Characterization of the Ca2+- or Mg2+-ATPase of transverse tubule membranes isolated from rabbit skeletal muscle

Transverse tubule membranes isolated from rabbit skeletal muscle have high levels of a Ca2+- or Mg2+-ATPase with Km values for Ca-ATP or Mg-ATP in the 0.2 mM range, but do not display detectable levels of ATPase activity activated by micromolar [Ca2+]. The transverse tubule enzyme is less temperature or pH dependent than the Ca2+-ATPase of sarcoplasmic reticulum and hydrolyzes equally well ATP, ITP, UTP, CTP, and GTP. Of several ionic, non-ionic, and zwitterionic detergents tested, only lysolecithin solubilizes the transverse tubule membrane while preserving ATPase activity. After extraction of about 50% of the transverse tubule proteins by solubilization with lysolecithin most of the ATPase activity remains membrane bound, indicating that the Ca2+- or Mg2+-ATPase is an intrinsic membrane enzyme. A second extraction of the remaining transverse tubule proteins with lysolecithin results in solubilization and partial purification of the enzyme. Sedimentation of the Ca2+- or Mg2+-ATPase, partially purified by lysolecithin solubilization, through a continuous sucrose gradient devoid of detergent leads to additional purification, with an overall 3- to 5-fold purification factor. The purified enzyme preparation contains two main protein components of molecular weights 107,000 and 30,000. Cholesterol, which is highly enriched in the transverse tubule membrane, copurifies with the enzyme. Transverse tubule membrane vesicles also display ATP-dependent calcium transport which is not affected by phosphate or oxalate. The possibility that the Ca2+- or Mg2+-ATPase is the enzyme responsible for the Ca2+ transport displayed by isolated transverse tubules is discussed.     

Effect of halothane on Ca2+-induced Ca2+ release from sarcoplasmic reticulum vesicles isolated from rat skeletal muscle

Halothane induces the release of Ca2+ from a subpopulation of sarcoplasmic reticulum vesicles that are derived from the terminal cisternae of rat skeletal muscle. Halothane-induced Ca2+ release appears to be an enhancement of Ca2+-induced Ca2+ release. The low-density sarcoplasmic reticulum vesicles which are believed to be derived from nonjunctional sarcoplasmic reticulum lack the capability of both Ca2+-induced and halothane-induced Ca2+ release. Ca2+ release from terminal cisternae vesicles induced by halothane is inhibited by Ruthenium red and Mg2+, and require ATP (or an ATP analogue), KCl (or similar salt) and extravesicular Ca2+. Ca2+-induced Ca2+ release has similar characteristics.     

Ca2+ and Mg2+-binding and a putative calmodulin type Ca2+-binding site in Synechococcus Photosystem II

Thylakoids and Photosystem II particles prepared from the cyanobacterium Synechococcus PCC 7942 washed with a HEPES/glycerol buffer exhibited low rates of light-induced oxygen evolution. Addition of either Ca²⁺ or Mg²⁺ to both thylakoids and Photosystem II particles increased oxygen evolution independently, maximal rates being obtained by addition of both ions. If either preparation was washed with NaCl, light induced O₂ evolution was completely inhibited, but re-activated in the same manner by Ca²⁺ and Mg²⁺ but to a lower level. In the presence of Mg²⁺, the reactivation of O₂ evolution by Ca²⁺ allowed sigmoid kinetics, implying co-operative binding. The results are interpreted as indicating that not only Ca²⁺, but also Mg²⁺, is essential for light-induced oxygen evolution in thylakoids and Photosystem II particles from Synechococcus PC 7942. The significance of the reactivation kinetics is discussed. Reactivation by Ca²⁺ was inhibited by antibodies to mammalian calmodulin, indicating that the binding site in Photosystem II may be analogous to that of this protein.Abbreviation HEPES n-2-Hydroxyethylpiperazine--2-ethane sulphonic acid     

Novel effects of calmodulin and calmodulin antagonists on the plasma membrane (Ca2+ + Mg2+)-ATPase from rabbit kidney proximal tubules.

