Toward a general method to observe the phosphate groups of phosphoenzymes with infrared spectroscopy

A general method to study the phosphate group of phosphoenzymes with infrared difference spectroscopy by helper enzyme-induced isotope exchange was developed. This allows the selective monitoring of the phosphate P-O vibrations in large proteins, which provides detailed information on several band parameters. Here, isotopic exchange was achieved at the oxygen atoms of the catalytically important phosphate group that transiently binds to the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a). [gamma-(18)O(3)]ATP phosphorylated the ATPase, which produced phosphoenzyme that was initially isotopically labeled. The helper enzyme adenylate kinase regenerated the substrate ATP from ADP (added or generated upon ATP hydrolysis) with different isotopic composition than used initially. With time this produced the unlabeled phosphoenzyme. The method was tested on the ADP-insensitive phosphoenzyme state of the Ca(2+)-ATPase for which the vibrational frequencies of the phosphate group are known, and it was established that the helper enzyme is effective in mediating the isotope exchange process.     

Inhibition of plasma membrane Ca-ATPase by CrATP. LaATP but not CrATP stabilizes the Ca-occluded state

The bidentate complex of ATP with Cr³⁺, CrATP, is a nucleotide analog that is known to inhibit the sarcoplasmic reticulum Ca²⁺-ATPase and the Na⁺,K⁺-ATPase, so that these enzymes accumulate in a conformation with the transported ion (Ca²⁺ and Na⁺, respectively) occluded from the medium. Here, it is shown that CrATP is also an effective and irreversible inhibitor of the plasma membrane Ca²⁺-ATPase. The complex inhibited with similar efficiency the Ca²⁺-dependent ATPase and the phosphatase activities as well as the enzyme phosphorylation by ATP. The inhibition proceeded slowly (T_1/2 = 30 min at 37 °C) with a K_i = 28 ± 9 μM. The inclusion of ATP, ADP or AMPPNP in the inhibition medium effectively protected the enzyme against the inhibition, whereas ITP, which is not a PMCA substrate, did not. The rate of inhibition was strongly dependent on the presence of Mg²⁺ but unaltered when Ca²⁺ was replaced by EGTA. In spite of the similarities with the inhibition of other P-ATPases, no apparent Ca²⁺ occlusion was detected concurrent with the inhibition by CrATP. In contrast, inhibition by the complex of La³⁺ with ATP, LaATP, induced the accumulation of phosphoenzyme with a simultaneous occlusion of Ca²⁺ at a ratio close to 1.5 mol/mol of phosphoenzyme. The results suggest that the transport of Ca²⁺ promoted by the plasma membrane Ca²⁺-ATPase goes through an enzymatic phospho-intermediate that maintains Ca²⁺ ions occluded from the media. This intermediate is stabilized by LaATP but not by CrATP.     

The effects of felodipine and bepridil on calcium-stimulated calmodulin binding and calcium pumping ATPase of cardiac sarcolemma before and after removal of endogenous calmodulin

