ATPase Of Sarcoplasmic Reticulum Research Articles

Functional role of "N" (nucleotide) and "P" (phosphorylation) domain interactions in the sarcoplasmic reticulum (SERCA) ATPase.

Experimental perturbations of the nucleotide site in the N domain of the SR Ca2+ ATPase were produced by chemical derivatization of Lys492 or/and Lys515, mutation of Arg560 to Ala, or addition of inactive nucleotide analogue (TNP-AMP). Selective labeling of either Lys492 or Lys515 produces strong inhibition of ATPase activity and phosphoenzyme intermediate formation by utilization of ATP, while AcP utilization and reverse ATPase phosphorylation by Pi are much less affected. Cross-linking of the two residues with DIDS, however, drastically inhibits utilization of both ATP and AcP, as well as of formation of phosphoenzyme intermediate by utilization of ATP, or reverse phosphorylation by Pi. Mutation of Arg560 to Ala produces strong inhibition of ATPase activity and enzyme phosphorylation by ATP but has a much lower effect on enzyme phosphorylation by Pi. TNP-AMP increases the ATPase activity at low concentrations (0.1-0.3 microM), but inhibits ATP, AcP, and Pi utilization at higher concentration (1-10 microM). Cross-linking with DIDS and TNP-AMP binding inhibits formation of the transition state analogue with orthovanadate. It is concluded that in addition to the binding pocket delimited by Lys 492 and Lys515, Arg560 sustains an important and direct role in nucleotide substrate stabilization. Furthermore, the effects of DIDS and TNP-AMP suggest that approximation of N (nucleotide) and P (phosphorylation) domains is required not only for delivery of nucleotide substrate, but also to favor enzyme phosphorylation by nucleotide and nonnucleotide substrates, in the presence and in the absence of Ca2+. Domain separation is then enhanced by secondary nucleotide binding to the phosphoenzyme, thereby favoring its hydrolytic cleavage.

Sarcolipin uncouples hydrolysis of ATP from accumulation of Ca2+ by the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum.

Sarcolipin (SLN) is a small peptide found in the sarcoplasmic reticulum of skeletal muscle. It is predicted to contain a single hydrophobic transmembrane alpha-helix. Fluorescence emission spectra for the single Trp residue of SLN suggest that SLN incorporates fully into bilayers of dioleoylphosphatidylcholine, but only partially into bilayers of phosphatidylcholines with long (C(22) or C(24)) fatty acyl chains. The fluorescence of SLN is quenched in bilayers of dibromostearoylphosphatidylcholine, also consistent with incorporation into the lipid bilayer. SLN was reconstituted with the Ca(2+)-ATPase of skeletal-muscle sarcoplasmic reticulum. Even at a 50:1 molar ratio of SLN/ATPase, SLN had no significant effect on the rate of ATP hydrolysis by the ATPase or on the Ca(2+)-dependence of ATP hydrolysis. However, at a molar ratio of SLN/ATPase of 2:1 or higher the presence of SLN resulted in a marked decrease in the level of accumulation of Ca(2+) by reconstituted vesicles. The effect of SLN was structurally specific and did not result from a breakdown in the vesicular structure or from the formation of non-specific ion channels. Vesicles were impermeable to Ca(2+) in the absence of ATP in the external medium. The effects of SLN on accumulation of Ca(2+) can be simulated assuming that SLN increases the rate of slippage on the ATPase and the rate of passive leak of Ca(2+) mediated by the ATPase. It is suggested that the presence of SLN could be important in non-shivering thermogenesis, a process in which heat is generated by hydrolysis of ATP by skeletal-muscle sarcoplasmic reticulum.

