Distances between the functional sites of the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

Luminescence energy transfer measurements have been used to determine the distances between the two high affinity Ca2+ binding-transport sites of the (Ca2+ + Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum. The lanthanide Tb3+ situated at one high affinity Ca2+ site was used as the transfer donor, and acceptors at the other Ca2+ site were the lanthanides Nd3+, Pr3+, Ho3+, or Er3+. Terbium bound to the enzyme was excited directly with a pulsed dye laser. Analysis of the changes in the terbium luminescence lifetime due to the presence of the acceptor indicates that the distance between the Ca2+ sites is 10.7 A. The distance between the Ca2+ sites and the nucleotide-binding catalytic site was determined using Tb3+ at the Ca2+ sites and either trinitrophenyl nucleotides (TNP-N) or fluorescein 5-isothiocyanate (FITC) in the catalytic site as energy acceptors. The R0 values for the Tb-acceptor pairs are approximately 30 and approximately 40 A for TNP-N and FITC, respectively. The distance between Tb3+ at the Ca2+ sites and TNP-ATP at the nucleotide site is approximately 35 A and that between the Ca2+ sites and the FITC labeling site is approximately 47 A. Considerations of the molecular dimensions of the ATPase polypeptide indicate that while the two Ca2+ sites are close to each other, the Ca2+ sites and the nucleotide site are quite remote in the three-dimensional structure of the enzyme.     

The effects of membrane potential and lanthanides on the conformation of the Ca2+-transport ATPase in sarcoplasmic reticulum.

The effects of Ca2+, lanthanide ions (Gd3+, La3+ and Pr3+) and membrane potential on the fluorescence of tryptophan and covalently bound fluorescein were analysed in native and fluorescein isothiocyanate (FITC)-labelled sarcoplasmic reticulum vesicles. The binding of Ca2+ and lanthanides to the Ca2+-ATPase increases the fluorescence intensity of tryptophan and decreases the fluorescence intensity of FITC; the dependence of these effects on cation concentration is consistent with the involvement of the high-affinity Ca2+-binding sites of the Ca2+-ATPase in the cation-induced fluorescence changes. The fluorescence of FITC-labelled sarcoplasmic reticulum vesicles is also influenced by membrane potential changes induced by ion substitution. Inside positive potential increases, while inside negative potential decreases, the fluorescence of bound FITC. Smaller potential-dependent changes in tryptophan fluorescence were also observed. The effects of Ca2+, lanthanides and membrane potential on the fluorescence of tryptophan and FITC are discussed in terms of the two major conformations of the Ca2+-ATPase (E1 and E2), that are assumed to alternate during Ca2+ transport. The observations support the suggestion [Dux, Taylor, Ting-Beall & Martonosi (1985) J. Biol. Chem. 260, 11730-11743] that the vanadate-induced crystals of Ca2+-ATPase represent the E2, while the Ca2+ and lanthanide-induced crystals the E1, conformation of the enzyme.     

Fluorescence energy transfer as an indicator of Ca2+-ATPase interactions in sarcoplasmic reticulum.

Ca2+-ATPase molecules were labeled in intact sarcoplasmic reticulum (SR) vesicles, sequentially with a donor fluorophore, fluorescein-5'-isothiocyanate (FITC), and with an acceptor fluorophore, eosin-5'-isothiocyanate (EITC), each at a mole ratio of 0.25-0.5 mol/mol of ATPase. The resonance energy transfer was determined from the effect of acceptor on the intensity and lifetime of donor fluorescence. Due to structural similarities, the two dyes compete for the same site(s) on the Ca2+-ATPase, and under optimal conditions each ATPase molecule is labeled either with donor or acceptor fluorophore, but not with both. There is only slight labeling of phospholipids and other proteins in SR, even at concentrations of FITC or EITC higher than those used in the reported experiments. Efficient energy transfer was observed from the covalently bound FITC to EITC that is assumed to reflect interaction between ATPase molecules. Protein denaturing agents (8 M urea and 4 M guanidine) or nonsolubilizing concentrations of detergents (C12E8 or lysolecithin) abolish the energy transfer. These results are consistent with earlier observations that a large portion of the Ca2+-ATPase is present in oligomeric form in the native membrane. The technique is suitable for kinetic analysis of the effect of various treatments on the monomer-oligomer equilibrium of Ca2+-ATPase. A drawback of the method is that the labeled ATPase, although it retains conformational responses, is enzymatically inactive.     

