Pathway for ATP synthesis by sarcoplasmic reticulum ATPase

Millisecond mixing and quenching experiments were performed in order to study the rate of phosphorylation by P_i of the Ca²⁺-dependent ATPase of sarcoplasmic reticulum vesicles. A rapid phosphoenzyme formation was observed when the vesicles were preincubated in the absence of Ca²⁺ prior to the addition of P_i and Mg²⁺ to the medium, the half-time being in the range of 6 to 10 ms. A lag phase and a 5- to 10-fold slower rate of phosphoenzyme formation were observed when the enzyme was preincubated with Ca²⁺ prior to the addition to the reaction mixture of P_i, Mg²⁺, and an excess of ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid. The rate of phosphoenzyme hydrolysis was measured either by the addition of Ca²⁺ or, in the absence of Ca²⁺, by tracing the hydrolysis of radioactive phosphoenzyme upon the addition of nonradioactive P_i. In the presence of Ca²⁺, the rate of phosphoenzyme hydrolysis was found to be one order of magnitude slower than the rate of hydrolysis measured in the absence of Ca²⁺. Different rates of phosphoenzyme formation and cleavage were found depending on whether sarcoplasmic reticulum vesicles or purified Ca²⁺-dependent ATPase were used. A transient phosphorylation by P_i was observed when the enzyme was preincubated in the absence of Ca²⁺ and then added to a medium containing P_i, Mg²⁺, and excess of Ca²⁺. The enzyme was phosphorylated during the initial 100 ms, the phosphoenzyme formed being slowly hydrolyzed in the subsequent incubation intervals. In these conditions ATP synthesis was observed if ADP was added to the mixture 100 ms after starting the reaction. No transient phosphorylation by P_i was observed when the enzyme was preincubated with Ca²⁺. Synthesis of a small but significant amount of ATP was observed when the enzyme was preincubated in the absence of Ca²⁺ and then added to a medium containing P_i, ADP, Mg²⁺, and 20 mm CaCl₂. This was not observed when the enzyme was preincubated in the presence of Ca²⁺.     

Regulation of ATP synthesis catalyzed by the calcium pump of sarcoplasmic reticulum

The role of pH, KCl, ATP, water activity, and temperature in ATP synthesis from ADP and Pi was investigated in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. In totally aqueous medium, the synthesis of ATP was inhibited by ATP, KCl, and pH values above 6.5. When the water activity of the medium was decreased by the addition of 30% (v/v) dimethyl sulfoxide, the synthesis of ATP was no longer inhibited by ATP; it was activated by KCl and the optimum pH changed from 6.5 to 7.5. In totally aqueous medium, the concentration of MgCl2 needed for half-maximal synthesis of ATP was found to vary with the temperature of the assay medium; at 35 degrees C it was 1 mM and increased to a value higher than 10 mM when the temperature was decreased to 15 degrees C. In the presence of 30% dimethyl sulfoxide, maximal synthesis of ATP was attained in presence of 0.05 mM MgCl2 at both 15 and 35 degrees C. The hypothesis is raised that in the living cell water structure may play a role in regulating the synthesis of ATP observed during the reversal of the Ca2+ pump of the sarcoplasmic reticulum.     

Interaction of spin-labeled nucleotides with sarcoplasmic reticulum adenosinetriphosphatase

Spin-labeled derivatives of AMP-PCP, ATP, and 2'-deoxy-ATP, with a nitroxide moiety attached to the ribose ring [3'-O-(1-oxy-2,2,5,5-tetramethylpyrroline-3-carbonyl)nucleotide], are used to study the nucleotide binding site stoichiometry of sarcoplasmic reticulum (SR) ATPase. With all derivatives, a maximal binding of 4.5 nmol/mg of SR protein is found, a value close to the number of phosphorylation sites obtained with ATP. The spin-labeled nucleotides cannot be utilized by the enzyme as substrates. Binding of spin-labeled nucleotides is inhibited by labeling the ATPase with fluorescein 5'-isothiocyanate, indicating that all the labeled nucleotide is located at the catalytic site. Additions of spin-labeled ATP to vesicle suspensions during steady turnover demonstrate competitive inhibition of both catalysis and the regulatory effect normally exhibited by ATP. As secondary binding of spin-labeled ATP is not detected at pertinent concentrations, it is suggested that both functions of ATP may be effected through a single site.     

