Ca(2+) oscillations regulated by Na(+)-Ca(2+) exchanger and plasma membrane Ca(2+) pump induce fluctuations of membrane currents and potentials in human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the potential to differentiate into several types of cells. We have demonstrated spontaneous [Ca(2+)](i) oscillations in hMSCs without agonist stimulation, which result primarily from release of Ca(2+) from intracellular stores via InsP(3) receptors. In this study, we further investigated functions and contributions of Ca(2+) transporters on plasma membrane to generate [Ca(2+)](i) oscillations. In confocal Ca(2+) imaging experiments, spontaneous [Ca(2+)](i) oscillations were observed in 193 of 280 hMSCs. The oscillations did not sustain in the Ca(2+) free solution and were completely blocked by the application of 0.1mM La(3+). When plasma membrane Ca(2+) pumps (PMCAs) were blocked with blockers, carboxyeosin or caloxin, [Ca(2+)](i) oscillations were inhibited. Application of Ni(2+) or KBR7943 to block Na(+)-Ca(2+) exchanger (NCX) also inhibited [Ca(2+)](i) oscillations. Using RT-PCR, mRNAs were detected for PMCA type IV and NCX, but not PMCA type II. In the patch clamp experiments, Ca(2+) activated outward K(+) currents (I(KCa)) with a conductance of 170+/-21.6pS could be recorded. The amplitudes of I(KCa) and membrane potential (V(m)) periodically fluctuated liked to [Ca(2+)](i) oscillations. These results suggest that in undifferentiated hMSCs both Ca(2+) entry through plasma membrane and Ca(2+) extrusion via PMCAs and NCXs play important roles for [Ca(2+)](i) oscillations, which modulate the activities of I(KCa) to produce the fluctuation of V(m).     

Synchronous Ca(2+) oscillation emerges from voltage fluctuations of Ca(2+) stores

Synchronous Ca(2+) oscillation occurs in various cell types to regulate cellular functions. However, the mechanism for synchronization of Ca(2+) increases between cells remains unclear. Recently, synchronous oscillatory changes in the membrane potential of internal Ca(2+) stores were recorded using an organelle-specific voltage-sensitive dye [Yamashita et al. (2006) FEBS J273, 3585-3597], and an electrical coupling model of the synchronization of store potentials and Ca(2+) releases has been proposed [Yamashita (2006) FEBS Lett580, 4979-4983]. This model is based on capacitative coupling, by which transient voltage changes can be synchronized, but oscillatory slow potentials cannot be communicated. Another candidate mechanism is synchronization of action potentials and ensuing Ca(2+) influx through voltage-dependent Ca channels. The present study addresses the question of whether Ca(2+) increases are synchronized by action potentials, and how oscillatory store potentials are synchronized across the cells. Electrophysiological and Ca(2+)-sensitive fluorescence measurements in early embryonic chick retina showed that synchronous Ca(2+) oscillation was caused by releases of Ca(2+) from Ca(2+) stores without any evidence of action potentials in retinal neuroepithelial cells or newborn neurons. High-speed fluorescence measurement of store membrane potential surprisingly revealed that the synchronous oscillatory changes in the store potential were periodic repeats of a burst of high-frequency voltage fluctuations. The burst coincided with a Ca(2+) increase. The present study suggests that synchronization of Ca(2+) release is mediated by the high-frequency fluctuation in the store potential. Close apposition of the store membrane and plasma membrane in an epithelial structure would allow capacitative coupling across the cells.     

