Calcium-dependent phosphorylation regulates the plasma-membrane H+-ATPase activity of maize (Zea mays L.) roots

Phosphorylation/dephosphorylation of the plasma-membrane H⁺-ATPase (EC 3.6.1.35) could act as a regulatory mechanism to control its activity. In this work, a plasmalemma-enriched fraction from maize roots and a partially purified H⁺-ATPase were used to investigate the effects of Ca²⁺ and calmodulin on the H⁺-ATPase activity and on its phosphorylation status. Both the hydrolytic and the proton-pumping activities were reduced approximately 50% by micromolar Ca²⁺ concentrations while calmodulin did not show any effect either alone or in the presence of Ca²⁺. The lack of effect of calmodulin antagonists indicated that calmodulin was not involved in this response. The addition of staurosporine, a kinase inhibitor, abolished the inhibitory effect of Ca²⁺. Phosphorylation of plasma membrane and partially purified H⁺-ATPase showed the same behavior. In the presence of Ca²⁺ a polypeptide of 100 kDa was phosphorylated. This polypeptide cross-reacted with antibodies raised against the H⁺-ATPase of maize roots. The autoradiogram of the immunodetected protein clearly showed that this polypeptide, which corresponds to the H⁺-ATPase, was phosphorylated. Additional clear evidence comes from the immunoprecipitation experiments: the data obtained show that the H⁺-ATPase activity is indeed influenced by its state of phosphorylation. Received: 19 October 1998 / Accepted: 23 February 1999     

Purification and characterization of a calcium-dependent protein kinase from beetroot plasma membranes

Several calcium-dependent protein kinases (CDPKs) are located in plant plasma membranes where they phosphorylate enzymes and transporters, like the H⁺-ATPase and water channels, thereby regulating their activities. In order to determine which kinases phosphorylate the H⁺-ATPase, a calcium-dependent kinase was purified from beetroot (Beta vulgaris L.) plasma membranes by anion-exchange chromatography, centrifugation in glycerol gradients and hydrophobic interaction chromatography. The kinetic parameters of this kinase were determined (V _max: 3.5 μmol mg⁻¹ min⁻¹, K _m for ATP: 67 μM, K _m for syntide 2: 15 μM). The kinase showed an optimum pH of 6.8 and a marked dependence on low-micromolar Ca²⁺ concentrations (K _d : 0.77 μM). During the purification procedure, a 63-kDa protein with an isoelectric point of 4.7 was enriched. However, this protein was shown not to be a kinase by mass spectrometry. Kinase activity gels showed that a 50-kDa protein could be responsible for most of the activity in purified kinase preparations. This protein was confirmed to be a CDPK by mass spectrometry, possibly the red beet ortholog of rice CDPK2 and Arabidopsis thaliana CPK9, both found associated with membranes. This kinase was able to phosphorylate purified H⁺-ATPase in a Ca²⁺-dependent manner.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Phosphorylation of the plasma-membrane H+-ATPase of oat roots by a calcium-stimulated protein kinase

When plasma-membrane vesicles isolated from oat (Avena sativa L.) root cells were incubated with [-³²P]ATP, the H⁺-ATPase was found to be phosphorylated at serine and threonine residues. Phosphotyrosine was not detected. Endogenous ATPase kinase activity was also observed in plasma-membrane vesicles isolated from potato (Solanum tuberosum L.) root cells as well as from yeast (Saccharomyces cerevisiae). Identity of the phosphorylated oat root M_r=100 000 polypeptide as the ATPase was confirmed using conventional glycerol density-gradient centrifugation to purify the native enzyme and by a new procedure for purifying the denatured polypeptide using reversephase high-performance liquid chromatography. Kinase-mediated phosphorylation of the oat root plasma-membrane H⁺-ATPase was stimulated by the addition of low concentrations of Ca²⁺ and by a decrease in pH, from 7.2 to 6.2. These results demonstrate that kinase-mediated phosphorylation of the H⁺-ATPase is a plausible mechanism for regulating activity. They further indicate that changes in the cytoplasmic [Ca²⁺] and pH are potentially important elements in modulating the kinase-mediated phosphorylation.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - M_r relative molecular mass - RP-HPLC reverse-phase high-performance liquid chromatography - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Regulation of reconstituted renal Na+/H+ exchanger by calcium-dependent protein kinases

