hydrolyzed by the Ca2+-ATPase, indicating that secondary changes in the system caused by the hydrolysis of ATE such as a decrease in pH, contribute only slightly, if at all, to the observed decrease in phosphorescence. The phosphorescence of parvalbumin, a Ca2÷-binding protein which does not bind ATP, was not quenched by ATP in this concentration range (unpublished observation). This is a further, indirect evidence, that ATP in solution is not responsible for the observed quenching of phosphorescence in the enzyme-ATP complex.
The effect of vanadate on the phosphorescence of sarcoplasmic reticulum and purified Ca 2 +-A TPase The tryptophan phosphorescence of sarcoplasmic reticulum vesicles was measured in a medium of 0.1 M KCI, 10 rnM Tris-HCl (pH 7.4), 5 mM MgCI 2 and either 0.5-1.0 mM CaC! 2 or 0.2 mM EGTA. Addition of 0.075-0.2 mM Na 2VO4 to the vesicles caused similar decrease in the intensity of phosphorescence in the presence of either Ca 2÷ or EGTA (Fig. 3). At a vanadate concentration of 0.05 mM, the quenching of phosphorescence by vanadate was frequently greater in the presence of EGTA than in the presence of Ca 2+, but these differences were not consistently reproducible. The effect of vanadate on the tryptophan phosphorescence is a property of the Ca 2÷ATPase, since the magnitude of the effect is similar in sarcoplasmic reticulum, in the purified Ca2÷-ATPase prepared according to Meissner et al. [29], and in sarcoplasmic reticulum preparations that were treated with 1 mM EDTA in a medium of 0.1 M KCI and 10 mM Tris-HCl (pH 7.9) overnight at 2°C to remove the extrinsic water soluble proteins from the membrane [34].
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Fig. 3. The effect of vanadate on the phosphorescence of sarcoplasmic reticulum. (A) The phosphorescence of sarcoplasmic reticulum was measured as described under Methods and Fig. 1, in a medium of 0.1 M KCI, 10 mM Tris (pH 7.4), 5 mM MgCI 2 and =10 -5 M CaCi2 (1). To sample 2, 0.1 mM EGTA, and to sample 3, 0.2 mM EGTA+0.1 mM vanadate were added. (B) Sample 1 has an identical composition to that of A (1). To sample 2, 1 mM CaC! 2, and to sample 3, 1 mM CaCi 2 + 0.1 mM vanadate were added.
Phosphorescence lifetime data on sarcoplasmic reticulum The phosphorescence decay was multiexponential (Fig. 4). Due o technical difficulties in the 0.2
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Fig. 4. Decay curve of phosphorescence. The phosphorescence decay was measured in a medium of 0.1 M KCi, 10 mM Tris-HCI buffer (pH 7.4) and 5 mM MgCl 2 at 24°C, as described in Methods. Excitation wavelength: 295 nm; emission wavelength: 440 nm.
234 TABLE I PHOSPHORESCENCE LIFETIME PLASMIC R E T I C U L U M
DATA
ON SARCO-
The phosphorescence decay measured between 1-150 ms after the exciting light flash was multiexponential. In the table, the lifetimes of the slowest component are given, based on an average of 200-500 scans. For other details, see text. The number of independent measurements are denoted by n. n
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activity of sarcop!asmic reticulum assayed either in the absence or in the presence of 10 ItM A23187. Methylvinyl ketone and trichloroacetate caused modest (20-30%) inhibitien of ATPase activity at 45.5 mM concentration, w~ale NaNO 2 was ineffective, even under these conditions. Therefore denaturation of the Ca2+-ATPase is not likely to contribute to the quenching of phosphorescence by either of these agents. Our observations on the effect of trichloroacetate on phosphorescence are in agreement with earlier fluorescence and kinetic data [35,36]. Discussion
measurement of phosphorescence decay at times immediately following the flash of exciting light, only data collected between 1 and 150 ms after the exciting flash of 5 Its duration were included in the evaluation of phosphorescence lifetime. The lifetime of long-lived components of phosphorescence calculated as the average of 19 experiments, was 22 ms (Table I). In spite of the significant quenching of phosphorescence by vanadate, the average lifetime measured under these conditions was only slightly reduced. Therefore the phosphorescence quenching by vanadate affects primarily the short-lived components of phosphorescence that are not included in the dataset samples for phosphorescence decay. The phosphorescence lifetime of sarcoplasmic reticulum was not affected significantly by 20 mM Ca z+ (Table I).
