Stimulatory effect of regucalcin on ATP-dependent Ca(2+) uptake activity in rat liver mitochondria

The effect of Ca(2+)-binding protein regucalcin on Ca(2+)-ATPase activity in isolated rat liver mitochondria was investigated. The presence of regucalcin (0.1, 0.25, and 0.5 microM) in the enzyme reaction mixture led to a significant increase in Ca(2+)-ATPase activity. Regucalcin significantly stimulated ATP-dependent (45)Ca(2+) uptake by the mitochondria. Ruthenium red (10(-5) M) or lanthanum chloride (10(-4) M), an inhibitor of mitochondrial Ca(2+) uptake, completely inhibited regucalcin (0.25 microM)-increased mitochondrial Ca(2+)-ATPase activity and (45)Ca(2+) uptake. The effect of regucalcin (0.25 microM) in increasing Ca(2+)-ATPase activity was completely inhibited by the presence of digitonin (10(-2)%), a solubilizing reagent of membranous lipids, or vanadate (10(-5) M), an inhibitor of phosphorylation of ATPase. The activatory effect of regucalcin (0.25 microM) on Ca(2+)-ATPase activity was not further enhanced in the presence of dithiothreitol (2.5 mM), a protecting reagent of the sulfhydryl (SH) group of the enzyme, or calmodulin (0.60 microM), a modulator protein of Ca(2+) action that could increase mitochondrial Ca(2+)-ATPase activity. The present study demonstrates that regucalcin can stimulate Ca(2+) pump activity in rat liver mitochondria, and that the protein may act on an active site (SH group)-related to phosphorylation of mitochondrial Ca(2+)-ATPase.     

Regucalcin increases Ca2+-ATPase activity in the mitochondria of brain tissues of normal and transgenic rats

The role of regucalcin, which is a regulatory protein in intracellular signaling, in the regulation of Ca(2+)-ATPase activity in the mitochondria of brain tissues was investigated. The addition of regucalcin (10(-10) to 10(-8) M), which is a physiologic concentration in rat brain tissues, into the enzyme reaction mixture containing 25 microM calcium chloride caused a significant increase in Ca(2+)-ATPase activity, while it did not significantly change in Mg(2+)-ATPase activity. The effect of regucalcin (10(-9) M) in increasing mitochondrial Ca(2+)-ATPase activity was completely inhibited in the presence of ruthenium red (10(-7) M) or lanthanum chloride (10(-7) M), both of which are inhibitors of mitochondrial uniporter activity. Whether the effect of regucalcin is modulated in the presence of calmodulin or dibutyryl cyclic AMP (DcAMP) was examined. The effect of regucalcin (10(-9) M) in increasing Ca(2+)-ATPase activity was not significantly enhanced in the presence of calmodulin (2.5 microg/ml) which significantly increased the enzyme activity. DcAMP (10(-6) to 10(-4) M) did not have a significant effect on Ca(2+)-ATPase activity. The effect of regucalcin (10(-9) M) in increasing Ca(2+)-ATPase activity was not seen in the presence of DcAMP (10(-4) M). Regucalcin levels were significantly increased in the brain tissues or the mitochondria obtained from regucalcin transgenic (RC TG) rats. The mitochondrial Ca(2+)-ATPase activity was significantly increased in RC TG rats as compared with that of wild-type rats. This study demonstrates that regucalcin has a role in the regulation of Ca(2+)-ATPase activity in the brain mitochondria of rats.     

Stimulatory effect of regucalcin on mitochondrial ATP‐dependent calcium uptake activity in rat kidney cortex