Summary Studies were performed to determine the effect of protein phosphorylation mediated by calcium-calmodulin-dependent multifunctional protein kinase II and calcium-phospholipid-dependent protein kinase on Na⁺/H⁺ exchange activity. Proteins from the apical membrane of the proximal tubule of the rabbit kidney were solubilized in octyl glucoside and incubated in phosphorylating solutions containing the protein kinase.²²Na⁺ uptake was determined subsequently after reconstitution of the proteins into proteoliposomes. Calcium-calmodulin-dependent multifunction protein kinase II inhibited the amiloride-sensitive component of proton gradient-stimulated Na⁺ uptake in a dose-dependent manner. The inhibitory effect of this kinase had an absolute requirement for calmodulin, Ca²⁺, and ATP. Calcium-phospholipid-dependent protein kinase stimulated the amiloride-sensitive component of proton gradient-stimulated Na⁺ uptake in a dose-dependent manner. The stimulating effect of this kinase had an absolute requirement for ATP, Ca²⁺, and an active phorbol ester. These experiments indicate that Na⁺/H⁺ exchange activity of proteoliposomes reconstituted with proteins from renal brush-border membranes are inhibited by protein phosphorylation mediated by calcium-calmodulin-dependent multifunctional protein kinase II and stimulated by that mediated by calcium-calmodulin-dependent protein kinase.     

Cold stress-induced calcium-dependent protein kinase(s) in rice (Oryza sativa L.) seedling stem tissues

Ca²⁺-dependent protein kinases (CDPKs) play an important role in plant signal transduction. Protein kinase(s) activities induced by 5°C cold stress in rice (Oryza sativa L.) seedlings were investigated in both leaf and stem tissues in an early (up to 45 min) and late (up to 12 h) response study. The leaf had 37-, 47- and 55-kDa protein kinase activities, and the stem had 37-, 47- and 55-kDa protein kinase activities. A 16-kDa protein showed constitutive kinase activity in the rice seedling leaf and stem. It was further identified that the 47-kDa protein kinase activity induced by cold in both the cytosolic and membrane fractions of the stem was strictly Ca²⁺-dependent. This CDPK activitiy increased in the presence of the Ca²⁺ ionophore A23187 in stem segments, whereas it was decreased by the Ca²⁺ channel blocker, LaCl₃, and the Ca²⁺ chelator, EGTA. The general protein kinase inhibitor, staurosporine, completely inhibited this CDPK activity in vitro, and both W7, a calmodulin antagonist, and H7, a protein kinase C inhibitor, could only partially decrease this activity. The protein phosphatase inhibitor, okadaic acid, increased CDPK activity. This CDPK activity was also induced by salt, drought stress and the phytohormone abscicic acid. Among the 18 rice varieties tested, this cold-induced 47-kDa CDPK activity was stronger in the cold-tolerant varieties than in the sensitive ones. Received: 13 August 1999 / Accepted: 24 January 2000     

Regulation of the skeletal sarcoplasmic reticulum Ca2+-ATPase by phospholamban and negatively charged phospholipids in reconstituted phospholipid vesicles