稀土元素Pr~(3+)、Nd~(3+)对蚕豆(Vicia feba)叶片原生质膜上Ca~(2+)·Mg~(2+)-ATPase活性的影响

本文介绍用二相分配法制备蚕豆叶片原生质膜上的Ca~(2+)·Mg~(2+)-ATPase,用以研究镧系,稀土离子对此酶活性的影响。初步证实Pr~(3+)、Nd~(3+)对依赖于CaM的以及不依赖于CaM的蚕豆叶片原生质膜上Ca~(2+)·Mg~(2+)-ATPase活性的抑制不是CaM专一的。     

Nd3+ and Co2+ binding to sarcoplasmic reticulum CaATPase. An estimation of the distance from the ATP binding site to the high-affinity calcium binding sites

Nd3+ binding to sarcoplasmic reticulum (SR) was detected by inhibition of ATPase activity and directly by a fluorimetric assay. Both methods indicated that Nd3+ inhibited the ATPase activity by binding in the high-affinity Ca2+ binding sites. The stoichiometry of binding was about 11 nmol of Nd3+ bound per mg of SR proteins at pNd = 6.5. At higher [Nd3+], substantial nonspecific binding occurred. The association constant for Nd3+ binding to the high-affinity Ca2+ binding sites was estimated to be near 2 X 10(9) M-1. When the CaATPase was inactivated with fluorescein isothiocyanate (FITC), 5.3 nmol were bound per mg of SR protein. This fluorescent probe is known to bind in the ATP binding site. The stoichiometry of Nd3+ binding to FITC-labeled CaATPase was the same, within experimental error, as to the unlabeled CaATPase. Fluorescence energy transfer between FITC in the ATP site and Nd3+ in the Ca2+ sites was found to be very small. This donor-acceptor pair has a critical distance of 0.93 nm and the distance between the ATP site and the closest Ca2+ was estimated to be greater than 2.1 nm. Parallel measurements with FITC-labeled SR and Co2+, an acceptor with a critical distance 1.2 nm, suggested the ATP and Ca2+ binding sites are greater than 2.6 nm apart.     

Conformational basis of the phospholipid requirement for the activity of SR Ca(2+)-ATPase

The delipidated sarcoplasmic reticulum (SR) Ca(2+)-ATPase was reconstituted into proteoliposomes containing different phospholipids. The result demonstrated the necessity of phosphatidylcholine (PC) for optimal ATPase activity and phosphatidylethanolamine (PE) for the optimal calcium transport activity. Fluorescence intensity of Fluorescein 5-isothiocyanate (FITC)-labeled enzyme at Lys515 as well as the measurement of the distance between 5-((2-[(iodoacetyl) amino] ethyl) amino)naphthalene-1-sulphonic acid (IAEDANS) label sites (Cys674/670) and Pr3+ demonstrated a conformational change of cytoplasmic domain, consequently, leading to the variation of the enzyme function with the proteoliposomes composition. Both the intrinsic fluorescence of Trp and its dynamic quenching by HB decreased with increasing PE content, revealing the conformational change of transmembrane domain. Time-resolved fluorescence study characterized three classes of Trp residues, which showed distinctive variation with the change in phospholipid composition. The phospholipid headgroup size caused the conformational change of SR Ca(2+)-ATPase, subsequent the ATPase activity and Ca2+ uptake.     

Resonance energy transfer between points in a reconstituted skeletal muscle thin filament. A conformational change of the thin filament in response to a change in Ca2+ concentration

