Fluorescence studies on calmodulin binding to erythrocyte Ca2(+)-ATPase in different oligomerization states

The fluorescent spinach calmodulin derivative 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin (MIANS-CaM) was used to investigate calmodulin interaction with the purified, detergent-solubilized erythrocyte Ca2(+)-ATPase. Previous studies have shown that the Ca2(+)-ATPase exists in equilibria between monomeric and oligomeric forms. We report here that MIANS-CaM binds to both enzyme forms in a Ca2(+)-dependent manner, with a approximately 50% fluorescence enhancement. These findings confirm our previous observation that enzyme oligomers retain their ability to bind calmodulin, even though they are fully activated in the absence of calmodulin. The Ca2+ dependence of MIANS-CaM binding to monomeric Ca2(+)-ATPase is of higher affinity (K 1/2 = 0.09 microM Ca2+) and less cooperative (nH = 1.1) than the Ca2+ dependence of enzyme activation by MIANS-CaM (K 1/2 = 0.26 microM Ca2+, nH = 2.8). These Ca2+ dependences and the order of events, in which calmodulin binding precedes enzyme activation, demonstrate that calmodulin indeed could be a physiological activator of the monomeric enzyme. The calcium dependence of calmodulin binding to oligomeric Ca2(+)-ATPase occurs at even lower levels of Ca2+ (K 1/2 = 0.04 microM Ca2+), in a highly cooperative fashion (nH = 2.3), and essentially in parallel with enzyme activation (K 1/2 = 0.05 microM Ca2+, nH = 2.9). The observed differences between monomers and oligomers suggest that the oligomerized Ca2(+)-ATPase is in a conformation necessary for efficient, cooperative calcium binding at nanomolar Ca2+, which the monomeric enzyme acquires only upon interaction with calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Persistent Ca2(+)-induced activation of erythrocyte membrane Ca2(+)-ATPase unrelated to calpain proteolysis

Preincubation of human erythrocyte membranes with calcium in the submillimolar to millimolar concentration range resulted in an increase of the Ca2+ affinity and apparent maximum velocity of the Ca2(+)-stimulated Mg2(+)-dependent ATPase (Ca2(+)-ATPase). The activation was persistent, as it was not reversed when the Ca2(+)-preincubated membranes were washed with ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid-containing buffers. Magnesium was not required for the activation, whereas greater than 2 mM Mg2+ partially antagonized the activation by Ca2+. In some membrane preparations ATP was required in addition to Ca2+ for activation of the Ca2(+)-ATPase, but nonhydrolyzable analogs of ATP had the same effect. Calmodulin prevented the activation by Ca2+ over the same concentration range in which it interacts with the Ca2(+)-ATPase. Taken together the results obtained provided strong evidence that the Ca2+ activation of the enzyme was not due to proteolytic cleavage by endogenous calpain. Thus, activation by Ca2+ was not blocked by leupeptin (100-200 microM), did not require dithiothreitol, and occurred at Ca2+ concentrations greater than those required for activation of calpain I. Furthermore, Ca2+ activation did not result in change in the mobility the native 136-kDa species of the Ca2(+)-ATPase on SDS-gel electrophoresis. Moreover, solubilization of the Ca2(+)-pretreated membranes with Triton X-100 reversed the Ca2+ activation of the Ca2(+)-ATPase. On the other hand, Ca2(+)-pretreatment of the membranes modified the susceptibility of the Ca2(+)-ATPase to both cleavage and activation by exogenously added calpain I. We conclude that pretreatment of Ca2(+)-ATPase in erythrocyte membranes with millimolar Ca2+ activates the enzyme by inducing a persistent conformational change of the enzyme which is, however, subsequently reversed by detergent solubilization.     

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.     