The Ca²⁺-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in proteoliposomes containing phosphatidylcholine (PC). When reconstitution occurred in the presence of PC and the acidic phospholipids, phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP), the Ca²⁺-uptake and Ca²⁺-ATPase activities were significantly increased (2–3 fold). The highest activation was obtained at a 50:50 molar ratio of PSYC and at a 10:90 molar ratio of PIP:PC. The skeletal SR Ca²⁺-ATPase, reconstituted into either PC or PC:PS proteoliposomes, was also found to be regulated by exogenous phospholamban (PLB), which is a regulatory protein specific for cardiac, slow-twitch skeletal, and smooth muscles. Inclusion of PLB into the proteoliposomes was associated with significant inhibition of the initial rates of Ca²⁺-uptake, while phosphorylation of PLB by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects. The effects of PLB on the reconstituted Ca²⁺-ATPase were similar in either PC or PC: PS proteoliposomes, indicating that inclusion of negatively charged phospholipid may not affect the interaction of PLB with the skeletal SR Ca²⁺-ATPase. Regulation of the Ca²⁺-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by crosslinking experiments, using a synthetic peptide which corresponded to amino acids 1–25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca²⁺-ATPase may be also regulated by phospholamban although this regulator is not expressed in fast-twitch skeletal muscles.     

The phosphorylation site of Ca2+-dependent protein kinase from alfalfa

A 50 kDa, calcium-dependent protein kinase (CDPK) was purified about 1000-fold from cultured cells of alfalfa (Medicago varia) on the basis of its histone H1 phosphorylation activity. The major polypeptide from bovine histone H1 phosphorylated by either animal protein kinase C (PK-C) or by the alfalfa CDPK gave an identical phosphopeptide pattern. The phosphoamino acid determination showed phosphorylation of serine residues in histone H1 by the plant enzyme. Histone-related oligopeptides known to be substrates for animal histone kinases also served as substrates for the alfalfa kinase. Both of the studied peptides (GKKRKRSRKA; AAASFKAKK) inhibited phosphorylation of H1 histones by bovine and alfalfa kinases. The results of competition studies with the nonapeptide (AAASFKAKK), which is a PK-C specific substrate, suggest common features in target recognition between the plant Ca²⁺-dependent kinase and animal protein kinase C. We also propose that synthetic peptides like AAASFKAKK can be used as a tool to study substrates of plant kinases in crude cell extracts.     

Protein kinase C–catalyzed calponin phosphorylation in swine carotid arterial homogenate

Calponin, a thin filament–associated protein, inhibits actin-activated myosin ATPase activity, and this inhibition is reversed by phosphorylation. Calponin phosphorylation by protein kinase C and Ca²⁺/calmodulin-dependent protein kinase II has been shown in purified protein systems but has been difficult to demonstrate in more physiological preparations. We have previously shown that calponin is phosphorylated in a cell-free homogenate of swine carotid artery. The goal of this study was to determine whether protein kinase C and/or Ca²⁺/calmodulin-dependent protein kinase II catalyzes calponin phosphorylation. Ca²⁺-dependent calponin phosphorylation was not inhibited by calmodulin antagonists. In contrast, both Ca²⁺- and phorbol dibutyrate/1-oleoyl-2-acetyl-sn-glycerol–dependent calponin phosphorylation were inhibited by the pseudosubstrate inhibitor of protein kinase C and staurosporine. Our results also demonstrate that stimulation with either Ca²⁺, phorbol dibutyrate, or 1-oleoyl-2-acetyl-sn-glycerol activates endogenous protein kinase C. We interpret our results as clearly demonstrating that the physiological kinase for calponin phosphorylation is protein kinase C and not Ca²⁺/calmodulin-dependent protein kinase II. We also present data showing that the direct measurement of ³²P incorporation into calponin and the indirect measurement of calponin phosphorylation using nonequilibrium pH gradient gel electrophoresis provide similar quantitative values of calponin phosphorylation. J. Cell. Physiol. 176:545–552, 1998. © 1998 Wiley-Liss, Inc.     