In this work we report an unusual pattern of activation by calmodulin on the (Ca2+ + Mg2+)-ATPase from basolateral membranes of kidney proximal tubule cells. The activity of the ATPase depleted of calmodulin is characterized by a high Ca2+ affinity (Km = 2.2-3.4 microM) and a biphasic dependence on ATP concentration. The preparation responded to the addition of calmodulin by giving rise to a new Ca2+ site of very high affinity (Km less than 0.05 microM). Calmodulin antagonists had diverse effects on ATPase activity. Compound 48/80 inhibited calmodulin-stimulated activity by 70%, whereas calmidazolium did not modify this component. In the absence of calmodulin, 48/80 still acted as an antagonist, increasing the Km for Ca2+ to 5.7 microM and reducing enzyme turnover by competing with ATP at the low affinity regulatory site. Calmidazolium did not affect Ca2+ affinity, but it did displace ATP from the regulatory site. At fixed Ca2+ (30 microM) and ATP (5 mM) concentrations, Pi protected against 48/80 and potentiated inhibition by calmidazolium. At 25 microM ATP, Pi protected against calmidazolium inhibition. We propose that the effects of ATP and Pi arise because binding of the drugs to the ATPase occurs mainly on the E2 forms.     

NMR analysis of the Mg2+-binding properties of aequorin, a Ca2+-binding photoprotein

Aequorin, which is a calcium-sensitive photoprotein and a member of the EF-hand superfamily, binds to Mg2+ under physiological conditions, which modulates its light emission. The Mg2+ binding site and its stabilizing influence were examined by NMR spectroscopy. The binding of Mg2+ to aequorin prevented the molecule from aggregating and stabilized it in the monomeric form. To determine the structural differences between Mg2+-bound and free aequorin, we have performed backbone NMR assignments of aequorin in the Mg2+-free state. Mg2+ binding induces conformational changes that are localized in the EF-hand loops. The chemical shift difference data indicated that there are two Mg2+-binding sites, EF-hands I and III. The Mg2+ titration experiment revealed that EF-hand III binds to Mg2+ with higher affinity than EF-hand I, and that only EF-hand III seems to be occupied by Mg2+ under physiological conditions.     

The effect of Mg2+ on the Ca2+-binding properties of non-activated phosphorylase kinase.

The calcium binding properties of non-activated phosphorylase kinase at pH 6.8 have been studied by the gel filtration technique at calcium concentrations from 50 nM to 50 muM. Taking into account the subunit structure alpha4beta4gamma4 the enzyme binds 12 mol Ca2+ per mol with an association constant of 6.0 X 10(7) M-1, 4 mol with an association constant of 1.7 X 10(6) M-1 and 36 mol with a binding constant of 3.9 X 10(4) M-1 at low ionic strength. In buffer of high ionic strength, i.e. 180 mM NH4Cl or 60 mM (NH4)2SO4, only a single set of eight binding sites with a binding constant of 5.5 X 10(7) M-1 is left. In a buffer containing 155 mM NH4Cl and 10 mM MgCl2, the calcium affinity of these sites is reduced to a KCa of 3.0 X 10(6) M-1, indicating competition between Ca2+ and Mg2+. From these measurements, the binding constant of Mg2+ for these sites is calculated to be 1.7 X 10(3) M-1 is left. In a buffer containing 155 mM NH4Cl and 10 mM MgCl2, the calcium affinity of these sites is reduced to a KCa of 3.0 X 10(6) M-1, indicating competition between Ca2+ and Mg2+. from these measurements, the binding constant of Mg2+ for these sites is calculated to be 1.7 X 10(3) M-1. Additionally, 10 mM Mg2+ induces a set of four new Ca2+ binding sites which show positive cooperativity. Their half-saturation constant under the conditions described is 3.5 X 10(5) M-1, and they, too, exhibit competition between Ca2+ and Mg2+. Since this set of sites is induced by Mg2+ a third group of binding sites for the latter metal must be postulated.     

