Polyphosphoinositide Phospholipase C in Plasma Membranes of Wheat (Triticum aestivum L.) : Orientation of Active Site and Activation by Ca and Mg

Polyphosphoinositide-specific phospholipase C activity was present in plasma membranes isolated from different tissues of several higher plants. Phospholipase C activities against added phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP₂) were further characterized in plasma membrane fractions isolated from shoots and roots of dark-grown wheat (Triticum aestivum L. cv Drabant) seedlings. In right-side-out (70-80% apoplastic side out) plasma membrane vesicles, the activities were increased 3 to 5 times upon addition of 0.01 to 0.025% (w/v) sodium deoxycholate, whereas in fractions enriched in inside-out (70-80% cytoplasmic side out) vesicles, the activities were only slightly increased by detergent. Furthermore, the activities of inside-out vesicles in the absence of detergent were very close to those of right-side-out vesicles in the presence of optimal detergent concentration. This verifies the general assumption that polyphosphoinositide phospholipase C activity is located at the cytoplasmic surface of the plasma membrane. PIP and PIP₂ phospholipase C was dependent on Ca²⁺ with maximum activity at 10 to 100 μm free Ca²⁺ and half-maximal activation at 0.1 to 1 μm free Ca²⁺. In the presence of 10 μm Ca²⁺, 1 to 2 mm MgCl₂ or MgSO₄ further stimulated the enzyme activity. The other divalent chloride salts tested (1.5 mm Ba²⁺, Co²⁺, Cu²⁺, Mn²⁺, Ni²⁺, and Zn²⁺) inhibited the enzyme activity. The stimulatory effect by Mg²⁺ was observed also when 35 mm NaCl was included. Thus, the PIP and PIP₂ phospholipase C exhibited maximum in vitro activity at physiologically relevant ion concentrations. The plant plasma membrane also possessed a phospholipase C activity against phosphatidylinositol that was 40 times lower than that observed with PIP or PIP₂ as substrate. The phosphatidylinositol phospholipase C activity was dependent on Ca²⁺, with maximum activity at 1 mm CaCl₂, and could not be further stimulated by Mg²⁺.     

Inhibition of Mung Bean UDP-Glucose: (1-->3)-beta-Glucan Synthase by UDP-Pyridoxal: Evidence for an Active-Site Amino Group

UDP-pyridoxal competitively inhibits the Ca²⁺-, cellobiose-activated (1→3)-β-glucan synthase activity of unfractionated mung bean (Vigna radiata) membranes, with a K_i of 3.8 ± 0.7 micromolar, when added simultaneously with the substrate UDP-glucose in brief (3 minute) assays. Preincubation of membranes with UDP-pyridoxal and no UDP-glucose, however, causes progressive reduction of the V_max of subsequently assayed enzyme and, after equilibrium is reached, 50% inhibition occurs with 0.84 ± 0.05 micromolar UDP-pyridoxal. This progressive inhibition is reversible provided that the UDP-pyridoxylated membranes are not treated with borohydride, indicating formation of a Schiff's base between the inhibitor and an enzyme amino group. Consistent with this, UDP-pyridoxine is not an inhibitor. The reaction of (1→3)-β-glucan synthase with UDP-pyridoxal is stimulated strongly by Ca²⁺ and, less effectively, by cellobiose or sucrose, and the enzyme is protected against UDP-pyridoxal by UDP-glucose or by other competitive inhibitors, implying that modification is occurring at the active site. Pyridoxal phosphate is a less potent and less specific inhibitor. Latent (1→3)-β-glucan synthase activity inside membrane vesicles can be unmasked and rendered sensitive to UDP-pyridoxal by the addition of digitonin. Treatment of membrane proteins with UDP-[³H]pyridoxal and borohydride labels a number of polypeptides but labeling of none of these specifically requires Ca²⁺ and sucrose; however, a polypeptide of molecular weight 42,000 is labeled by UDP-[³H]pyridoxal in the presence of Mg²⁺ and copurifies with (1→3)-β-glucan synthase activity.     