Summary The Ca²⁺ channel blockers felodipine and bepridil are known to affect selectively functions of calmodulin. We studied their effects on calmodulin binding and ATPase activities of calmodulin-containing and calmodulin-depleted rabbit heart sarcolemma. Both drugs as well as the specific anti-calmodulin drug calmidazolium at a concentration of 50 µM, inhibited the Ca²⁺-stimulated calmodulin binding to calmodulin-depleted sarcolemma. Within the concentration range of 3 to 100 µM all three drugs also progressively inhibited Ca²⁺ pumping ATPase in calmodulin containing sarcolemma, although the enzyme was assayed at saturating Ca²⁺ (100 µM). The inhibitory potency of calmidazolium and bepridil, but not that of felodipine, increased when the membrane protein concentration in the ATPase assay was lowered. At low membrane protein concentration 30 µM calmidazolium completely blocked calmodulin-dependent Ca²⁺ pumping ATPase, whereas the inhibition caused by 30 µM felodipine or bepridil remained partially. A similar inhibition pattern of the drugs was found in the calmodulin binding experiments. Within a concentration range of 3 to 30 µM, all three drugs had negligible effects on the basal Ca²⁺ pumping ATPase which was measured in calmodulin-depleted sarcolemma. In conclusion, the characteristics of the anti-calmodulin action of felodipine on the rabbit heart sarcolemmal Ca²⁺ pumping ATPase are not different from those of bepridil. Both drugs may inhibit the enzyme by interference with the Ca²⁺-stimulated binding of calmodulin.Abbreviations Ca²⁺ pumping ATPase Ca²⁺ stimulated Mg²⁺-dependent ATP hydrolyzing activity - Na⁺ pumping ATPase Na⁺-stimulated K⁺- and Mg²⁺-dependent ATP hydrolyzing activity - Tris-maleate tris (hydroxymethyl) aminomethane hydrogen maleate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Mes 2-(N-morpholino) ethane sulfonic acid and Egta, ethylene glycol bis (p-amino ethylether)-N,N,N,N tetraacetic acid     

Ca2+/H+ exchange,lumenal Ca2+ release and Ca2+/ATP coupling ratios in the sarcoplasmic reticulum ATPase

The Ca²⁺ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca²⁺ in intracellular membrane bound compartments, thereby lowering cytosolic Ca²⁺ to induce relaxation. The stored Ca²⁺ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca²⁺, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca²⁺. We review here biochemical and biophysical evidence demonstrating that release of bound Ca²⁺ into the lumen of SR requires Ca²⁺/H⁺ exchange at the low affinity Ca²⁺ sites. Rise of lumenal Ca²⁺ above its dissociation constant from low affinity sites, or reduction of the H⁺ concentration by high pH, prevent Ca²⁺/H⁺ exchange. Under these conditions Ca²⁺ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca²⁺pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.     

Mitochondrial inhibitors activate influx of external Ca in sea urchin sperm

Sea urchin sperm have a single mitochondrion which, aside from its main ATP generating function, may regulate motility, intracellular Ca²⁺ concentration ([Ca²⁺]_i) and possibly the acrosome reaction (AR). We have found that acute application of agents that inhibit mitochondrial function via differing mechanisms (CCCP, a proton gradient uncoupler, antimycin, a respiratory chain inhibitor, oligomycin, a mitochondrial ATPase inhibitor and CGP37157, a Na⁺/Ca²⁺ exchange inhibitor) increases [Ca²⁺]_i with at least two differing profiles. These increases depend on the presence of extracellular Ca²⁺, which indicates they involve Ca²⁺ uptake and not only mitochondrial Ca²⁺ release. The plasma membrane permeation pathways activated by the mitochondrial inhibitors are permeable to Mn²⁺. Store-operated Ca²⁺ channel (SOC) blockers (Ni²⁺, SKF96365 and Gd²⁺) and internal-store ATPase inhibitors (thapsigargin and bisphenol) antagonize Ca²⁺ influx induced by the mitochondrial inhibitors. The results indicate that the functional status of the sea urchin sperm mitochondrion regulates Ca²⁺ entry through SOCs. As neither CCCP nor dicycloexyl carbodiimide (DCCD), another mitochondrial ATPase inhibitor, eliminate the oligomycin induced increase in [Ca²⁺]_i, apparently oligomycin also has an extra mitochondrial target.     

