The tandemly repeated domains of a β-propeller phytase act synergistically to increase catalytic efficiency

β-Propeller phytases (BPPs) with tandemly repeated domains are abundant in nature. Previous studies have shown that the intact domain is responsible for phytate hydrolysis, but the function of the other domain is relatively unknown. In this study, a new dual-domain BPP (PhyH) from Bacillus sp. HJB17 was identified to contain an incomplete N-terminal BPP domain (PhyH-DI, residues 41-318) and a typical BPP domain (PhyH-DII, residues 319-644) at the C-terminus. Purified recombinant PhyH and PhyH-DII required Ca(2+) for phytase activity, showed activity at low temperatures (0-35 °C) and pH 6.0-8.0, and remained active (at 37 °C) after incubation at 60 °C and pH 6.0-12.0. Compared with PhyH-DII, PhyH is catalytically more active against phytate (catalytic constant 27.72 versus 4.17 s(-1)), which indicates the importance of PhyH-DI in phytate degradation. PhyH-DI was found to hydrolyze phytate intermediate D-Ins(1,4,5,6) P(4), and to act synergistically (a 1.2-2.5-fold increase in phosphate release) with PhyH-DII, other BPPs (PhyP and 168PhyA) and a histidine acid phosphatase. Furthermore, fusion of PhyH-DI with PhyP or 168PhyA significantly enhanced their catalytic efficiencies. This is the first report to elucidate the substrate specificity of the incomplete domain and the functional relationship of tandemly repeated domains in BPPs. We conjecture that dual-domain BPPs have succeeded evolutionarily because they can increase the amount of available phosphate by interacting together. Additionally, fusing PhyH-DI to a single-domain phytase appears to be an efficient way to improve the activity of the latter.     

RyR1/SERCA1 cross-talk regulation of calcium transport in heavy sarcoplasmic reticulum vesicles

We investigated the functional interdependence of sarco-endoplasmic reticulum Ca2+ ATPase isoform 1 and ryanodine receptor isoform 1 in heavy sarcoplasmic reticulum membranes by synchronous fluorescence determination of extravesicular Ca2+ transients and catalytic activity. Under conditions of dynamic Ca2+ exchange ATPase catalytic activity was well coordinated to ryanodine receptor activation/inactivation states. Ryanodine-induced activation of Ca2+ release channel leaks also produced marked ATPase activation in the absence of measurable increases in bulk free extravesicular Ca2+. This suggested that Ca2+ pumps are highly sensitive to Ca2+ release channel leak status and potently buffer Ca2+ ions exiting cytoplasmic openings of ryanodine receptors. Conversely, ryanodine receptor activation was dependent on Ca2+-ATPase pump activity. Ryanodine receptor activation by cytosolic Ca2+ was (i) inversely proportional to luminal Ca2+ load and (ii) dependent upon the rate of presentation of cytosolic Ca2+. Progressive Ca2+ filling coincided with progressive loss of Ca2+ sequestration rates and at a threshold loading, ryanodine-induced Ca2+ release produced small transient reversals of catalytic activity. These data indicate that attainment of threshold luminal Ca2+ loads coordinates sensitization of Ca2+ release channels with autogenic inhibition of Ca2+ pumping. This suggests that Ca2+-dependent control of Ca2+ release in intact heavy sarcoplasmic reticulum membranes involves a Ca2+-mediated "cross-talk" between sarco-endoplasmic reticulum Ca2+ ATPase isoform 1 and ryanodine receptor isoform 1.     

Post-translational processing of chicken bone phosphoproteins. Identification of bone (phospho)protein kinase.

We have detected a protein kinase which phosphorylates bone phosphoproteins (BPPs) in the detergent extract of the membranous fractions in the periosteal bone strips of 12-day-embryonic-chick tibia. This enzyme, tentatively named BPP kinase, has a catalytic subunit of Mr approximately 39,000, utilizes GTP as well as ATP as a phospho-group donor, is inhibited by 2,3-bisphosphoglycerate and heparin, and is therefore similar to casein kinase II. The enzyme can phosphorylate dephosphorylated proteins such as casein, phosvitin and chicken BPPs, but the last-named are preferred substrates. The in vitro-phosphorylation-assay products of this enzyme in the extract were indistinguishable on an SDS/polyacrylamide gel from the major [32P]phosphoproteins metabolically labelled in the embryonic-chick bone tissue. The regulatory mechanisms of the phosphorylation process of BPPs by BPP kinase as well as the potential role of this enzyme in mineralization are discussed.     