Sarcolipin uncouples hydrolysis of ATP from accumulation of Ca2+ by the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum

Sarcolipin (SLN) is a small peptide found in the sarcoplasmic reticulum of skeletal muscle. It is predicted to contain a single hydrophobic transmembrane α-helix. Fluorescence emission spectra for the single Trp residue of SLN suggest that SLN incorporates fully into bilayers of dioleoylphosphatidylcholine, but only partially into bilayers of phosphatidylcholines with long (C22 or C24) fatty acyl chains. The fluorescence of SLN is quenched in bilayers of dibromostearoylphosphatidylcholine, also consistent with incorporation into the lipid bilayer. SLN was reconstituted with the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum. Even at a 50:1 molar ratio of SLN/ATPase, SLN had no significant effect on the rate of ATP hydrolysis by the ATPase or on the Ca2+-dependence of ATP hydrolysis. However, at a molar ratio of SLN/ATPase of 2:1 or higher the presence of SLN resulted in a marked decrease in the level of accumulation of Ca2+ by reconstituted vesicles. The effect of SLN was structurally specific and did not result from a breakdown in the vesicular structure or from the formation of non-specific ion channels. Vesicles were impermeable to Ca2+ in the absence of ATP in the external medium. The effects of SLN on accumulation of Ca2+ can be simulated assuming that SLN increases the rate of slippage on the ATPase and the rate of passive leak of Ca2+ mediated by the ATPase. It is suggested that the presence of SLN could be important in non-shivering thermogenesis, a process in which heat is generated by hydrolysis of ATP by skeletal-muscle sarcoplasmic reticulum.

Molecular mechanism of the P-type ATPases.

The recent determination of the structure of the Ca2+-ATPase of sarcoplasmic reticulum to atomic resolution in the Ca2+-bound state and to near atomic resolution in the Ca2+-free, decavanadate-bound state has paved the way for an ultimate complete understanding of the molecular mechanism of the P-type ATPases. Analysis of this new structure information together with the large amount of biochemical information about these enzymes that preceded it has produced important new revelations about how the P-type ATPases work. Most models propose that these transporters operate by a strictly conformational energy coupling mechanism in which conformational changes in the large cytoplasmic head region mechanically drive the ions to be transported from their binding sites in the transmembrane helix region 50 A away. However, while these enzymes do indeed undergo profound conformational changes, the available evidence suggests that they do not mechanically transduce the chemical energy of ATP hydrolysis into transmembrane ion gradients via these conformational changes. As an alternative, it is proposed that the effects of the chemical events that occur at the phosphorylation/dephosphorylation site in the cytoplasmic region are exerted on the ion-binding sites via two well-defined charge transfer pathways that electronically connect the chemical reaction site with the site of ion binding. The recognition of these charge transfer pathways provides rational explanations of all of the key biochemical features of the P-type ATPase catalytic cycle. Thus, although a few details await elucidation, a nearly complete understanding of the P-type ATPase reaction mechanism may be at hand.

Curcumin, a Molecule That Inhibits the Ca2+-ATPase of Sarcoplasmic Reticulum but Increases the Rate of Accumulation of Ca2+

Curcumin, an important inhibitor of carcinogenesis, is an inhibitor of the ATPase activity of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR). Inhibition by curcumin is structurally specific, requiring the presence of a pair of -OH groups at the 4-position of the rings. Inhibition is not competitive with ATP. Unexpectedly, addition of curcumin to SR vesicles leads to an increase in the rate of accumulation of Ca(2+), unlike other inhibitors of the Ca(2+)-ATPase that result in a reduced rate of accumulation. An increase in the rate of accumulation of Ca(2+) is seen in the presence of phosphate ion, which lowers the concentration of free Ca(2+) within the lumen of the SR, showing that the effect is not passive leak across the SR membrane. Rather, simulations suggest that the effect is to reduce the rate of slippage on the ATPase, a process in which a Ca(2+)-bound, phosphorylated intermediate releases its bound Ca(2+) on the cytoplasmic rather than on the lumenal side of the membrane. The structural specificity of the effects of curcumin on ATPase activity and on Ca(2+) accumulation is the same, and the apparent dissociation constants for the two effects are similar, suggesting that the two effects of curcumin could follow from binding to a single site on the ATPase.

Gene expression of SERCA2a and L- and T-type Ca channels during human heart development.

In this study we report, for the first time, on the gene expression of human cardiac SERCA2a, L-type (alpha(1C)) and T-type (alpha(1H)) Ca channels during development, using RNase protection assay, relative quantitative RT-PCR and Western blot. Human hearts during early gestation (8- to 20-wk gestation), neonatal (1- to 4-d-old) and adult (18- to 48-year-old) stages were used. The results show that T-type Ca channel alpha(1H) subunit mRNA decreased and that L-type Ca channel alpha(1C) subunit mRNA increased with development. While the levels of sarcoplasmic reticulum ATPase (SERCA2a) mRNA did not significantly change with development, its protein levels increased with development. In conclusion, SERCA2a, L-type and T-type Ca channel transcripts were detected as early as 8-wk gestation. Defining the profile of Ca handling proteins during development is important to the understanding of excitation-contraction (EC)-coupling of the developing human heart.