Calcium Regulates Molecular Interactions of Otoferlin with Soluble NSF Attachment Protein Receptor (SNARE) Proteins Required for Hair Cell Exocytosis

Mutations in otoferlin, a C2 domain-containing ferlin family protein, cause non-syndromic hearing loss in humans (DFNB9 deafness). Furthermore, transmitter secretion of cochlear inner hair cells is compromised in mice lacking otoferlin. In the present study, we show that the C2F domain of otoferlin directly binds calcium (K_D = 267 μm) with diminished binding in a pachanga (D1767G) C2F mouse mutation. Calcium was found to differentially regulate binding of otoferlin C2 domains to target SNARE (t-SNARE) proteins and phospholipids. C2D–F domains interact with the syntaxin-1 t-SNARE motif with maximum binding within the range of 20–50 μm Ca²⁺. At 20 μm Ca²⁺, the dissociation rate was substantially lower, indicating increased binding (K_D = ∼10⁻⁹) compared with 0 μm Ca²⁺ (K_D = ∼10⁻⁸), suggesting a calcium-mediated stabilization of the C2 domain·t-SNARE complex. C2A and C2B interactions with t-SNAREs were insensitive to calcium. The C2F domain directly binds the t-SNARE SNAP-25 maximally at 100 μm and with reduction at 0 μm Ca²⁺, a pattern repeated for C2F domain interactions with phosphatidylinositol 4,5-bisphosphate. In contrast, C2F did not bind the vesicle SNARE protein synaptobrevin-1 (VAMP-1). Moreover, an antibody targeting otoferlin immunoprecipitated syntaxin-1 and SNAP-25 but not synaptobrevin-1. As opposed to an increase in binding with increased calcium, interactions between otoferlin C2F domain and intramolecular C2 domains occurred in the absence of calcium, consistent with intra-C2 domain interactions forming a “closed” tertiary structure at low calcium that “opens” as calcium increases. These results suggest a direct role for otoferlin in exocytosis and modulation of calcium-dependent membrane fusion.     

Extracellular Zinc Ion Regulates Transient Receptor Potential Melastatin 5 (TRPM5) Channel Activation through Its Interaction with a Pore Loop Domain

The transient receptor potential melastatin 5 (TRPM5) channel is a monovalent cation channel activated by intracellular Ca²⁺. Expression of this channel is restricted to taste cells, the pancreas and brainstem, and is thought to be involved in controlling membrane potentials. Its endogenous ligands are not well characterized. Here, we show that extracellular application of Zn²⁺ inhibits TRPM5 activity. In whole-cell patch-clamp recordings, extracellular application of ZnCl₂ inhibited step-pulse-induced TRPM5 currents with 500 nm free intracellular Ca²⁺ in a dose-dependent manner (IC₅₀ = 4.3 μm at −80 mV). ZnSO₄ also inhibited TRPM5 activity. Extracellular application of ZnCl₂ inhibited TRPM5 activation at several temperatures. Furthermore, inhibition by 30 μm ZnCl₂ was impaired in TRPM5 mutants in which His at 896, and Glu at 926 and/or Glu at 939 in the outer pore loop were replaced with Gln. From these results, we conclude that extracellular Zn²⁺ inhibits TRPM5 channels, and the residues in the outer pore loop of TRPM5 are critically involved in the inhibition.     

The Ca-Transport ATPase of Plant Plasma Membrane Catalyzes a nH/Ca Exchange

Microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings accumulate Ca²⁺ upon addition of MgATP. MgATP-dependent Ca²⁺ uptake co-migrates with the plasma membrane H⁺-ATPase on a sucrose gradient. Ca²⁺ uptake is insensitive to oligomycin, inhibited by vanadate (IC₅₀ 40 micromolar) and erythrosin B (IC₅₀ 0.2 micromolar) and displays a pH optimum between pH 6.6 and 6.9. MgATP-dependent Ca²⁺ uptake is insensitive to protonophores. These results indicate that Ca²⁺ transport in these microsomal vesicles is catalyzed by a Mg²⁺-dependent ATPase localized on the plasma membrane. Ca²⁺ strongly reduces ΔpH generation by the plasma membrane H⁺-ATPase and increases MgATP-dependent membrane potential difference (Δψ) generation. These effects of Ca²⁺ on ΔpH and Δψ generation are drastically reduced by micromolar erythrosin B, indicating that they are primarily a consequence of Ca²⁺ uptake into plasma membrane vesicles. The Ca²⁺-induced increase of Δψ is collapsed by permeant anions, which do not affect Ca²⁺-induced decrease of ΔpH generation by the plasma membrane H⁺-ATPase. The rate of decay of MgATP-dependent ΔpH, upon inhibition of the plasma membrane H⁺-ATPase, is accelerated by MgATP-dependent Ca²⁺ uptake, indicating that the decrease of ΔpH generation induced by Ca²⁺ reflects the efflux of H⁺ coupled to Ca²⁺ uptake into plasma membrane vesicles. It is therefore proposed that Ca²⁺ transport at the plasma membrane is mediated by a Mg²⁺-dependent ATPase which catalyzes a nH⁺/Ca²⁺ exchange.     