2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine mono-, di-, and triphosphates as photoaffinity probes of the Ca2+-ATPase of sarcoplasmic reticulum. Regulatory/superfluorescent nucleotides label the catalytic site with high efficiency

We have synthesized a new class of ATP photo-affinity analogs, 2',3'-O-(2,4,6-trinitrophenyl)-8-azido (TNP-8N3)-ATP, -ADP, and -AMP, and their radiolabeled derivatives, and characterized their interaction with sarcoplasmic reticulum vesicles. The nucleotides bind with high affinity (Kd = 0.04-0.4 microM) to the catalytic site of the Ca2+-ATPase. TNP-8N3-ATP and TNP-8N3-ADP, at low concentrations (less than 10 microM), accelerate ATPase activity 1.5- and 1.4-fold, respectively, indicating that they bind to a regulatory site. In the same concentration range, they all undergo a large increase in fluorescence ("superfluorescence") during enzyme turnover in the presence of ATP and Ca2+, or on phosphorylation from Pi in a Ca2+-depleted medium. Irradiation at alkaline pH results in specific covalent incorporation of the nucleotide at the catalytic site on the A1 tryptic subfragment. The efficiency of catalytic site labeling is greatest (up to 80% of available sites/irradiation period) in the presence of ATP, Ca2+, and Mg2+, conditions in which the probe binds only to the regulatory and superfluorescent sites. The covalently attached nucleotide exhibits fluorescence enhancement on enzyme turnover in the presence of acetyl phosphate plus Ca2+ or on phosphorylation from Pi in a Ca2+-depleted medium, but not in the presence of ATP plus Ca2+. The results suggest that the catalytic, regulatory, and superfluorescent nucleotide sites are at the same locus and that the binding domain includes portions of the A1 subfragment. The high efficiency with which the site is photolabeled during turnover is ascribed to water exclusion and possibly cleft closure in E2-P.     

Novel type of signaling molecules: protein kinases covalently linked to ion channels

Recently we identified a new class of protein kinases with a novel type of catalytic domain structurally and evolutionarily unrelated to the conventional eukaryotic protein kinases. This new class, which we named alpha-kinases, is represented by eukaryotic elongation factor-2 kinase and the Dictyostelium myosin heavy chain kinases. Here we cloned, sequenced and analyzed the tissue distribution of five new putative mammalian alpha-kinases: melanoma alpha-kinase, kidney alpha-kinase, heart alpha-kinase, skeletal muscle alpha-kinase, and lymphocyte alpha-kinase. All five are large proteins of more than 1000 amino acids with an alpha-kinase catalytic domain located at the very carboxyl-terminus. We expressed the catalytic domain of melanoma alpha-kinase in Escherichia coli, and found that it autophosphorylates on threonine residues, demonstrating that it is a genuine protein kinase. Unexpectedly, we found that the long amino-terminal portions of melanoma and kidney alpha-kinases represent new members of the transient receptor potential (TRP) ion channel family, which are implicated in the mediation of capacitative Ca2+ entry in nonexcitable mammalian cells. This suggests that melanoma and kidney alpha-kinases, which represent a novel type of signaling molecule, are involved in the regulation of Ca2+ influx in mammalian cells.     

Distances between the functional sites of the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

Luminescence energy transfer measurements have been used to determine the distances between the two high affinity Ca2+ binding-transport sites of the (Ca2+ + Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum. The lanthanide Tb3+ situated at one high affinity Ca2+ site was used as the transfer donor, and acceptors at the other Ca2+ site were the lanthanides Nd3+, Pr3+, Ho3+, or Er3+. Terbium bound to the enzyme was excited directly with a pulsed dye laser. Analysis of the changes in the terbium luminescence lifetime due to the presence of the acceptor indicates that the distance between the Ca2+ sites is 10.7 A. The distance between the Ca2+ sites and the nucleotide-binding catalytic site was determined using Tb3+ at the Ca2+ sites and either trinitrophenyl nucleotides (TNP-N) or fluorescein 5-isothiocyanate (FITC) in the catalytic site as energy acceptors. The R0 values for the Tb-acceptor pairs are approximately 30 and approximately 40 A for TNP-N and FITC, respectively. The distance between Tb3+ at the Ca2+ sites and TNP-ATP at the nucleotide site is approximately 35 A and that between the Ca2+ sites and the FITC labeling site is approximately 47 A. Considerations of the molecular dimensions of the ATPase polypeptide indicate that while the two Ca2+ sites are close to each other, the Ca2+ sites and the nucleotide site are quite remote in the three-dimensional structure of the enzyme.     