The spatial relationships between Lys-61, Cys-374 on actin or SH1 on myosin subfragment-1 (S1) and Cys-190 on tropomyosin or Cys-133 on troponin-I (TnI) in a reconstituted thin filament were studied by fluorescence resonance energy transfer. 5-(2-Iodoacetylaminoethyl)aminonaphthalene 1-sulfonic acid (IAEDANS) attached to Lys-190 on tropomyosin or to Cys-133 on TnI was used as a donor. Fluorescein 5-isothiocyanate (FITC) attached to Lys-61 or 5-(iodoacetoamido)fluorescein (IAF) attached to Cys-374 on actin and 4-dimethylaminophenyl-azophenyl 4'-maleimide (DABMI) attached to SH1 on S1 were used as an acceptor. The transfer efficiency between AEDANS attached to Cys-190 on tropomyosin and FITC attached to Lys-61 on actin was 0.42 in the absence of troponin, 0.46 in the presence of troponin and Ca2+ and 0.55 in the presence of troponin and absence of Ca2+. The corresponding distances between the probes were calculated to be 4.7 nm, 4.6 nm and 4.3 nm respectively, assuming a random orientation factor K2 = 2/3. A large difference in the transfer efficiency from AEDANS attached to Cys-133 on TnI to FITC attached to Lys-61 on actin was observed between in the presence (0.52) and absence (0.70) of Ca2+. The corresponding distances between the probes were calculated to be 4.5 nm in the presence of Ca2+ and 3.9 nm in the absence of Ca2+. The distance between Cys-190 on tropomyosin and Cys-374 on actin was measured to be 5.1 nm and the transfer efficiency (0.35) did not change upon addition of troponin whether Ca2+ is present or not, in agreement with the previous report [Tao, T., Lamkin, M. & Lehrer, S. S. (1983) Biochemistry 22, 3059-3064]. The distance between Cys-133 on TnI and Cys-374 on actin was measured to be 4.4 nm. No detectable change in transfer efficiency (0.58) was observed between values in the presence and absence of Ca2+. These results suggest that a relative movement of the two domains of actin monomer in a reconstituted thin filament occurs in response to a change in Ca2+ concentration. The transfer efficiencies between DABMI attached to SH1 on S1 and AEDANS attached to Cys-190 on tropomyosin or Cys-133 on TnI were too small (less than 2%) for an accurate estimation of the distances, suggesting the distances are longer than 7.3 nm.     

(Iodoacetamido)fluorescein labels a pair of proximal cysteines on the Ca2+-ATPase of sarcoplasmic reticulum

Previous energy transfer studies [Squier, T. C., Bigelow, D. J., de Ancos, J. G., & Inesi, G. (1987) J. Biol. Chem. 262, 4748-4754] have utilized fluorescent iodoacetamide derivatives covalently bound to the Ca2+-ATPase of sarcoplasmic reticulum (SR), using labeling conditions that completely modify the most reactive of the protein's surface sulfhydryls to a final level of 9 nmol/mg of SR protein. Unambiguous interpretation of these results requires localization of these labeling sites with respect to the primary structure of the Ca2+-ATPase. In the present study, we have used the probe 6-(iodoacetamido)fluorescein (IAF) as a marker for these sites. The IAF-labeled Ca2+-ATPase was completely proteolyzed with trypsin, followed by centrifugation to remove (unlabeled) membrane-associated portions. The soluble IAF-labeled tryptic peptides were purified by size-exclusion and reverse-phase high-performance liquid chromatography. Two IAF-peptides resulted. The major (4.1 nmol of IAF/mg of starting protein) and minor (1.9 nmol/mg) IAF-peptides were sequenced and were identified, respectively, as Ala673-IAF-Cys674-Cys675-Phe676-Ala677+ ++-Arg678 and as Glu668-Ala669-IAF-Cys670-Arg671. A model is proposed to explain the selectivity of IAF for Cys670 and Cys674 of the approximately 14 surface sulfhydryls of the Ca2+-ATPase. The labeling region, Arg667 through Arg678, has been predicted to be alpha-helical; Cys670 and Cys674 would be adjacent in the helix and imbedded in an Arg cluster. The Arg residues would both attract the anionic IAF and enhance sulfhydryl reactivities by lowering their pK values.     