The quenching of phosphorescence by NaNO 2, methyivinlpi ketone and trichloroacetate The intensity and lifetime of the phosphorescence of sarcoplasmic reticulum vesicles was reduced by NaNO 2 and trichloroacetate at submillimolar, and by methylvinyl ketone at millimolar concentrations. The quenching did not show evidence of saturation (data not shown), and it is therefore likely to be collisional. At concentrations up to 4.5 mM neither of the three quenchers had significant effect on the CaZ+-activated ATPase
Room temperature phosphorescence was detected and analyzed in sarcoplasmic reticulum vesicles. A major component of phosphorescence is attributed to tryptophan residues of the Ca 2+ATPase, but other protein components of the sarcoplasmic reticulum may also contribute. Only slight phosphorescence was observed after excitation at 295 nm in the isolated sarcoplasmic reticulum lipids. ATP at millimolar concentrations diminished the phosphorescence intensity by as much as 25-30%; ADP was less effective and AMP was essentially without effect. The ATP effect was observed even after the removal of Ca z+ by EGTA, suggesting that the phosphorylation of Ca 2÷ATPase is not required for the quenching of phosphorescence. Therefore the effect of ATP is presumed to be due to the formation of an enzymeATP complex. The phosphorescence quenching by ATP (25-30%) is an order of magnitude greater than the ATP-induced change in tryptophan fluorescence [24]. This may imply greater sensitivity of the long-lived triplet state, as compared with singlet state, to quenching by ATE Alternatnvely, the phosphorescence emission may involve a ~;:abpopulation of tryptophan residues that includes a relatively large proportion of the tryptophan:~ affected by ATP. The phosphorescet:ce emission in intact sarcoplasmic reticulum vesicles and in the purified Ca2+-ATPase preparations was also quer~ched by vanadate with only slight effect on the lifetime of the long-lived components of the phosphorescence
235
emission. The quenching of phosphorescence by vanadate was observed in the presence of either Ca 2+ 10.5-1.0 mM) or EGTA (0.2 mM). Since the stabilization of E~ conformation by vanadate is observed only at submicromolar Ca 2÷ concentration, these observations suggest that a localized change in the environment of tryptophan caused by the binding of vanadate to the CaZ+-ATPase, rather than a vanadate-induced shift in the conformation of the enzyme from the E 1 to the E 2 state is involved in the phosphorescence quenching. In addition to the indirect effects of ATP or vanadate on the environment and emission characteristics of protein tryptophan(s), a more direct static quenching by the bound ATP or vanadate of the tryptophan residues located near the substrate binding site remains a possibility. The fast and slow isoenzymes of the Ca 2÷ATPase of sarcoplasmic reticulum contain 13 tryptophans [37]. Twelve of these are located in hydrophobic fragments of Ca 2+-ATPase, and these are assigned in the predicted secondary structure of the enzyme to the membrane-spanning and stalk helices near the surface of the bilayer. Their relationship to the vanadate binding site is unknown. One tryptophan (residue 552) is assigned to the nucleotide binding region of the cytoplasmic, domain, not far from the exposed T 1 tryptic cleavage site (residue 505) and from the binding site for fluorescein 5'-isothiocyanate (residue 515). This tryptophan residue is a plausible candidate for direct quenching by ATP bound at the nucleotide binding site.
Acknowledgements Supported by research grants GM34448 and AM26545 from the National Institutes of Health, PCM84-03679 and Int. 86-17848 from the National Science Foundation, and by a grant-in-aid from the Muscular Dystrophy Association and grant OTKA 665 from Hungary.