A gibberellin-regulated protein phosphorylated by a putative Ca2+-dependent protein kinase is G-protein mediated in rice root

The GA-signal transduction pathways downstream to the Gα protein in rice seedling root were investigated using in-gel kinase assay and in vitro protein phosphorylation techniques with a Gα protein defective mutant, d1. A 50-kDa protein kinase was detected downstream to Gα protein in the membrane fraction of rice seedling roots using an in-gel kinase assay with histone III-S as a substrate. The activity of a 50-kDa protein kinase increased in the wild-type rice by gibberellin (GA₃) treatment, but did not change in the d1 mutant. This protein kinase activity was inhibited by the Ca²⁺ chelator ethyleneglycol-bis-(beta-aminoethylether)-N,N,N ¹,N ¹-tetraacetic acid (EGTA), protein kinase inhibitors, staurosporine and H7, and calmodulin antagonist, trifluoperazine, suggesting that the 50-kDa protein kinase is a putative plant Ca²⁺-dependent protein kinase (CDPK). The activity of the 50-kDa putative CDPK reached its highest level at 3 h after GA₃ treatment and then gradually declined with time. In order to identify the endogenous substrate for 50-kDa putative CDPK, two-dimensional polyacrylamide gel electrophoresis followed by in vitro protein phosphorylation was carried out. The phosphorylation activity of an endogenous protein PP30, identified as an unknown protein having molecular weight 30 kDa and isoelectric point 5.8 was increased in the wild-type rice by GA₃ treatment, compared with the d1 mutant. The addition of GA₃ treated membrane fraction, which predominantly represent a 50-kDa putative CDPK further increased the phosphorylation of PP30. Almost similar to GA₃ treatment, phosphorylation activity of PP30 was also increased by the treatment with cholera toxin in the wild-type rice but not in d1 mutant. These results suggest that the 50-kDa putative CDPK and an unknown protein, PP30 promoted by GA₃ treatment are G-protein mediated in rice seedling roots.     

Phosphorylation of a putative myosin light chain inChara by calcium-dependent protein kinase

Summary Ca²⁺-dependent protein kinase (CDPK) has been proposed to mediate inhibition by Ca²⁺ of cytoplasmic streaming in the green algaChara. We have identified the in vivo substrate(s) of CDPK inChara by using vacuolar perfusion of individual internodal cells with [-³²P]ATP. Phosphorylation of several polypeptides is enhanced when perfusions are performed at 10^–4M free Ca²⁺ compared to <10^–9M free Ca²⁺. The Ca²⁺-stimulated phosphorylation of these proteins is inhibited by the presence of a monoclonal antibody to soybean CDPK. One of these proteins is 16 to 18kDa and is recognized by an antibody against gizzard myosin light chains. These results demonstrate that inChara, several polypeptides are phophorylated by CDPK and one of these proteins has been tentatively identified as a myosin light chain. These observations support the hypothesis that Ca²⁺-regulated phosphorylation of myosin is involved in the regulation of cytoplasmic streaming.Abbreviations CDPK calcium-dependent protein kinase - mAb monoclonal antibody     

Protein kinase activities in tonoplast and plasmalemma membranes from corn roots

Protein kinase and phosphatase activities were studied in plasmalemma and tonoplast membrane fractions from corn (Zea mays L.) roots in order to test the hypothesis that the tonoplast H⁺-ATPase is regulated by intrinsic protein phosphorylation (G Zocchi, SA Rogers, JB Hanson 1983 Plant Sci Lett 31: 215-221), and to facilitate future purification of kinase activities from these membranes. Kinase activity in the plasmalemma was about three-fold higher than in the tonoplast, and displayed Michaelis Menten-type behavior with a K_m value for MgATP²⁻ of about 50 micromolar. Both activities were optimal at 3 millimolar free Mg²⁺ and had pH optima at 6.6 and 7.0 for the plasmalemma and tonoplast, respectively. Kinase activities in both fractions were stimulated by 1 micromolar free Ca²⁺, but calmodulin had no stimulatory effect, and chlorpromazine was inhibitory only at high concentrations. The pattern of phosphopeptides on SDS polyacrylamide gel electrophoresis was similar in both fractions except for one band of 50 kilodaltons that was present only in the tonoplast. A partially purified H⁺-ATPase fraction was prepared from tonoplast membranes, incubated under conditions optimal for protein phosphorylation. The three polypeptides (of 67, 57, and 36 kilodaltons), enriched in this fraction, did not become phosphorylated, suggesting that this protein is not regulated by endogenous protein phosphorylation. Protein phosphatase activity was detected only in the plasmalemma fraction. These results indicate that a regulatory cycle of protein phosphorylation and dephosphorylation may operate in the plasmalemma. The activity in the tonoplast appears to originate from plasmalemma contamination.     