Importance of Thr-353 of the Conserved Phosphorylation Loop of the Sarcoplasmic Reticulum Ca2+-ATPase in MgATP Binding and Catalytic Activity

Mutants in which Thr-353 of the Ca(2+)-ATPase of sarcoplasmic reticulum had been replaced with alanine, serine, glutamine, cysteine, valine, aspartate, or tyrosine were analyzed functionally. All the mutations severely affected MgATP binding, whereas ATP binding was close to normal in the alanine, serine, glutamine, and valine mutants. In the serine and valine mutants, the maximum rate of phosphorylation from MgATP was 8- and 600-fold lower, respectively, compared with wild type. Replacement of Mg(2+) with Mn(2+) led to a 1.5-fold enhancement of the maximum phosphorylation rate in the valine mutant and a 5-fold reduction in the wild type. The turnover of the phosphoenzyme formed from MgATP was slowed 1-2 orders of magnitude relative to wild type in the alanine, serine, and valine mutants, but was close to normal in the aspartate and cysteine mutants. Only the serine mutant formed a phosphoenzyme in the backward reaction with P(i), and the hydrolysis of this intermediate was greatly enhanced. Analysis of the functional changes in the mutants in the light of the recent high resolution structure of the Ca(2+)-ATPase crystallized without the MgATP substrate suggests that, in the native activated state of the enzyme, the side chain hydroxyl of Thr-353 participates in important interactions with nucleotide and phosphate, possibly in catalysis, whereas the main chain carbonyl of Thr-353, but not the side chain, may coordinate the catalytic Mg(2+).

Smooth Muscle Cell Response to Mechanical Injury Involves Intracellular Calcium Release and ERK1/ERK2 Phosphorylation

We have investigated possible signaling pathways coupled to injury-induced ERK1/2 activation and the subsequent initiation of vascular rat smooth muscle cell migration and proliferation. Aortic smooth muscle cells were cultured to confluency and subjected to in vitro injury under serum-free conditions. In fluo-4-loaded cells, injury induced a rapid wave of intracellular Ca2+ release that propagated about 200 μm in radius from the injured zone, reached a peak in about 20 s, and subsided to the baseline within 2 min. The wave was abolished by prior treatment with the sarcoplasmic reticulum ATPase inhibitor thapsigargin, but not by omission of extracellular Ca2+. ERK1/2 activation reached a peak at 10 min after injury and was inhibited by the MEK1 inhibitor PD98059, as well as by thapsigargin, fluphenazine, genistein, and the Src inhibitor PP2. These inhibitors also reduced [3H]thymidine incorporation and migration of cells into the injured area determined at 48 h after injury. These results show that mechanical injury to vascular smooth muscle cells induces a Ca2+ wave which is dependent on intracellular Ca2+ release. Furthermore, the injury activates ERK1/2 phosphorylation as well as cell migration and replication.

What the structure of a calcium pump tells us about its mechanism.

The report of the crystal structure of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum in its Ca(2+)-bound form [Toyoshima, Nakasako and Ogawa (2000) Nature (London) 405, 647-655] provides an opportunity to interpret much kinetic and mutagenic data on the ATPase in structural terms. There are no large channels leading from the cytoplasmic surface to the pair of high-affinity Ca(2+) binding sites within the transmembrane region. One possible access pathway involves the charged residues in transmembrane alpha-helix M1, with a Ca(2+) ion passing through the first site to reach the second site. The Ca(2+)-ATPase also contains a pair of binding sites for Ca(2+) that are exposed to the lumen. In the four-site model for transport, phosphorylation of the ATPase leads to transfer of the two bound Ca(2+) ions from the cytoplasmic to the lumenal pair of sites. In the alternating four-site model for transport, phosphorylation leads to release of the bound Ca(2+) ions directly from the cytoplasmic pair of sites, linked to closure of the pair of lumenal binding sites. The lumenal pair of sites could involve a cluster of conserved acidic residues in the loop between M1 and M2. Since there is no obvious pathway from the high-affinity sites to the lumenal surface of the membrane, transport of Ca(2+) ions must involve a significant change in the packing of the transmembrane alpha-helices. The link between the phosphorylation domain and the pair of high-affinity Ca(2+) binding sites is probably provided by two small helices, P1 and P2, in the phosphorylation domain, which contact the loop between transmembrane alpha-helices M6 and M7.