Effects of ATP on the interaction of Ca++, Mg++, and K+ with fragmented sarcoplasmic reticulum isolated from rabbit skeletal muscle

Fragmented sarcoplasmic reticulum isolated from skeletal muscle of the rabbit has a cation-binding capacity of about 350 µeq/g of protein at neutral pH. The same binding sites bind Ca, Mg, K, and H ions and, consequently, the selective binding of Ca induced by ATP releases an amount of the other cations equivalent to the Ca taken up. At pH values below 6.2, an increasing number of binding sites are associated with H⁺, and ATP induces exchange of Ca mostly for H⁺. At pH values above 6.2, the binding sites exist in the form of Mg and K, and Ca is bound in exchange for these cations. The total bound Ca + Mg + K, expressed in microequivalents of cations bound per gram of protein, is approximately constant at various pCa values, which indicates a stoichiometric exchange of Ca for the other cations. To accomplish the same degree of exchange of Ca for other cations bound, in the absence of ATP, concentrations of free Ca⁺⁺ of about 1000-fold higher than those needed in the presence of ATP are required in the medium. We cannot distinguish between a mechanism whereby Ca actively transported into a compartment of the microsomal vesicles containing also the binding sites is bound passively to these sites in exchange for Mg, K, and H and another in which ATP selectively increases the affinity of surface-binding sites for Ca. Irrespective of the mechanism of accumulation, the Ca retained does not contribute to the activity of the cation in the membrane fraction. Caffeine (10 mM) has no effect on the binding of Ca, but releases a more labile fraction of Ca, which presumably accumulates in excess of the bound Ca. Procaine (5 mM) antagonizes the effect of caffeine. Acetylcholine and epinephrine have no effect on the binding of Ca.     

Ca2+ release by inositol-trisphosphorothioate in isolated triads of rabbit skeletal muscle.

The effectiveness of the nonmetabolizable second messenger analogue DL-myo-inositol 1,4,5-trisphosphorothioate (IPS3) described by Cooke, A. M., R. Gigg, and B. V. L. Potter, (1987b. Jour. Chem. Soc. Chem. Commun. 1525-1526.) was examined in triads purified from rabbit skeletal muscle. A Ca2+ electrode uptake-release assay was used to determine the size and sensitivity of the IPS3-releasable pool of Ca2+ in isolated triads. Uptake was initiated by 1 mM MgATP, pCa 5.8, pH 7.5 Release was initiated when the free Ca2+ had lowered to pCa approximately 7. We found that 5-25 microM myo-inositol 1,4,5-trisphosphate (IP3), and separately IPS3, consistently released 5-20% of the Ca2+ pool actively loaded into triads. Single channel recording was used to determine if ryanodine receptor Ca2+ release channels were affected by IPS3 at the same myoplasmic Ca2+ and IPS3 concentrations. Open probability of ryanodine receptor Ca2+ release channels was monitored in triads fused to bilayers over long periods (200 s) in the absence and following addition of 30 microM IPS3 to the same channel. At myoplasmic pCa approximately 7, IPS3 had no effect in the absence of MgATP (Po = 0.0094 +/- 0.001 in control and Po = 0.01 +/- 0.006 after IPS3) and slightly increased activity in the presence of 1 mM MgATP (Po = 0.024 +/- 0.03 in control and Po = 0.05 +/- 0.03 after IPS3). Equally small effects were observed at higher myoplasmic Ca2+. The onset of channel activation by IPS3 or IP3 was slow, on the time scale 20-60 s. We suggest that in isolated triads of rabbit skeletal muscle, IP3-induced release of stored Ca2+ is probably not mediated by the opening of Ca2+ release channels.     

Ca 2+ -specific removal of Z lines from rabbit skeletal muscle

Removal of rabbit psoas strips immediately after death and incubation in a saline solution containing 1 mM Ca²⁺ and 5 nM Mg²⁺ for 9 hr at 37°C and pH 7.1 causes complete Z-line removal but has no ultrastructurally detectable effect on other parts of the myofibril. Z lines remain ultrastructurally intact if 1 mM 1,2-bis-(2-dicarboxymethylaminoethoxy)-ethane (EGTA) is substituted for 1 mM Ca²⁺ and the other conditions remain unchanged. Z lines are broadened and amorphous but are still present after incubation for 9 hr at 37°C if 1 mM ethylenediaminetetraacetate (EDTA) is substituted for 1 mM Ca²⁺ and 5 mM Mg²⁺ in the saline solution. A protein fraction that causes Z-line removal from myofibrils in the presence of Ca²⁺ at pH 7.0 can be isolated by extraction of ground muscle with 4 mM EDTA at pH 7.0–7.6 followed by isoelectric precipitation and fractionation between 0 and 40% ammonium sulfate saturation. Z-line removal by this protein fraction requires Ca²⁺ levels higher than 0.1 mM, but Z lines are removed without causing any other ultrastructurally detectable degradation of the myofibril. This is the first report of a protein endogenous to muscle that is able to catalyze degradation of the myofibril. The very low level of unbound Ca²⁺ in muscle cells in vivo may regulate activity of this protein fraction, or alternatively, this protein fraction may be localized in lysosomes.     