Electrical pump currents generated by the Ca-ATPase of sarcoplasmic reticulum vesicles adsorbed on black lipid membranes

Sarcoplasmic reticulum vesicles adsorbed on a black lipid membrane generate an electrical current after a fast increment of the concentration of ATP. This demonstrates directly that the sarcoplasmic Ca²⁺-ATPase from skeletal muscle acts as an electrogenic ion pump. The increment of the concentration of ATP is achieved by the photolysis of caged ATP (P³-1-(2-nitro)phenylethyl adenosine 5′-triphosphate) a protected analogue of ATP (Kaplan, J.H. et al. (1978) Biochemistry 17, 1929–1935), which is split into ATP and 2-nitroso acetophenone. The release of ATP leads to a transient current flow across the lipid membrane indicating that the vesicles are capacitatively coupled to the underlying lipid membrane. In addition to this transient signal, a stationary current flow is obtained in the presence of ionophores which increase the conductance of the bilayer system and prevent the accumulation of Ca²⁺ in the lumen of the vesicles. The direction of the transient and the stationary current is in accordance with the concept that Ca²⁺ is pumped into the lumen of the vesicles. The transient current depends on the concentration of ATP, Ca²⁺ and Mg²⁺ as would be the case for a current generated by the sarcoplasmic Ca²⁺-ATPase. Its amplitude is half-maximal at 10 μM ATP and 1 μM Ca²⁺. At Ca²⁺ concentrations above 0.1 mM the amplitude of the current signal declines again. The Mg²⁺ concentration dependence of the current amplitude at a constant ATP concentration indicates that the MgATP complex is the substrate for the activation of the current. The pump current is inhibited by vanadate and ADP. No current signal is observed if caged ATP is replaced by caged ADP. However, the release of ADP from caged ADP generates a pump current in the presence of an ATP generating system such as creatine phosphate and creatine kinase.     

Relationship between Ca2+-transport and ATP hydrolytic activities in guinea-pig pancreatic acinar plasma membranes

Partially purified plasma membrane fractions were prepared from guinea-pig pancreatic acini. These membrane preparations were found to contain an ATP-dependent Ca²⁺-transporter as well as a heterogenous ATP-hydrolytic activity. The Ca²⁺-transporter showed high affinity for Ca²⁺ (K_Ca ²⁺ = 0.04 ± 0.01 M), an apparent requirement for Mg²⁺ and high substrate specificity. The major component of ATPase activity could be stimulated by either Ca²⁺ or Mg²⁺ but showed a low affinity for these cations. At low concentrations, Mg²⁺ appeared to inhibit the Ca²⁺-dependent ATPase activity expressed by these membranes. However, in the presence of high Mg²⁺ concentration (0.5–1 mM), a high affinity Ca²⁺-dependent ATPase activity was observed (K_Ca ²⁺ = 0.08 ± 0.02 M). The hydrolytic activity showed little specificity towards ATP. Neither the Ca²⁺-transport nor high affinity Ca²⁺-ATPase activity were stimulated by calmodulin. The results demonstrate, in addition to a low affinity Ca²⁺ (or Mg⁺)-ATPase activity, the presence of both a high affinity Ca²⁺-pump and high affinity Ca²⁺-dependent ATPase. However, the high affinity Ca²⁺-ATPase activity does not appear to be the biochemical expression of the Ca²⁺-pump.Abbreviations Ca²⁺-ATPase calcium-activated, magnesium-dependent adenosine triphosphatase - CaM calmodulin - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate - EDTA ethylene-diaminetetraacetate - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetate - NADPH reduced form of nicotinamide adenine dinucleotide phosphate     

Adenosine 5'-O(3-thiotriphosphate) in the control of phosphorylase activity

Rabbit muscle phosphorylase b (EC 2.4.1.1) is converted to a thio-analog of phosphorylase a by phosphorylase kinase, Mg²⁺ and adenosine 5′-O(3-thiotriphosphate)(ATPγS). Conversion proceeds at one-fifth the rate obtained with ATP though the extent of reaction and final level of activation of the enzyme are the same. However, the thiophosphorylase a produced is resistant to phosphorylase phosphatase and, therefore, behaves as a competitive inhibitor with a K_I of 3 μM, similar to the K_M obtained with normal phosphorylase a. ATPγS can also be utilized by protein kinase in the activation of phosphorylase kinase at a rate similar to that obtained with ATP. It is hydrolyzed at 5 to 10 times the normal rate by the sarcoplasmic reticulum ATPase. When added to a muscle glycogen-particulate complex in the presence of Ca²⁺ and Mg²⁺, ATPγS triggers an activation of phosphorylase with simultaneous inhibition of phosphorylase phosphatase as previously observed with ATP.     