What the structure of a calcium pump tells us about its mechanism

The report of the crystal structure of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum in its Ca2+-bound form [Toyoshima, Nakasako and Ogawa (2000) Nature (London) 405, 647–655] provides an opportunity to interpret much kinetic and mutagenic data on the ATPase in structural terms. There are no large channels leading from the cytoplasmic surface to the pair of high-affinity Ca2+ binding sites within the transmembrane region. One possible access pathway involves the charged residues in transmembrane α-helix M1, with a Ca2+ ion passing through the first site to reach the second site. The Ca2+-ATPase also contains a pair of binding sites for Ca2+ that are exposed to the lumen. In the four-site model for transport, phosphorylation of the ATPase leads to transfer of the two bound Ca2+ ions from the cytoplasmic to the lumenal pair of sites. In the alternating four-site model for transport, phosphorylation leads to release of the bound Ca2+ ions directly from the cytoplasmic pair of sites, linked to closure of the pair of lumenal binding sites. The lumenal pair of sites could involve a cluster of conserved acidic residues in the loop between M1 and M2. Since there is no obvious pathway from the high-affinity sites to the lumenal surface of the membrane, transport of Ca2+ ions must involve a significant change in the packing of the transmembrane α-helices. The link between the phosphorylation domain and the pair of high-affinity Ca2+ binding sites is probably provided by two small helices, P1 and P2, in the phosphorylation domain, which contact the loop between transmembrane α-helices M6 and M7.

Cytoplasmic interactions between phospholamban residues 1–20 and the calcium-activated ATPase of the sarcoplasmic reticulum

Phospholamban regulates the activity of the calcium-activated ATPase (CaATPase) of cardiac sarcoplasmic reticulum. Equilibrium fluorescence studies have shown that the N-terminal cytoplasmic region of phospholamban (residues 1-20, domain 1) causes a decrease in the intrinsic tryptophan fluorescence of the CaATPase. The interaction of phospholamban residues 1-20 with the CaATPase also results in spectral changes for the extrinsic chromophore FITC covalently attached to the cytoplasmic region of the calcium pump. The fluorescence changes for both reporter groups correlate with a dissociation constant of approximately 40 microM for the complex between phospholamban residues 1-20 and the CaATPase. Complex formation is notably weaker when phospholamban 1-20 is titrated into the CaATPase in the presence of calcium, with altered conformational effects resulting from binding. The interaction of domain 1 of phospholamban with the CaATPase is also reduced upon phosphorylation of phospholamban 1-20 at Ser-16. This region of phospholamban 1-20 is shown by isotope-edited NMR study to be involved in interaction with the CaATPase. Binding of the phosphorylated peptide is not abolished, however, indicating that phospholamban 1-20 remains associated with the CaATPase even after phosphorylation. The data provide direct evidence for the interaction between the cytoplasmic regions of phospholamban and the pump, and are discussed in the context of the mechanism for inhibition of cardiac CaATPase activity by phospholamban.