References 1 Calhoun, D.B., Vanderkooi, J.M., Woodrow, G.V. II1 and Englander, S.W. (1983) Biochemistry 22, 1526-1532. 2 Calhoun, D.B., Vanderkooi, J.M. and Englander, S.W. (1983) Biochemistry 22, 1533-1539.
3 Calhoun, D.B., Vanderkooi, J.M., Holtom, G.R. and Englander, S.W. (1986) Proteins: Structure, Function and Genetics 1,109-115. 4 Englander, S.W., Calhoun, D.B. and Englander, J.J. (1987) Anal. Biochem. 161,300-306. 5 Strambini, G.B. (1987) Biophys. J. 52, 23-28. 6 Vanderkooi, J.M., Calhoun, D.B. and Englander, S.W. (1987) Science 236, 568-569. 7 Vanderkooi, J.M., Maniara, G., Green, T.J. and Wilson, D.F. (1987) J. Biol. Chem. 262, 5476-5482. 8 Saviotti, M.L. and Galley, W.C. (1974) Proc. Natl. Acad. Sci. USA 71, 4154-4158. 9 Eftink, M.R. and Ghiron, C.A. (1981) Anal. Biochem. 114, 199-227. 10 Lakowicz, J.R. (1983) Principles of Fluorescence Spectroscopy, Plenum Press, New York. 11 Martonosi, A. and Beeler, T.J. (1983) in Handbook of Physiology. Section 10. Skeletal Muscle (Peachey, L.D. and Adrian, R.H., eds.), pp. 417-485, American Physiological Society, Bethesda. 12 lnesi, G. and de Meis, L. (1985) in The Enzymes of Biological Membranes, 2nd Edn. (Martonosi, A., ed.), Vol. 3, pp. 157-191, Plenum Press, New York. 13 Martonosi, A., Taylor, K.A., Varga, S. and Ting-Beall, H.P. (1987) in Electron Microscopy of Proteins, Vol. 6, Membranous Structures (Harris, J.R. and Home, R.W., eds.), pp. 254-376, Academic Press, London. 14 Jona, I. and Martonosi, A. (1986) Biochem. J. 234, 363-371. 15 Dux, L., Taylor, K.A., Ting-Beall, H.P. and Martonosi, A. (1985) J. Biol. Chem. 260, 11730-11743. 16 Dux, L. and Martonosi, A. (1983) J. Biol. Chem. 258, 2599-2603. 17 Dux, L. and Martonosi, A. (1983) J. Biol. Chem. 258, 11896-11902. 18 Taylor, K.A., Dux, L. and Martonosi, A. (1984) J. Moi. Biol. 174, 193-204. 19 Taylor, K.A., Dux, L. and Martonosi, A. (1986) J. Mol. Biol. 187, 417-427. 20 Taylor, K.A., Ho, M.H. and Martonosi, A. (1986) Ann. NY Acad. Sci. 483, 31-43. 21 Dupont, Y. (1976) Biochem. Biophys. Res. Commun. 71, 544-550. 22 Dupont, Y. 11978) Biochem. Biophys. Res. Commun. 82, 893-900. 23 Dupont, Y. and Leigh. J.B. 11978) Nature 273, 396-398. 24 Dupont, Y., Bennett, N. and Lacapere, J.-J. 11982) Ann. NY Acad. Sci. 402, 569-572. 25 Pick, U. and Karlish, S.J.D. 11982) J. Biol. Chem. 257, 6120-6126. 26 Medda, P. and Hasselbach, W. 11983) Eur. J. Biochem. 137, 7-14. 27 Markus, S., Priel, Z. and Chipman, D.M. 11986) Biochim. Biophys. Acta 874, 128-135. 28 Varga, S., Mullner, N., Pikula, S., Papp, S., Varga, K. and Martonosi, A. 11986) J. Biol. Chem. 261, 13943-13956. 29 Meissner, G., Conner, G.E. and Fleischer, S. 11973) Biochim. Biophys. Acta 298, 246-269. 30 Folch, J., Lees, M. and Sloane Stanley, G.IH. 11957) J. Biol. Chem. 226, 497-509.