Ca2+/H+ exchange in the plasma membrane of Arabidopsis thaliana leaves

A large number of plant Ca²⁺/H⁺ exchangers have been identified in endomembranes, but far fewer have been studied for Ca²⁺/H⁺ exchange in plasma membrane so far. To investigate the Ca²⁺/H⁺ exchange in plasma membrane here, inside-out plasma membrane vesicles were isolated from Arabidopsis thaliana leaves using aqueous two-phase partitioning method. Ca²⁺/H⁺ exchange in plasma membrane vesicles was measured by Ca²⁺-dependent dissipation of a pre-established pH gradient. The results showed that transport mediated by the Ca²⁺/H⁺ exchange was optimal at pH 7.0, and displayed transport specificity for Ca²⁺ with saturation kinetics at K _m = 47 μM. Sulfate and vanadate inhibited pH gradient across vesicles and decreased the Ca²⁺-dependent transport of H⁺ out of vesicles significantly. When the electrical potential across plasma membrane was dissipated with valinomycin and potassium, the rate of Ca²⁺/H⁺ exchange increased comparing to control without valinomycin effect, suggesting that the Ca²⁺/H⁺ exchange generated a membrane potential (interior negative), i.e. that the stoichiometric ratio for the exchange is greater than 2H⁺:Ca²⁺. Eosin Y, a Ca²⁺-ATPase inhibitor, drastically inhibited Ca²⁺/H⁺ exchange in plasma membrane as it does for the purified Ca²⁺-ATPase in proteoliposomes, indicating that measured Ca²⁺/H⁺ exchange activity is mainly due to a plasma membrane Ca²⁺ pump. These suggest that calcium (Ca²⁺) is transported out of Arabidopsis cells mainly through a Ca²⁺-ATPase-mediated Ca²⁺/H⁺ exchange system that is driven by the proton-motive force from the plasma membrane H⁺-ATPase.     

Reconstitution of cAMP-Dependent protein kinase regulated renal Na+−H+ exchanger

Summary Studies were performed to determine if the Na⁺–H⁺ exchanger, solubilized from renal brush border membranes from the rabbit and assayed in reconstituted artificial proteoliposomes, could be regulated by cAMP-dependent protein kinase. Octyl glucoside solubilized renal apical membrane proteins from the rabbit kidney were phosphorylated by incubation with ATP and highly purified catalytic subunit of cAMP-dependent kinase.²²Na⁺ uptake was determined subsequently after reconstitution of the proteins into proteoliposomes. cAMP-dependent protein kinase resulted in sustained protein phosphorylation and a concentration-dependent decrease in the amiloride-sensitive component of pH gradient-stimulated sodium uptake. The inhibitory effect of cAMP-dependent protein kinase demonstrated an absolute requirement for ATP and was blocked by the specific protein inhibitor of this kinase. cAMP-dependent protein kinase also inhibited²²Na⁺ uptake in the absence of a pH gradient (pH_in 6.0. pH_out 6.0) and the inhibitory effect was blocked by the specific inhibitor of the kinase. Solubilized membrane proteins exhibited little endogenous protein kinase or protein phosphatase activity.These studies indicate that Na⁺–H⁺ exchange activity of proteoliposomes reconstituted with proteins from renal brush border membranes is inhibited by phosphorylation of selected proteins by cAMP-dependent protein kinase. These findings also indicate that the regulatory components of the Na⁺–H⁺ exchanger remain active during the process of solubilization and reconstitution of renal apical membrane proteins.     