Effect of acylphosphatase on human erythrocyte membrane Ca2(+)-ATPase

We studied the effect of human acylphosphatase on the activity of human erythrocyte membrane Ca2(+)-ATPase. Both the acylphosphatase that is contained in hemolysate and the purified enzyme isolated from red blood cells were able to stimulate Ca2(+)-ATPase activity in erythrocyte membranes. Given the same acylphosphatase activity, however, the hemolysate showed higher stimulatory effect than the purified enzyme. Acylphosphatase stimulation was additive to that induced by calmodulin, thus indicating that acylphosphatase acts in a calmodulin-independent manner. Trifluoperazine, a calmodulin antagonist, did not inhibit acylphosphatase-induced stimulation of Ca2(+)-ATPase activity. Acylphosphatase significantly decreased the rate of Ca2+ influx into inside-out erythrocyte membrane vescicles, thus acting as Ca2+ pump inhibitor. Taken together these findings indicate that acylphosphatase is a soluble, non-calmodulin activator of erythrocyte membrane Ca2(+)-ATPase and might be involved in the control of calcium transport across the plasma membrane.     

Effects of fatty acids on activity and calmodulin binding of Ca2+-ATPase of human erythrocyte membranes

Activation and inhibition of Ca2+-ATPase of calmodulin-depleted human erythrocyte membranes by oleic acid and a variety of other fatty acids have been measured. Low concentrations of oleic acid stimulate the enzyme activity, both in the presence and in the absence of calmodulin. Concomitantly, the affinity of the membrane bound enzyme to calmodulin progressively decreases due to competitive interactions of calmodulin and oleic acid with the enzyme. Removal of oleic acid from the membrane by serum albumin extinguishes the activating effect of oleic acid and restores the ability of the enzyme to bind calmodulin with high affinity. High concentrations of oleic acid induce an almost complete and irreversible loss of enzyme activity which cannot be abolished by removal of oleic acid. Despite a complete loss of enzyme activity, binding of calmodulin to membranes is approximately normal after removal of oleic acid. Activities of (Na+ + K+)-ATPase, Mg2+-ATPase and acetylcholine esterase, as well as the total protein content, show no gross changes upon treatment of membranes with increasing amounts of oleic acid, which seems to exclude that membrane solubilisation by oleic acid causes an inactivation of the enzyme.     

Relation between Ca2+-ATPase and endogenous calmodulin of human erythrocyte membranes

Short incubation of erythrocyte membranes with oleic acid releases Ca2+-independently bound endogenous calmodulin together with a minor fraction of membrane-associated proteins without destruction of the membranes. The released endogenous calmodulin is similar if not identical to cytosolic calmodulin reversibly bound to ghosts in a Ca2+-dependent manner. The release of endogenous calmodulin proceeds without affecting the activity of Ca2+-ATPase when ghosts are incubated with oleic acid in the presence of Ca2+ plus ATP and thereafter freed from oleic acid by washings with serum albumin. Kinetic parameters of Ca2+-ATPase of ghosts with and without endogenous calmodulin are identical as are amounts of exogenous calmodulin bound to these ghosts. Thus, endogenous calmodulin does not function as an essential part of Ca2+-ATPase.     

Ca2+-mediated activation of human erythrocyte membrane Ca2+-ATPase

Ca2+-ATPase of human erythrocyte membranes, after being washed to remove Ca2+ after incubation with the ion, was found to be activated. Stimulation of the ATPase was related neither to fluidity change nor to cytoskeletal degradation of the membranes mediated by Ca2+. Activation of the transport enzyme was also unaffected by detergent treatment of the membrane, but was suppressed when leupeptin was included during incubation of the membranes with Ca2+. Stimulation of the ATPase by a membrane-associated Ca2+-dependent proteinase was thus suggested. Much less 138 kDa Ca2+-ATPase protein could be harvested from a Triton extract of membranes incubated with Ca2+ than without Ca2+. Activity of the activated enzyme could not be further elevated by exogenous calpain, even after treatment of the membranes with glycodeoxycholate. There was also an overlap in the effect of calmodulin and the Ca2+-mediated stimulation of membrane Ca2+-ATPase. While Km(ATP) of the stimulated ATPase remained unchanged, a significant drop in the free-Ca2+ concentration for half-maximal activation of the enzyme was observed.     