The effect of regucalcin, which is a regulatory protein of Ca²⁺ signaling, on Ca²⁺‐ATPase activity in isolated rat renal cortex mitochondria was investigated. The presence of regucalcin (50, 100, and 250 nM) in the enzyme reaction mixture led to a significant increase in Ca²⁺‐ATPase activity. Regucalcin significantly stimulated ATP‐dependent ⁴⁵Ca²⁺ uptake by the mitochondria. Ruthenium red (10⁻⁶ M) or lanthunum chloride (10⁻⁶ M), an inhibitor of mitochondrial Ca²⁺ uptake, markedly inhibited regucalcin (100 nM)‐increased mitochondrial Ca²⁺‐ATPase activity and ⁴⁵Ca²⁺ uptake. The effect of regucalcin (100 nM) in elevating Ca²⁺‐ATPase activity was completely prevented by the presence of digitonin (10⁻²%), a solubilizing reagent of membranous lipids, vanadate, an inhibitor of phosphorylation of ATPase, or dithiothreitol (50 mM), a protecting reagent of the sulfhydryl (SH) group of the enzyme. The activating effect of regucalcin (100 nM) on Ca²⁺‐ATPase activity was not further enhanced by calmodulin (0.30 μM) or dibutyryl cyclic AMP (10⁻⁴ M), which could increase Ca²⁺‐ATPase activity. Trifluoperazine (TFP; 50 μM), an antagonist of calmodulin, significantly decreased Ca²⁺‐ATPase activity. The activating effect of regucalcin on the enzyme was also seen in the presence of TFP, indicating that regucalcin's effect is not involved in mitochondrial calmodulin. The present study demonstrates that regucalcin can stimulate Ca²⁺‐pump activity in rat renal cortex mitochondria, and that the protein may act on an active site (SH group) related to phosphorylation of mitochondrial Ca²⁺‐ATPase. J. Cell. Biochem. 80:285–292, 2000. © 2000 Wiley‐Liss, Inc.     

Role of regucalcin as an activator of Ca(2+)-ATPase activity in rat liver microsomes.

The effect of Ca(2+)-binding protein regucalcin on Ca(2+)-ATPase activity in isolated rat liver microsomes was investigated. The presence of regucalcin (0.1-1.0 microM) in the enzyme reaction mixture led to a significant increase in Ca(2+)-ATPase activity. Regucalcin significantly stimulated ATP-dependent (45)Ca(2+) uptake by the microsomes. Thapsigargin (10(-6) M), a specific inhibitor of microsomal Ca(2+) pump enzyme (Ca(2+)-ATPase), clearly inhibited regucalcin (0.5 microM)-increased microsomal Ca(2+)-ATPase activity. Liver microsomal Ca(2+)-ATPase activity was markedly decreased by N-ethylmaleimide (NEM; 2.5 mM), while the activity was clearly elevated by dithiothreitol (DTT; 2.5 mM), indicating that the sulfhydryl (SH) group of the enzyme is an active site. The effect of regucalcin (0.5 microM) in increasing Ca(2+)-ATPase activity was completely inhibited by the presence of NEM (2.5 mM) or digitonin (10(-2) %), a solubilizing reagent of membranous lipids. Moreover, the effect of regucalcin on enzyme activity was seen in the presence of Ca(2+) ionophore (A23187; 10(-7) M). The present study demonstrates that regucalcin can stimulate Ca(2+) pump activity in rat liver microsomes, and that the protein may act the SH groups of microsomal Ca(2+)-ATPase.     

Role of endogenous regucalcin in the regulation of Ca(2+)-ATPase activity in rat liver nuclei

The role of endogenous regucalcin in the regulation of Ca(2+)-ATPase, a Ca(2+) sequestrating enzyme, in rat liver nuclei was investigated. Nuclear Ca(2+)-ATPase activity was significantly reduced by the addition of regucalcin (0.1-0.5 microM) into the enzyme reaction mixture. The presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) caused a significant elevation of Ca(2+)-ATPase activity; this effect was completely abolished by the addition of regucalcin (0.1 microM). The effect of anti-regucalcin antibody (50 ng/ml) in increasing Ca(2+)-ATPase activity was completely prevented by the presence of thapsigargin (10(-6) M), an inhibitor of Ca(2+) sequestrating enzyme, N-ethylmaleimide (1 mM), a modifying reagent of thiol groups, or vanadate (10(-5) M), an inhibitor of phosphorylation of the enzyme by ATP, which revealed an inhibitory effect on nuclear Ca(2+)-ATPase activity. Meanwhile, the effect of anti-regucalcin antibody (50 ng/ml) was significantly enhanced by the addition of calmodulin (5 microg/ml), which could increase nuclear Ca(2+)-ATPase activity. In addition, the effect of antibody (50 ng/ml) was significantly reduced by the presence of trifluoperazine (20 microM), an antagonist of calmodulin. These results suggest that the endogenous regucalcin in liver nuclei has a suppressive effect on nuclear Ca(2+)-ATPase activity, and that regucalcin can inhibit an activating effect of calmodulin on the enzyme.     