Activation of skeletal muscle myosin light chain kinase by calcium(2+) and calmodulin

Many biological processes are now known to be regulated by Ca2+ via calmodulin (CM). Although a general mechanistic model by which Ca2+ and calmodulin modulate many of these activities has been proposed, an accurate quantitative model is not available. A detailed analysis of skeletal muscle myosin light chain kinase activation was undertaken in order to determine the stoichiometries and equilibrium constants of Ca2+, calmodulin, and enzyme catalytic subunit in the activation process. The analysis indicates that activation is a sequential, fully reversible process requiring both Ca2+ and calmodulin. The first step of the activation process appears to require binding of Ca2+ to all four divalent metal binding sites on calmodulin for form the complex, Ca42+-calmodulin. This complex then interacts with the inactive catalytic subunit of the enzyme to form the active holoenzyme complex, Ca42+-calmodulin-enzyme. Formation of the holoenzyme follows simply hyperbolic kinetics, indicating 1:1 stoichiometry of Ca42+-calmodulin to catalytic subunit. The rate equation derived from the mechanistic model was used to determine the values of KCa2+ and KCM, the intrinsic activation constants for each step of the activation process. KCa2+ and KCM were found to have values of 10 microM and 0.86 nM, respectively, at 10 mM Mg2+. The rate equation using these equilibrium constants accurately predicts the extent of enzyme activation over a wide range of Ca2+ and calmodulin concentrations. The kinetic model and analytical techniques employed herein may be generally applicable to other enzymes with similar regulatory schemes.     

Identification of Ca2+-pump-related phosphoprotein in plasma membrane vesicles of Ehrlich ascites carcinoma cells

Plasma membrane vesicles of Ehrlich ascites carcinoma cells have been isolated to a high degree of purity. In the presence of Mg2+, the plasma membrane preparation exhibits a Ca2+-dependent ATPase activity of 2 mumol Pi per h per mg protein. It is suggested that this (Ca2+ + Mg2+)-ATPase activity is related to the measured Ca2+ transport which was characterized by Km values for ATP and Ca2+ of 44 +/- 9 microM and 0.25 +/- 0.10 microM, respectively. Phosphorylation of plasma membranes with [gamma-32P]ATP and analysis of the radioactive species by polyacrylamide gel electrophoresis revealed a Ca2+-dependent hydroxylamine-sensitive phosphoprotein with a molecular mass of 135 kDa. Molecular mass and other data differentiate this phosphoprotein from the catalytic subunit of (Na+ + K+)-ATPase and from the catalytic subunit of (Ca2+ + Mg2+)-ATPase of endoplasmic reticulum. It is suggested that the 135 kDa phosphoprotein represents the phosphorylated catalytic subunit of the (Ca2+ + Mg2+)-ATPase of the plasma membrane of Ehrlich ascites carcinoma cells. This finding is discussed in relation to previous attempts to identify a Ca2+-pump in plasma membranes isolated from nucleated cells.     

Functional characterization of lanthanide binding sites in the sarcoplasmic reticulum Ca(2+)-ATPase: do lanthanide ions bind to the calcium transport site?

Gd3+ binding sites on the purified Ca(2+)-ATPase of sarcoplasmic reticulum were characterized at 2 and 6 degrees C and pH 7.0 under conditions in which 45Ca2+ and 54Mn2+ specifically labeled the calcium transport site and the catalytic site of the enzyme, respectively. We detected several classes of Gd3+ binding sites that affected enzyme function: (a) Gd3+ exchanged with 54Mn2+ of the 54MnATP complex bound at the catalytic site. This permitted slow phosphorylation of the enzyme when two Ca2+ ions were bound at the transport site. The Gd3+ ion bound at the catalytic site inhibited decomposition of the ADP-sensitive phosphoenzyme. (b) High-affinity binding of Gd3+ to site(s) distinct from both the transport site and the catalytic site inhibited the decomposition of the ADP-sensitive phosphoenzyme. (c) Gd3+ enhanced 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence in NBD-modified enzyme by probably binding to the Mg2+ site that is distinct from both the transport site and the catalytic site. (d) Gd3+ inhibited high-affinity binding of 45Ca2+ to the transport site not by directly competing with Ca2+ for the transport site but by occupying site(s) other than the transport site. This conclusion was based mainly on the result of kinetic analysis of displacement of the enzyme-bound 45Ca2+ ions by Gd3+ and vice versa, and the inability of Gd3+ to phosphorylate the enzyme under conditions in which GdATP served as a substrate. These results strongly suggest that Ln3+ ions cannot be used as probes to structurally and functionally characterize the calcium transport site on the Ca(2+)-ATPase.     