The effect of Ca2+ and calmodulin on the inhibition of Ca2(+)+Mg2(+)-ATPase in erythrocyte ghost membranes by nonpolar and polar carbodiimides

N,N'-dicyclohexylcarbodiimide (DCCD) and 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide (CMCD) inhibited calmodulin-dependent Ca2(+)+Mg2(+)-ATPase activity in erythrocyte ghost membranes. The extent of the inhibition caused by carbodiimides strongly depended on their hydrophobicity. Hydrophobic DCCD was a more potent inhibitor then hydrophilic CMCD. Calmodulin (CaM) protected the enzyme against the former carbodiimide, whereas Ca2+ did the same against the latter. In contrast to previous observations made by Villalobo et al., on the purified enzyme, neither carbodiimide affected the calmodulin-independent ATPase activity in ghost membranes. Inhibition of the calmodulin-dependent ATPase activity was due to a decrease of the maximum activity, whereas the Km value for Ca2+ remained unchanged. Titration of erythrocyte ghost membranes with CaM revealed a biphasic response of ATPase to this activator. Two affinity constants were found for CaM, 0.64 nM and 14 nM. DCCD affected the interaction with CaM at high- and low-affinity binding sites in a competitive manner. CMCD acted as a noncompetitive inhibitor for CaM low-affinity sites, whereas it behaved in a competitive way against CaM interaction with high-affinity sites. In E2 form (stabilized by vanadate and EGTA) ATPase was more sensitive to carbodiimides than in E1 form (induced by La3+).     

Enhanced activation of human erythrocyte Ca2+-ATPase by calmodulin after storage or brief exposure to disulfite

Ca2+-ATPase of human erythrocyte membranes which are prepared from freshly drawn human blood can be activated by the calmodulin present in the hemolysate to 1.5-times the basal level. However, when the membranes are prepared from blood stored for 5-14 days the activation by calmodulin reaches 2.5-times the basal level. An enhanced reactivity to calmodulin of similar magnitude was produced by brief exposure of fresh erythrocytes to 25 mM Na2S2O5 prior to isolation of the membranes. Reincubation of the activated cells in a disulfite-free medium restored the membrane-bound Ca2+-ATPase to a state of normal reactivity to calmodulin. It is hypothesized that these results are related to the level of cytoplasmic Ca2+ which is partly controlled by complex formation with 2,3-diphosphoglycerate, the concentration of which is diminished when its specific phosphatase is activated by Na2S2O5.     

Rate constants for calmodulin binding to Ca2+-ATPase in erythrocyte membranes

The Ca2+-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes, which is part of the Ca2+ pump, can be activated by binding of calmodulin. Rate constants (k1) for association of calmodulin and enzyme, which depends on the Ca2+ concentration, have been determined by the aid of an enzyme model. k1 increased from 0.25 . 10(6) to 17.3 . 10(6) M-1 . min-1 (70 times) when the free Ca2+ concentration was raised from 0.7 to 20 microM. The binding of calmodulin to the Ca2+-ATPase is reversible. The rate constants (k-1) for dissociation of enzyme-calmodulin complex decreased from 6.0 to 0.044 min-1 (135 times) when the free Ca2+ concentration was increased from 0.1 to 2-20 microM. The apparent dissociation constant Kd = k-1/k1 accordingly increased from 2.5 nM to 25 microM (or higher) when the Ca2+ concentration was reduced from 20 to 0.1 microM. Therefore, at 10(-7) M free Ca2+ most of the Ca2+-pump enzyme will not bind calmodulin. For the intact cell the time dependences of activation and deactivation of the Ca2+-pump enzyme have been estimated from the rate constants above. The results suggest that the Ca2+ pump is well suited to maintain a cytosolic concentration of 10(-7) M free Ca2+ (or lower) in the unstimulated cell and, when the cell is stimulated, to allow transient Ca2+ signals up to approx. 10(-5) M in the cytosol.     