Activatory effect of calcium-binding protein regucalcin on ATP-dependent calcium transport in the basolateral membranes of rat kidney cortex

The effect of regucalcin, a calcium-binding protein, on ATP-dependent Ca2+ transport in the basolateral membranes isolated from rat kidney cortex was investigated. The prepared membranes were in inside-out oriented and membrane vesicles. Ca2+-ATPase activity in the basolateral membranes was progressively elevated by increasing concentrations of regucalcin (10-8 to 10-6 M) in the reaction mixture. This increase was dependent on Ca2+ addition. The activatory effect of regucalcin on the enzyme is inhibited by the presence of digitonin (5 × 10-6%) which can solubilize the membranous lipids. Moreover, the regucalcin effect was clearly abolished by the presence of vanadate (0.1 mM) or N-ethylmaleimide (5.0 mM). However, the effect of calmodulin (6 × 10-7 M) to increase Ca2+-ATPase activity was not significantly inhibited by vanadate or N-ethylmaleimide, indicating that the action mode of regucalcin differs from that of calmodulin. Also, the activatory effect of regucalcin on Ca2+-ATPase was appreciably inhibited by addition of dibutyryl cAMP (10-5 and 10-3 M), while inositol 1,4,5-trisphosphate (10-7 and 10-5 M) had no effect. Dibutyryl cAMP itself did not have an effect on the enzyme activity. Furthermore, the 45Ca2+ uptake by the basolateral membranes was clearly increased by the presence of regucalcin (10-7 and 10-6 M). This increase was completely blocked by the presence of vanadate (0.1 mM), N-ethylmaleimide (5.0 mM) or dibutyryl cAMP (10-4 and 10-3 M) in the reaction mixture. These results clearly demonstrate that regucalcin, which is expressed in rat kidney cortex, can increase Ca2+-ATPase activity and Ca2+ uptake in the basolateral membranes. Regucalcin may play a cell physiologic role as an activator in the ATP-dependent Ca2+ pumps in the basolateral membranes from rat kidney cortex.     

Suppressive effect of endogenous regucalcin on nitric oxide synthase activity in cloned rat hepatoma H4-II-E cells overexpressing regucalcin

The role of endogenous regucalcin, which is a regulatory protein in calcium signaling, in the regulation of nitric oxide (NO) synthase activity in the cloned rat hepatoma H4-II-E cells was investigated. Hepatoma cells were cultured for 24-72 h in the presence of fetal bovine serum (FBS; 10%). NO synthase activity in the 5,500 g supernatant of cell homogenate was significantly increased by the addition of calcium chloride (10 microM) and calmodulin (2.5 microg/ml) in the enzyme reaction mixture. The presence of trifluoperazine (TFP; 50 microM), an antagonist of calmodulin, inhibited the effect of calcium (10 microM) addition in increasing NO synthase activity, indicating the existence of Ca(2+)/calmodulin-dependent NO synthase in hepatoma cells. NO synthase activity was significantly decreased by the addition of regucalcin (10(-8) or 10(-7) M) in the reaction mixture without or with Ca(2+)/calmodulin addition. The effect of regucalcin (10(-7) M) in decreasing NO synthase activity was also seen in the presence of TFP (50 microM) or EGTA (1 mM). The presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture caused a significant elevation of NO synthase activity. NO synthase activity was significantly suppressed in the hepatoma cells (transfectants) overexpressing regucalcin. This decrease was completely abolished in the presence of anti-regucalcin monoclonal antibody (50 ng/ml) in the reaction mixture. Moreover, the effect of Ca(2+)/calmodulin addition in increasing NO synthase activity in the hepatoma cells (wild-type) was completely prevented in transfectants. The present study demonstrates that endogenous regucalcin has a suppressive effect on NO synthase activity in the cloned rat hepatoma H4-II-E cells.     