Hg(2+) affects the intracellular free Ca(2+) oscillatory pattern and the correlated membrane conductance changes in Mg(2+)-stimulated oocytes of the prawn Palaemon serratus

The impact of mercuric ions (Hg(2+)) on prawn oocytes was examined. Prawn oocytes constitute an unusual system in that they are activated at spawning by seawater Mg(2+), which mediates correlated dynamic changes in intracellular free calcium concentration [(Ca(2+))(i)] and membrane conductance associated with the meiosis resumption. Using a voltage clamp technique and intracellular calcium measurements, we observed that treatment with Hg(2+) (5, 10, and 20 microM) resulted in simultaneous impairments of both (Ca(2+))(i) and membrane current responses to external Mg(2+). Treatment with Hg(2+) also resulted in a gradual dose-dependent slow increase in the baseline level of both (Ca(2+))(i) and membrane conductance, independent of stimulation with external Mg(2+). The effect of Hg(2+) on (Ca(2+))(i) and membrane conductance changes resulted from a block of the signal transduction pathway at some point before the InsP(3) receptor channel involved in Ca(2+) release from the endoplasmic reticulum (ER) stocks. The Hg(2+)-dependent gradual increase in both (Ca(2+))(i) and membrane conductance baseline levels may potentially result from a slow permeabilization of the ER membrane, resulting in Ca(2+) leaking into the cytosol. Indeed, this effect could be blocked with the cell permeable Hg(2+) competitor dithiothreitol, which was able to displace Hg(2+) from its intracellular target regardless of whether external Ca(2+) was present or not.     

Electric field increases the phase transition temperature in the bilayer membrane of phosphatidic acid

The effect of the electric field on the phase transition temperature (Tc) of acidic 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) and 1,2-dipalmitoyl-sn-glycero-3-thionphosphate (thion-DPPA) and zwitterion, i.e. 1,2-dipalmitoyl-rac-3-phosphocholine and 1,2-distearoyl-rac-glycero-3-phosphocholine (DPPC and DSPC), lipids has been investigated. The phase transition was detected using the jump-like increase effect in the conductance of the planar bilayer membrane. A voltage increase to 150 mV has been shown to increase the phase transition temperature in a bilayer lipid membrane (BLM) of phosphatidic acids (DPPA and thion-DPPA) by 8-12 degrees C while the transition temperature in the bilayer of zwitterion lipids (DPPC and DSPC) increases insignificantly. The increasing of Tt in BLM of acidic lipids is attributed to the voltage-induced changes in the molecule packing density.     

Annexins in cell membrane dynamics. Ca(2+)-regulated association of lipid microdomains

The sarcolemma of smooth muscle cells is composed of alternating stiff actin-binding, and flexible caveolar domains. In addition to these stable macrodomains, the plasma membrane contains dynamic glycosphingolipid- and cholesterol-enriched microdomains, which act as sorting posts for specific proteins and are involved in membrane trafficking and signal transduction. We demonstrate that these lipid rafts are neither periodically organized nor exclusively confined to the actin attachment sites or caveolar regions. Changes in the Ca²⁺ concentration that are affected during smooth muscle contraction lead to important structural rearrangements within the sarcolemma, which can be attributed to members of the annexin protein family. We show that the associations of annexins II, V, and VI with smooth muscle microsomal membranes exhibit a high degree of Ca²⁺ sensitivity, and that the extraction of annexins II and VI by detergent is prevented by elevated Ca²⁺ concentrations. Annexin VI participates in the formation of a reversible, membrane–cytoskeleton complex (Babiychuk, E.B., R.J. Palstra, J. Schaller, U. Kämpfer, and A. Draeger. 1999. J. Biol. Chem. 274:35191–35195). Annexin II promotes the Ca²⁺-dependent association of lipid raft microdomains, whereas annexin V interacts with glycerophospholipid microcompartments. These interactions bring about a new configuration of membrane-bound constituents, with potentially important consequences for signaling events and Ca²⁺ flux.     

Effect of cell membrane lipid peroxidation on Ca(2+)-ATPase activity]

Effects of non-enzymic peroxidation of lipids of thymocytes plasmic membranes on the activity of Ca(2+)-ATPase was studied. Calculations were made of kinetic and thermodynamic parameters of enzymic reaction and changes of the membrane potential during peroxidation of lipids.     