Stimulation of the purified erythrocyte Ca2+-ATPase by tryptic fragments of calmodulin

Highly purified tryptic peptides of calmodulin have been obtained by high-performance liquid chromatography. Tryptic cleavage of calmodulin in the presence of Ca2+ results in two main fragments which have been identified by analysis of the amino acid composition as 1-77 and 78-148. In the absence of Ca2+, trypsin cleavage yields fragments 1-106, 1-90, and 107-148. Only fragments 78-148 and 1-106 are still able to stimulate the purified Ca2+-ATPase of erythrocytes, albeit much less efficiently on a molar basis, than intact calmodulin. On the other hand, the same fragments were unable to stimulate the calmodulin-dependent cyclic nucleotide phosphodiesterase, even at 1000-fold molar excess (shown also by Newton, D.L., Oldewurtel, M.D., Krinks, M.H., Shiloach, J., and Klee, C.B. (1984) J. Biol. Chem. 259, 4419-4426). This points to the importance of the carboxyl-terminal half of calmodulin and especially of Ca2+-binding region III in the interaction of calmodulin with the Ca2+-ATPase and provides clear evidence that calmodulin interacts differently with different targets. Oxidation of methionine(s) of fragment 78-148 with N-chlorosuccinimide removes the ability of this fragment to stimulate the ATPase.     

Troponin C/calmodulin chimeras as erythrocyte plasma membrane Ca2+-ATPase activators

Calmodulin (CaM) and troponin C (TnC) are EF-hand proteins that play fundamentally different roles in animal physiology. TnC has a very low affinity for the plasma membrane Ca2+-ATPase and is a poor substitute for CaM in increasing the enzyme's affinity for Ca2+ and the rate of ATP hydrolysis. We use a series of recombinant TnC (rTnC)/CaM chimeras to clarify the importance of the CaM carboxyl-terminal domain in the activation of the plasma membrane Ca2+-ATPase. The rTnC/CaM chimera, in which the carboxyl-terminal domain of TnC is replaced by that of CaM, has the same ability as CaM to bind and transmit the signal to Ca2+ sites on the enzyme. There is no further functional gain when the amino-terminal domain is modified to make the rTnC/CaM chimera more CaM-like. To identify which regions of the carboxyl-terminal domain of CaM are responsible for these effects, we constructed the chimeras rTnC/3CaM and rTnC/4CaM, where only one-half of the C-terminal domain of CaM (residues 85-112 or residues 113-148) replaces the corresponding region in rTnC. Neither rTnC/3CaM nor rTnC/4CaM can mimic CaM in its affinity for the enzyme. Nevertheless, with respect to the signal transduction process, rTnC/4CaM, but not rTnC/3CaM, shows the same behaviour as CaM. We conclude that the whole C-terminal domain is required for binding to the enzyme while Ca2+-binding site 4 of CaM bears all the requirements to increase Ca2+ binding at PMCA sites. Such mechanism of binding and activation is distinct from that proposed for most other CaM targets. Furthermore, we suggest that Ala128 and Met124 from CaM site 4 may play a crucial role in discriminating CaM from TnC.     

Effects of phospholipids on binding of calcium to (Ca2(+)-Mg2(+)-ATPase

The (Ca2(+)-Mg2(+)-ATPase purified from skeletal muscle sarcoplasmic reticulum binds two Ca2+ ions per ATPase molecule. On reconstitution into bilayers of dioleoylphosphatidylcholine [C18:1)PC) or dinervonylphosphatidylcholine [C24:1)PC) the stoichiometry of binding remains unchanged, but when the ATPase is reconstituted into bilayers of dimyristoleoylphosphatidylcholine [C14:1)PC) the stoichiometry changes to one Ca2+ ion per ATPase molecule. Nevertheless, the level of phosphorylation is the same for the ATPase reconstituted with (C18:1)PC or (C14:1)PC. The effect of (C14:1)PC on the stoichiometry of Ca2+ binding is prevented by androstenol at a 1:1 molar ratio with the phospholipid.     