Stimulatory effect of regucalcin on ATP-dependent calcium transport in rat liver plasma membranes

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytoplasm, on ATP-dependent calcium transport in the plasma membrane vesicles of rat liver was investigated. (Ca2+-Mg2+)-ATPase activity in the liver plasma membranes was significantly increased by the presence of regucalcin (0.1-0.5 \sgmaelig;M) in the enzyme reaction mixture. This increase was completely inhibited by the presence of sulfhydryl group modifying reagent Nethylmaleimide (5.0 mM NEM) or digitonin (0.04%), which can solubilize the membranous lipids. When ATP-dependent calcium uptake by liver plasma membrane vesicles was measured by using 45CaCl2, the presence of regucalcin (0.1-0.5 \sgmaelig;M) in the reaction mixture caused a significant increase in the 45Ca2+ uptake. This increase was about 2-fold with 0.5 \sgmaelig;M regucalcin addition. An appreciable increase was seen by 5 min incubation with regucalcin addition. The regucalcin-enhanced ATP-dependent 45Ca2+ uptake by the plasma membrane vesicles was completely inhibited by the presence of NEM (5.0 mM) or digitonin (0.04%). These results demonstrate that regucalcin activates (Ca2+-Mg2+)-ATPase in the liver plasma membranes and that it can stimulate ATP-dependent calcium transport across the plasma membranes.     

Suppressive role of endogenous regucalcin in the enhancement of nitric oxide synthase activity in liver cytosol of normal and regucalcin transgenic rats

The suppressive role of endogenous regucalcin, which is a regulatory protein of calcium signaling, in the enhancement of nitric oxide (NO) synthase activity in the liver cytosol of rats was investigated. The enzyme activity was measured in a reaction mixture containing either vehicle or calcium chloride (1-20 microM) in the absence or presence of regucalcin (0.1, 0.25, or 0.5 microM). NO synthase activity was significantly increased by the addition of calcium (5-20 microM). This increase was completely abolished in the presence of trifluoperazine (TFP; 10-50 microM), an antagonist of Ca(2+)/calmodulin. The addition of regucalcin (0.1-0.5 microM) caused a significant fall in the calcium-increased enzyme activity. The effect of regucalcin (0.25 microM) in decreasing NO synthase activity was seen in the presence of ethylene glycol bis-(2-aminoethylether) N,N,N',N'-tetraacetic acid (EGTA, 1 mM) or TFP (20 microM), indicating that regucalcin acts independent on Ca(2+)/calmodulin. NO synthase activity was significantly raised in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture. The effect of the antibody (50 ng/ml) or calcium (10 microM) in elevating NO synthase activity in the liver cytosol of normal rats was not seen in the liver cytosol obtained from regucalcin transgenic rats. Moreover, the increase in NO synthase activity in the liver cytosol of normal rats induced by a single intraperitoneal administration of calcium (5.0 mg/100 g body weight) was significantly enhanced in the presence of anti-regucalcin monoclonal antibody (50 ng/ml) in the reaction mixture. The administration of calcium caused a significant increase in regucalcin level in the liver cytosol of normal rats. The present study demonstrated that endogenous regucalcin plays a suppressive role in the enhancement of NO synthase activity in the liver cytosol of rats.     

Allosteric modulation of Leishmania donovani plasma membrane Ca(2+)-ATPase by endogenous calmodulin.

The plasma membrane of the human pathogen Leishmania donovani possesses a high-affinity transmembrane Ca(2+)-ATPase that has its catalytic site oriented toward the cytoplasmic milieu (Ghosh, J., Ray, M., Sarkar, S., and Bhaduri, A. (1990) J. Biol. Chem. 265, 11345-11351). When the enzyme is studied in its more authentic, physiologically relevant, membrane-associated form, it exhibits pronounced sigmoidal kinetics with Ca2+ (K0.5 approximately 700 nM) in a trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid buffering system that effectively complexes all available Mg2+. Addition of exogenous Mg2+ (60 microM) completely abolishes sigmoidicity and establishes strictly hyperbolic kinetics, and the Km for Ca2+ reduces to 100 nM. Mg2+ can be replaced by heterologous calmodulin. The exclusive dependence of the enzyme on only Ca2+ for its activity and its positive allosteric modulation by Mg2+ distinguish this enzyme from other well-characterized plasma membrane Ca(2+)-ATPases. Employing this Ca(2+)-ATPase as the assay system, a soluble endogenous activating protein factor was purified that, by several criteria, corresponds to authentic calmodulin. The parasite calmodulin shifts the kinetics to hyperbolic kinetics, increases the Vmax 2-fold, and most important lowers the Km (approximately 100 nM) to a physiological level. The interaction with endogenous calmodulin thus converts the enzyme from a totally inactive to a fully active state.     

Regulation of the erythrocyte Ca(2+)-ATPase by mutant calmodulins with Glu----Ala substitutions in the Ca(2+)-binding domains.

