Time-resolved FRET reveals the structural mechanism of SERCA–PLB regulation

We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to characterize the interaction between phospholamban (PLB) and the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA) under conditions that relieve SERCA inhibition. Unphosphorylated PLB inhibits SERCA in cardiac SR, but inhibition is relieved by either micromolar Ca²⁺ or PLB phosphorylation. In both cases, it has been proposed that inhibition is relieved by dissociation of the complex. To test this hypothesis, we attached fluorophores to the cytoplasmic domains of SERCA and PLB, and reconstituted them functionally in lipid bilayers. TR-FRET, which permitted simultaneous measurement of SERCA–PLB binding and structure, was measured as a function of PLB phosphorylation and [Ca²⁺]. In all cases, two structural states of the SERCA–PLB complex were resolved, probably corresponding to the previously described T and R structural states of the PLB cytoplasmic domain. Phosphorylation of PLB at S16 completely relieved inhibition, partially dissociated the SERCA–PLB complex, and shifted the T/R equilibrium within the bound complex toward the R state. Since the PLB concentration in cardiac SR is at least 10 times that in our FRET measurements, we calculate that most of SERCA contains bound phosphorylated PLB in cardiac SR, even after complete phosphorylation. 4 μM Ca²⁺ completely relieved inhibition but did not induce a detectable change in SERCA–PLB binding or cytoplasmic domain structure, suggesting a mechanism involving structural changes in SERCA’s transmembrane domain. We conclude that Ca²⁺ and PLB phosphorylation relieve SERCA–PLB inhibition by distinct mechanisms, but both are achieved primarily by structural changes within the SERCA–PLB complex, not by dissociation of that complex.     

The potassium channel opener NS1619 modulates calcium homeostasis in muscle cells by inhibiting SERCA

NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazole-2-one) is widely used as a large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel opener. It was previously reported that activation of BK_Ca channels by NS1619 could protect the cardiac muscle against ischaemia and reperfusion injury. This study reports the effects of NS1619 on intracellular Ca²⁺ homeostasis in H9C2 and C2C12 cells as well as its molecular mechanism of action. The effects of NS1619 on Ca²⁺ homeostasis in C2C12 and H9C2 cells were assessed using the Fura-2 fluorescence method. Ca²⁺ uptake by sarcoplasmic reticulum (SR) vesicles isolated from rat skeletal muscles and sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) activity were measured. The effect of NS1619 on the isometric force of papillary muscle contraction in the guinea pig heart was also examined. H9C2 and C2C12 cells treated with NS1619 released Ca²⁺ from internal stores in a concentration-dependent manner. Ca²⁺ accumulation by the SR vesicles was inhibited by NS1619 treatment. NS1619 also decreased the activity of SERCA derived from rat skeletal muscle. The calcium release from cell internal stores and inhibition of SERCA by NS1619 are pH dependent. Finally, NS1619 had a profound effect on the isometric force of papillary muscle contraction in the guinea pig heart. These results indicate that NS1619 is a potent modulator of the intracellular Ca²⁺ concentration in H9C2 and C1C12 cells due to its interaction with SRs. The primary target of NS1619 is SERCA, which is located in SR vesicles. The effect of NS1619-mediated SERCA inhibition on cytoprotective processes should be considered.     

Regulation of SERCA pumps expression in diabetes

Cytosolic calcium concentration ([Ca²⁺]_c) is fundamental for regulation of many cellular processes such metabolism, proliferation, muscle contraction, cell signaling and insulin secretion. In resting conditions, the sarco/endoplasmic reticulum (ER/SR) Ca²⁺ ATPase's (SERCA) transport Ca²⁺ from the cytosol to the ER or SR lumen, maintaining the resting [Ca²⁺]_c about 25–100 nM. A reduced activity and expression of SERCA2 protein have been described in heart failure and diabetic cardiomyopathy, resulting in an altered Ca²⁺ handling and cardiac contractility. In the diabetic pancreas, there has been reported reduction in SERCA2b and SERCA3 expression in β-cells, resulting in diminished insulin secretion. Evidence obtained from different diabetes models has suggested a role for advanced glycation end products formation, oxidative stress and increased O-GlcNAcylation in the lowered SERCA2 expression observed in diabetic cardiomyopathy. However, the role of SERCA2 down-regulation in the pathophysiology of diabetes mellitus and diabetic cardiomyopathy is not yet well described. In this review, we make a comprehensive analysis of the current knowledge of the role of the SERCA pumps in the pathophysiology of insulin-dependent diabetes mellitus type 1 (TIDM) and type 2 (T2DM) in the heart and β-cells in the pancreas.     