Activation of the erythrocyte Ca2+-ATPase by either self-association or interaction with calmodulin

The octaethyleneglycol mono-n-dodecyl ether solubilized Ca2+-ATPase purified from human erythrocytes has been studied to determine the physical mechanism of its activation by calmodulin. The dependence of Ca2+-ATPase activity on the enzyme concentration shows a transformation from a calmodulin-dependent to a fully active calmodulin-independent form. The transformation is cooperative with a half-maximal activation at 10-20 nM enzyme. This suggests that at higher enzyme concentrations interactions between Ca2+-ATPase polypeptide chains substitute for calmodulin-enzyme interactions, resulting in activation. In support of this interpretation, the inclusion of higher octaethyleneglycol mono-n-dodecyl ether concentrations shifts the half-maximal transformation to higher enzyme concentrations. Regardless of the detergent concentration, calmodulin decreases by about 2-fold the enzyme concentration required to observe half-maximal Ca2+-ATPase activation, without affecting the maximal velocity or cooperativity. This indicates that calmodulin facilitates interactions between enzyme molecules. The fluorescein-5'-isothiocyanate-modified Ca2+-ATPase shows an increase in fluorescence polarization which occurs over the same narrow concentration range that is seen with the Ca2+-ATPase activity, confirming association of enzyme molecules. Stimulation of the Ca2+-ATPase activity by calmodulin has revealed a stoichiometry of 0.73, with a dissociation constant of 1.6 nM calmodulin. We have demonstrated by use of calmodulin-Sepharose chromatography that both the calmodulin-dependent and independent Ca2+-ATPase forms bind calmodulin, even though stimulation of activity is seen only with the former one. Our data suggest the following two mechanisms for the Ca2+-ATPase activation: self-association of enzyme molecules or interaction with calmodulin.     

Single-molecule dynamics of the calcium-dependent activation of plasma-membrane Ca2+-ATPase by calmodulin

The plasma membrane calcium-ATPase (PMCA) helps to control cytosolic calcium levels by pumping out excess Ca2+. PMCA is regulated by the Ca2+ signaling protein calmodulin (CaM), which stimulates PMCA activity by binding to an autoinhibitory domain of PMCA. We used single-molecule polarization methods to investigate the mechanism of regulation of the PMCA by CaM fluorescently labeled with tetramethylrhodamine. The orientational mobility of PMCA-CaM complexes was determined from the extent of modulation of single-molecule fluorescence upon excitation with a rotating polarization. At a high Ca2+ concentration, the distribution of modulation depths reveals that CaM bound to PMCA is orientationally mobile, as expected for a dissociated autoinhibitory domain of PMCA. In contrast, at a reduced Ca2+ concentration a population of PMCA-CaM complexes appears with significantly reduced orientational mobility. This population can be attributed to PMCA-CaM complexes in which the autoinhibitory domain is not dissociated, and thus the PMCA is inactive. The presence of these complexes demonstrates the inadequacy of a two-state model of Ca2+ pump activation and suggests a regulatory role for the low-mobility state of the complex. When ATP is present, only the high-mobility state is detected, revealing an altered interaction between the autoinhibitory and nucleotide-binding domains.     

Modulation of human erythrocyte Ca2+-ATPase activity by glycerol: The role of calmodulin

The effect of an intracellular cryoprotectant glycerol on human erythrocyte Ca2+-ATPase activity and possible involvement of calmodulin in the regulation of Ca2+-pump under these conditions were investigated. The experiments were carried out using saponin-permeabilized cells and isolated erythrocyte membrane fractions (white ghosts). Addition of rather low concentrations of glycerol to the medium increased Ca2+-ATPase activity in the saponin-permeabilized cells; the maximal effect was observed at 10% glycerol. Subsequent increase in glycerol concentrations above 20% was accompanied by inhibition of Ca2+-ATPase activity. Lack of stimulating effect of glycerol on white ghost Ca2+-ATPase may be attributed to removal of endogenous compounds regulating activity of this ion transport system. Inhibitory analysis using R24571 revealed that activation of Ca2+-ATPase by 10% glycerol was observed only in the case of inhibitor administration after modification of cells with glycerol; in the case of inhibitor addition before erythrocyte contact with glycerol, this phenomenon disappeared. These data suggest the possibility of regulation of human erythrocyte Ca2+-ATPase by glycerol; this regulatory effect may be attributed to both glycerol-induced structural changes in the membrane and also involvement of calmodulin in modulation of catalytic activity of the Ca2+-pump.     