We have used four mutant calmodulins to study the regulation of human erythrocyte Ca(2+)-ATPase by the calmodulin-dependent pathway; the conserved Glu at position 12 in each of the four Ca(2+)-binding domains of calmodulin (Glu31, Glu67, Glu104, or Glu140) was replaced by Ala. At pCa 7, where unmodified calmodulin maximally activates the erythrocyte Ca(2+)-ATPase, all four mutants stimulated Ca(2+)-ATPase activity to the same maximal velocity. However, the concentrations of mutant calmodulins required for half-maximal activation (KCaM) were significantly higher than that for unmodified calmodulin and were strongly dependent on the domain in which the mutated Glu was located; substitution in either the first or second Ca(2+)-binding domain had little effect (2-3-fold increase in KCaM), whereas substitution in either the third or fourth domain resulted in a dramatic, 25-71-fold increase in KCaM. The same order of sensitivity was observed when the Ca2+ dependence of enzyme activation was measured at a constant 100 nM concentration of mutant calmodulin. These data point to dramatic differences in the functional significance of the replacement of the Glu at position 12 in each of the four Ca(2+)-binding domains for activation of the Ca(2+)-ATPase. The 2 Glu residues located in the carboxyl-terminal half of calmodulin (particularly Glu140) are crucial for activation of the Ca(2+)-ATPase at physiologically significant Ca2+ concentrations.     

The regulation of Ca2+ transport by fast skeletal muscle sarcoplasmic reticulum. Role of calmodulin and of the 53,000-dalton glycoprotein

Ca2+ uptake and Ca2+-dependent ATP hydrolysis of fast skeletal muscle sarcoplasmic reticulum (SR) are strongly inhibited by trifluoperazine (TFP). Inhibition, which is Ca2+-dependent, is 90% with 14 microM TFP and 0.2 microM Ca2+. TFP interacts strongly, in a Ca2+-dependent way, with two SR proteins, calmodulin and the 53,000-dalton glycoprotein. The two proteins were purified by TFP affinity chromatography. The inhibition of SR activity by TFP was correlated with the interaction of the drug with the glycoprotein, rather than with calmodulin. The main effect was a shift of the (Ca2+-Mg2+)-ATPase from a high to a low affinity form. Calmodulin-dependent phosphorylation of three proteins (Mr = 57,000, 35,000, and 20,000) of the SR membrane of fast skeletal muscle was also demonstrated. Phosphorylation of these three proteins plays no role in the regulation of the active Ca2+-uptake reaction.     

Inhibition of Ca2+/calmodulin-dependent phosphatase activity by regucalcin in rat liver cytosol: Involvement of calmodulin binding

The regulatory effect of regucalcin on Ca²⁺/calmodulin-dependent phosphatase activity and the binding of regucalcin to calmodulin was investigated. Phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine in rat liver cytosol was significantly increased by the addition of Ca²⁺ (100 μM) and calmodulin (0.30 μM). Thess increases were clearly inhibited by the addition of regucalcin (0.50–1.0 μM) into the enzyme reaction mixture. The cytosolic phosphoamino acid phosphatase activity was significantly elevated by the presence of anti-regucalcin monoclonal antibody (0.2 μg/ml), suggesting that endogenous regucalcin in the cytosol has an inhibitory effect on the enzyme activity. This elevation was prevented by the addition of regucalcin (0.50 μM). Purified calcineurin phosphatase activity was significantly increased by the addition of calmodulin (0.12 μM) in the presence of Ca²⁺ (1 and 10 μM). This increase was completely inhibited by the presence of regucalcin (0.12 μM). The inhibitory effect of regucalcin was reversed by the addition of calmodulin with the higher concentration (0.36 μM). Regucalcin has been demonstrated to bind on calmodulin-agarose beads by analysis with sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The present study demonstrates that regucalcin inhibits Ca²⁺/calmodulin-dependent protein phosphatase activity in rat liver cytosol, and that regucalcin can bind to calmodulin. J. Cell. Biochem. 71:140–148, 1998. © 1998 Wiley-Liss, Inc.     