Caloxin: a novel plasma membrane Ca2+ pump inhibitor

Plasma membrane (PM) Ca2+ pump is a Ca+-Mg2+-ATPase that expels Ca2+ from cells to help them maintain low concentrations of cytosolic Ca2+ . There are no known extracellularly acting PM Ca2+ pump inhibitors, as digoxin and ouabain are for Na+ pump. In analogy with digoxin, we define caloxins as extracellular PM Ca2+ pump inhibitors and describe caloxin 2A1. Caloxin 2A1 is a peptide obtained by screening a random peptide phage display library for binding to the second extracellular domain (residues 401-413) sequence of PM Ca2+ pump isoform 1b. Caloxin 2A1 inhibits Ca2+-Mg2+-ATPase in human erythrocyte leaky ghosts, but it does not affect basal Mg2+-ATPase or Na+-K+-ATPase in the ghosts or Ca2+-Mg2+-ATPase in the skeletal muscle sarcoplasmic reticulum. Caloxin 2A1 also inhibits Ca2+-dependent formation of the 140-kDa acid-stable acylphosphate, which is a partial reaction of this enzyme. Consistent with inhibition of the PM Ca2+ pump in vascular endothelium, caloxin 2A1 produces an endothelium-dependent relaxation that is reversed by N(G)-nitro-L-arginine methyl ester. Thus caloxin 2A1 is a novel PM Ca2+ pump inhibitor selected for binding to an extracellular domain.

The effect of Mg2+ on cardiac muscle function: is CaATP the substrate for priming myofibril cross-bridge formation and Ca2+ reuptake by the sarcoplasmic reticulum?

Kinetics are established for the activation of the myofibril from the relaxed state [Smith, Dixon, Kirschenlohr, Grace, Metcalfe and Vandenberg (2000) Biochem. J. 346, 393–402]. These require two troponin Ca2+-binding sites, one for each myosin head, to act as a single unit in initial cross-bridge formation. This defines the first, or activating, ATPase reaction, as distinct from the further activity of the enzyme that continues when a cross-bridge to actin is already established. The pairing of myosin heads to act as one unit suggests a possible alternating mechanism for muscle action. A large positive inotropic (contraction-intensifying) effect of loading the Mg2+ chelator citrate, via its acetoxymethyl ester, into the heart has confirmed the competitive inhibition of the Ca2+ activation by Mg2+, previously seen in vitro. In the absence of a recognized second Ca2+-binding site on the myofibril, with appropriate binding properties, the bound ATP is proposed as the second activating Ca2+-binding site. As ATP, free or bound to protein, can bind either Mg2+ or Ca2+, this leads to competitive inhibition by Mg2+. Published physico-chemical studies on skeletal muscle have shown that CaATP is potentially a more effective substrate than MgATP for cross-bridge formation. The above considerations allow calculation of the observed variation of fractional activation by Ca2+ as a function of [Mg2+] and in turn reveal simple Michaelis–Menten kinetics for the activation of the ATPase by sub-millimolar [Mg2+]. Furthermore the ability of bound ATP to bind either cation, and the much better promotion of cross-bridge formation by CaATP binding, give rise to the observed variation of the Hill coefficient for Ca2+ activation with altered [Mg2+]. The inclusion of CaADP within the initiating cross-bridge and replacement by MgADP during the second cycle is consistent with the observed fall in the rate of the myofibril ATPase that occurs after two phosphates are released. The similarity of the kinetics of the cardiac sarcoplasmic reticulum ATPase to those of the myofibril, in particular the positive co-operativity of both Mg2+ inhibition and Ca2+ activation, leads to the conclusion that this ATPase also has an initiation step that utilizes CaATP. The first-order activation by sub-millimolar [Mg2+], similar to that of the myofibril, may be explained by Mg2+ involvement in the phosphate-release step of the ATPase. The inhibition of both the myofibril and sarcoplasmic reticulum Ca2+-transporting ATPases by Mg2+ offers an explanation for the specific requirement for phosphocreatine (PCr) for full activity of both enzymes in situ and its effect on their apparent affinities for ATP. This explanation is based on the slow diffusion of Mg2+ within the myofibril and on the contrast of PCr with both ATP and phosphoenolpyruvate, in that PCr does not bind Mg2+ under physiological conditions, whereas both the other two bind it more tightly than the products of their hydrolysis do. The switch to supply of energy by diffusion of MgATP into the myofibril when depletion of PCr raises [ATP]/[PCr] greatly, e.g. during anoxia, results in a local [Mg2+] increase, which inhibits the ATPase. It is possible that mechanisms similar to those described above occur in skeletal muscle but the Ca2+ co-operativity involved would be masked by the presence of two Ca2+-binding sites on each troponin.

The effect of Mg2+ on cardiac muscle function: Is CaATP the substrate for priming myofibril cross-bridge formation and Ca2+ reuptake by the sarcoplasmic reticulum?