Activation of brain calcineurin phosphatase towards nonprotein phosphoesters by Ca2+, calmodulin, and Mg2+

Calcineurin purified from bovine brain was found to be active towards beta-naphthyl phosphate greater than p-nitrophenyl phosphate greater than alpha-naphthyl phosphate much greater than phosphotyrosine. In its native state, calcineurin shows little activity. It requires the synergistic action of Ca2+, calmodulin, and Mg2+ for maximum activation. Ca2+ and Ca2+ X calmodulin exert their activating effects by transforming the enzyme into a potentially active form which requires Mg2+ to express the full activity. Ni2+, Mn2+, and Co2+, but not Ca2+ or Zn2+, can substitute for Mg2+. The pH optimum, and the Vm and Km values of the phosphatase reaction are characteristics of the divalent cation cofactor. Ca2+ plus calmodulin increases the Vm in the presence of a given divalent cation, but has little effect on the Km for p-nitrophenyl phosphate. The activating effects of Mg2+ are different from those of the transition metal ions in terms of effects on Km, Vm, pH optimum of the phosphatase reaction and their affinity for calcineurin. Based on the Vm values determined in their respective optimum conditions, the order of effectiveness is: Mg2+ greater than or equal to Ni2+ greater than Mn2+ much greater than Co2+. The catalytic properties of calcineurin are markedly similar to those of p-nitrophenyl phosphatase activity associated with protein phosphatase 3C and with its catalytic subunit of Mr = 35,000, suggesting that there are common features in the catalytic sites of these two different classes of phosphatase.     

Reversible activation of human neutrophil calpain promoted by interaction with plasma membranes

Human neutrophil calpain is a monomer of 85 kDa molecular weight. The proteinase shows an absolute requirement for Ca2+ with maximal catalytic activity at 0.1-0.2 mM Ca2+ and negligible activity at 1-5 microM Ca2+. At this concentration of Ca2+ neutrophil calpain becomes active and reaches 65% of its maximal catalytic activity following interaction with plasma membranes. The activation is fully reversible since the enzyme returns to its native, high Ca2+ requiring form following removal of the membranes. Membrane phospholipids appear to be the physiological compounds responsible for the promotion of such reversible activation. Unlike other Ca2+ dependent proteinases, neutrophil calpain does not undergo conversion to a low Ca2+ requiring form by limited autoproteolysis.     

Comparison of the catalytic and structural properties of Ca2+-ATPase in longitudinal tubules and terminal cisternae of the sarcoplasmic reticulum

The catalytic behavior and structural features of Ca2+-ATPase in the vesicles of longitudinal tubules and terminal cisternae of the sarcoplasmic reticulum isolated from rabbit skeletal muscles was analysed. pH measurements have shown under optimal conditions Ca2+-ATPase has similar catalytic behavior both in the fractions of longitudinal tubules and terminal cisternae. Under non-optimal conditions, the behavior similarity was not observed. The specific activity of the ATPase enzyme under optimal conditions was shown to be much higher in the fraction of longitudinal tubules than in the fraction of terminal cisternae. Caffeine added to both fractions had no effect on the catalytic behavior of Ca2+-ATPase. As judged from fluorescence analysis, the structure of Ca2+-ATPase of longitudinal tubules differs from that structure of terminal cisternae. In sarcoplasmic reticulum membrane, at least half of the tryptophan residues of Ca2+-ATPase was shown to be buried in the lipid bilayer. Our findings suggest that in terminal cisternae some of the Ca2+-ATPase molecules exist as an oligomeric protein and do not participate in ATP hydrolysis (named "silent" Ca2+-ATPase).     

Macrophage arachidonate-mobilizing phospholipase A2: role of Ca2+ for membrane binding but not for catalytic activity.