Genetic modulation of the SERCA activity does not affect the Ca leak from the cardiac sarcoplasmic reticulum

The Ca²⁺ content in the sarcoplasmic reticulum (SR) determines the amount of Ca²⁺ released, thereby regulating the magnitude of Ca²⁺ transient and contraction in cardiac muscle. The Ca²⁺ content in the SR is known to be regulated by two factors: the activity of the Ca²⁺ pump (SERCA) and Ca²⁺ leak through the ryanodine receptor (RyR). However, the direct relationship between the SERCA activity and Ca²⁺ leak has not been fully investigated in the heart. In the present study, we evaluated the role of the SERCA activity in Ca²⁺ leak from the SR using a novel saponin-skinned method combined with transgenic mouse models in which the SERCA activity was genetically modulated. In the SERCA overexpression mice, the Ca²⁺ uptake in the SR was significantly increased and the Ca²⁺ transient was markedly increased. However, Ca²⁺ leak from the SR did not change significantly. In mice with overexpression of a negative regulator of SERCA, sarcolipin, the Ca²⁺ uptake by the SR was significantly decreased and the Ca²⁺ transient was markedly decreased. Again, Ca²⁺ leak from the SR did not change significantly. In conclusion, the selective modulation of the SERCA activity modulates Ca²⁺ uptake, although it does not change Ca²⁺ leak from the SR.     

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.     

Store-operated Ca entry and depolarization explain the anomalous behaviour of myometrial SR: Effects of SERCA inhibition on electrical activity,Ca and force

In the myometrium SR Ca²⁺ depletion promotes an increase in force but unlike several other smooth muscles, there is no Ca²⁺ sparks-STOCs coupling mechanism to explain this. Given the importance of the control of contractility for successful parturition, we have examined, in pregnant rat myometrium, the effects of SR Ca²⁺-ATPase (SERCA) inhibition on the temporal relationship between action potentials, Ca²⁺ transients and force. Simultaneous recording of electrical activity, calcium and force showed that SERCA inhibition, by cyclopiazonic acid (CPA 20 μM), caused time-dependent changes in excitability, most noticeably depolarization and elevations of baseline [Ca²⁺]_i and force. At the onset of these changes there was a prolongation of the bursts of action potentials and a corresponding series of Ca²⁺ spikes, which increased the amplitude and duration of contractions. As the rise of baseline Ca²⁺ and depolarization continued a point was reached when electrical and Ca²⁺ spikes and phasic contractions ceased, and a maintained, tonic force and Ca²⁺ was produced. Lanthanum, a non-selective blocker of store-operated Ca²⁺ entry, but not the L-type Ca²⁺ channel blocker nifedipine (1–10 μM), could abolish the maintained force and calcium. Application of the agonist, carbachol, produced similar effects to CPA, i.e. depolarization, elevation of force and calcium. A brief, high concentration of carbachol, to cause SR Ca²⁺ depletion without eliciting receptor-operated channel opening, also produced these results. The data obtained suggest that in pregnant rats SR Ca²⁺ release is coupled to marked Ca²⁺ entry, via store operated Ca²⁺ channels, leading to depolarization and enhanced electrical and mechanical activity.     