Rate of calcium release and ATP synthesis in sarcoplasmic reticulum vesicles

Sarcoplasmic reticulum vesicles can catalyze the synthesis of ATP coupled to the efflux of calcium. The rate of this reaction is much faster when the vesicles are loaded in a medium containing phosphate than when oxalate is the precipitating agent. Two components of ATP synthesis can be observed when vesicles loaded with calcium phosphate are used. In the millisecond range and when the loaded vesicles are phosphorylated by Pi, the addition of ADP leads to an initial burst of ATP synthesis and after 50 ms approximately 3.0 nmol of ATP/mg protein are synthesized. This burst is not inhibited by ATP and is enhanced by physiological concentrations of KCl. The slow component of ATP synthesis is inhibited by both ATP and 100 mM KCl. In the physiological pH range, betaine, a trimethylamine present in different tissues, increases the level of phosphoenzyme formed by Pi and enhances the amount of ATP synthesized during the first turn of the reversal of the calcium pump.     

Kinetic control of mitochondrial ATP synthesis

In order to gain a clearer understanding of the kinetic control of ATP synthesis, rat liver and rat heart mitochondria were incubated under conditions that resulted in various rates of net ATP synthesis or ATP hydrolysis. Radiolabeled phosphate was included in the incubation media, and exchange rates between phosphate and ATP were determined as a function of rates of net ATP synthesis. Since ATP synthase is a highly reversible enzyme, the catalyzed reaction was expected to approach equilibrium especially at low rates of respiration and net ATP synthesis. Thus ADP + Pi V1 in equilibrium V2 ATP. If V1 is the rate of incorporation of radiolabeled phosphate into ATP, then net ATP synthesis (or hydrolysis) is V1 - V2. Since V1 and V1 - V2 could be measured, it was possible to calculate V2. V1 doubled in the transition from zero to maximal net ATP synthesis, whereas V2 decreased by over 90% when the rate of ATP synthesis was high due to high-media ADP. In heart mitochondria at 37 degrees C when respiration increased from 104 +/- 10 to 842 +/- 51 nanoatoms of O2/(min X mg), incorporation of [33P]phosphate into ATP (V1) increased from 1,100 +/- 60 to 1,978 +/- 121 and V2 decreased from 1,100 to near zero. These data demonstrate that mitochondrial ATP synthesis does not occur near equilibrium under physiological conditions and relatively high rates of ATP synthesis. A reaction with a high ratio of forward to reverse flux is obviously not near equilibrium. The important most sensitively controlled reaction appears to be V2, ATP hydrolysis. Possible mechanisms of kinetic control of V2 are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversible loss of cooperative calcium ion binding by sarcoplasmic reticulum calcium adenosinetriphosphatase

Incubation of rabbit skeletal muscle sarcoplasmic reticulum vesicles in solutions of very low [Ca2+] caused Ca2+ to bind noncooperatively, as determined by the dependence of the intrinsic tryptophan fluorescence intensity on added increments of Ca2+. Cooperative Ca2+ binding was obtained if the ATPase was incubated in [Ca2+] high enough (25 microM) to saturate the two high-affinity Ca2+ binding sites and then titrated with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. The cooperative binding had an apparent association constant of 6.3 X 10(6) M-1 and a Hill coefficient of 2.6; these constants for the noncooperative binding case were 5.0 X 10(5) M-1 and 1.2, respectively. The transitions from the noncooperative to the cooperative Ca2+ binding forms of the enzyme were slow compared to the time required for Ca2+ binding to reach equilibrium. Thus, it appears that sarcoplasmic reticulum CaATPase is a hysteretic enzyme. Intrinsic association constants for Ca2+ binding and equilibrium constants for the transitions between the two forms in low and high [Ca2+] were estimated from analyses of a general scheme for cooperative and noncooperative binding.     

Patterns of proteolytic cleavage and carbodiimide derivatization in sarcoplasmic reticulum adenosinetriphosphatase

Two series of experiments were carried out to characterize (a) peptide fragments of sarcoplasmic reticulum (SR) ATPase, based on proteolysis with different enzymes and distribution of known labels, and (b) specific labeling and functional inactivation patterns, following ATPase derivatization with dicyclohexylcarbodiimide (DCCD) under various conditions. Digestion with trypsin or chymotrypsin results in the initial cleavage of the SR ATPase in two fragments of similar size and then into smaller fragments, while subtilisin and thermolysin immediately yield smaller fragments. Peptide fragments were assigned to segments of the protein primary structure and to functionally relevant domains, such as those containing the 32P at the active site and the fluorescein isothiocyanate at the nucleotide site. ATPase derivatization with [14C]DCCD under mild conditions produced selective inhibition of ATPase hydrolytic catalysis (EP + H2O in equilibrium E + Pi) without significant incorporation of the 14C radioactive label. This effect is attributed to blockage of catalytically active residues by reaction of the initial DCCD adduct with endogenous or exogenous nucleophiles. ATPase derivatization with [14C]DCCD under more drastic conditions produced inhibition of calcium binding, 14C radioactive labeling of tryptic fragments A1 and A2 (but not of B), and extensive cross-linking. Intermolecular and, to some extent, intramolecular cross-linking were prevented by exogenous nucleophiles. The presence of calcium during derivatization prevented functional inactivation, radioactive labeling of fragment A2, and internal cross-linking of fragment A1. It is proposed that both A1 and A2 fragments participate in formation of the calcium binding domain and that the labeled residues of fragment A2 are directly involved in calcium complexation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Roles of phosphorylation and nucleotide binding domains in calcium transport by sarcoplasmic reticulum adenosinetriphosphatase