The effects of Ca2+ binding on the dynamic properties of a designed Ca2+-binding protein

The effects of Ca(2+) binding on the dynamic properties of Ca(2+)-binding proteins are important in Ca(2+) signaling. To understand the role of Ca(2+) binding, we have successfully designed a Ca(2+)-binding site in the domain 1 of rat CD2 (denoted as Ca.CD2) with the desired structure and retained function. In this study, the backbone dynamic properties of Ca.CD2 have been investigated using (15)N spin relaxation NMR spectroscopy to reveal the effect of Ca(2+) binding on the global and local dynamic properties without the complications of multiple interactive Ca(2+) binding and global conformational change. Like rat CD2 (rCD2) and human CD2 (hCD2), residues involved in the recognition of the target molecule CD48 exhibit high flexibility. Mutations N15D and N17D that introduce the Ca(2+) ligands increase the flexibility of the neighboring residues. Ca(2+)-induced local dynamic changes occur mainly at the residues proximate to the Ca(2+)-binding pocket or the residues in loop regions. The beta-strand B of Ca.CD2 that provides two Asp for the Ca(2+) undergoes an S(2) decrease upon the Ca(2+) binding, while the DE-loop that provides one Asn and one Asp undergoes an S(2) increase. Our study suggests that Ca(2+) binding has a differential effect on the rigidity of the residues depending on their flexibility and location within the secondary structure.     

Inactivation of a Ca2+-induced Ca2+ release channel from skeletal muscle sarcoplasmic reticulum during active Ca2+ transport

ATP-dependent Ca2+ uptake by subfractions of skeletal muscle sarcoplasmic reticulum (SR) was studied with the Ca2+ indicator dye, antipyrylazo III. Ca2+ uptake by heavy SR showed two phases, a slow uptake phase and a fast uptake phase. By contrast, Ca2+ uptake by light SR exhibited a monophasic time course. In both fractions a steady state of Ca2+ uptake was observed when the concentration of free Ca2+ outside the vesicles was reduced to less than 0.1 microM. In the steady state, the addition of 5 microM Ca2+ to the external medium triggered rapid Ca2+ release from heavy SR but not from light SR, indicating that the heavy fraction contains a Ca2+-induced Ca2+ release channel. During Ca2+ uptake, heavy SR showed a constant Ca2+-dependent ATPase activity (1 mumol/mg protein X min) which was about 150 times higher than the rate of Ca2+ uptake in the slow uptake phase. Ruthenium red, an inhibitor of Ca2+-induced Ca2+ release, enhanced the rate of Ca2+ uptake during the slow phase without affecting Ca2+-dependent ATPase activity. Adenine nucleotides, activators of Ca2+ release, reduced the Ca2+ uptake rate. These results suggest that the rate of Ca2+ accumulation by heavy SR is not proportional to ATPase activity during the slow uptake phase due to the activation of the channel for Ca2+-induced Ca2+ release. In addition, they suggest that the release channel is inactivated during the fast Ca2+ uptake phase.     

Apocalmodulin and Ca2+ calmodulin bind to the same region on the skeletal muscle Ca2+ release channel.

The skeletal muscle Ca2+ release channel (RYR1) is regulated by calmodulin in both its Ca2+-free (apocalmodulin) and Ca2+-bound (Ca2+ calmodulin) states. Apocalmodulin is an activator of the channel, and Ca2+ calmodulin is an inhibitor of the channel. Both apocalmodulin and Ca2+ calmodulin binding sites on RYR1 are destroyed by a mild tryptic digestion of the sarcoplasmic reticulum membranes, but calmodulin (either form), bound to RYR1 prior to tryptic digestion, protects both the apocalmodulin and Ca2+ calmodulin sites from tryptic destruction. The protected sites are after arginines 3630 and 3637 on RYR1. These studies suggest that both Ca2+ calmodulin and apocalmodulin bind to the same or overlapping regions on RYR1 and block access of trypsin to sites at amino acids 3630 and 3637. This sequence is part of a predicted Ca2+ CaM binding site of amino acids 3614-3642 [Takeshima, H., et al. (1989) Nature 339, 439-445].     