236 31 Bartlett, G.R. (1959) J. Biol. Chem. 234, 446-468. 32 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 33 Gornall, A.G., Bardawill, C.J. and David, M.M. (1949) J. Biol. Chem. 177, 751-766. 34 Duggan, P.F. and Martonosi, A. (1970) J. Gen. Physiol. 56, 147-167.
35 The, R. and Hasselbach, W. (1975) Eur. J. Biochem. 53, 105-113. 36 Ludi, H. and Hasseibach, W. (1983) Biochim. Biophys. Acta 732, 479-482. 37 Brandi, C.J., Green, N.M., Korczak, B. and MacLennan, D.H. (1986) Cell 44, 597-607.
230
BBA 33260
Tryptophan phosphorescence of the Ca2+-ATPase of sarcoplasmic reticulum Jane M. Vanderkooi a Sandor Papp b, Slawomir Pikula b and Anthony Martonosi b a Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA and b Department of Biochemistry and Molecular Biology, State University of New York, Health Science Center, Syracuse N Y (U. S. A.)
(Received 30 March 1988)
Key words: Phosphorescence; Calcium ion ATPase; Tryptophan residue; Sarcoplasmic reticulum
Phosphorescence of protein tryptophan was analyzed in sarcoplasmic reticulum vesicles, and in the purified Ca 2+ transport ATPase in deoxygenated aqueous solutions at room temperature. Upon excitation with light of 295 nm wavelength, the emission maxima of fluorescence and phosphorescence were at 330 nm and at 445 nm, respectively. The phosphorescence decay was multiexponential; the lifetime of the long-lived component of phosphorescence was --22 ms. ATP and vandate significantly reduced the phosphorescence in the presence of either Ca 2+ or EGTA; ADP was less effective, while AMP was without effect. The quenching by ATP showed saturation consistent with the idea that the ATP-enzyme complex had a lower phosphotcscetlce yield. Upon exhaustion of ATP, the phosphorescence returned to starting level. Significant quenching of phosphorescence with a decrease in phosphorescence lifetime was also caused by NaNO2, methylvinyl ketone and trichioroacetate, without effect on ATPase activity; this quenching did not show saturation and was therefore probably collisional in nature.
Introduction Protein phosphorescence has received little attention so far, perhaps because oxygen at the usual concentration of --250 /tM, reduces the lifetime and intensity of protein phosphorescence, rendering it difficult to detect [1-3]. The powerful quenching of phosphorescence by 02 is due to its ability to penetrate through the proteins, reaching even masked tryptophans with rate constants near the diffusional limit (Ref. 4 see, however, Ref. 5). For reliable measurement of tryptophan phosphorescence the oxygen concentration of the soluCorrespondence: J.M. Vanderkooi, Department of Biochemistry and Biophysics, University School of Medicine, Philadelphia, PA 19104, U.S.A.
tion must be maintained at submicromolar levels by flushing the system with argon, sealing the solutions from atmospheric oxygen with an overlay of heavy mineral oil, and by removing oxygen traces from the solutions with oxygen scavengers, such as glucose oxidase and dithionite [4,6,7]. The relatively easy detection of tryptophan phosphorescence under these conditions in several water-soluble proteins [6,8,9] opened a new approach for the investigation of protein conformational dynamics on a time-scale much longer than that permitted by tryptophan fluorescence. While the photon emission from the singlet state in tryptophan fluorescence occurs within nanoseconds [10], the lifetime of triplet state involved in phosphorescence emission is of the order of 1-1000
ms [6].