The use of reconstitution and inhibitor/ion interaction assays to distinguish between Ca2+/H+and Cd2+/H+ antiporter activities of oat and tobacco tonoplast vesicles

Tonoplast, ion antiport activities are critical to ion homeostasis and sequestration in plants. The biochemical properties of these activities, and the enzymes that catalyse them, are little characterized. Here we applied biochemical approaches to study some characteristics and to distinguish between Ca²⁺/H⁺ and Cd²⁺/H⁺ antiporter activities of tonoplast vesicles from non‐transformed, wild‐type plants. Solubilization and reconstitution of oat‐seedling (Avena sativa L.) root tonoplast vesicles resulted in about a 6‐fold loss of protein, about a 6‐fold enhancement of Cd²⁺/H⁺ antiport specific activity (at 10 µM Cd²⁺), and almost complete loss of Ca²⁺/H⁺ antiport activity. Similar results were found for vesicles from mature tobacco (Nicotiana tabacum) roots. Cd²⁺ concentration‐dependent proton efflux was similar and linear with both oat vesicles and proteoliposomes. In contrast, Ca²⁺ concentration‐dependent proton efflux of oat vesicles was easily observed while that with proteoliposomes was minimal and non‐linear. Cd²⁺ pre‐treatment of oat vesicles reduced verapamil inhibition of Cd²⁺/H⁺ activity and verapamil binding to vesicles, while Ca²⁺ pre‐treatment was much less protective of Ca^2+/H⁺ activity and verapamil binding. Results show the usefulness of reconstitution, and also inhibitor/ion interaction assays for distinguishing between transporter activities in vitro, but they do not resolve the question of whether there are separate enzymes for Cd²⁺/H⁺ and Ca²⁺/H⁺. Our observation that solubilization and reconstitution have similar effects on both Cd²⁺/H⁺ and Ca²⁺/H⁺ activities of root tonoplast vesicles from immature oat and mature tobacco roots suggests that the transporters involved are similar in young and mature roots, and in roots of different species.     

Ca2+-dependent protein kinase from alfalfa (Medicago varia): Partial purification and autophosphorylation

A calcium-dependent protein kinase (CDPK) was purified to 1400-fold from the soluble fraction of alfalfa (Medicago varia) cells by ammonium sulfate fractionation, Sephacryl-300, DEAE-Sephacel, Phenyl-Sepharose and Hydroxylapatite column chromatography. The enzyme is mainly monomeric. During the course of the purification steps a 50 kDa phosphoprotein doublet and a 56 kDa phosphoprotein copurified with the CDPK activity. Mobility shift of these proteins have been shown by SDS PAGE in Ca²⁺ free conditions. Tests on enzyme activity after separation by native gel electrophoresis revealed two protein kinase activities in our enzyme preparation and the phosphorylation of the 50 kDa and 56 kDa proteins. We suggest that these proteins are the autophosphorylated forms of calcium dependent protein kinases. Preincubation of the CDPK in ATP resulted in a marked increase in enzyme activity, but did not alter the Ca²⁺ sensitivity of the protein kinase.     

Characterisation of a Ca2+-dependent ATP hydrolysis associated with the vacuolar membrane of wheat roots

Microsomal fractions from wheat tissues exhibit a higher level of ATP hydrolytic activity in the presence of Ca²⁺ than Mg²⁺. Here we characterise the Ca²⁺-dependent activity from roots of Triticum aestivum lev. Troy) and investigate its possible function. Ca²⁺-dependent ATP hydrolysis in the microsomal fraction occurs over a wide pH range with two slight optima at pH 5.5 and 7.5. At these pHs the activity co-migrates with the major peak of nitrate-inhibited Mg²⁺. Cl-ATPase on continuous sucrose gradients indicating that it is associated with the vacuolar membrane. Ca²⁺-dependent ATP hydrolysis can be distinguished from an inhibitory effect of Ca²⁺ on the plasma membrane K⁺, Mg²⁺-ATPase following microsomal membrane separation using aqueous polymer two phase partitioning. The Ca²⁺-dependent activity is stimulated by free Ca²⁺ with a K_m of 8.1 μM in the absence of Mg²⁺ ([CaATP] = 0.8 mM). Vacuoiar membrane vacuolar preparations contain a higher Ca²⁺-dependent than Mg²⁺-dependent ATP hydrolysis, although the two activities are not directly additive. The nucleotide specificity of the divalent ion-dependent activities in vacuolar membrane-enriched fractions was low. hydrolysis of CTP and UTP being greater than ATP hydrolysis with both Ca²⁺ and Mg²⁺ The Ca²⁺-dependent activity did discriminate against dinucleotides, and mononucleotides. and failed to hydrolyse phosphatase substrates. Despite low nucleotide specificity the Mg²⁺-dependent activity functioned as a bafilomycin sensitive H⁺-pump in vacuolar membrane vesicles. Ca²⁺-dependent ATP hydrolysis was not inhibited by the V-, P-, or F-type ATPase inhibitors bafilomycin. vanadate and azide, respectively. nor by the phosphatase inhibitor molybdate, but was inhibited 20% at pH 7.5 by K⁺. Possible functions of Ca²⁺-dependent hydrolysis as a H⁺-pump or a Ca²⁺-pump was investigated using vacuolar membrane vesicles. No H⁺ or Ca²⁺ translocating activity was observed under conditions when the Ca²⁺-dependent ATP hydrolysis was active.     