Oxidation of the skeletal muscle Ca2+ release channel alters calmodulin binding

This study presents evidence for a close relationship betweenthe oxidation state of the skeletal muscleCa²⁺ release channel (RyR1) andits ability to bind calmodulin (CaM). CaM enhances the activity of RyR1in low Ca²⁺ and inhibits itsactivity in high Ca²⁺. Oxidation,which activates the channel, blocks the binding of ¹²⁵I-labeled CaM at bothmicromolar and nanomolar Ca²⁺concentrations. Conversely, bound CaM slows oxidation-induced cross-linking between subunits of the RyR1 tetramer. Alkylation ofhyperreactive sulfhydryls (<3% of the total sulfhydryls) on RyR1with N-ethylmaleimide completelyblocks oxidant-induced intersubunit cross-linking and inhibitsCa²⁺-free¹²⁵I-CaM but notCa²⁺/¹²⁵I-CaMbinding. These studies suggest that1) the sites on RyR1 for bindingapocalmodulin have features distinct from those of theCa²⁺/CaM site,2) oxidation may alter the activityof RyR1 in part by altering its interaction with CaM, and3) CaM may protect RyR1 fromoxidative modifications during periods of oxidative stress.

     

Use of site-directed mutations in the individual Ca2(+)-binding sites of calmodulin to examine Ca2(+)-induced conformational changes

Mutant versions of the calmodulin of Drosophila melanogaster have been prepared for use in the study of Ca2+ binding and Ca2(+)-induced conformational changes. In each mutant, a conserved glutamic acid residue indicated to play a critical role in Ca2+ binding has been mutated to glutamine in one of the Ca2(+)-binding sites. Thus a series of four proteins, each with an analogous mutation in one of the four binding sites, has been generated. Here the Ca2(+)-induced conformational changes in these proteins have been examined by use of the fluorescent hydrophobic reporter molecule, 9-anthroyl choline. These studies confirm earlier work which indicates that the carboxyl-terminal pair of Ca2(+)-binding sites shows cooperative Ca2+ binding to produce a major conformational change in the protein. However, these studies provide evidence that the sites of the amino-terminal pair are more independent in their Ca2+ binding properties and contribute individually to the conformational changes associated with Ca2+ binding in the amino-terminal half of the protein. This work also indicates that mutation of either of the amino-terminal Ca2(+)-binding sites can influence the conformational change produced by Ca2+ binding to the carboxyl-terminal sites.     

Ca2+-induced Ca2+ release in skinned single smooth muscle cells isolated from guinea-pig taenia caeci

The Ca2+ release from intracellular Ca2+ storage sites of skinned single smooth muscle cells isolated from guinea-pig taenia caeci was studied. The Ca2+ release from intracellular Ca2+ storage sites of the skinned single cells was enhanced by the presence of submicromolar concentrations of Ca2+ in the solution. The Ca2+ release was enhanced by caffeine and adenine, and suppressed by Mg2+ and procaine. These results suggest that the Ca2+-induced Ca2+ release mechanism may play an important role in the release of Ca2+ from intracellular storage sites of guinea-pig taenia caeci smooth muscle cells.     

Sarcoplasmic reticulum Ca2+-ATPase from rabbit skeletal muscle modified by peroxynitrite

Abstract

Objective: Effect of peroxynitrite on SERCA1 activity was studied in correlation with enzyme carbonylation. Kinetic parameters and location of peroxynitrite effect on SERCA1 were determined.

Methods: Carbonyls were determined by immunoblotting. FITC, NCD-4 and Trp fluorescence were used to indicate changes in cytosolic and transmembrane regions of SERCA1.

Results: Peroxynitrite-concentration-dependent decrease of SERCA1 activity was associated with elevation of protein carbonyls. 4-HNE was not involved in carbonylation of SERCA1. Increased FITC fluorescence in the presence of peroxynitrite correlated with the decrease of the enzyme affinity to ATP.

Discussion and conclusion: Peroxynitrite-induced SERCA1 carbonylation that was not accompanied with the formation of 4-HNE-SERCA1 adducts is indicative of direct oxidation of SERCA1. As assessed by FITC fluorescence and decreased affinity of the enzyme to ATP, peroxynitrite impairment was found to occur in the cytosolic ATP-binding region of SERCA1.