The roles of the phosphorylation (phosphorylated enzyme intermediate) and nucleotide binding domains in calcium transport were studied by comparing acetyl phosphate and ATP as substrates for the Ca2+-ATPase of sarcoplasmic reticulum vesicles. We found that the maximal level of phosphoenzyme obtained with either substrate is approximately 4 nmol/mg of protein, corresponding to the stoichiometry of catalytic sites in our preparation. The initial burst of phosphoenzyme formation observed in the transient state, following addition of either substrate, is accompanied by internalization of 2 mol of calcium per mole of phosphoenzyme. The internalized calcium is then translocated with a sequential pattern, independent of the substrate used. Following a rate-limiting step, the phosphoenzyme undergoes hydrolytic cleavage and proceeds to the steady-state activity which is soon "back inhibited" by the rise of Ca2+ concentration in the lumen of the vesicles. When the "back inhibition" is released by the addition of oxalate, substrate utilization and calcium transport occur with a ratio of 1:2, independent of the substrate and its concentration. When the nucleotide binding site is derivatized with FITP, the enzyme can still utilize acetyl phosphate (but not ATP) for calcium transport. No secondary activation of acetyl phosphate utilization by the FITC-enzyme was obtained with millimolar nucleotide. These observations demonstrate that the basic coupling mechanism of catalysis and calcium transport involves the phosphorylation and calcium binding domains, and not the nucleotide binding domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

A spectrophotometric assay of ATP synthesized by sarcoplasmic reticulum.

The problems encountered with a coupled enzyme assay for ATP using glucose, hexokinase and glucose-6-phosphate dehydrogenase are discussed and a modification where fructose and glucosephosphate isomerase were substituted for glucose is described. This modified assay was used successfully to measure the ATP synthesized by reversal of the sarcoplasmic reticulum ATPase. ATP synthesized by adenylate kinase contaminating the sarcoplasmic reticulum was easily corrected for by a subtraction procedure.     

Binding of Ca2+ to the calcium adenosinetriphosphatase of sarcoplasmic reticulum

The binding of Ca2+ and the resulting change in catalytic specificity that allows phosphorylation of the calcium ATPase of sarcoplasmic reticulum by ATP were examined by measuring the amount of phosphoenzyme formation from [32P]ATP, or 45Ca incorporation into vesicles, after the simultaneous addition of ATP and EGTA at different times after mixing enzyme and Ca2+ (25 degrees C, pH 7.0, 5 mM MgSO4, 0.1 M KCl). A "burst" of calcium binding in the presence of high [Ca2+] gives approximately 12% phosphorylation and internalization of two Ca2+ at very short times after the addition of Ca2+ with this assay. This shows that calcium binding sites are available on the cytoplasmic-facing side of the free enzyme. Calcium binding to these sites induces the formation of cE.Ca2, the stable high-affinity form of the enzyme, with k = 40 s-1 at saturating [Ca2+] and a half-maximal rate at approximately 20 microM Ca2+ (from Kdiss = 7.4 X 10(-7) M for Ca.EGTA). The formation of cE.Ca2 through a "high-affinity" pathway can be described by the scheme E 1 in equilibrium cE.Ca1 2 in equilibrium cE.Ca2, with k1 = 3 X 10(6) M-1 s-1, k2 = 4.3 X 10(7) M-1 s-1, k-1 = 30 s-1, k-2 = 60 s-1, K1 = 9 X 10(-6) M, and K2 = 1.4 X 10(-6) M. The approach to equilibrium from E and 3.2 microM Ca2+ follows kobsd = kf + kr = 18 s-1 and gives kf = kr = 9 s-1. The rate of exchange of 45Ca into the inner position of cE.Ca2 shows an induction period and is not faster than the approach to equilibrium starting with E and 45Ca. The dissociation of 45Ca from the inner position of cE.45Ca.Ca in the presence of 3.2 microM Ca2+ occurs with a rate constant of 7 s-1. These results are inconsistent with a slow conformational change of free E to give cE, followed by rapid binding-dissociation of Ca2+.     