Role of calcium and calmodulin in the regulation of the rabbit ileal brush-border membrane Na+/H+ antiporter

Summary In rabbit ileum, Ca²⁺/calmodulin (CaM) appears to be involved in physiologically inhibiting the linked NaCl absorptive process, since inhibitors of Ca²⁺/CaM stimulate linked Na⁺ and Cl^– absorption. The role of Ca²⁺/CaM-dependent phosphorylation in regulation of the brush-border Na⁺/H⁺ antiporter, which is believed to be part of the neutral linked NaCl absorptive process, was studied using purified brush-border membrane vesicles, which contain both the Na⁺/H⁺ antiporter and Ca²⁺/CaM-dependent protein kinase(s) and its phosphoprotein substrates. Rabbit ileal villus cell brush-border membrane vesicles were prepared by Mg precipitation and depleted of ATP. Using a freezethaw technique, the ATP-depleted vesicles were loaded with Ca²⁺, CaM, ATP and an ATP-regenerating system consisting of creatine kinase and creatine phosphate. The combination of Ca²⁺/CaM and ATP inhibited Na⁺/H⁺ exchange by 45±13%. This effect was specific since Ca²⁺/CaM and ATP did not alter diffusive Na⁺ uptake, Na⁺-dependent glucose entry, or Na⁺ or glucose equilibrium volumes. The inhibition of the Na⁺/H⁺ exchanger by Ca²⁺/CaM/ATP was due to an effect on theV _max and not on theK _m for Na⁺. In the presence of CaM and ATP, Ca²⁺ caused a concentration-dependent inhibition of Na⁺ uptake, with an effect 50% of maximum occurring at 120nm. This Ca²⁺ concentration dependence was similar to the Ca²⁺ concentration dependence of Ca²⁺/CaM-dependent phosphorylation of specific proteins in the vesicles. The Ca²⁺/CaM/ATP-inhibition of Na⁺/H⁺ exchange was reversed by W₁₃, a Ca²⁺/CaM antagonist, but not by a hydrophobic control, W₁₂, or by H-7, a protein kinase C antagonist. we conclude that Ca²⁺, acting through CaM, regulates ileal brush-border Na⁺/H⁺ exchange, and that this may be involved in the regulation of neutral linked NaCl absorption.     

The Ca2+ Pump of the Plasma Membrane of Arabidopsis thaliana: Characteristics and Sensitivity to Fluorescein Derivatives