Kinetics are established for the activation of the myofibril from the relaxed state [Smith, Dixon, Kirschenlohr, Grace, Metcalfe and Vandenberg (2000) Biochem. J. 346, 393-402]. These require two troponin Ca2+-binding sites, one for each myosin head, to act as a single unit in initial cross-bridge formation. This defines the first, or activating, ATPase reaction, as distinct from the further activity of the enzyme that continues when a cross-bridge to actin is already established. The pairing of myosin heads to act as one unit suggests a possible alternating mechanism for muscle action. A large positive inotropic (contraction-intensifying) effect of loading the Mg2+ chelator citrate, via its acetoxymethyl ester, into the heart has confirmed the competitive inhibition of the Ca2+ activation by Mg2+, previously seen in vitro. In the absence of a recognized second Ca2+ binding site on the myofibril, with appropriate binding properties, the bound ATP is proposed as the second activating Ca2+-binding site. As ATP, free or bound to protein, can bind either Mg2+ or Ca2+, this leads to competitive inhibition by Mg2+. Published physico-chemical studies on skeletal muscle have shown that CaATP is potentially a more effective substrate than MgATP for cross-bridge formation. The above considerations allow calculation of the observed variation of fractional activation by Ca2+ as a function of [Mg2+] and in turn reveal simple Michaelis-Menten kinetics for the activation of the ATPase by sub-millimolar [Mg2+]. Furthermore the ability of bound ATP to bind either cation, and the much better promotion of cross-bridge formation by CaATP binding, give rise to the observed variation of the Hill coefficient for Ca2+ activation with altered [Mg2+]. The inclusion of CaADP within the initiating cross-bridge and replacement by MgADP during the second cycle is consistent with the observed fall in the rate of the myofibril ATPase that occurs after two phosphates are released. The similarity of the kinetics of the cardiac sarcoplasmic reticulum ATPase to those of the myofibril, in particular the positive co-operativity of both Mg2+ inhibition and Ca2+ activation, leads to the conclusion that this ATPase also has an initiation step that utilizes CaATP. The first-order activation by sub-millimolar [Mg2+], similar to that of the myofibril, may be explained by Mg2+ involvement in the phosphate-release step of the ATPase. The inhibition of both the myofibril and sarcoplasmic reticulum Ca2+ transporting ATPases by Mg2+ offers an explanation for the specific requirement for phosphocreatine (PCr) for full activity of both enzymes in situ and its effect on their apparent affinities for ATP. This explanation is based on the slow diffusion of Mg2+ within the myofibril and on the contrast of PCr with both ATP and phosphoenolpyruvate, in that PCr does not bind Mg2+ under physiological conditions, whereas both the other two bind it more tightly than the products of their hydrolysis do. The switch to supply of energy by diffusion of MgATP into the myofibril when depletion of PCr raises [ATP]/[PCr] greatly, e.g. during anoxia, results in a local [Mg2+] increase, which inhibits the ATPase. It is possible that mechanisms similar to those described above occur in skeletal muscle but the Ca2+ co-operativity involved would be masked by the presence of two Ca2+ binding sites on each troponin.

ATP utilization for calcium uptake and force production in different types of human skeletal muscle fibres.

The contractile properties and ATPase activity of skinned human skeletal muscle fibres from vastus lateralis were examined. Fibre types were resolved from single fibre segments by SDS-polyacrylamide gel electrophoresis. ATPase activity was determined by enzymatic coupling of ATP resynthesis to the oxidation of NADH. The partitioning of ATPase activity into (a) calcium-activated activity due to actomyosin (AM) interaction, (b) calcium-activated activity of the sarcoplasmic reticular (SR) calcium pump, and (c) basal (calcium independent) activity was investigated by comparing ATP utilization before and after exposure of the preparations for 30 min to a solution containing 0.5 % Triton X-100, which effectively abolished the SR ATPase activity. Partitioning of ATPase activity was also determined by measuring ATP utilization and force at different concentrations of butanedione monoxime (BDM), which inhibits AM interaction. The results obtained with Triton X-100 and BDM were similar. At saturating Ca2+ concentrations and 20 degrees C, the AM, SR and basal ATPase activities per litre cell volume (+/- S.E.M.) amounted to 46 +/- 4, 51 +/- 4 and 19 +/- 2 muM s-1 in type I fibres (n = 21), 139 +/- 14, 69 +/- 8 and 30 +/- 3 muM s-1 in type IIA fibres (n = 25), 137 +/- 22, 175 +/- 28 and 26 +/- 8 muM s-1 in type IIA/B fibres (n = 4) and 108 +/- 13, 169 +/- 42 and 32 +/- 8 muM s-1 in type IIB fibres (n = 2). These results indicate that ATP utilization for SR Ca2+ pumping in fast fibres is considerably larger than in slow fibres. The SR ATPase activity in human muscle represents a considerable fraction of the total (AM + SR + basal) ATPase activity.