Characteristics of Ca2+-stimulated ATPase in rat heart sarcolemma in the presence of dithiothreitol and alamethicin

We have studied the activities of Ca²⁺-stimulated ATPase in rat heart sarcolemma upon modulating the redox state of membrane thiol groups with dithiothreitol (DTT). The suitability of alamethicin to unmask the latent activity of this enzyme was also investigated. The Ca²⁺-stimulated ATPase in sarcolemma exhibited two activation sites — one with low affinity (Km = 0.70 ± 0.2 mM; Vmax = 10.0 ± 2.2 mol Pi/mg/h) and the other with high affinity (Km = 0.16 ± 0.7 mM; Vmax = 4.6 ± 0.8 mol Pi/mg/h) for Mg²⁺ATP. Alamethicin at a ratio of 1:1 with the sarcolemmal protein caused a 3-fold activation of Ca²⁺-stimulated ATPase without affecting its sensitivity to Ca²⁺ or Mg²⁺ATP. Treatment of sarcolemma with deoxycholate or sodium dodecyl sulfate resulted in a total loss of the enzyme activity; high concentrations of alamethicin also showed a detergent-like action on the sarcolemmal vesicles. DTT at 5–10 mM concentrations caused a 4–5 fold activation of Ca²⁺-stimulated ATPase in sarcolemma and this effect was observed to be dependent on the concentration of Mg²⁺ATP. DTT increased the affinity of the enzyme to Mg²⁺ATP at the high affinity site and enhanced the Vmax at the low affinity site in addition to increasing the sensitivity of Ca²⁺-stimulated ATPase to Ca²⁺. DTT protected the Ca²⁺-stimulated ATPase against deterioration by detergents and restored the enzyme activity after treatment with N-ethylmaleimide. The mechanism of action of DTT on Ca²⁺-stimulated ATPase may involve the reduction of essential thiols at the active site of the enzyme or its interaction with specific DTT-dependent inhibitor protein. No changes in the sensitivity of sarcolemmal Ca²⁺-stimulated ATPase to orthovanadate was evident in the absence or presence of DTT and alamethicin. The results suggest the use of both DTT and alamethicin for the determination of Ca²⁺-stimulated ATPase activity in sarcolemmal preparations.     

ATP-Activated Nonselective Cation Current in NG108-15 Cells

Abstract: ATP (1 mM) induced a biphasic increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), i.e., an initial transient increase decayed to a level of sustained increase, in NG108-15 cells. The transient increase was inhibited by a phospholipase C inhibitor, 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), whereas the sustained increase was abolished by removal of external Ca²⁺. We examined the mechanism of the ATP-elicited sustained [Ca²⁺]_i increase using the fura-2 fluorescent method and the whole-cell patch clamp technique. ATP (1 mM) induced a membrane current with the reversal potential of 12.5 ± 0.8 mV (n = 10) in Tyrode external solution. The EC₅₀ of ATP was ～0.75 mM. The permeability ratio of various cations carrying this current was Na⁺ (defined as 1) > Li⁺ (0.92 ± 0.01; n = 5) > K⁺ (0.89 ± 0.03; n = 6) > Rb⁺ (0.55 ± 0.02; n = 6) > Cs⁺ (0.51 ± 0.01; n = 5) > Ca²⁺ (0.22 ± 0.03; n = 3) > N-methyl-d -glucamine (0.13 ± 0.01; n = 5), suggesting that ATP activated a nonselective cation current. The ATP-induced current was larger at lower concentrations of external Mg²⁺. ATP analogues that induced the current were 2-methylthio-ATP (2MeSATP), benzoylbenzoic-ATP, adenosine 5′-thiotriphosphate (ATPγS), and adenosine 5′-O-(2-thiodiphosphate), but not adenosine, ADP, α,β-methylene-ATP (AMPCPP), β,γ-methylene-ATP (AMPPCP), or UTP. Concomitant with the current data, 2MeSATP and ATPγS, but not AMPCPP or AMPPCP, increased the sustained [Ca²⁺]_i increase. We conclude that ATP activates a class of Ca²⁺-permeable nonselective cation channels via the P_2z receptor in NG108-15 cells.     