Structural effects of substrate utilization on the adenosinetriphosphatase chains of sarcoplasmic reticulum

Addition of ATP to suspensions of fragmented sarcoplasmic reticulum (SR) containing low concentrations of a detergent that does not by itself produce major vesicular disruption is followed by a transient reduction in turbidity accompanied by solubilization of the vesicles. The effect of ATP is Ca2+ dependent and proceeds in parallel with utilization of the nucleotide as a substrate for the SR ATPase. Analogous effects are observed with other substrates producing enzyme phosphorylation at the catalytic site. The effect of ATP can also be detected in studies of fluorescence energy transfer between enzyme chains, by using the technique of Vanderkooi et al. [Vanderkooi, J., Ierokomas, A., Nakamura, H., & Martonosi, A. (1977) Biochemistry 16, 1262]. For this purpose, ATPase chains are labeled separately with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) and 6-(iodoacetamido) fluorescein (IAF). Samples of vesicles uniformly labeled with either IAEDANS or IAF, mixtures of two populations of vesicles uniformly labeled with either fluorophore, and vesicles containing randomized chains labeled with either fluorophore are used as experimental systems. In the last system, significant energy transfer from IAEDANS (donor) to IAF (acceptor) is revealed by fluorescence spectra and measurements of donor fluorescence intensity and lifetime. This is attributed to close interactions between ATPase chains within the membrane bilayer. It is then found that in the presence of low detergent concentrations, ATP changes the extent of energy transfer between labeled ATPase chains, consistent with destabilization of the interaction of chains. The observed effects are attributed to a reversible structural transition concomitant with enzyme phosphorylation and related to catalytic and transport function.     

Capsaicin stimulates uncoupled ATP hydrolysis by the sarcoplasmic reticulum calcium pump

In muscle cells the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) couples the free energy of ATP hydrolysis to pump Ca(2+) ions from the cytoplasm to the SR lumen. In addition, SERCA plays a key role in non-shivering thermogenesis through uncoupled reactions, where ATP hydrolysis takes place without active Ca(2+) translocation. Capsaicin (CPS) is a naturally occurring vanilloid, the consumption of which is linked with increased metabolic rate and core body temperature. Here we document the stimulation by CPS of the Ca(2+)-dependent ATP hydrolysis by SERCA without effects on Ca(2+) accumulation. The stimulation by CPS was significantly dependent on the presence of a Ca(2+) gradient across the SR membrane. ATP activation assays showed that the drug reduced the nucleotide affinity at the catalytic site, whereas the affinity at the regulatory site increased. Several biochemical analyses indicated that CPS stabilizes an ADP-insensitive E(2)P-related conformation that dephosphorylates at a higher rate than the control enzyme. Under conditions where uncoupled SERCA was specifically inhibited by the treatment with fluoride, low temperatures, or dimethyl sulfoxide, CPS had no stimulatory effect on ATP hydrolysis by SERCA. It is concluded that CPS stabilizes a SERCA sub-conformation where Ca(2+) is released from the phosphorylated intermediate to the cytoplasm instead of the SR lumen, increasing ATP hydrolysis not coupled with Ca(2+) transport. To the best of our knowledge CPS is the first natural drug that augments uncoupled SERCA, presumably resulting in thermogenesis. The role of CPS as a SERCA modulator is discussed.     

Steady state kinetics of ATP synthesis and hydrolysis coupled calcium transport catalyzed by the reconstituted sarcoplasmic reticulum ATPase

The steady state kinetics of calcium transport driven by ATP hydrolysis and ATP synthesis catalyzed by purified, reconstituted calcium ATPase has been investigated as a function of the transmembrane calcium gradient. Purified calcium ATPase was reconstituted into phospholipid vesicles enabling control of the transmembrane calcium gradient. Calcium transport was monitored spectrophotometrically by the calcium indicator, Arsenazo III. Thus, only the enzymatic activity of coupled transport was measured. It was shown under conditions of low external calcium that ATP hydrolysis and synthesis follow simple Michaelis-Menten kinetics and that Michaelis constants obtained for both processes appear independent of the calcium gradient. The maximum velocities for both hydrolysis and synthesis strongly depend on the transmembrane calcium gradient. Based on these results, a mechanism is proposed in which a random addition of substrates for ATP synthesis is followed by random release of ATP and calcium. By measuring the ATP hydrolysis and synthesis under identical conditions, determination of the equilibrium constant for ATP hydrolysis as a function of the transmembrane calcium gradient was possible. Our results indicate that the thermodynamics of the catalytic cycle can be totally accounted for by the energetics of transport of 2.2 +/- 0.3 calciums and the hydrolysis of 1 ATP. An equilibrium constant for ATP hydrolysis in the absence of a calcium gradient was determined to be 4.0 X 10(4).