Characterization of the tryptophan fluorescence from sarcoplasmic reticulum adenosinetriphosphatase by frequency-domain fluorescence spectroscopy

We examined the tryptophan decay kinetics of sarcoplasmic reticulum Ca2+-ATPase using frequency-domain fluorescence. Consistent with earlier reports on steady-state fluorescence intensity, our intensity decays reveal a reproducible and statistically significant 2% increase in the mean decay time due to calcium binding to specific sites involved in enzyme activation. This Ca2+ effect could not be eliminated with acrylamide quenching, which suggests a global effect of calcium on the Ca2+-ATPase, as opposed to a specific effect on a single water-accessible tryptophan residue. The tryptophan anisotropy decays indicate substantial rapid loss of anisotropy, which can be the result of either intramolecular energy transfer or a change in segmental flexibility of the ATPase protein. Energy transfer from tryptophan to TNP-ATP in the nucleotide binding domain, or to IEADANS on Cys-670 and -674, indicates that most tryptophan residues are 30 A or further away from these sites and that this distance is not decreased by Ca2+. In light of known structural features of the Ca2+-ATPase, the tryptophan fluorescence changes are attributed to stabilization of clustered transmembrane helices resulting from calcium binding.     

Ischemia-induced structural change in SR Ca2+-ATPase is associated with reduced enzyme activity in rat muscle

In this study, we employed an in vivo model of prolonged ischemia in rat skeletal muscle to investigate the hypothesis that structural modifications to the sarcoplasmic reticulum (SR) Ca2+-ATPase can explain the alterations in Ca2+-ATPase activity that occur with ischemia. To induce total ischemia, a tourniquet was placed around the upper hindlimb in 27 female Sprague-Dawley rats weighing 256 +/- 6.7 g (mean +/- SE) and was inflated to 350 mmHg for 4 h. The contralateral limb served as control (C) to the ischemic limb (I), and the limbs of animals killed immediately after anesthetization served as a double control (CC). Mixed gastrocnemius and tibialis anterior muscles were sampled and used for SR vesicle preparation. Maximal Ca2+-ATPase activity (micromol x g protein(-1) x min(-1)) of C (15,802 +/- 1,246) and I (11,609 +/- 1,029) was 90 and 73% (P < 0.05) of CC (17,562 +/- 1,682), respectively. No differences were found between groups in either the Hill coefficient or the free Ca2+ at half-maximal activity. The fluorescent probes, FITC and N-cyclohexyl-N'-(dimethylamino-alpha-naphthyl) carbodiimide, used to assess structural alterations in the regions of the ATP binding site and the Ca2+ binding sites of the Ca2+-ATPase, respectively, indicated a 26% reduction (P < 0.05) in FITC binding capacity (absolute units) in I (0.22 +/- 0.01) compared with CC (0.29 +/- 0.02) and C (0.29 +/- 0.03). Our results suggest that the reduction in maximal SR Ca2+-ATPase activity in SR vesicles with ischemia is related to structural modification in the region of the nucleotide binding domain by mechanisms that are as yet unclear.     

Effect of calcium on the interactions between Ca2+-ATPase molecules in sarcoplasmic reticulum

The interaction between Ca2+-ATPase molecules in the native sarcoplasmic reticulum membrane and in detergent solutions was analyzed by chemical crosslinking, high performance liquid chromatography (HPLC), and by the polarization of fluorescence of fluorescein 5'-isothiocyanate (FITC) covalently attached to the Ca2+-ATPase. Reaction of sarcoplasmic reticulum vesicles with glutaraldehyde causes the crosslinking of Ca2+-ATPase molecules with the formation of dimers, tetramers and higher oligomers. At moderate concentrations of glutaraldehyde solubilization of sarcoplasmic reticulum by C12 E8 or Brij 36T (approximately equal to 4 mg/mg protein) decreased the formation of higher oligomers without significant interference with the appearance of crosslinked ATPase dimers. These observations are consistent with the existence of Ca2+-ATPase dimers in detergent-solubilized sarcoplasmic reticulum. Ca2+ (2-20 mM) and glycerol (10-20%) increased the degree of crosslinking at pH 6.0 both in vesicular and in solubilized sarcoplasmic reticulum, presumably by promoting interactions between ATPase molecules; at pH 7.5 the effect of Ca2+ was less pronounced. In agreement with these observations, high performance liquid chromatography of sarcoplasmic reticulum proteins solubilized by Brij 36T or C12 E10 revealed the presence of components with the expected elution characteristics of Ca2+-ATPase oligomers. The polarization of fluorescence of FITC covalently attached to the Ca2+-ATPase is low in the native sarcoplasmic reticulum due to energy transfer, consistent with the existence of ATPase oligomers (Highsmith, S. and Cohen, J.A. (1987) Biochemistry 26, 154-161); upon solubilization of the sarcoplasmic reticulum by detergents, the polarization of fluorescence increased due to dissociation of ATPase oligomers. Based on its effects on the fluorescence of FITC-ATPase, Ca2+ promoted the interaction between ATPase molecules, both in the native membrane and in detergent solutions.     