Inorganic mercury (Hg2+) transport through lipid bilayer membranes

Summary Diffusion of inorganic mercury (Hg²⁺) through planar lipid bilayer membranes was studied as a function of chloride concentration and pH. Membranes were made from egg lecithin plus cholesterol in tetradecane. Tracer (²⁰³Hg) flux and conductance measurements were used to estimate the permeabilities to ionic and nonionic forms of Hg. At pH 7.0 and [Cl^–] ranging from 10–1000mm, only the dichloride complex of mercury (HgCl₂) crosses the membrane at a significant rate. However, several other Hg complexes (HgOHCl, HgCl ₃ ^– and HgCl ₄ ^2– ) contribute to diffusion through the aqueous unstirred layer adjacent to the membrane. The relation between the total mercury flux (J _Hg), Hg concentrations, and permeabilities is: 1/J _Hg=1/P ^ul[Hg ^t ]+1/P ^m [HgCl₂], where [Hg ^t ] is the total concentration of all forms of Hg,P ^ul is the unstirred layer permeability, andP ^m is the membrane permeability to HgCl₂. By fitting this equation to the data we find thatP ^m =1.3×10^–2 cm sec^–1. At Cl^– concentrations ranging from 1–100mm, diffusion of Hg ^t through the unstirred layer is rate limiting. At Cl^– concentrations ranging from 500–1000mm, the membrane permeability to HgCl₂ becomes rate limiting because HgCl₂ comprises only about 1% of the total Hg. Under all conditions, chemical reactions among Hg²⁺, Cl^– and/or OH^– near the membrane surface play an important role in the transport process. Other important metals, e.g., Zn²⁺, Cd²⁺, Ag⁺ and CH₃Hg⁺, form neutral chloride complexes under physiological conditions. Thus, it is likely that chloride can facilitate the diffusion of a variety of metals through lipid bilayer and biological membranes.     

A requirement for lipid rafts in B cell receptor induced Ca(2+) flux

Although the major biochemical events triggered by ligation of the B-cell receptor (BCR) have been well defined [1] [2], little is known about the spatio-temporal organization of BCR signaling components within the cell membrane and the mechanisms by which signaling specificity is achieved. Partitioning of signaling complexes into specialized domains in the plasma membrane may provide a mechanism for channeling specific stimuli into distinct signaling pathways. Here, we report that multiple tyrosine-phosphorylated proteins accumulate transiently upon BCR activation in detergent-insoluble membrane microdomains known as lipid rafts. We found an activation-dependent translocation to the rafts of the BCR itself, as well as phospholipase Cgamma2 (PLCgamma2), an enzyme critical for BCR-induced Ca(2+) flux in B cells. An intact raft structure was required for BCR-induced tyrosine phosphorylation of PLCgamma2 and the induction of Ca(2+) flux. Taken together, these data provide a functional role for lipid rafts in BCR signaling.     

Arachidonic acid in astrocytes blocks Ca(2+) oscillations by inhibiting store-operated Ca(2+) entry,and causes delayed Ca(2+) influx

ATP-elicited oscillations of the concentration of free intracellular Ca(2+) ([Ca(2+)](i)) in rat brain astrocytes were abolished by simultaneous arachidonic acid (AA) addition, whereas the tetraenoic analogue 5,8,11,14-eicosatetraynoic acid (ETYA) was ineffective. Inhibition of oscillations is due to suppression by AA of intracellular Ca(2+) store refilling. Short-term application of AA, but not ETYA, blocked Ca(2+) influx, which was evoked by depletion of stores with cyclopiazonic acid (CPA) or thapsigargin (Tg). Addition of AA after ATP blocked ongoing [Ca(2+)](i) oscillations. Prolonged AA application without or with agonist could evoke a delayed [Ca(2+)](i) increase. This AA-induced [Ca(2+)](i) rise developed slowly, reached a plateau after 5 min, could be reversed by addition of bovine serum albumin (BSA), that scavenges AA, and was blocked by 1 microM Gd(3+), indicative for the influx of extracellular Ca(2+). Specificity for AA as active agent was demonstrated by ineffectiveness of C16:0, C18:0, C20:0, C18:2, and ETYA. Moreover, the action of AA was not affected by inhibitors of oxidative metabolism of AA (ibuprofen, MK886, SKF525A). Thus, AA exerted a dual effect on astrocytic [Ca(2+)](i), firstly, a rapid reduction of capacitative Ca(2+) entry thereby suppressing [Ca(2+)](i) oscillations, and secondly inducing a delayed activation of Ca(2+) entry, also sensitive to low Gd(3+) concentration.     