A recently purified Ca(2+)-dependent intracellular phospholipase A2 from spleen, kidney and macrophage cell lines is activated by Ca2+ at concentrations achieved intracellularly. Using enzyme from the murine cell line J774 we here demonstrate the formation of a ternary complex of phospholipase, 45Ca2+ and phospholipid vesicle, and provide evidence for a single Ca(2+)-binding site on the enzyme involved in its vesicle binding. Although Ca2+ binds to and functions as an activator of the enzyme, this ion does not appear to be involved in its catalytic mechanism, since enzyme brought to the phospholipid vesicle by molar concentrations of NaCl or NH4+ salts exhibited Ca(2+)-independent catalytic activity.     

Functional evidence of a transmembrane channel within the Ca2+ transport ATPase of sarcoplasmic reticulum.

Ca2+ efflux can be studied conveniently following dilution of sarcoplasmic reticulum (SR) vesicles preloaded with 45Ca2+ by active transport. The rates of efflux are highly dependent on ATPase substrates and cofactors (Pi, Mg2+, Ca2+ and ADP) in the efflux medium. On the other hand, phenothiazines stimulate efflux through a passive permeability channel with no coupled catalytic events. Efflux activation by manipulation of catalytically active ATPase ligands, as well as by the catalytically inactive phenothiazines, can be prevented by thapsigargin, which is a highly specific inhibitor of the Ca(2+)-ATPase. This demonstrates that the passive channel activated by phenothiazines is an integral part of the ATPase, and can operate either uncoupled or coupled to catalytic events.     

The O2- generating oxidoreductase of human neutrophils: evidence of an obligatory requirement for calcium and magnesium for expression of catalytic activity

NADPH-dependent O2- generating oxidoreductase activity recovered from cell lysates of phorbol myristate acetate-stimulated human neutrophils exhibits dependence on Ca+2 and Mg+2 for full expression of its catalytic activity. O2- generating activity was completely abolished by exposure of the oxidoreductase to EDTA, then reconstituted by exposure of the enzyme to Ca+2 and Mg+2 in excess of the EDTA concentration used to block catalytic activity. The oxidoreductase responded maximally to either 0.25 mM Ca+2 or 0.80 mM Mg+2. The pH optimum of the oxidoreductase exposed to Ca+2 and Mg+2 is between pH 7.0 and 7.6. The molar ratio of NADPH oxidation to O2- production determined at pH 7.6 in the presence of Ca+2 and Mg+2 is 0.49, indicating 1 mole of NADPH oxidized per 2 moles of O2- formed. Particulate fractions recovered from cell lysates of resting neutrophils exhibited no oxidoreductase activity under the same conditions.     

Characterization of a calcium-modulated adenylate cyclase from abalone spermatozoa

Abalone spermatozoa contain a particulate adenylate cyclase that displays maximal catalytic activity when Mn2+ is present as a metal cofactor in excess of ATP. Unlike other sperm adenylate cyclases, the abalone enzyme displays a high Mg2+-supported catalytic activity (Mg2+/Mn2+ activity ratio = 0.8). Kinetics analyses demonstrate that the enzyme contains both a MgATP catalytic site and a separate Mg2+ regulatory site. Mg2+-supported enzyme activity, however, is not stimulated by guanine nucleotides, NaF, cholera toxin, forskolin, or a variety of hormones. The enzyme from unfractionated sperm homogenates is inhibited by added Ca2+ in a concentration-dependent manner, when EGTA is not present in the assay. Methylxanthines, such as 1-methyl-3-isobutylxanthine and theophylline, also inhibit enzyme activity in a concentration-dependent manner through a noncompetitive mechanism. On the other hand, when intact cells are preincubated with Ca2+ prior to breakage and assayed for enzyme activity, Ca2+ stimulates enzyme activity at low concentrations. Enzyme activity of intact sperm preincubated with methylxanthines, in either the absence or presence of added Ca2+, is also stimulated. This effect is expressed via an effect on the velocity of the enzyme. A-23187 has similar stimulatory effects on the enzyme under these conditions. These data provide further support for the role of Ca2+ conductance in modulating sperm adenylate cyclase activity. The abalone sperm enzyme also appears to have regulatory properties that are unique among other sperm types.     