Chlorpromazine and dimethyl sulfoxide modulate the catalytic activity of the plasma membrane Ca<Superscript>2+</Superscript>-ATPase from human erythrocyte

The plasma membrane Ca²⁺-ATPase (PMCA) removes Ca²⁺ from the cytosol into the extracellular space. Its catalytic activity can be stimulated by calmodulin (CaM) or by limited proteolysis. We evaluated the effect of chlorpromazine (CPZ) and dimethyl sulfoxide (DMSO) over the hydrolytic activity of PMCA. Activity was monitored in three different forms: native, CaM-activated and proteolyzed by trypsin. CPZ appears to inhibit PMCA without directly interfering with the C-terminal site, since it is affected by CaM and proteolysis. Although the treatment of PMCA with trypsin and CaM produces an activation, it also produces an enzymatic form that is more sensitive to inhibition by CPZ. The same case was observed in the DMSO inhibition experiments. In the absence of CPZ, DMSO produces a progressive loss of activity, but in the presence of CPZ the profile of activity against DMSO changes and produces a recovery of activity, indicating a possible partition of CPZ by the solvent. Increasing Ca²⁺ concentrations indicated that CPZ interacts with PMCA rather than with CaM. This observation is supported by docking analysis that suggests that the CPZ-PMCA interaction is non-competitive. We propose that CPZ interacts with the state of lower affinity for Ca^{2 +}.     

Effect of Chitosan on Membrane Permeability of Suspension-Cultured Glycine max and Phaseolus vulgaris Cells

Treatment of suspension-cultured Glycine max cv Harosoy 63 cells with soluble chitosan (20-500 micrograms per milliliter) increased membrane permeability as shown by leakage of electrolytes, protein, and UV absorbing material. Severe damage to the cell membrane by chitosan (100 and 500 μg/ml) was also indicated by reduced staining with fluorescein diacetate and the leakage of fluorescein from preloaded cells. Other basic polymers (poly-l-lysine, histone, DEAE-dextran, protamine sulfate, and glycol chitosan) also increased permeability, whereas the basic monomers l-lysine and d-glucosamine, and acidic or neutral polymers were not active. Chitosan-induced leakage was inhibited by divalent cations, the order of effectiveness being Ba²⁺ > Ca²⁺ > Sr²⁺ > Mg²⁺. Na polygalacturonate and Na poly-l-aspartate also reduced polycation-induced leakage, probably by formation of polycation-polyanion complexes. A chitosan-polygalacturonate complex precipitated on mixing solutions of the two polymers containing approximately equal numbers of galacturonate and glucosamine residues, but not with either polymer in excess. A similar concentration-dependent precipitation of chitosan by Na poly-l-aspartate was found. Leakage from Phaseolus vulgaris cv Grandessa cells was also induced by chitosan, and was inhibited by Ca²⁺ and Na polygalacturonate.     

L-Type Ca2+ Channel Sparklets Revealed by TIRF Microscopy in Mouse Urinary Bladder Smooth Muscle