Thermogenic activity of Ca-ATPase from skeletal muscle heavy sarcoplasmic reticulum: The role of ryanodine Ca channel

The sarcoplasmic reticulum Ca²⁺ ATPase 1 (SERCA 1) is able to handle the energy derived from ATP hydrolysis in such a way as to determine the parcel of energy that is used for Ca²⁺ transport and the fraction that is converted into heat. In this work we measured the heat production by SERCA 1 in the two sarcoplasmic reticulum (SR) fractions: the light fraction (LSR), which is enriched in SERCA and the heavy fraction (HSR), which contains both the SERCA and the ryanodine Ca²⁺ channel. We verified that although HSR cleaved ATP at faster rate than LSR, the amount of heat released during ATP hydrolysis by HSR was smaller than that measured by LSR. Consequently, the amount of heat released per mol of ATP cleaved (ΔH^cal) by HSR was lower compared to LSR. In HSR, the addition of 5 mM Mg²⁺ or ruthenium red, conditions that close the ryanodine Ca²⁺ channel, promoted a decrease in the ATPase activity, but the amount of heat released during ATP hydrolysis remained practically the same. In this condition, the ΔH^cal values of ATP hydrolysis increased significantly. Neither Mg²⁺ nor ruthenium red had effect on LSR. Thus, we conclude that heat production by SERCA 1 depends on the region of SR in which the enzyme is inserted and that in HSR, the ΔH^cal of ATP hydrolysis by SERCA 1 depends on whether the ryanodine Ca²⁺ channel is opened or closed.     

The binding of ATP to the catalytic and the regulatory site of Ca2+,Mg2+-dependent ATPase of the sarcoplasmic reticulum

Two kinds of ATP binding sites were found on the ATPase molecule in deoxycholic acid-treated sarcoplasmic reticulum. One was the catalytic site (1 mol/mol active site) and its affinity was high. Upon addition of Ca²⁺, all the ATP bound to the catalytic site disappeared at 75 mM KCl, while a significant amount of ATP remained bound to the site at 0–2 mM KCl. The latter binding was found to be due to the formation of a slowly exchanging enzyme-ATP complex, which is in equilibrium with phosphoenzyme + ADP. The other binding site was the regulatory one (1 mol/mol active site) and its affinity was low, changing only insignificantly upon addition of Ca²⁺. The ATP binding to the regulatory site shifted the equilibrium between the slowly exchanging complex and EP toward EP.     

Uncoupling of Occlusion From ATP Hydrolysis Activity in Sarcoplasmic Reticulum (Ca2++Mg2+)-ATPase