Calcium-dependent catalytic activity of a novel phytase from Bacillus amyloliquefaciens DS11

The thermostable phytase from Bacillus amyloliquefaciens DS11 hydrolyzes phytate (myo-inositol hexakisphosphate, IP6) to less phosphorylated myo-inositol phosphates in the presence of Ca2+. In this report, we discuss the unique Ca2+-dependent catalytic properties of the phytase and its specific substrate requirement. Initial rate kinetic studies of the phytase indicate that the enzyme activity follows a rapid equilibrium ordered mechanism in which binding of Ca2+ to the active site is necessary for the essential activation of the enzyme. Ca2+ turned out to be also required for the substrate because the phytase is only able to hydrolyze the calcium-phytate complex. In fact, both an excess amount of free Ca2+ and an excess of free phytate, which is not complexed with each other, can act as competitive inhibitors. The Ca2+-dependent catalytic activity of the enzyme was further confirmed, and the critical amino acid residues for the binding of Ca2+ and substrate were identified by site-specific mutagenesis studies. Isothermal titration calorimetry (ITC) was used to understand if the decreased enzymatic activity was related to poor Ca2+ binding. The pH dependence of the Vmax and Vmax/Km consistently supported these observations by demonstrating that the enzyme activity is dependent on the ionization of amino acid residues that are important for the binding of Ca2+ and the substrate. The Ca2+-dependent activation of enzyme and substrate was found to be different from other histidine acid phytases that hydrolyze metal-free phytate.     

Reaction mechanism of (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum. The role of Mg2+ that activates hydrolysis of the phosphoenzyme

The role of Mg2+ in the activation of phosphoenzyme hydrolysis has been investigated with the (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles. The enzyme of the native and solubilized vesicles was phosphorylated with ATP at 0 degrees C, pH 7.0, in the presence of Ca2+ and Mg2+. When Ca2+ and Mg2+ in the medium were chelated, phosphoenzyme hydrolysis continued for about 15 s and then ceased. The extent of this hydrolysis increased with increasing concentrations of Mg2+ added before the start of phosphorylation. This shows that the hydrolysis was activated by the Mg2+ added. The Mg2+ which activated phosphoenzyme hydrolysis was distinct from Mg2+ derived from MgATP bound to the substrate site. The Mg2+ site at which Mg2+ combined to activate phosphoenzyme hydrolysis was located on the outer surface of the vesicular membranes. During the catalytic cycle, Mg2+ combined with the Mg2+ site before Ca2+ dissociated from the Ca2+ transport site of the ADP-sensitive phosphoenzyme with bound Ca2+. This Mg2+ did not activate hydrolysis of the ADP-sensitive phosphoenzyme with bound Ca2+, but markedly activated hydrolysis of the ADP-insensitive phosphoenzyme without bound Ca2+. It is concluded that during the catalytic cycle, Mg2+ activates phosphoenzyme hydrolysis only after Ca2+ has dissociated from the Ca2+ transport site of phosphoenzyme.     

Crystal structure of a micro-like calpain reveals a partially activated conformation with low Ca2+ requirement

The two Ca2+-dependent cysteine proteases, micro- and m-calpain, are involved in various Ca2+-linked signal pathways but differ markedly in their Ca2+ requirements for activation. We have determined the structure of a micro-like calpain, which has 85% micro-calpain sequence (the first 48 and the last 62 residues of the large subunit are those from m-calpain) and a low Ca2+ requirement. This construct was used because micro-calpain itself is too poorly expressed. The structure of micro-like calpain is very similar in overall fold to that of m-calpain as expected, but differs significantly in two aspects. In comparison with m-calpain, the catalytic triad residues in micro-like calpain, His and Cys, are much closer together in the absence of Ca2+, and significant portions of the Ca2+ binding EF-hand motifs are disordered and more flexible. These structural differences imply that Ca2+-free micro-calpain may represent a partially activated structure, requiring lower Ca2+ concentration to trigger its activation.     

Reaction cycle of solubilized monomeric Ca2+-ATPase of sarcoplasmic reticulum is the same as that of the membrane form

Monomeric Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum dispersed in Triton X-100 is stoichiometrically phosphorylated from Pi in a Ca2+-depleted medium containing dimethyl sulfoxide and catalyzes efficient (80%) phosphoryl transfer to ADP following a jump in water activity in the presence of Ca2+. The Ca2+ concentration dependence of ATP synthesis was sigmoidal (nH = 1.7) and in the millimolar range (K0.5 = 0.3 mM), indicating the involvement of at least two low affinity Ca2+ binding sites. These results, taken together with the properties of the monomer in the forward direction of catalysis, show that the catalytic cycle of the detergent-solubilized monomer is essentially the same as that of the membrane enzyme. The substrate and ion specificity of the catalytic intermediates suggest that the monomer is capable of coupled vectorial transport of Ca2+.