Inhibitory effect of regucalcin on protein phosphatase activity in the nuclei of rat kidney cortex

The role of regucalcin, which is a regulatory protein of calcium signaling, in the regulation of protein phosphatase activity in the nuclei of rat kidney cortex was investigated. Protein phosphatase activity towards phosphotyrosine, phosphoserine, and phosphothreonine was found in the nuclei. The enzyme activity towards three phosphoamino acids was significantly increased by the addition of calcium chloride (10-50 microM) in the enzyme reaction mixture. This increase was significantly inhibited by trifluoperazine (25 or 50 microM), an antagonist of calmodulin. The presence of regucalcin (50 or 100 nM) in the enzyme reaction mixture caused a significant decrease in protein phosphatase activity towards three phosphoamino acids. This effect was also seen in the presence of calcium (25 microM) and/or calmodulin (5 microg/ml). Protein phosphatase activity towards three phosphoamino acids was significantly increased in the presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) in the enzyme reaction mixture. This effect was completely blocked by the addition of regucalcin (100 nM). The effect of antibody (25 ng/ml) in increasing protein phosphatase activity towards phosphotyrosine was significantly inhibited by vanadate (10(-4) M). Also, the antibody's effect towards phosphoserine and phosphothreonine was significantly inhibited by cyclosporin A (10(-5) M). Endogenous regucalcin was found in the nuclei of rat kidney cortex using Western blot analysis. Nuclear regucalcin level was significantly reduced by the administration of saline (0.9% NaCl) for seven days in rats. Protein phosphatase activity towards three phosphoamino acids was significantly decreased by saline administration. The effect of anti-regucalcin monoclonal antibody (25 ng/ml) in increasing protein phosphatase activity towards three phosphoamino acids was weakened in the renal cortex nuclei of saline-administrated rats. The present study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in the nuclei of rat kidney cortex cells.     

Dynamics of matrix-free Ca2+ in cardiac mitochondria: two components of Ca2+ uptake and role of phosphate buffering

Mitochondrial Ca(2+) uptake is thought to provide an important signal to increase energy production to meet demand but, in excess, can also trigger cell death. The mechanisms defining the relationship between total Ca(2+) uptake, changes in mitochondrial matrix free Ca(2+), and the activation of the mitochondrial permeability transition pore (PTP) are not well understood. We quantitatively measure changes in [Ca(2+)](out) and [Ca(2+)](mito) during Ca(2+) uptake in isolated cardiac mitochondria and identify two components of Ca(2+) influx. [Ca(2+)](mito) recordings revealed that the first, MCU(mode1), required at least 1 μM Ru360 to be completely inhibited, and responded to small Ca(2+) additions in the range of 0.1 to 2 μM with rapid and large changes in [Ca(2+)](mito). The second component, MCU(mode2), was blocked by 100 nM Ru360 and was responsible for the bulk of total Ca(2+) uptake for large Ca(2+) additions in the range of 2 to 10 μM; however, it had little effect on steady-state [Ca(2+)](mito). MCU(mode1) mediates changes in [Ca(2+)](mito) of 10s of μM, even in the presence of 100 nM Ru360, indicating that there is a finite degree of Ca(2+) buffering in the matrix associated with this pathway. In contrast, the much higher Ca(2+) loads evoked by MCU(mode2) activate a secondary dynamic Ca(2+) buffering system consistent with calcium-phosphate complex formation. Increasing P(i) potentiated [Ca(2+)](mito) increases via MCU(mode1) but suppressed [Ca(2+)](mito) changes via MCU(mode2). The results suggest that the role of MCU(mode1) might be to modulate oxidative phosphorylation in response to intracellular Ca(2+) signaling, whereas MCU(mode2) and the dynamic high-capacity Ca(2+) buffering system constitute a Ca(2+) sink function. Interestingly, the trigger for PTP activation is unlikely to be [Ca(2+)](mito) itself but rather a downstream byproduct of total mitochondrial Ca(2+) loading.     