0167-4838/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
231 The Ca2+-ATPase of sarcoplasmic reticulum alternates between two major conformations, designated E 1 and E 2, during Ca 2+ transport [11,12]. The equilibrium between the El and E 2 states is influenced by ligands that preferentially react with the enzyme in either of the two states (for review, see Ref. 13). The E1 conformation is stabilized by Ca 2+ or lanthanides [14], leading to the formation of Ca2+-ATPase crystals, with a two-sided plane group of pl [15]. In the absence of Ca 2+, the Ca2+-ATPase reacts with inorganic phosphate or vanadate (V) in the E 2 conformation, inducing the formation of E 2 type Ca 2+-ATPase crystals [16,17], with a two-sided plane group of p2 [18-20]. The tryptophan fluorescence of the Ca 2+ATPase is greater in the E~ state stabilized by Ca 2+ [21-24] or lanthanides [14] than in the E 2 state stabilized by vanadate or inorganic phosphate [25]. Increasing the Ca 2+ concentration in the presence of vanadate disrupts the vanadate-induced p2 crystals of Ca2+-ATPase [17] and increases the intensity of tryptophan fluorescence [25] by shifting the equilibrium from the E 2 toward the E~ state. Although stabilization of the E 2 conformation by vanadate is observed only at submicromolar Ca 2+ concentrations, r,nd Ca 2+ decreases the affinity of vanadate for the Ca 2+ATPase [26], simultaneous binding of Ca 2+ and vanadate to the Ca2+-ATPase with an apparent dissociation constant of = 7 #M has been observed [27]. In this report observations are presented on the phosphorescence of tryptophan in the Ca 2+ATPase of sarcoplasmic reticulum. The intensity of phosphorescence is influenced by ATP, vanadate and water-soluble quenchers, such as NaNO 2, methylvinyl ketone and trichloroacetate. Materials and Methods
Materials Glucose oxidase, catalase, lactic dehydrogenase (rabbit muscle), pyruvate kinase (rabbit muscle), EGTA, NaNO 2, methylvinyl ketone, adenosine 5'-triphosphate, adenosine 5'-diphosphate, adenosine 5'-monophosphate and phosphoenolpyruvate were obtained from Sigma Chemical Co. (St. Louis, MO), sodium dithionite from Virginia Chemical Co. (Portsmouth, VA) and grade 5 argon
gas from Airco (Murray Hill, N J). Sodium vanadate and trichloroacetic acid were supplied by Fisher Scientific (Fairlawn, N J). A23187 was the product of Behring Diagnostics (Calbiochem), La Jolla, CA.
Methods Sarcoplasmic reticulum vesicles were isolated from predominantly white muscles of rabbits, as described earlier [28]. The purified Ca2+-ATPase was prepared according to Meissner et al. [29]. The sarcoplasmic reticulum lipids were extracted with chloroform/methanol (2:1, v/v) according to Folch et al. [30]. The organic solvent phase was evaporated to dryness under a nitrogen stream at 35-40°C. The phospholipid phosphorus was determined according to Bartlett [31]. The Lowry method [32] or the biuret procedure [33] were used for the determination of proteins. For phosphorescence measurements, the samples were prepared under the conditions described by Englander et al. [4] to exclude oxygen. Sarcoplasmic reticulum or purified ATPase preparations (1.4-1.6 mg protein/ml) were dispersed in a medium of argon-saturated 0.1 M KCI, 0.01 M Tris-HCl (pH 7.4), 5 mM MgCI 2 containing the deoxygenating system of glucose oxidase (80 nM), catalase (16 riM) and glucose (3 mg/ml). Other additions are indicated in the figure legends. After incubation at room temperature for 10 min, sodium dithionite was added to a final concentration of 80 /zM, the samples were overlaid with argon-saturated mineral oil, and the cuvette was closed with a glass stopper. The phosphorescence spectra were measured at 350-650 nm in a Perkin Elmer model LS-5 phosphorescence spectrometer using light of 295 nm wavelength for excitation with a time delay of 0.5 ms and a gate time of 2 ms. For measurement of phosphorescence lifetimes a Perkin Elmer MPF-2A fluorometer was used, interfaced with an A T & T 6300 computer. The CW lamp of the fluorometer was replaced by a xenon flash lamp [7]. Data were analyzed using the Asystant software package (MacMillan Software Company, New York, NY). The phosphorescence lifetime was measured at an emission wavelength of 440 nm, using light of 295 nm for e~citation. The lifetimes were calculated from the average of 200-500 decay curves from data col-
232 lected between 1-150 ms after the light flash of --5 /ts duration. Since emissions that occurred within 1 ms after the exciting light flash were not included in the evaluation, the estimated lifetimes reflect the long-lived components of the phosphorescence.