Calcium binding studies of peptides of human phospholipid scramblases 1 to 4 suggest that scramblases are new class of calcium binding proteins in the cell

Background

Phospholipid scramblases are a group of four homologous proteins conserved from C. elegans to human. In human, two members of the scramblase family, hPLSCR1 and hPLSCR3 are known to bring about Ca²⁺ dependent translocation of phosphatidylserine and cardiolipin respectively during apoptotic processes. However, affinities of Ca²⁺/Mg²⁺ binding to human scramblases and conformational changes taking place in them remains unknown.

Methods

In the present study, we analyzed the Ca²⁺ and Mg²⁺ binding to the calcium binding motifs of hPLSCR1–4 and hPLSCR1 by spectroscopic methods and isothermal titration calorimetry.

Results

The results in this study show that (i) affinities of the peptides are in the order hPLSCR1  > hPLSCR3 > hPLSCR2 > hPLSCR4 for Ca²⁺ and in the order hPLSCR1 > hPLSCR2 > hPLSCR3 > hPLSCR4 for Mg²⁺, (ii) binding of ions brings about conformational change in the secondary structure of the peptides. The affinity of Ca²⁺ and Mg²⁺ binding to protein hPLSCR1 was similar to that of the peptide I. A sequence comparison shows the existence of scramblase-like motifs among other protein families.

Conclusions

Based on the above results, we hypothesize that the Ca²⁺ binding motif of hPLSCR1 is a novel type of Ca²⁺ binding motif.

General significance

Our findings will be relevant in understanding the calcium dependent scrambling activity of hPLSCRs and their biological function.     

Heparin-derived oligosaccharides interact with the phospholamban cytoplasmic domain and stimulate SERCA function

The association between the cardiac transmembrane proteins phospholamban and sarcoplasmic reticulum Ca²⁺ ATPase (SERCA2a) regulates the active transport of Ca²⁺ into the sarcoplasmic reticulum (SR) lumen and controls the contraction and relaxation of the heart. Heart failure (HF) and cardiac hypertrophy have been linked to defects in Ca²⁺ uptake by the cardiac SR and stimulation of calcium transport by modulation of the PLB-SERCA interaction is a potential therapy. This work is part of an effort to identify compounds that destabilise the PLB-SERCA interaction in well-defined membrane environments. It is shown that heparin-derived oligosaccharides (HDOs) interact with the cytoplasmic domain of PLB and consequently stimulate SERCA activity. These results indicate that the cytoplasmic domain of PLB is functionally important and could be a valid target for compounds with drug-like properties.     

The use of small subcutaneous transponders for quantifying thermal biology and torpor in small mammals

Remote measurements of body temperature (T_b) in animals require implantation of relatively large temperature-sensitive radio-transmitters or data loggers, whereas rectal temperature (T_rec) measurements require handling and therefore may bias the results. We investigated whether ∼0.1 g temperature-sensitive subcutaneously implanted transponders can be reliably used to quantify thermal biology and torpor use in small mammals. We examined (i) the precision of transponder readings as a function of temperature and (ii) whether subcutaneous transponders can be used to remotely record subcutaneous temperature (T_sub). Five adult male dunnarts (Sminthopsis macroura, body mass 24 g) were implanted with subcutaneous transponders to determine T_sub as a function of time and ambient temperature (T_a), and in comparison to thermocouple readings of T_rec. Transponder temperature was highly correlated with water bath temperature (r²=0.96–0.99) over a range of approximately 10.0–40.0 °C. Transponders provided reliable data (±0.6 °C) over the T_sub of 21.4–36.9 °C and could be read from a distance of up to 5 cm. Below 21.4 °C, accuracy was reduced to ±2.8 °C, but individual transponder accuracy varied. Consequently, small subcutaneous transponders are useful to remotely quantify thermal physiology and torpor patterns without having to disturb the animal and disrupt torpor. Even at T_sub<21.4 °C where the accuracy of the temperature readings was reduced, transponders do provide reliable data on whether and when torpor is used.     