Decrease of apparent calmodulin affinity of erythrocyte (Ca2+ + Mg2+)-ATPase at low Ca2+ concentrations

The calmodulin activation of the (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied in the range of 1 nM to 40 microM of purified calmodulin. The apparent calmodulin-affinity of the ATPase was strongly dependent on Ca2+ and decreased approx. 1000-times when the Ca2+ concentration was reduced from 112 to 0.5 microM. The data of calmodulin (Z) activation were analyzed by the aid of a kinetic enzyme model which suggests that 1 molecule of calmodulin binds per ATPase unit and that the affinities of the calcium-calmodulin complexes (CaiZ) decreases in the order of Ca3Z greater than Ca4Z greater than Ca2Z greater than or equal to CaZ. Furthermore, calmodulin dissociates from the calmodulin-saturated Ca2+-ATPase in the range of 10(-7)-10(-6) M Ca2+, even at a calmodulin concentration of 5 microM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 microM, corresponding to 50-80-times the cellular concentration of Ca2+-ATPase, estimated to be approx. 10 nmol/h membrane protein. We therefore conclude that most of the calmodulin is dissociated from the Ca2+-transport ATPase in erythrocytes at the prevailing Ca2+ concentration (probably 10(-7)-10(-8) M) in vivo, and that the calmodulin-binding and subsequent activation of the Ca2+-ATPase requires that the Ca2+ concentration rises to 10(-6)-10(-5) M.     

Ca(2+)-dependent inhibition of actin-activated myosin ATPase activity by S100C (S100A11), a novel member of the S100 protein family

S100C (S100A11, calgizzarin) inhibits the actin-activated myosin Mg(2+)-ATPase activity of smooth muscle in a dose-dependent manner: its half-maximal effect occurs at a S100C/actin molar ratio of 0.05 and its maximal effect occurs at a ratio of 0.20. Furthermore, S100C was found to bind to actin with a stoichiometry of 1:6-7 in the presence of Ca(2+), with an affinity of 1 x 10(-6) M determined by cosedimentation assays. Other Ca(2+)-binding proteins such as S100A1, S100A2, S100B, and calmodulin did not inhibit actin-activated myosin Mg(2+)-ATPase activity. Calmodulin, S100A1, and S100B reversed the inhibitory effect of calponin in a Ca(2+)-dependent manner, S100A2 had no effect, and S100C had additional inhibitory effects. The results suggest that S100C might be involved in the regulation of actin-activated myosin Mg(2+)-ATPase activity through its Ca(2+)-dependent interaction with actin filaments.     

Does calmodulin mediate inhibition of human erythrocyte Ca(2+)-pumping ATPase by myricetin?

Myricetin, a flavonoid, potently inhibits human erythrocyte plasma membrane Ca(2+)-pumping ATPase activity: half-maximal inhibition of the basal and calmodulin-stimulated activity is obtained with 6 microM myricetin. Inhibition of the Ca(2+)-ATPase by myricetin is observed at all concentrations of free Ca2+ from 10(-8)M to 10(-5)M. The extent of inhibition of the Ca(2+)-ATPase is dependent on the time of preincubation of the plasma membranes with myricetin. Inhibition of the Ca(2+)-ATPase by myricetin is not reversed by excess calmodulin. It is concluded that calmodulin does not mediate myricetin's inhibition of human erythrocyte plasma membrane Ca(2+)-pumping ATPase.     

Regulation of protein phosphatase activity by regucalcin localization in rat liver nuclei.

The regulatory role of regucalcin on protein phosphatase activity in isolated rat liver nuclei was investigated. Phosphatase activity toward phosphotyrosine and phosphoserine was significantly increased by the addition of CaCl(2) (10(-5) and 10(-4) M) in the enzyme reaction mixture. Trifluoperazine (25 and 50 microM), an antagonist of calmodulin, significantly inhibited protein phosphatase activity toward phosphoserine, while it had no effect on the enzyme activity toward phosphotysine and phosphothreonine. Cyclosporin A (10(-6)-10(-4) M), an inhibitor of Ca(2+)/calmodulin-dependent protein phosphatase activity toward phosphoserine, but not phosphotyrosine and phosphoserine. Thus, Ca(2+)/calmodulin-dependent phosphatases were present in liver nuclei. Regucalcin (0.25 and 0.5 microM) had an inhibitory effect on liver nuclear phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine. The presence of anti-regucalcin monoclonal antibody (25 and 50 ng/ml) in the enzyme reaction mixture caused a significant elevation of nuclear phosphatase activity toward three phosphoaminoacids. An analysis with sodium sulfate-polyacrylamide gel electrophoresis suggested a possibility of localization of regucalcin in liver nuclei. Moreover, regucalcin was determined in liver nuclei using enzyme-linked immunoadsorbent assay. The present study demonstrates that the endogenous regucalcin inhibits phosphatase activity in the liver nuclei.