Mitochondrial Ca(2+)-induced Ca(2+) release mediated by the Ca(2+) uniporter

We have reported that a population of chromaffin cell mitochondria takes up large amounts of Ca(2+) during cell stimulation. The present study focuses on the pathways for mitochondrial Ca(2+) efflux. Treatment with protonophores before cell stimulation abolished mitochondrial Ca(2+) uptake and increased the cytosolic [Ca(2+)] ([Ca(2+)](c)) peak induced by the stimulus. Instead, when protonophores were added after cell stimulation, they did not modify [Ca(2+)](c) kinetics and inhibited Ca(2+) release from Ca(2+)-loaded mitochondria. This effect was due to inhibition of mitochondrial Na(+)/Ca(2+) exchange, because blocking this system with CGP37157 produced no further effect. Increasing extramitochondrial [Ca(2+)](c) triggered fast Ca(2+) release from these depolarized Ca(2+)-loaded mitochondria, both in intact or permeabilized cells. These effects of protonophores were mimicked by valinomycin, but not by nigericin. The observed mitochondrial Ca(2+)-induced Ca(2+) release response was insensitive to cyclosporin A and CGP37157 but fully blocked by ruthenium red, suggesting that it may be mediated by reversal of the Ca(2+) uniporter. This novel kind of mitochondrial Ca(2+)-induced Ca(2+) release might contribute to Ca(2+) clearance from mitochondria that become depolarized during Ca(2+) overload.     

Functional coupling of the beta(1) subunit to the large conductance Ca(2+)-activated K(+) channel in the absence of Ca(2+). Increased Ca(2+) sensitivity from a Ca(2+)-independent mechanism

Coexpression of the beta(1) subunit with the alpha subunit (mSlo) of BK channels increases the apparent Ca(2+) sensitivity of the channel. This study investigates whether the mechanism underlying the increased Ca(2+) sensitivity requires Ca(2+), by comparing the gating in 0 Ca(2+)(i) of BK channels composed of alpha subunits to those composed of alpha+beta(1) subunits. The beta(1) subunit increased burst duration approximately 20-fold and the duration of gaps between bursts approximately 3-fold, giving an approximately 10-fold increase in open probability (P(o)) in 0 Ca(2+)(i). The effect of the beta(1) subunit on increasing burst duration was little changed over a wide range of P(o) achieved by varying either Ca(2+)(i) or depolarization. The effect of the beta(1) subunit on increasing the durations of the gaps between bursts in 0 Ca(2+)(i) was preserved over a range of voltage, but was switched off as Ca(2+)(i) was increased into the activation range. The Ca(2+)-independent, beta(1) subunit-induced increase in burst duration accounted for 80% of the leftward shift in the P(o) vs. Ca(2+)(i) curve that reflects the increased Ca(2+) sensitivity induced by the beta(1) subunit. The Ca(2+)-dependent effect of the beta(1) subunit on the gaps between bursts accounted for the remaining 20% of the leftward shift. Our observation that the major effects of the beta(1) subunit are independent of Ca(2+)(i) suggests that the beta(1) subunit mainly alters the energy barriers of Ca(2+)-independent transitions. The changes in gating induced by the beta(1) subunit differ from those induced by depolarization, as increasing P(o) by depolarization or by the beta(1) subunit gave different gating kinetics. The complex gating kinetics for both alpha and alpha+beta(1) channels in 0 Ca(2+)(i) arise from transitions among two to three open and three to five closed states and are inconsistent with Monod-Wyman-Changeux type models, which predict gating among only one open and one closed state in 0 Ca(2+)(i).     

The behaviors of Ca(2+)-ATPase embedded in interdigitated bilayer.