Characterisation of thapsigargin-releasable Ca2+ from the Ca2+-ATPase of sarcoplasmic reticulum at limiting [Ca2+

The Ca2+ binding sites of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum (SR) have been identified as two high-affinity sites orientated towards the cytoplasm, two sites of low affinity facing the lumen, and a transient occluded species that is isolated from both membrane surfaces. Binding and release studies, using 45Ca2+, have invoked models with sequential binding and release from high- and low-affinity sites in a channel-like structure. We have characterised turnover conditions in isolated SR vesicles with oxalate in a Ca2+-limited state, [Ca2]lim, where both high- and low-affinity sites are vacant in the absence of chelators (Biochim. Biophys. Acta 1418 (1999) 48–60). Thapsigargin (TG), a high-affinity specific inhibitor of the Ca2+-ATPase, released a fraction of total Ca2+ at [Ca2+]lim that accumulated during active transport. Maximal Ca2+ release was at 2:1 TG/ATPase. Ionophore, A23187, and Triton X-100 released the rest of Ca2+ resistant to TG. The amount of Ca2+ released depended on the incubation time at [Ca2+]lim, being 3.0 nmol/mg at 20 s and 0.42 nmol/mg at 1000 s. Rate constants for release declined from 0.13 to 0.03 s−1. The rapidly released early fraction declined with time and k=0.13 min−1. Release was not due to reversal of the pump cycle since ADP had no effect; neither was release impaired with substrates acetyl phosphate or GTP. A phase of reuptake of Ca2+ followed release, being greater with shorter delay (up to 200 s) following active transport. Reuptake was minimal with GTP, with delays more than 300 s, and was abolished by vanadate and at higher [TG], >5 μM. Ruthenium red had no effect on efflux, indicating that ryanodine-sensitive efflux channels in terminal cisternal membranes are not involved in the Ca2+ release mechanism. It is concluded that the Ca2+ released by TG is from the occluded Ca2+ fraction. The Ca2+ occlusion sites appear to be independent of both high-affinity cytoplasmic and low-affinity lumenal sites, supporting a multisite ‘in line’ sequential binding mechanism for Ca2+ transport.

Labeling the Ca2+-ATPase of Skeletal Muscle Sarcoplasmic Reticulum with Maleimidylsalicylic Acid

Maleimidylsalicylic acid reacts with the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum with high affinity and inhibits the ATPase activity following a pseudo-first-order kinetic with a rate constant of 8.3 m(-1) s(-1). Calcium binding remains unaffected in the maleimide-inhibited ATPase. However, the presence of ATP, ADP, and, to a lesser extent, AMP protects the enzyme against inhibition. Furthermore, ATPase inhibition is accompanied by a concomitant decrease in ATP binding. The stoichiometry of the nucleotide-dependent maleimidylsalicylic acid binding is 6-10 nmol/mg ATPase, which corresponds to the binding of up to one molecule of maleimide/molecule of ATPase. The stoichiometry of maleimide binding is decreased in the presence of nucleotides and in the ATPase previously labeled with fluorescein-5'-isothiocyanate or N-ethylmaleimide A fluorescent peptide was isolated by high performance liquid chromatography after trypsin digestion of the maleimide-labeled ATPase. Analysis of the sequence and mass spectrometry of the peptide leads us to propose Cys(344) as the target for maleimidylsalicylic acid in the inhibition reaction. The effect of Cys(344) modification on the nucleotide site is discussed.