Role of disulfide bonds in conformational stability and folding of 5′-deoxy-5′-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus

Sulfolobus solfataricus 5′-deoxy-5′-melthylthioadenosine phosphorylase II (SsMTAPII), is a hyperthermophilic hexameric protein with two intrasubunit disulfide bonds (C138–C205 and C200–C262) and a CXC motif (C259–C261). To get information on the role played by these covalent links in stability and folding, the conformational stability of SsMTAPII and C262S and C259S/C261S mutants was studied by thermal and guanidinium chloride (GdmCl)-induced unfolding and analyzed by fluorescence spectroscopy, circular dichroism, and SDS-PAGE. No thermal unfolding transition of SsMTAPII can be obtained under nonreducing conditions, while in the presence of the reducing agent Tris-(2-carboxyethyl) phosphine (TCEP), a Tm of 100 °C can be measured demonstrating the involvement of disulfide bridges in enzyme thermostability. Different from the wild-type, C262S and C259S/C261S show complete thermal denaturation curves with sigmoidal transitions centered at 102 °C and 99 °C respectively. Under reducing conditions these values decrease by 4 °C and 8 °C respectively, highlighting the important role exerted by the CXC disulfide on enzyme thermostability. The contribution of disulfide bonds to the conformational stability of SsMTAPII was further assessed by GdmCl-induced unfolding experiments carried out under reducing and nonreducing conditions. Thermal unfolding was found to be reversible if the protein was heated in the presence of TCEP up to 90 °C but irreversible above this temperature because of aggregation. In analogy, only chemical unfolding carried out in the presence of reducing agents resulted in a reversible process suggesting that disulfide bonds play a role in enzyme denaturation. Thermal and chemical unfolding of SsMTAPII occur with dissociation of the native hexameric state into denatured monomers, as indicated by SDS-PAGE.     

SERCA2a: a prime target for modulation of cardiac contractility during heart failure

Heart failure is one of the leading causes of sudden death in developed countries. While current therapies are mostly aimed at mitigating associated symptoms, novel therapies targeting the subcellular mechanisms underlying heart failure are emerging. Failing hearts are characterized by reduced contractile properties caused by impaired Ca²⁺ cycling between the sarcoplasm and sarcoplasmic reticulum (SR). Sarcoplasmic/endoplasmic reticulum Ca²⁺ATPase 2a (SERCA2a) mediates Ca²⁺ reuptake into the SR in cardiomyocytes. Of note, the expression level and/or activity of SERCA2a, translating to the quantity of SR Ca²⁺ uptake, are significantly reduced in failing hearts. Normalization of the SERCA2a expression level by gene delivery has been shown to restore hampered cardiac functions and ameliorate associated symptoms in pre-clinical as well as clinical studies. SERCA2a activity can be regulated at multiple levels of a signaling cascade comprised of phospholamban, protein phosphatase 1, inhibitor-1, and PKCα. SERCA2 activity is also regulated by post-translational modifications including SUMOylation and acetylation. In this review, we will highlight the molecular mechanisms underlying the regulation of SERCA2a activity and the potential therapeutic modalities for the treatment of heart failure. [BMB Reports 2013; 46(5): 237-243]     

Purification and characterisation of a bifunctional alginate lyase from novel Isoptericola halotolerans CGMCC 5336

A novel halophilic alginate-degrading microorganism was isolated from rotten seaweed and identified as Isoptericola halotolerans CGMCC5336. The lyase from the strain was purified to homogeneity by combining of ammonium sulfate fractionation and anion-exchange chromatography with a specific activity of 8409.19 U/ml and a recovery of 25.07%. This enzyme was a monomer with a molecular mass of approximately 28 kDa. The optimal temperature and pH were 50 °C and pH 7.0, respectively. The lyase maintained stability at neutral pH (7.0–8.0) and temperatures below 50 °C. Metal ions including Na⁺, Mg²⁺, Mn²⁺, and Ca²⁺ notably increased the activity of the enzyme. With sodium alginate as the substrate, the K_m and V_max were 0.26 mg/ml and 1.31 mg/ml min, respectively. The alginate lyase had substrate specificity for polyguluronate and polymannuronate units in alginate molecules, indicating its bifunctionality. These excellent characteristics demonstrated the potential applications in alginate oligosaccharides production with low polymerisation degrees.     