The transport and hydrolytic activities of the plasma membrane (PM) Ca²⁺ pump were characterized in a PM fraction purified from seedlings of Arabidopsis thaliana by the aqueous two-phase partitioning technique. Ca²⁺ uptake could be energized by ATP and by ITP (at about 70% the rate sustained by ATP). This characteristic was used to measure the hydrolytic activity of the enzyme as Ca²⁺-dependent ITPase activity. The PM Ca²⁺ pump displayed a broad pH optimum around pH 7.2, was drastically inhibited by erythrosin B (EB), and was half-saturated by 60 μM ITP. It was stimulated by CaM, specially at low, non-saturating Ca²⁺ concentrations. All of these characteristics closely resemble those of the PM Ca²⁺ pump in other plant materials. Analysis of the effects of EB and other fluorescein derivatives (eosin Y and rose bengal) showed that: i) EB behaved as a competitive inhibitor with respect to ITP; ii) the PM Ca²⁺ pump was drastically inhibited by concentrations of fluorescein derivatives (submicromolar), much lower than those required to inhibit the PM H⁺-ATPase; iii) the different fluorescein derivatives were diversely efficient in inhibiting the activities of the Ca²⁺ pump and of the H⁺-ATPase of the PM (eosin Y was about 10000-fold, EB 1000-fold and rose bengal only 50-fold more active on the Ca²⁺ pump than on the H⁺-ATPase); and iv) the effectiveness of EB in inhibiting the Ca²⁺ pump was strongly affected by the protein concentration in the assay medium.     

Syringomycin-Stimulated Phosphorylation of the Plasma Membrane H-ATPase from Red Beet Storage Tissue

The syringomycin-stimulated in vitro protein phosphorylation of the plasma membrane H⁺-ATPase of red beet (Beta vulgaris L.) storage tissue was investigated. Peptides representing the H⁺-ATPase N and C termini and nucleotide binding site (P-2, P-3, and P-1, respectively) were synthesized, and rabbit antisera against each were produced. In western immunoblots of purified plasma membranes, these antisera immunoreacted with the 100-kilodalton polypeptide of the H⁺-ATPase and with other smaller polypeptides. The smaller polypeptides appeared to be degraded forms of the intact 100-kilodalton polypeptide. Immunoprecipitation experiments showed that plasma membranes treated with syringomycin had increased protein phosphorylation rates of the 100-kilodalton polypeptide. Optimal phosphorylation levels were achieved with 25 micromolar free Ca²⁺. Phosphoserine and phosphothreonine were detected in the immunoprecipitates. Washed immunoprecipitates generated with anti-P-1 possessed protein phosphorylation activity. This immunoprecipitate activity was not stimulated by syringomycin, but it was inhibited when plasma membranes were treated with sodium deoxycholate before immunoprecipitation. The findings show that syringomycin stimulates the phosphorylation of the plasma membrane H⁺-ATPase and that specific protein kinase(s) are probably associated with the enzyme.     

ATPase and phosphatase activities from human red cell membranes: I. The effects of N-ethylmaleimide

Summary (i) In human red cell membranes the sensitivity to N-ethylmaleimide of Ca²⁺-dependent ATPase and phosphatase activities is at least ten times larger than the sensitivity to N-ethylmaleimide of (Na⁺+K⁺)-ATPase and K⁺-activated phosphatase activities. All activities are partially protected against N-ethylmaleimide by ATP but not by inorganic phosphate or byp-nitrophenylphosphate. (ii) Protection by ATP of (Na⁺+K⁺)-ATPase is impeded by either Na⁺ or K⁺ whereas only K⁺ impedes protection by ATP of K⁺-activated phosphatase. On the other hand, Na⁺ or K⁺ slightly protects Ca²⁺-dependent activities against N-ethylmaleimide, this effect being independent of ATP. (iii) The sensitivity to N-ethylmaleimide of Ca²⁺-dependent ATPase and phosphatase activities is markedly enhanced by low concentrations of Ca²⁺. This effect is half-maximal at less than 1 m Ca²⁺ and does not require ATP, which suggests that sites with high affinity for Ca²⁺ exist in the Ca²⁺-ATPase in the absence of ATP. (iv) Under all conditions tested the response to N-ethylmaleimide of the ATPase and phosphatase activites stimulated by K⁺ or Na⁺ in the presence of Ca²⁺ parallels that of the Ca²⁺-dependent activities, suggesting that the Ca²⁺-ATPase system possesses sites at which monovalent cations bind to increase its activity.