Calcium is a ubiquitous second messenger in urinary bladder smooth muscle (UBSM). In this study, small discrete elevations of intracellular Ca²⁺, referred to as Ca²⁺ sparklets have been detected in an intact detrusor smooth muscle electrical syncytium using a TIRF microscopy Ca²⁺ imaging approach. Sparklets were virtually abolished by the removal of extracellular Ca²⁺ (0.035±0.01 vs. 0.23±0.07 Hz/mm²; P<0.05). Co-loading of smooth muscle strips with the slow Ca²⁺ chelator EGTA-AM (10 mM) confirmed that Ca²⁺ sparklets are restricted to the cell membrane. Ca²⁺ sparklets were inhibited by the calcium channel inhibitors R-(+)-Bay K 8644 (1 μM) (0.034±0.02 vs. 0.21±0.08 Hz/mm²; P<0.05), and diltiazem (10 μM) (0.097±0.04 vs. 0.16±0.06 Hz/mm²; P<0.05). Ca²⁺ sparklets were unaffected by inhibition of P2X₁ receptors α,β-meATP (10 μM) whilst sparklet frequencies were significantly reduced by atropine (1 μM). Ca²⁺ sparklet frequency was significantly reduced by PKC inhibition with Gö6976 (100 nM) (0.030±0.01 vs. 0.30±0.1 Hz/mm²; P<0.05), demonstrating that Ca²⁺ sparklets are PKC dependant. In the presence of CPA (10 μM), there was no apparent change in the overall frequency of Ca²⁺ sparklets, although the sparklet frequencies of each UBSM became statistically independent of each other (Spearman''s rank correlation 0.2, P>0.05), implying that Ca²⁺ store mediated signals regulate Ca²⁺ sparklets. Under control conditions, inhibition of store operated Ca²⁺ entry using ML-9 (100 μM) had no significant effect. Amplitudes of Ca²⁺ sparklets were unaffected by any agonists or antagonists, suggesting that these signals are quantal events arising from activation of a single channel, or complex of channels. The effects of CPA and ML-9 suggest that Ca²⁺ sparklets regulate events in the cell membrane, and contribute to cytosolic and sarcoplasmic Ca²⁺ concentrations.     

Electrophysiological Characterization of the Rat Epithelial Na+ Channel (rENaC) Expressed in MDCK Cells : Effects of Na+ and Ca2+

The epithelial Na⁺ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na⁺ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na⁺ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na⁺ conductance was Li⁺ > Na⁺ >> K⁺ = N-methyl-d-glucamine⁺ (NMDG⁺). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na⁺ channel current, was found to be ∼5 and 8 pS when Na⁺ and Li⁺ were used as a charge carrier, respectively. K⁺ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P _o), was increased by membrane hyperpolarization. Both whole-cell Na⁺ current and conductance were saturated with increased extracellular Na⁺ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP _o) was significantly decreased when cytosolic Na⁺ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na⁺ conductance (with Li⁺ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca²⁺ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca²⁺ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca²⁺ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca²⁺ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca²⁺ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na⁺ and Ca²⁺.     

Calcium ion transport by pig erythrocyte membrane vesicles

Preincubating pig erythrocyte membranes with ATP enhances their ability to accumulate Ca²⁺ against a concentration gradient. The extent of this increase is dependent on preincubation time over the period 0–60min. As the accessibility of outside membrane markers is decreased by preincubation and as accumulated Ca²⁺ is not removed by EGTA [ethanedioxybis(ethylamine)tetra-acetate], it is suggested that ATP causes the formation of sealed inside-out vesicles which can transport Ca²⁺ inward. The transport system requires ATP and Mg²⁺ and exhibits an apparent dissociation constant for Ca²⁺ of approx. 100μm. Since the dissociation constant for Ca²⁺-sensitive ATPase (adenosine triphosphatase) in these preparations is similar, it is concluded that this ATPase is responsible for Ca²⁺ transport. Polyphosphoinositide concentrations are also increased during incubation with ATP; however, there is no change in their rate of synthesis or breakdown during Ca²⁺ transport.     

The metabolism of cyclic nucleotides in the guinea-pig pancreas. Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase

Both cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase were recovered mainly from the supernatant fractions of guinea-pig pancreas, but a higher proportion of the activity of the former was associated with the pellet fractions. The activities in the supernatant were not separated by gel filtration, but were clearly separated by subsequent chromatography on an anion-exchange resin. The activities of cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase had high-affinity (K_m 6.5±1.1μm and 31.9±3.9μm respectively) and low-affinity (K_m 0.56±0.05mm and 0.32±0.03mm respectively) components. The activity of neither enzyme was affected by the pancreatic secretogens, cholecystokinin-pancreozymin, secretin and carbachol. Removal of ions by gel filtration resulted in a marked reduction in cyclic nucleotide phosphodiesterase activity, which could be restored by addition of Mg²⁺. Mn²⁺ (3mm) was as effective as Mg²⁺ (3mm) in the case of cyclic AMP phosphodiesterase, but was less than half as effective in the case of cyclic GMP phosphodiesterase. The metal-ion chelators, EDTA and EGTA, also decreased activity. Ca²⁺ (1mm) did not affect the activity of cyclic nucleotide phosphodiesterase when the concentration of Mg²⁺ was 3mm. At concentrations of Mg²⁺ between 0.1 and 1mm, 1mm-Ca²⁺ was activatory, and at concentrations of Mg²⁺ below 0.1mm, 1mm-Ca²⁺ was inhibitory. These results are discussed in terms of the possible significance of cyclic nucleotide phosphodiesterase in the physiological control of cyclic nucleotide concentrations during stimulus–secretion coupling.     

Inability of tributyltin-induced chloride/hydroxyl exchange to stimulate calcium transport in mitochondria isolated from flight muscle of the sheep blowfly Lucilia cuprina

Tributyltin in the concentration range 1–4μm failed to stimulate Ca²⁺ transport by Lucilia flight-muscle mitochondria in a medium containing KCl and respiratory substrate but devoid of P_i, despite its promotion of a rapid Cl⁻/OH⁻ exchange. When 2mm-P_i was present, concentrations of tributyltin greater than 1μm inhibited the initial rate of Ca²⁺ transport and induced efflux of the ion from the mitochondria in Cl⁻- or NO₃⁻-containing media. Lower concentrations had little effect. Oligomycin added at up to 10μg/mg of mitochondrial protein had no effect on Ca²⁺ transport. By contrast, approx. 0.3μm-tributyltin completely inhibited respiration supported by α-glycerophosphate in either the presence or absence of added ADP. The data suggest that tributyltin can inhibit Ca²⁺ transport in Lucilia flight-muscle mitochondria other than by facilitating a Cl⁻/OH⁻ exchange or producing an oligomycin-like effect.     

Ca-stimulated, Mg-independent ATP hydrolysis and the high affinity Ca-pumping ATPase. Two different activities in rat kidney basolateral membranes

The Mg²⁺-dependency of Ca²⁺-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg²⁺- and Ca²⁺-buffering ligands. ATP hydrolysis is strongly stimulated by Mg²⁺ with a K_m of 13 μ M in the absence or presence of 1 μ M free Ca²⁺. At free Mg²⁺ concentrations of 1 μ M and lower, ATP hydrolysis is Mg²⁺ -independent, but is strongly stimulated by submicromolar Ca²⁺ concentrations K_m = 0.25 μM, V_max = 24 μmol P_i/h per mg protein). The Ca²⁺-stimulated ATP hydrolysis strongly decreases at higher Mg²⁺ concentrations. The Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg²⁺ concentrations calmodulin and trifluoperazine affect the high affinity Ca²⁺-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca²⁺-pumping in renal basolaterals and on Ca²⁺-ATPase in erythrocyte ghosts suggest that the Ca²⁺-pumping enzyme requires Mg²⁺. In contrast, a role of the Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis in active Ca²⁺ transport across basolateral membranes is rather unlikely.     

Deoxycytidylate deaminase. Properties of the enzyme from cultured kidney cells of baby hamster

dCMP deaminase was partially purified from BHK-21/C13 cells grown in culture. The molecular weight of the enzyme was estimated by gel filtration and gradient centrifugation to be 130000 and 115000 respectively. The enzyme had a pH optimum of 8.4. Its activity versus substrate concentration curve was sigmoid, the substrate concentration at half-maximal velocity being 4.4mm. dCTP activated the deaminase maximally at 40μm, gave a hyperbolic curve for activity versus dCMP concentration and a K_m value for dCMP of 0.91mm. dCTP activation required the presence of Mg²⁺ or Mn²⁺ ions. dTTP inhibited the deaminase maximally at 15μm; the inhibition required the presence of Mg²⁺ or Mn²⁺ ions. The enzyme was very heat-labile but could be markedly stabilized by dCTP at 0.125mm and ethylene glycol at 20% (v/v).     