Emerging views on the structure and dynamics of the Ca2(+)-ATPase in sarcoplasmic reticulum

The ATP-dependent Ca2+ transport in sarcoplasmic reticulum involves transitions between several structural states of the Ca2(+)-ATPase, that occur without major changes in the secondary structure. The rates of these transitions are modulated by the lipid environment and by interactions between ATPase molecules. Although the Ca2(+)-ATPase restricts the rotational mobility of a population of lipids, there is no evidence for specific interaction of the Ca2(+)-ATPase with phospholipids. Fluorescence polarization and energy transfer (FET) studies, using site specific fluorescent indicators, combined with crystallographic, immunological and chemical modification data, yielded a structural model of Ca2(+)-ATPase in which the binding sites of Ca2+ and ATP are tentatively identified. The temperature dependence of FET between fluorophores attached to different regions of the ATPase indicates the existence of 'rigid' and 'flexible' regions within the molecule characterized, by different degrees of thermally induced structural fluctuations.     

Steroid-induced conformational changes of FITC-labelled sarcoplasmic reticulum Ca2+-ATPase

Interactions between transmembrane and cytoplasmic domains of Ca2+-ATPase from sarcoplasmic reticulum (SR) have been studied. To affect the hydrophobic transmembrane domain, we used four amphiphilic steroids - esters of a dibasic acid and 20-oxypregnene. All four steroids contained cholesterol-like nuclei and differed by the structure of side chains. Steroids with carboxyl groups in the side chains inhibited the rates of ATP hydrolysis and Ca2+ transport, whereas a steroid without the carboxyl group did not appreciably affect Ca2+-ATPase function. Fluorimetric titration of FITC-labelled Ca2+-ATPase in SR vesicles by Nd3+ showed that steroids increased the apparent dissociation constant for Nd3+ bound to the hydrolytic site, the potency order of the steroids being the same as for the sterol-induced inhibition of the hydrolytic activity of Ca2+-ATPase. These results suggest structural changes in the active site. Ca2+ transport was inhibited more efficiently by steroids than the hydrolytic activity of the enzyme. This could be partially due to the increase of the membrane passive permeability induced by steroids, which, in turn, reflected the efficiency of the interaction of the steroids with lipid bilayers. The effects of the steroids were largely dependent on their amphiphilicity (the availability of polar groups in regions A and D), the structure of the side chains, and, possibly, on the distance between the molecular polar groups. We suggest that the inhibition of hydrolytic and transport functions of Ca2+-ATPase in the SR membrane is due to the interaction of the steroids with the transmembrane alpha-helical segments.     

Catalytic and regulatory ATP-binding sites of the red cell Ca2+ pump studied by irreversible modification with fluorescein isothiocyanate

Catalytic and regulatory binding sites for ATP on the red cell Ca2+ pump have been investigated using fluorescein isothiocyanate (FITC). Both (Ca2+ + Mg2+)-ATPase activity and ATP-dependent Ca2+ flux are selectively and irreversibly inactivated by FITC and the pump is protected from FITC by the presence of ATP. The time course of inactivation by FITC is characteristically biphasic. Analysis of the kinetics of inactivation by FITC and protection by ATP reveals the participation of both high and low affinity binding sites for ATP and FITC. The sites binding ATP or reacting with FITC do not, however, appear to co-exist on the same enzyme molecules. Thus, "flip-flop" mechanisms for (Ca2+ + Mg2+)-ATPase, involving negative interactions between high and low affinity ATP sites, are considered unlikely. The two affinities for ATP are most simply explained by assuming that the Ca2+ pump protein exists in alternative conformational forms, E1 having a high affinity for ATP and E2 having a low affinity for ATP. Ca2+ pumping and (Ca2+ + Mg2+)-ATPase involve interconversion between these forms. It is suggested that regulation of Ca2+ pump activity by Mg-ATP reflects acceleration of the conformational transition between the E1 and E2 forms, as well as a previously described acceleration of phosphoenzyme hydrolysis (Muallem, S., and Karlish, S. J. D. (1981) Biochim. Biophys. Acta 647, 73-86; Garrahan, P. J., and Rega, A. F. (1978) Biochim. Biophys. Acta 513, 59-65).     