Baicalin prevents Candida albicans infections via increasing its apoptosis rate

Background

These experiments were employed to explore the mechanisms underlying baicalin action on Candida albicans.

Methodology and principal findings

We detected the baicalin inhibition effects on three isotope-labeled precursors of ³H-UdR, ³H-TdR and ³H-leucine incorporation into C. albicans using the isotope incorporation technology. The activities of Succinate Dehydrogenase (SDH), cytochrome oxidase (CCO) and Ca²⁺–Mg²⁺ ATPase, cytosolic Ca²⁺ concentration, the cell cycle and apoptosis, as well as the ultrastructure of C.albicans were also tested. We found that baicalin inhibited ³H-UdR, ³H-TdR and ³H-leucine incorporation into C.albicans (P < 0.005). The activities of the SDH and Ca²⁺–Mg²⁺ ATPase of C.albicans in baicalin groups were lower than those in control group (P < 0.05). Ca²⁺ concentrations of C. albicans in baicalin groups were much higher than those in control group (P < 0.05). The ratio of C.albicans at the G0/G1 stage increased in baicalin groups in dose dependent manner (P < 0.01). There were a significant differences in the apoptosis rate of C.albicans between baicalin and control groups (P < 0.01). After 12–48 h incubation with baicalin (1 mg/ml), C. albicans shown to be markedly damaged under transmission electron micrographs.

Innovation and significance

Baicalin can increase the apoptosis rate of C. albicans. These effects of Baicalin may involved in its inhibiting the activities of the SDH and Ca²⁺–Mg²⁺ ATPase, increasing cytosolic Ca²⁺ content and damaging the ultrastructure of C. albicans.     

New N-aryl-N-alkyl-thiophene-2-carboxamide compound enhances intracellular Ca2+ dynamics by increasing SERCA2a Ca2+ pumping

The type 2a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) plays a central role in the intracellular Ca²⁺ homeostasis of cardiac myocytes, pumping Ca²⁺ from the cytoplasm into the sarcoplasmic reticulum (SR) lumen to maintain relaxation (diastole) and prepare for contraction (systole). Diminished SERCA2a function has been reported in several pathological conditions, including heart failure. Therefore, development of new drugs that improve SERCA2a Ca²⁺ transport is of great clinical significance. In this study, we characterized the effect of a recently identified N-aryl-N-alkyl-thiophene-2-carboxamide (or compound 1) on SERCA2a Ca²⁺-ATPase and Ca²⁺ transport activities in cardiac SR vesicles, and on Ca²⁺ regulation in a HEK293 cell expression system and in mouse ventricular myocytes. We found that compound 1 enhances SERCA2a Ca²⁺-ATPase and Ca²⁺ transport in SR vesicles. Fluorescence lifetime measurements of fluorescence resonance energy transfer between SERCA2a and phospholamban indicated that compound 1 interacts with the SERCA-phospholamban complex. Measurement of endoplasmic reticulum Ca²⁺ dynamics in HEK293 cells expressing human SERCA2a showed that compound 1 increases endoplasmic reticulum Ca²⁺ load by enhancing SERCA2a-mediated Ca²⁺ transport. Analysis of cytosolic Ca²⁺ dynamics in mouse ventricular myocytes revealed that compound 1 increases the action potential-induced Ca²⁺ transients and SR Ca²⁺ load, with negligible effects on L-type Ca²⁺ channels and Na⁺/Ca²⁺ exchanger. However, during adrenergic receptor activation, compound 1 did not further increase Ca²⁺ transients and SR Ca²⁺ load, but it decreased the propensity toward Ca²⁺ waves. Suggestive of concurrent desirable effects of compound 1 on RyR2, [³H]-ryanodine binding to cardiac SR vesicles shows a small decrease in nM Ca²⁺ and a small increase in μM Ca²⁺. Accordingly, compound 1 slightly decreased Ca²⁺ sparks in permeabilized myocytes. Thus, this novel compound shows promising characteristics to improve intracellular Ca²⁺ dynamics in cardiomyocytes that exhibit reduced SERCA2a Ca²⁺ uptake, as found in failing hearts.     