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Fluorescence and phosphorescence spectra of sarcoplasmic reticulum Excitation of sarcoplasmic reticulum vesicles with light of 295 nm wavelength in a medium of 0.1 M KCI, 10 mM Tris-HCl buffer (pH 7.4), and 5 mM MgCI ~, causes the emission of fluorescence and phosphorescence (Fig. 1). The emission maximum of fluorescence is near 340 nm (Fig. 1A), and that of the much weaker phosphorescence at
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Fig. 1. Fluorescence and phosphorescence spectra of sarcoplasmic reficulum vesicles, purified Ca2+-ATPase, and extracted lipids. The sarcoplasmic reticulum (sample 1), purified ATPase (sample 2) and the iiposomesprepared from extracted lipids (sample 3) weredispersed in 0.1 M KCi, 10 mM Tris-HC! (pH 7.4), 5 mM MgCI, and prepared for deoxygenation, as described under Methods. The final protein concentrations in A and B were 1.6 mg protein/ml; the concentration of lipids in C was 1.5 mg/ml. Excitation wavelength: 295 nm. Temperature: 24°C. The fluorescence (A) and phosphorescence (B) spectra were measured as described under Methods.
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Fig. 2. The effect of ATP on the phosphorescence of sarcoplasmic reticulum vesicles. The phosphorescence of sarcoplasmic reticulum vesicles (protein concentration 1.46 mg/ml) was measured at 20°C in a medium of 0.1 M KCI, 10 mM Tris-HCI (pH 7.4), 5 mM MgCI2, and =10 -5 M CaC!2, without further addition (line 1), and after the addition of 3 mM ATP (line 2) or 6 mM ATP (line 3).
445 nm (Fig. 1B). The intensity ratio of fluorescence/phosphorescence emission is greater than 100. The sharp band in Fig. 1B near 590 nm is due to second-order scattering of the exciting light by the turbid vesicle suspension. The phosphorescence emission of purified Ca 2+-ATPase is weaker than that of the sarcoplasmic reticulum vesicles (Fig. 1B). Thi~ difference is probably due to the presence of highly phosphorescent accessory proteins in the sarcoplasmic reticulum, that were removed during the purification of the Ca 2+ATPase. The extracted sarcoplasmic reticulum lipids have only slight phosphorescence in the wavelength range of 350-550 nm (Fig. 1B). Solubilization of sarcoplasmic reticulum with deoxycholate (1 mg per mg protein) did not alter significantly the wavelength distribution or intensity of phosphorescence. Therefore the lower phosphorescence emission of purified ATPase, as compared with sarcoplasmic reticulum (Fig. 1), is not attributable to the use of deoxycholate for the purification of the Ca2+-ATPase. Deoxycholate (1 m g / m g phospholipid) also had no influence on the phosphorescence of extracted sarcoplasmic reticulum lipids.
The effect of.4 TP on phosphore.~cence The phosphorescence was significantly reduced by 3 - 6 mM ATP in the presence of ---10 -5 M Ca 2+ (Fig. 2) or 1 m M EGTA (not shown). Since
233
lowering the Ca 2+ concentration by EGTA inhibits the phosphorylation of Ca2+-ATPase by ATP, these observations suggest that the decrease in phosphorescence caused by ATP is due to the formation of enzyme-ATP complex. Under similar conditions ADP (3-6 mM) had only slight effect on the phosphorescence, while AMP was ineffective. Therefore the phosphorescence quenching by ATP cannot be due to absorption of the exciting light or to quenching by free nucleotides in solution. The phosphorescence returned to starting level after the added ATP was hydrolyzed by the Ca2+-ATPase, indicating that secondary changes in the system caused by the hydrolysis of ATE such as a decrease in pH, contribute only slightly, if at all, to the observed decrease in phosphorescence. The phosphorescence of parvalbumin, a Ca2÷-binding protein which does not bind ATP, was not quenched by ATP in this concentration range (unpublished observation). This is a further, indirect evidence, that ATP in solution is not responsible for the observed quenching of phosphorescence in the enzyme-ATP complex.