Target sizes of human erythrocyte membrane Ca2+-ATPase and Mg2+-ATPase activities in the presence and absence of calmodulin

We have investigated the subunit structure of Ca2+-transport ATPase in human erythrocyte membranes using radiation inactivation analysis. All inactivation data were linear on a semilog plot down to at least 20% of the control activity. We found a target size for the calmodulin-dependent Ca2+-ATPase activity of 331 kDa, consistent with the presence of this enzyme as a dimer in calmodulin-depleted ghosts. Membranes which had been saturated with calmodulin before irradiation yield a a similar size of 317 kDa, implying that activation of Ca2+-transport ATPase by calmodulin does not involve significant change in oligomeric structure. Basal (calmodulin-independent) Ca2+-ATPase activity corresponded to a size of 290 kDa, suggesting that this activity resides in the same, or similar-sized, complex as the calmodulin-dependent activity. Mg2+-ATPase activity, however, was found to reside in a smaller complex of 224 kDa, which proved to be statistically distinct from the target size of Ca2+-ATPase activity. It would appear that Mg2+-ATPase is a distinct entity whose function is likely unrelated to the Ca2+-transport ATPase.     

Decrease of apparent calmodulin affinity of erythrocyte (Ca2+ + Mg2+)-ATPase at low Ca2+ concentrations

The calmodulin activation of the (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied in the range of 1 nM to 40 microM of purified calmodulin. The apparent calmodulin-affinity of the ATPase was strongly dependent on Ca2+ and decreased approx. 1000-times when the Ca2+ concentration was reduced from 112 to 0.5 microM. The data of calmodulin (Z) activation were analyzed by the aid of a kinetic enzyme model which suggests that 1 molecule of calmodulin binds per ATPase unit and that the affinities of the calcium-calmodulin complexes (CaiZ) decreases in the order of Ca3Z greater than Ca4Z greater than Ca2Z greater than or equal to CaZ. Furthermore, calmodulin dissociates from the calmodulin-saturated Ca2+-ATPase in the range of 10(-7)-10(-6) M Ca2+, even at a calmodulin concentration of 5 microM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 microM, corresponding to 50-80-times the cellular concentration of Ca2+-ATPase, estimated to be approx. 10 nmol/h membrane protein. We therefore conclude that most of the calmodulin is dissociated from the Ca2+-transport ATPase in erythrocytes at the prevailing Ca2+ concentration (probably 10(-7)-10(-8) M) in vivo, and that the calmodulin-binding and subsequent activation of the Ca2+-ATPase requires that the Ca2+ concentration rises to 10(-6)-10(-5) M.     

Time-resolved fluorescence of erythrocyte plasma membrane Ca2(+)-ATPase in different functional states.

The fluorescence decay of the plasma membrane calmodulin-activated Ca2(+)-ATPase from the erythrocyte was measured for the first time. The availability of a novel procedure for on-line blank subtraction in frequency-domain lifetime data acquisition (G.G. Reinhart, B. Feddersen, D. Jameson and E. Gratton, Biophys. J. 57 (1990) 189a) permitted the elimination of background interference from detergent-solubilized purified plasma membrane ATPase samples. The fluorescence decay of the erythrocyte Ca2(+)-ATPase was measured in the absence of Ca2+, or in the presence of Ca2+ or Ca2+ plus calmodulin. In the three different experimental conditions the fluorescence decay was very heterogeneous and could be best described by Lorentzian distributions of lifetime values. In the absence of Ca2+ the decay was described by a broad lifetime distribution centered at 4.4 ns with a width of 3.2 ns, indicating heterogeneity of tryptophan microenvironments in the ATPase. Calcium ion binding promoted an 11% increase in the center and a 27% decrease in the width of the distribution. By contrast, addition of calmodulin in the presence of Ca2+ caused a 15% decrease in the center of the distribution, revealing structural difference between calmodulin-activated and Ca2(+)-activated states of the ATPase. These results indicate the usefulness of on-line blank subtraction in frequency-domain lifetime measurements to investigate conformational changes in detergent-solubilized membrane protein samples.