Biosynthesis of homoeriodictyol from eriodictyol by flavone 3′-O-methyltransferase from recombinant Yarrowia lioplytica: Heterologous expression,biochemical characterization,and optimal transformation

In this work, we attempted to synthesize homoeriodictyol by transferring one methyl group of S-adenosyl-l-methionine (SAM) to eriodictyol using flavone 3′-O-methyltransferase ROMT-9, which was produced by recombinant Yarrowia lipolytica. Specifically, the ROMT-9 gene from rice was synthesized and cloned into the multi-copy integrative vector pINA1297, and was further expressed in Y. lipolytica with a growth phase-dependent constitutive promoter hp4d. The highest ROMT-9 activity reached 5.53 U/L after 4 days of culture in shake flask. The optimal pH and temperature of the purified ROMT-9 were 8.0 and 37 °C, respectively. The purified enzyme was stable up to 40 °C, and retained more than 80% of its maximal activity between pH 6.5 and 9.0. The recombinant ROMT-9 did not require Mg²⁺ for catalysis, while was completely inhibited in the presence of 5 mM Zn²⁺, Cu²⁺, Ba²⁺, Al³⁺, or Ni²⁺. The purified ROMT-9 was used to synthesize homoeriodictyol, and the maximal transformation ratio reached 52.4% at 16 h under the following conditions: eriodictyol 0.2 g/L, ROMT-9 0.16 g/L, SAM 0.2 g/L, CH₃OH 6% (v/v), temperature 37 °C, and pH 8.0. This work provides an alternative strategy for efficient synthesis of homoeriodictyol and compared to the traditional plant extraction or chemical synthesis, the biotransformation approach generates less environmental pollution and has a great potential for the sustainable production of homoeriodictyol.     

Extracellular expression and biochemical characterization of α-cyclodextrin glycosyltransferase from Paenibacillus macerans

The cgt gene encoding α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans strain JFB05-01 was expressed in Escherichia coli as a C-terminal His-tagged protein. After 90 h of induction, the activity of α-CGTase in the culture medium reached 22.5 U/mL, which was approximately 42-fold higher than that from the parent strain. The recombinant α-CGTase was purified to homogeneity through either nickel affinity chromatography or a combination of ion-exchange and hydrophobic interaction chromatography. Then, the purified enzyme was characterized in detail with respect to its cyclization activity. It is a monomer in solution. Its optimum reaction temperature is 45 °C, and half-lives are approximately 8 h at 40 °C, 1.25 h at 45 °C and 0.5 h at 50 °C. The recombinant α-CGTase has an optimum pH of 5.5 with broad pH stability between pH 6 and 9.5. It is activated by Ca²⁺, Ba²⁺, and Zn²⁺ in a concentration-dependent manner, while it is dramatically inhibited by Hg²⁺. The kinetics of the α-CGTase-catalyzed cyclization reaction could be fairly well described by the Hill equation.     

Effects of monovalent cations on Ca uptake by skeletal and cardiac muscle sarcoplasmic reticulum

Ca²⁺ transport by the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) is sensitive to monovalent cations. Possible K⁺ binding sites have been identified in both the cytoplasmic P-domain and the transmembrane transport-domain of the protein. We measured Ca²⁺ transport into SR vesicles and SERCA ATPase activity in the presence of different monovalent cations. We found that the effects of monovalent cations on Ca²⁺ transport correlated in most cases with their direct effects on SERCA. Choline⁺, however, inhibited uptake to a greater extent than could be accounted for by its direct effect on SERCA suggesting a possible effect of choline on compensatory charge movement during Ca²⁺ transport. Of the monovalent cations tested, only Cs⁺ significantly affected the Hill coefficient of Ca²⁺ transport (n_H). An increase in n_H from ∼2 in K⁺ to ∼3 in Cs⁺ was seen in all of the forms of SERCA examined. The effects of Cs⁺ on the maximum velocity of Ca²⁺ uptake were also different for different forms of SERCA but these differences could not be attributed to differences in the putative K⁺ binding sites of the different forms of the protein.     