The uncoupling of Ca²⁺ transport from ATP hydrolysis in the sarcoplasmic reticulum (Ca²⁺ + Mg²⁺)-ATPase by trypsin digestion was re-investigated by comparing ATPase activity with the ability of the enzyme to occlude Eu³⁺ (a transport parameter) after various tryptic digests. With this method, re-examination of uncoupling by tryptic digest of the ATPase revealed that TD2 cleavage (Arg-198) had no effect on either occlusion or ATPase activity. Digestion past TD2 in the presence of 5 mM Co²⁺ and at 25°C resulted in the loss of about 70% of the ATPase activity, but no loss of occlusion. Digestion past TD2 in the presence of 5 mM Ca²⁺, 3 mM ATP, and at 25°C resulted in a partially uncoupled enzyme complex which retained about 50% of the ATPase activity, but completely lost the ability to occlude Eu³⁺. Digest past TD2 in the presence of 5 mM Ca²⁺ and 3 mM AMP-PNP. (a non-hydrolyzable ATP analog) at 25°C resulted in no loss of occlusion, thus revealing the absolute requirement of ATP during the digest to eliminate occlusion. From these findings we conclude that uncoupling of Ca²⁺ transport from ATPase activity is possible by tryptic digestion of the (Ca²⁺ + Mg²⁺)-ATPase. Interestingly, only after phosphorylation of the enzyme do the susceptible bond(s) which lead to the loss of occlusion become exposed to trypsin.     

Structures of the Ca2+-ATPase complexes with ATP, AMPPCP and AMPPNP. An FTIR study

We studied binding of ATP and of the ATP analogs adenosine 5'-(beta,gamma-methylene)triphosphate (AMPCP) and beta,gamma-imidoadenosine 5'-triphosphate (AMPPNP) to the Ca(2+)-ATPase of the sarcoplasmic reticulum membrane (SERCA1a) with time-resolved infrared spectroscopy. In our experiments, ATP reacted with ATPase which had AMPPCP or AMPPNP bound. These experiments monitored exchange of ATP analog by ATP and phosphorylation to the first phosphoenzyme intermediate Ca(2)E1P. These reactions were triggered by the release of ATP from caged ATP. Only small differences in infrared absorption were observed between the ATP complex and the complexes with AMPPCP and AMPPNP indicating that overall the interactions between nucleotide and ATPase are similar and that all complexes adopt a closed conformation. The spectral differences between ATP and AMPPCP complex were more pronounced at high Ca(2+) concentration (10 mM). They are likely due to a different position of the gamma-phosphate which affects the beta-sheet in the P domain.     

Opiates inhibit calmodulin activation of a high-affinity Ca2+-stimulated Mg2+-dependent ATPase in synaptic membranes

A high affinity Ca²⁺/Mg²⁺ ATPase has been identified and localized in synaptic membrane subfractions. This enzyme is stimulated by low concentrations of Ca²⁺ (1 M) believed to approximate the range of Ca²⁺ in the synaptosomal cytosol (0.1 to 5.0 M). The opiate agonist levorphanol, in a concentration-dependent fashion, inhibited Ca²⁺-stimulated ATP hydrolysis in lysed synaptic membranes. This inhibition was reversed by naloxone, while dextrorphan, the inactive opiate isomer, was without effect. Inhibition by levorphanol was most pronounced in a subfraction of synaptic membranes (SPM-1). The inhibition of Ca²⁺-stimulated ATP hydrolysis was characterized by a reduction inV _max for Ca²⁺. Levorphanol pretreatment reduced the Hill coefficient (H_N) of 1.5 to 0.7, suggesting cooperative interaction between the opiate receptor and the enzyme protein. Levorphanol, but not dextrorphan, also inhibited (28%) ATP-dependent Ca²⁺ uptake by synaptic membranes. Opiate ligand stereoisomers were tested for their effects on calmodulin stimulating of high affinity Ca²⁺/Mg²⁺ ATPase in synaptic membranes. Levorphanol (10 M), but not the inactive stereoisomer (+)dextrorphan, significantly inhibited (35%) the calmodulin-activated Ca²⁺-dependent ATP hydrolysis activity in a preparation of lysed synaptic membranes. Both Ca²⁺-dependent and calmodulin-dependent stimulation of the enzyme in the presence of optimal concentrations of the other co-substrate were inhibited by levorphanol (35–40%) but not dextrorphan. Inhibition of ATP hydrolysis was characterized by a reduction inV _max for both Ca²⁺ and calmodulin stimulation of the enzyme. Calmodulin stimulation of enzyme activity was most pronounced in SPM-1, the membrane fraction which also exhibits the maximal opiate inhibition (40%) of the Ca²⁺-ATPase. The results demonstrate that opiate receptor activation inhibits a high affinity Ca²⁺/Mg²⁺ ATPase in synaptic plasma membranes in a stereospecific fashion. The inhibition of the enzyme may occur by a mechanism involving both Ca²⁺ and calmodulin. Inhibition of calmodulin activation may contribute to the mechanism by which opiate ligands disrupt synaptosomal Ca²⁺ buffering mechanisms. Changes in the cytosolic distribution of synaptosomal Ca²⁺ following inhibition of Ca²⁺/Mg²⁺ ATPase may underlie some of the pharmacological effects of opiate drugs.     