Action mechanisms of a new erythrocyte-derived depressing factor

To investigate the action mechanisms of a new erythrocyte-derived depressing factor (EDDF), the focus is placed on the effect of EDDF on both cytosolic and nuclear free calcium (Ca2+) transportation in vascular smooth muscle cell (VSMC), as well as the apoptosis and cell cycle of VSMC of rats. EDDF has been extracted from human erythrocytes. The changes of Ca2+ levels in cytoplasm ([Ca2+]i) and nucleus ([Ca2+]n) have been observed using a laser scanning confocal microscope together with fluo-3/AM as a calcium indicator. Flow cytometric technique was used to study the effect of EDDF on cell cycle and apoptosis of VSMC. [Ca2+], and [Ca2+]n were significantly decreased through several different pathways: ( i ) it reduced the Ca2+ influx by blocking L-type voltage-dependent calcium channel (L-VDC) and R-type voltage-dependent calcium channel (R-VDC); (ii) it inhibited the Ca2+ release from inositol 1, 4, 5-trisphosphate (IP3) sensitive calcium store; and (iii) activated Ca2+-ATPase of sarcoplasmic reticulum (SR) and promoted the transportation of Ca2+ from cytoplasm to SR. However, EDDF seemed to have little inhibitory effect on the Ca2+ release from ryonodine sensitive calcium pool. It was also found that EDDF (10−4 g/mL) significantly decreased the proportion of S phase of human umbilical vein (HUV) and inhibited the proliferation of VSMC induced by angiotensin II (Angll, 10−5 mol/L). The apopotosis did not occur when VSMC was cultured under normal condition. While VSMC apoptosis was induced by Angll (10−5 mol/L) and EDDF (10−4 g/mL) seemed to have little effect on it. The inhibitory effect of EDDF on the elevation of [Ca2+]i and [Ca2+]n of VSMC might play an essential role in its action mechanisms and the ways it affects the Ca2+ handling of VSMC demonstrate that EDDF was different from other endogenous blood pressure regulators and some known antihypertensive drugs. EDDF could inhibit the proliferation of VSMC, which indicated that it might be beneficial to the prevention and treatment of hypertension and arteriosclerosis.

Congestive Heart Failure: Fifty Years of Progress

Congestive Heart Failure: Fifty Years of Progress

Distinct Topologies of Mono- and Decavanadate Binding and Photo-oxidative Cleavage in the Sarcoplasmic Reticulum ATPase

UV irradiation of the sarcoplasmic reticulum (SR) ATPase in the presence of vanadate cleaves the enzyme at either of two different sites. Under conditions favoring the presence of monovanadate, and in the presence of Ca(2+), ADP, and Mg(2+), cleavage results in two fragments of 71- and 38-kDa electrophoretic mobility. On the other hand, under conditions permitting formation of decavanadate, and in the absence of Ca(2+) and ADP, cleavage results in two fragments of 88- and 21-kDa electrophoretic mobility. The amino terminus resulting from cleavage is blocked and resistant to Edman degradation. However, the initial photo-oxidation product can be reduced with NaB(3)H(4,) resulting in incorporation of radioactive (3)H label. Extensive digestion of the labeled protein with trypsin then yields labeled peptides that are specific for the each of the photo-oxidation conditions, and can be sequenced after purification. Collection of the Edman reaction fractional products reveals the radioactive label and demonstrates that Thr(353) is the residue oxidized by monovanadate at the phosphorylation site (i.e. Asp(351)). Correct positioning of monovanadate at the phosphorylation site requires binding of Mg(2+) and ADP to the Ca(2+)-dependent conformation of the enzyme. Subsequent hydrolytic cleavage is likely assisted by the neighboring Asp(601), and yields the 71- and 38-kDa fragments. On the other hand, Ser(186) (and possibly the following three residues: Val(187), Ile(188), and Lys(189)) is the residue that is photo-oxidized by decavanadate in the absence of ADP. Hydrolytic cleavage of the oxidized product at this site is likely assisted by neighboring acidic residues, and yields the 88- and 21-kDa fragments. The bound decavanadate, which we find to produce steric interference with TNP-AMP binding, must therefore extend to the A domain (i.e. small cytosolic loop) in order to oxidize Ser(186). This protein conformation is only obtained in the absence of Ca(2+).