Metal Ions and Electrolytes Regulate the Dissociation of Heme from Human Hemopexin at Physiological pH

The stability of the hemopexin-heme (Hx-heme) complex to dissociation of the heme prosthetic group has been examined in bicarbonate buffers in the presence and absence of various divalent metal ions. In NH₄HCO₃ buffer (pH 7.4, 20 mm, 25 °C) containing Zn²⁺ (100 μm), 14% of the heme dissociates from this complex (4.5 μm) within 10 min, and 50% dissociates within 2 h. In the absence of metal ions, the rate of dissociation of this complex is far lower, is decreased further in KHCO₃ solution, and is minimal in NaHCO₃. In NH₄HCO₃ buffer, dissociation of the Hx-heme complex is accelerated by addition of divalent metals with decreasing efficiency in the order Zn²⁺ > Cu²⁺ ≫ Ni²⁺ > Co²⁺≫Mn²⁺. Addition of Ca²⁺ prior to addition of Zn²⁺ stabilizes the Hx-heme complex to dissociation of the heme group, and addition of Ca²⁺ after Zn²⁺-induced dissociation of the Hx-heme complex results in re-formation of the Hx-heme complex. These effects are greatly accelerated at 37 °C and diminished in other buffers. Overall, the solution conditions that promote formation of the Hx-heme complex are similar to those found in blood plasma, and conditions that promote release of heme are similar to those that the Hx-heme complex should encounter in endosomes following endocytosis of the complex formed with its hepatic receptor.     

Immunolocalization of the sarcolemmal Ca2+/Mg2+ ecto-ATPase (myoglein) in rat myocardium

Cardiac plasma membrane Ca²⁺/Mg²⁺ ecto-ATPase (myoglein) requires millimolar concentrations of either Ca²⁺ or Mg²⁺ for maximal activity. In this paper, we report its localization by employing an antiserum raised against the purified rat cardiac Ca²⁺/Mg²⁺ ATPase. As assessed by Western blot analysis, the antiserum and the purified immunoglobulin were specific for Ca²⁺/Mg²⁺ ecto-ATPase; no cross reaction was observed towards other membrane bound enzymes such as cardiac sarcoplasmic reticulum Ca²⁺-pump ATPase or sarcolemmal Ca²⁺-pump ATPase. On the other hand, the cardiac Ca²⁺/Mg²⁺ ecto-ATPase was not recognized by antibodies specific for either cardiac sarcoplasmic reticulum Ca²⁺-pump ATPase or plasma membrane Ca²⁺-pump ATPase. Furthermore, the immune serum inhibited the Ca²⁺/Mg²⁺ ecto-ATPase activity of the purified enzyme preparation. Immunofluorescence of cardiac tissue sections and neonatal cultured cardiomyocytes with the Ca²⁺/Mg²⁺ ecto-ATPase antibodies indicated the localization of Ca²⁺/Mg²⁺ ecto-ATPase in association with the plasma membrane of myocytes, in areas of cell-matrix or cell-cell contact. Staining for the Ca²⁺/Mg²⁺ ecto-ATPase was not cardiac specific since the antibodies detected the presence of membrane proteins in sections from skeletal muscle, brain, liver and kidney. The results indicate that Ca²⁺/Mg²⁺ ecto-ATPase is localized to the plasma membranes of cardiomyocytes as well as other tissues such as brain, liver, kidney and skeletal muscle.     

Cluster of Differentiation 38 (CD38) Mediates Bile Acid-induced Acinar Cell Injury and Pancreatitis through Cyclic ADP-ribose and Intracellular Calcium Release