Different sensitivity of the sarcoplasmic reticulum Ca(2+)-ATPase enzyme to fluorescein-isothiocyanate in rabbit and carp muscles.

1. Carp and rabbit sarcoplasmatic reticulum Ca(2+)-ATPase enzymes were compared with respect to their sensitivity to FITC labelling. 2. The carp enzyme showed much lower sensitivity to FITC in the Ca(2+)-Mg2+ activated ATPase activity. Fifty percent inhibition was observed at 20 microM labelling FITC concentration; in rabbit enzyme this inhibition was already achieved at 2 microM FITC. 3. The tryptic cleavage products of the carp enzyme identified with immunoblot analysis as well as with FITC fluorescence, suggest multiple cleavage, yielding different fragments from the ones well known in rabbit and in rat enzyme. 4. The present results indicates major structural differences with respect to the FITC binding, and tryptic cleavage between the SR Ca(2+)-ATPase enzymes from carp and rabbit, despite the cross-reactivity with polyclonal antibodies.     

Distances between the FITC-binding site in the (Ca2++Mg2+)-ATPase from sarcoplasmic reticulum and fluorescent probes located in the membrane

Fluorescence energy transfer measurements have been performed to investigate the distances between the FITC-binding site in the (Ca2++Mg2+)-ATPase from sarcoplasmic reticulum and 8-aniline-1-naftalenesulfonate (ANS-) and diphenylhexatriene (DPH) localized in different sites of the membrane. The distances calculated between FITC bound to the ATPase and ANS- and DPH in the membrane, were approx. 51 A and 60 A, respectively.     

Fluorescence energy transfer studies of purified erythrocyte Ca2+-ATPase. Ca2+-regulated activation by oligomerization

Fluorescence resonance energy transfer has been used to study oligomerization of the purified erythrocyte Ca2+-ATPase. The energy transfer efficiency has been measured at different enzyme concentrations, from fluorescein 5'-isothiocyanate attached on one enzyme molecule to eosin 5-maleimide or tetramethylrhodamine 5-isothiocyanate attached on another enzyme molecule. The energy transfer efficiency showed a sigmoid dependence on enzyme concentration and was half-maximal at 10-12 nM enzyme; this dependence on enzyme concentration closely resembled previously demonstrated dependence of Ca2+-ATPase activity and polarization of the fluorescein 5'-isothiocyanate enzyme (Kosk-Kosicka, D., and Bzdega, T. (1988) J. Biol. Chem. 263, 18184-18189). Thus, the three independent methods establish that enzyme concentration-dependent oligomerization is a mechanism of activation of the erythrocyte Ca2+-ATPase. Further energy transfer studies demonstrated that enzyme oligomerization required calcium. This calcium dependence was characterized by high affinity (half-maximal energy transfer at pCa 7.15) and cooperativity (Hill coefficient of 2.36), being very similar in both respects to the Ca2+ dependence of the Ca2+-ATPase activity. The data indicated that the oligomerization process produced a highly cooperative, Ca2+-regulated activation of the enzyme at physiologically relevant Ca2+ concentrations. These studies show that the Ca2+-ATPase can be fully activated by a Ca2+-dependent oligomerization mechanism, which is independent of the previously described activation by calmodulin. We propose two pathways for the activation of the Ca2+-ATPase, taking into account the interdependencies between the Ca2+, calmodulin, and enzyme concentrations.     

Interaction of calcium and vanadate with fluorescein isothiocyanate labeled Ca2+-ATPase from sarcoplasmic reticulum: kinetics and equilibria

The interaction of Ca2+ and vanadate with fluorescein isothiocyanate (FITC) labeled sarcoplasmic reticulum (SR) Ca2+-ATPase has been studied by following the kinetics of changes in the reporter group fluorescence and equilibrium fluorescence levels. The vanadate species bound to the enzyme is clearly monomeric orthovanadate, probably H2VO4-. Vanadate binding is noncooperative, suggesting an absence of interactions between the Ca2+-ATPase subunits. The fluorescence experiments confirm the existence of a calcium-enzyme-vanadate complex (in the presence of magnesium). On the basis of the fluorescence properties of this complex, it is similar in its conformation to the calcium-enzyme complex, i.e., "E1-like" rather than "E2-like". However, Ca2+ binds to the enzyme-vanadate complex via sites that are only accessible from the interior of the SR vesicles. The complex Ca2E*Van, which is rapidly formed, isomerizes very slowly (t1/2 approximately 1 min) to the stable ternary complex. The mutual destabilization between bound vanadate and two bound Ca2+ ions is only 1.6 kcal/mol, much smaller than that produced by the interaction of calcium and phosphate.     