Characterization of Calumenin-SERCA2 Interaction in Mouse Cardiac Sarcoplasmic Reticulum

Calumenin is a multiple EF-hand Ca²⁺-binding protein localized in the sarcoplasmic reticulum (SR) with C-terminal SR retention signal HDEF. Recently, we showed evidence that calumenin interacts with SERCA2 in rat cardiac SR (Sahoo, S. K., and Kim, D. H. (2008) Mol. Cells 26, 265–269). The present study was undertaken to further characterize the association of calumenin with SERCA2 in mouse heart by various gene manipulation approaches. Immunocytochemical analysis showed that calumenin and SERCA2 were partially co-localized in HL-1 cells. Knockdown (KD) of calumenin was conducted in HL-1 cells and 80% reduction of calumenin did not induce any expressional changes of other Ca²⁺-cycling proteins. But it enhanced Ca²⁺ transient amplitude and showed shortened time to reach peak and decreased time to reach 50% of baseline. Oxalate-supported Ca²⁺ uptake showed increased Ca²⁺ sensitivity of SERCA2 in calumenin KD HL-1 cells. Calumenin and SERCA2 interaction was significantly lower in the presence of thapsigargin, vanadate, or ATP, as compared with 1.3 μm Ca²⁺, suggesting that the interaction is favored in the E1 state of SERCA2. A glutathione S-transferase-pulldown assay of calumenin deletion fragments and SERCA2 luminal domains suggested that regions of 132–222 amino acids of calumenin and 853–892 amino acids of SERCA2-L4 are the major binding partners. On the basis of our in vitro binding data and available information on three-dimensional structure of Ca²⁺-ATPases, a molecular model was proposed for the interaction between calumenin and SERCA2. Taken together, the present results suggest that calumenin is a novel regulator of SERCA2, and its expressional changes are tightly coupled with Ca²⁺-cycling of cardiomyocytes.     

Inositol-1 (or 4)-monophosphatase from Glycine max embryo axes is a phosphatase with broad substrate specificity that includes phytate dephosphorylation

A phosphate-hydrolyzing activity from Glycine max embryo axes was purified by a series of chromatographic steps and electroelution from activity gels, and demonstrated to be an inositol-1 (or 4)-monophosphatase by partial internal amino acid sequence. This enzyme hydrolyzed ATP, sodium pyrophosphate (NaPPi), inositol hexakisphosphate, and inositol 1-monophosphate, but not p-nitrophenyl phosphate, ADP, AMP or glucose 6-P. Using NaPPi as substrate, the highly purified protein hydrolyzed up to 0.4 mmol phosphate min^− 1 mg^− 1 protein and had a Km_avg of 235 μM for NaPPi. Since NaPPi is relatively inexpensive and readily available, we used this as substrate for the subsequent characterization. We observed the following: (a) specific inhibition by Li and NaF but not by butanedione monoxime, or orthovanadate; (b) activation by Cu²⁺ and Mg²⁺; (c) optimum activity at pH 7.4; and (d) temperature stability after 1-h incubations at 37–80 °C, with maximum activity at 37 °C. The partially purified protein was detected by in-gel activity assays and the band was electroeluted to yield a highly purified protein. Analysis by SDS-PAGE and native IEF-PAGE yielded a single major polypeptide of 29 kDa and pI ∼ 5.9, respectively. In addition, in-gel activity from embryo axes and whole hypocotyls at early germination times revealed one high and one intermediate molecular weight isoform, but only the intermediate one corresponded to IMPase. Throughout the post-imbibition period, the activity of the high molecular weight isoform disappeared and IMPase increased, indicating an increasing expression of the enzyme as germination and growth proceeded. These data indicate that the inositol-1 (or 4)-monophosphatase present in the embryo axis of G. max has a wide phosphate substrate specificity, and may play an important role in phosphate metabolism during the germination process.