The effect of vanadate on the phosphorescence of sarcoplasmic reticulum and purified Ca 2 +-A TPase The tryptophan phosphorescence of sarcoplasmic reticulum vesicles was measured in a medium of 0.1 M KCI, 10 rnM Tris-HCl (pH 7.4), 5 mM MgCI 2 and either 0.5-1.0 mM CaC! 2 or 0.2 mM EGTA. Addition of 0.075-0.2 mM Na 2VO4 to the vesicles caused similar decrease in the intensity of phosphorescence in the presence of either Ca 2÷ or EGTA (Fig. 3). At a vanadate concentration of 0.05 mM, the quenching of phosphorescence by vanadate was frequently greater in the presence of EGTA than in the presence of Ca 2+, but these differences were not consistently reproducible. The effect of vanadate on the tryptophan phosphorescence is a property of the Ca 2÷ATPase, since the magnitude of the effect is similar in sarcoplasmic reticulum, in the purified Ca2÷-ATPase prepared according to Meissner et al. [29], and in sarcoplasmic reticulum preparations that were treated with 1 mM EDTA in a medium of 0.1 M KCI and 10 mM Tris-HCl (pH 7.9) overnight at 2°C to remove the extrinsic water soluble proteins from the membrane [34].
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Fig. 3. The effect of vanadate on the phosphorescence of sarcoplasmic reticulum. (A) The phosphorescence of sarcoplasmic reticulum was measured as described under Methods and Fig. 1, in a medium of 0.1 M KCI, 10 mM Tris (pH 7.4), 5 mM MgCI 2 and =10 -5 M CaCi2 (1). To sample 2, 0.1 mM EGTA, and to sample 3, 0.2 mM EGTA+0.1 mM vanadate were added. (B) Sample 1 has an identical composition to that of A (1). To sample 2, 1 mM CaC! 2, and to sample 3, 1 mM CaCi 2 + 0.1 mM vanadate were added.
Phosphorescence lifetime data on sarcoplasmic reticulum The phosphorescence decay was multiexponential (Fig. 4). Due o technical difficulties in the 0.2
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Fig. 4. Decay curve of phosphorescence. The phosphorescence decay was measured in a medium of 0.1 M KCi, 10 mM Tris-HCI buffer (pH 7.4) and 5 mM MgCl 2 at 24°C, as described in Methods. Excitation wavelength: 295 nm; emission wavelength: 440 nm.
234 TABLE I PHOSPHORESCENCE LIFETIME PLASMIC R E T I C U L U M
DATA
ON SARCO-
The phosphorescence decay measured between 1-150 ms after the exciting light flash was multiexponential. In the table, the lifetimes of the slowest component are given, based on an average of 200-500 scans. For other details, see text. The number of independent measurements are denoted by n. n
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activity of sarcop!asmic reticulum assayed either in the absence or in the presence of 10 ItM A23187. Methylvinyl ketone and trichloroacetate caused modest (20-30%) inhibitien of ATPase activity at 45.5 mM concentration, w~ale NaNO 2 was ineffective, even under these conditions. Therefore denaturation of the Ca2+-ATPase is not likely to contribute to the quenching of phosphorescence by either of these agents. Our observations on the effect of trichloroacetate on phosphorescence are in agreement with earlier fluorescence and kinetic data [35,36]. Discussion
measurement of phosphorescence decay at times immediately following the flash of exciting light, only data collected between 1 and 150 ms after the exciting flash of 5 Its duration were included in the evaluation of phosphorescence lifetime. The lifetime of long-lived components of phosphorescence calculated as the average of 19 experiments, was 22 ms (Table I). In spite of the significant quenching of phosphorescence by vanadate, the average lifetime measured under these conditions was only slightly reduced. Therefore the phosphorescence quenching by vanadate affects primarily the short-lived components of phosphorescence that are not included in the dataset samples for phosphorescence decay. The phosphorescence lifetime of sarcoplasmic reticulum was not affected significantly by 20 mM Ca z+ (Table I).