Aberrant Ca²⁺ signals within pancreatic acinar cells are an early and critical feature in acute pancreatitis, yet it is unclear how these signals are generated. An important mediator of the aberrant Ca²⁺ signals due to bile acid exposure is the intracellular Ca²⁺ channel ryanodine receptor. One putative activator of the ryanodine receptor is the nucleotide second messenger cyclic ADP-ribose (cADPR), which is generated by an ectoenzyme ADP-ribosyl cyclase, CD38. In this study, we examined the role of CD38 and cADPR in acinar cell Ca²⁺ signals and acinar injury due to bile acids using pharmacologic inhibitors of CD38 and cADPR as well as mice deficient in Cd38 (Cd38^−/−). Cytosolic Ca²⁺ signals were imaged using live time-lapse confocal microscopy in freshly isolated mouse acinar cells during perifusion with the bile acid taurolithocholic acid 3-sulfate (TLCS; 500 μm). To focus on intracellular Ca²⁺ release and to specifically exclude Ca²⁺ influx, cells were perifused in Ca²⁺-free medium. Cell injury was assessed by lactate dehydrogenase leakage and propidium iodide uptake. Pretreatment with either nicotinamide (20 mm) or the cADPR antagonist 8-Br-cADPR (30 μm) abrogated TLCS-induced Ca²⁺ signals and cell injury. TLCS-induced Ca²⁺ release and cell injury were reduced by 30 and 95%, respectively, in Cd38-deficient acinar cells compared with wild-type cells (p < 0.05). Cd38-deficient mice were protected against a model of bile acid infusion pancreatitis. In summary, these data indicate that CD38-cADPR mediates bile acid-induced pancreatitis and acinar cell injury through aberrant intracellular Ca²⁺ signaling.     

Reconstitution of the energy-linked transhydrogenase activity in membranes from a mutant strain of Escherichia coli K12 lacking magnesium ion- or calcium ion-stimulated adenosine triphosphatase

1. We have isolated a mutant of Escherichia coli K12 (strain AN295) that forms de-repressed amounts of Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase. 2. The Mg²⁺,Ca²⁺-stimulated triphosphatase activity was separated from membrane preparations from strain AN295 by extraction with 5mm-Tris–HCl buffer containing EDTA and dithiothreitol, resulting in a loss of the ATP-dependent transhydrogenase activity. The non-energy-linked transhydrogenase activity remained in the membrane residue. 3. The solubilized Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase activity from strain AN295 was partially purified by repeated gel filtration. The addition of the purified Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase to the membrane residue from strain AN295 reactivated the ATP-dependent transhydrogenase activity. 4. Strain AN296, lacking Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase activity, was derived by transducing the mutant allele, uncA401, into strain AN295. The ATP-dependent transhydrogenase activity was lost but the non-energy linked transhydrogenase was retained. 5. The ATP-dependent transhydrogenase activity in membrane preparations from strain AN296 (uncA⁻) could not be re-activated by the purified Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase from strain AN295. However, after extraction by 5mm-Tris–HCl buffer containing EDTA and dithiothreitol, the ATP-dependent transhydrogenase activity could be re-activated by the addition of the purified Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase from strain AN295 to the membrane residue from strain AN296 (uncA⁻).     

Protein 4.2 Binds to the Carboxyl-terminal EF-hands of Erythroid ��-Spectrin in a Calcium- and Calmodulin-dependent Manner

Spectrin and protein 4.1 cross-link F-actin protofilaments into a network called the membrane skeleton. Actin and 4.1 bind to one end of β-spectrin. The adjacent end of α-spectrin, called the EF-domain, is calmodulin-like, with calcium-dependent and calcium-independent EF-hands. It has no known function. However, the sph^1J/sph^1J mouse has very fragile red cells and lacks the last 13 amino acids in the EF-domain, suggesting the domain is critical for skeletal integrity. Using pulldown binding assays, we find the α-spectrin EF-domain either alone or incorporated into a mini-spectrin binds native and recombinant protein 4.2 at a previously identified region of 4.2 (G₃ peptide). Native 4.2 binds with an affinity comparable with other membrane skeletal interactions (K_d = 0.30 μm). EF-domains bearing the sph^1J mutation are inactive. Binding of protein 4.2 to band 3 (K_d = 0.45 μm) does not interfere with the spectrin-4.2 interaction. Spectrin-4.2 binding is amplified by micromolar concentrations of Ca²⁺ (but not Mg²⁺) by three to five times. Calmodulin also binds to the EF-domain (K_d = 17 μm), and Ca²⁺-calmodulin blocks Ca²⁺-dependent binding of protein 4.2 but not Ca²⁺-independent binding. The data suggest that protein 4.2 is located near protein 4.1 at the spectrin-actin junctions. Because proteins 4.1 and 4.2 also bind to band 3, the erythrocyte anion channel, we suggest that one or both of these proteins cause a portion of band 3 to localize near the spectrin-actin junctions and provide another point of attachment between the membrane skeleton and the lipid bilayer.