The plasma membrane Ca pump catalyzes the hydrolysis of ATP at low rate in the absence of Ca

The plasma membrane Ca²⁺ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca²⁺ at a rate near 1.5% of that attained at saturating concentrations of Ca²⁺. Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca²⁺-independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca²⁺-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca²⁺ and is catalyzed by E₂-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.     

Modulation of calcium signalling by mitochondria

In this review we will attempt to summarise the complex and sometimes contradictory effects that mitochondria have on different forms of calcium signalling. Mitochondria can influence Ca²⁺ signalling indirectly by changing the concentration of ATP, NAD(P)H, pyruvate and reactive oxygen species — which in turn modulate components of the Ca²⁺ signalling machinery i.e. buffering, release from internal stores, influx from the extracellular solution, uptake into cellular organelles and extrusion by plasma membrane Ca²⁺ pumps. Mitochondria can directly influence the calcium concentration in the cytosol of the cell by importing Ca²⁺ via the mitochondrial Ca²⁺ uniporter or transporting Ca²⁺ from the interior of the organelle into the cytosol by means of Na⁺/Ca²⁺ or H⁺/Ca²⁺ exchangers. Considerable progress in understanding the relationship between Ca²⁺ signalling cascades and mitochondrial physiology has been accumulated over the last few years due to the development of more advanced optical techniques and electrophysiological approaches.     

Differential alterations in ATP-supported calcium transport activities of sarcoplasmic reticulum and sarcolemma of aging myocardium