The quenching of phosphorescence by NaNO 2, methyivinlpi ketone and trichloroacetate The intensity and lifetime of the phosphorescence of sarcoplasmic reticulum vesicles was reduced by NaNO 2 and trichloroacetate at submillimolar, and by methylvinyl ketone at millimolar concentrations. The quenching did not show evidence of saturation (data not shown), and it is therefore likely to be collisional. At concentrations up to 4.5 mM neither of the three quenchers had significant effect on the CaZ+-activated ATPase
Room temperature phosphorescence was detected and analyzed in sarcoplasmic reticulum vesicles. A major component of phosphorescence is attributed to tryptophan residues of the Ca 2+ATPase, but other protein components of the sarcoplasmic reticulum may also contribute. Only slight phosphorescence was observed after excitation at 295 nm in the isolated sarcoplasmic reticulum lipids. ATP at millimolar concentrations diminished the phosphorescence intensity by as much as 25-30%; ADP was less effective and AMP was essentially without effect. The ATP effect was observed even after the removal of Ca z+ by EGTA, suggesting that the phosphorylation of Ca 2÷ATPase is not required for the quenching of phosphorescence. Therefore the effect of ATP is presumed to be due to the formation of an enzymeATP complex. The phosphorescence quenching by ATP (25-30%) is an order of magnitude greater than the ATP-induced change in tryptophan fluorescence [24]. This may imply greater sensitivity of the long-lived triplet state, as compared with singlet state, to quenching by ATE Alternatnvely, the phosphorescence emission may involve a ~;:abpopulation of tryptophan residues that includes a relatively large proportion of the tryptophan:~ affected by ATP. The phosphorescet:ce emission in intact sarcoplasmic reticulum vesicles and in the purified Ca2+-ATPase preparations was also quer~ched by vanadate with only slight effect on the lifetime of the long-lived components of the phosphorescence
235
emission. The quenching of phosphorescence by vanadate was observed in the presence of either Ca 2+ 10.5-1.0 mM) or EGTA (0.2 mM). Since the stabilization of E~ conformation by vanadate is observed only at submicromolar Ca 2÷ concentration, these observations suggest that a localized change in the environment of tryptophan caused by the binding of vanadate to the CaZ+-ATPase, rather than a vanadate-induced shift in the conformation of the enzyme from the E 1 to the E 2 state is involved in the phosphorescence quenching. In addition to the indirect effects of ATP or vanadate on the environment and emission characteristics of protein tryptophan(s), a more direct static quenching by the bound ATP or vanadate of the tryptophan residues located near the substrate binding site remains a possibility. The fast and slow isoenzymes of the Ca 2÷ATPase of sarcoplasmic reticulum contain 13 tryptophans [37]. Twelve of these are located in hydrophobic fragments of Ca 2+-ATPase, and these are assigned in the predicted secondary structure of the enzyme to the membrane-spanning and stalk helices near the surface of the bilayer. Their relationship to the vanadate binding site is unknown. One tryptophan (residue 552) is assigned to the nucleotide binding region of the cytoplasmic, domain, not far from the exposed T 1 tryptic cleavage site (residue 505) and from the binding site for fluorescein 5'-isothiocyanate (residue 515). This tryptophan residue is a plausible candidate for direct quenching by ATP bound at the nucleotide binding site.
Acknowledgements Supported by research grants GM34448 and AM26545 from the National Institutes of Health, PCM84-03679 and Int. 86-17848 from the National Science Foundation, and by a grant-in-aid from the Muscular Dystrophy Association and grant OTKA 665 from Hungary.
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