Distances between functional sites in cardiac sarcoplasmic reticulum (Ca2+ +Mg2+)-ATPase. Inter-lanthanide energy transfer

The high-affinity Ca2+-binding sites of cardiac sarcoplasmic reticulum (Ca2+ +Mg2+)-ATPase have been probed using trivalent lanthanide ions. Non-radiative energy-transfer studies, using luminescent probe Eu3+ as a donor and Nd3+ or Pr3+ as acceptor, were carried out to estimate the distance between two high-affinity Ca2+-binding/transport sites. Eu3+ was excited directly with pulsed laser light and the energy-transfer efficiency to Nd3+ or Pr3+ was measured, under the conditions in which most donor-acceptor pairs occupied the high-affinity Ca2+ sites. The distance between two high-affinity Ca2+ sites is about 0.89 nm. In the presence of ATP the distance between the high-affinity sites is about 0.855 nm, whereas in the presence of adenosine 5'-[beta, gamma-methylene]triphosphate or adenosine 5'-[beta, gamma-imino]triphosphate the distance is about 0.895 nm. To estimate the distance between the high-affinity Ca2+ sites and ATP-binding/hydrolytic site, we have measured the energy-transfer efficiency between Eu3+ and Cr3+-ATP with Eu3+ at the high-affinity Ca2+ sites and Cr3+-ATP at the ATP-binding/hydrolytic site. Our results show that ATP-binding/hydrolytic site is separated by about 2.2 nm from each high-affinity Ca2+ site.     

An investigation of the SH1-SH2 and SH1-ATPase distances in myosin subfragment-1 by resonance energy transfer using nanosecond fluorimetry

The separation between the two reactive thiols SH1 (Cys-704) and SH2 (Cys-694) and that between SH1 and the active site of myosin subfragment-1 were further investigated by F?rster energy transfer techniques. The SH1-SH2 distance was determined with the probe 5-[[2-[(iodoacetyl)amino]ethyl] amino]naphthalene-1-sulfonic acid (AEDANS) attached to SH1 as the energy donor and 5-(iodoacetamido)fluorescein (IAF) attached to SH2 as energy acceptor. The results derived from measurements of donor lifetimes yielded a donor-acceptor separation in the range 26-52 A, with the distance R(2/3) based on rapid and isotropic probe motions being 40 A. These parameters were not sensitive to added MgADP, in agreement with previous results obtained by using the steady-state method. The SH1-SH2 distance was also determined with AEDANS attached to SH1 and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) attached to SH2. The range in R for the AEDANS/DDPM pair was 12-36 A, with R(2/3) equal to 27 A. The transfer efficiency between these two probes increased by an average of 38% upon addition of MgADP. These results are in agreement with those previously reported (Dalbey, R.E., Weiel, J. and Yount, R.G. (1983) Biochemistry 22, 4696-4706), but the uncertainty in choosing an appropriate value of the orientation factor to describe the AEDANS-DDPM separation does not allow a unique interpretation of the observed increase in energy transfer because it could reflect either an increase in the average orientation factor or a decrease in the donor-acceptor separation. Nevertheless, the results are consistent with the notion that nucleotide binding induces structural perturbations that can be sensed by SH1 and SH2. The distance between SH1 and the ATPase site was determined with AEDANS linked to SH1 and the nucleotide analogue 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) noncovalently bound to the active site as energy acceptor. The bound TNP-ADP was highly immobilized, with a depolarization factor approaching unity. The separation between AEDANS at SH1 and TNP-ADP at the active site was in the range 15-44 A. The actual minimal separation between SH1 and the active site is probably less than 15 A, which suggests that direct interaction between the two sites cannot be ruled out from energy transfer results.