Two membrane fractions, one enriched in sarcoplasmic reticulum and the other enriched in sarcolemma, were isolated from the myocardium of young (3–4-months-old) and aged (24–25-months old) rats. ATP-supported Ca²⁺ binding and accumulating activities as well as (Mg²⁺ + Ca²⁺)-ATPase activities of these membrane fractions were studied in an effort to determine the influence of age on the Ca²⁺ pump function of the two myocardial membrane systems. Sarcoplasmic reticulum from aged hearts showed significantly reduced (approx. 50%) rates of ATP-supported (oxalate-facilitated) Ca²⁺ accumulation compared to sarcoplasmic reticulum from young hearts; the amount of Ca²⁺ accumulated by this membrane of aged heart at steady state was also lower. On the other hand, sarcolemma from aged hearts displayed 2-fold higher rates of ATP-supported Ca²⁺ accumulation compared to sarcolemma from young hearts; at steady state, sarcolemma from aged hearts accumulated significantly higher amounts of Ca²⁺ than did sarcolemma from young hearts. Similar age-related differences were also observed in the ATP-dependent Ca²⁺ binding activities of the two membranes, determined in the absence of oxalate. The divergent age-associated changes in Ca²⁺ binding and accumulating activities of sarcoplasmic reticulum and sarcolemma were seen at varying Ca²⁺ concentrations (0.24–39.1 μM).With either membrane, kinetic analysis showed 2-fold age-related differences in the V values for ATP-supported Ca²⁺ accumulation (V (nmol Ca²⁺/mg protein per min): sarcoplasmic reticulum — young, 119 ± 8; aged, 59 ± 5; sarcolemma — young, 11 ± 2; aged, 21 ± 3); the concentrations of Ca²⁺ required for half-maximal velocities did not differ significantly with age (K_0.5 for Ca²⁺ (μM): sarcoplasmic reticulum — young, 2.5 ± 0.20; aged, 2.9 ± 0.25; sarcolemma — yount, 2.7 ± 0.25; aged, 3.2 ± 0.30). Kinetic parameters of ATP-dependent Ca²⁺ binding also indicated that the velocity of Ca²⁺ binding but not the concentration of Ca²⁺ required for half-maximal binding was altered due to aging. At identical Ca²⁺ concentrations, the combined Ca²⁺ accumulating activity of sarcoplasmic reticulum and sarcolemma from aged hearts was significantly lower (38–47%) than the combined Ca²⁺ accumulating activity of the two membranes from young hearts. No significant age-related differences were observed in the ATP-independent (passive) Ca²⁺ binding (or accumulation) by sarcoplasmic reticulum and sarcolemma, the (Mg²⁺ + Ca²⁺)-ATPase activities of these membranes, their polypeptide composition or relative purity. These results indicate that differential alterations occur in the ATP-supported Ca²⁺ pump activities of sarcoplasmic reticulum and sarcolemma in aging myocardium and such alterations may be due to age-associated changes in the efficacy of coupling ATP hydrolysis to Ca²⁺ transport. Further, the age-related increment in the Ca²⁺ pump activity of sarcolemma is inadequate to fully compensate for the diminished Ca²⁺ pump activity of sarcoplasmic reticulum. It is, therefore, suggested that deterioration of the Ca²⁺ pump function of sarcoplasmic reticulum may contribute to the increased relaxation time observed in aging heart.     

Ion transport and energy transduction of P-type ATPases: Implications from electrostatic calculations

This paper summarizes our present electrostatic calculations on P-type ATPases and their contribution to understand the molecular details of the reaction mechanisms. One focus was set on analyzing the proton countertransport of the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA1a). Protonation of acidic residues was calculated in dependence of pH for different enzyme states in the reaction cycle of the Ca²⁺-ATPase. We proposed that the acidic Ca²⁺ ligands Glu 771, Asp 800 and Glu 908 participate in the proton countertransport whereas Glu 309 is more likely to serve as a proton shuttle between binding site I and the cytoplasm. Complementary to infrared measurements, we assigned infrared bands to specific Ca²⁺ ligands that are hydrogen bonded. Ion pathways were proposed based on the calculations and structural data. Another focus was set on analyzing the energy transduction mechanism of P-type ATPases. In accordance to electrophysiological experiments, we simulated an electric field across the membrane. The impact of the electric field was studied by an accumulated number of residue conformational and ionization changes on specific transmembrane helices. Our calculations on the Ca²⁺-ATPase and the Na⁺/K⁺-ATPase indicated that the highly conserved transmembrane helix M5 is one structural element that is likely to act as energy transduction element in P-type ATPases. Perspectives and limitations of the electrostatic calculations for future computational studies are pointed out.     

Modulation of the kinetic characteristics of the sarcoplasmic reticulum ATPase by membrane fluidity

(Ca²⁺ + Mg²⁺)-ATPase from sarcoplasmic reticulum has been reconstituted with dipalmitoylphosphatidylcholine, and the activating effect of ATP and Ca²⁺ on this enzyme has been studied at different temperatures. It has been found that two kinetic forms of the enzyme are interconverted at about 31°C, and this is possibly related to a phase change in the phospholipid which is more directly associated with the protein. Above 31°C the enzyme is less dependent on ATP activation at high ATP concentrations but shows positive cooperativity for Ca²⁺ activation. On the other hand, below 31°C, the reconstituted enzyme is more dependent on ATP for activation at high ATP concentrations than the purified ATPase and does not show cooperativity for Ca²⁺ activation.