We investigated the behavior of a membrane protein, Ca(2+)-ATPase, in interdigitated phospholipid bilayers. The results showed that Ca(2+)-ATPase does not cause significant alterations in the interdigitation of 16:0 LPC/DPPC (27.0 mol% LPC) vesicles when it is reconstituted with lipids. Intrinsic fluorescence, acrylodan fluorescent adducts, and CD spectra indicated that Ca(2+)-ATPase, when embedded in interdigitated bilayer structures, is more exposed to the hydrophilic environment and has a looser structure than when embedded in non-interdigitated bilayers. The interdigitation of acyl chains induces a rapid loss of enzyme activity. It is suggested that interdigitated bilayer structures may play an important role as negative regulatory factors in physiological functions.     

Mechanism of modulation of the plasma membrane Ca(2+)-ATPase by arachidonic acid

The intracellular level of long chain fatty acids controls the Ca(2+) concentration in the cytoplasm. The molecular mechanisms underlying this Ca(2+) mobilization are not fully understood. We show here that the addition of low micromolar concentrations of fatty acids directly to the purified plasma membrane Ca(2+)-ATPase enhance ATP hydrolysis, while higher concentration decrease activity, exerting a dual effect on the enzyme. The effect of arachidonic acid is similar in the presence or absence of calmodulin, acidic phospholipids or ATP at the regulatory site, thereby precluding these sites as probable acid binding sites. At low arachidonic acid concentrations, neither the affinity for calcium nor the phosphoenzyme levels are significantly modified, while at higher concentrations both are decreased. The action of arachidonic acid is isoenzyme specific. The increase on ATP hydrolysis, however, is uncoupled from calcium transport, because arachidonic acid increases the permeability of erythrocyte membranes to calcium. Oleic acid has no effect on membrane permeability while linoleic acid shows an effect similar to that of arachidonic acid. Such effects might contribute to the entry of extracellular Ca(2+) following to fatty acid release.     

Variations in Ca(2+)-mediated activation of Ca(2+)-ATPase and its associated inhibitor in erythrocyte membrane.

1. Two distinct patterns of Ca(2+)-mediated activation of Ca(2+)-ATPase were identified in calmodulin-depleted membranes. 2. In membranes showing no activation (type A), preincubation with micromolar concentration of cyclic AMP and ATP made possible stimulation of the enzyme while in membranes already exhibiting activation (type B), preincubation with cyclic AMP and ATP abolished the activation. 3. ATPase stimulation in type A membranes was suppressible by leupeptin. 4. Triton extractable inhibitor isolated from type A membranes was as active as that derived from type B membranes only after preincubating the membranes with cyclic AMP and ATP. 5. The inhibitor could be inactivated by alkaline phosphatase.     

Ca(2+)-induced recruitment of the secretory vesicle protein DOC2B to the target membrane

Ca(2+)-dependent fusion of transport vesicles at their target can be enhanced by intracellular Ca2+ and diacylglycerol. Diacylglycerol induces translocation of the vesicle priming factor Munc13 and association of the secretory vesicle protein DOC2B to the membrane. Here we demonstrate that a rise in intracellular Ca2+ is sufficient for a Munc13-independent recruitment of DOC2B to the target membrane. This novel mechanism occurred readily in the absence of Munc13 and was not influenced by DOC2B mutations that abolish Munc13 binding. Purified DOC2B (expressed as a bacterial fusion protein) bound phospholipids in a Ca(2+)-dependent way, suggesting that the translocation is the result of a C2 domain activation mechanism. Ca(2+)-induced translocation was also observed in cultured neurons expressing DOC2B-enhanced green fluorescent protein. In this case, however, various degrees of membrane association occurred under resting conditions, suggesting that physiological Ca2+ concentrations modulate DOC2B localization. Depolarization of the neurons induced a complete translocation of DOC2B-enhanced green fluorescent protein to the target membrane within 5 s. We hypothesize that this novel Ca(2+)-induced activity of DOC2B functions synergistically with diacylglycerol-induced Munc13 binding to enhance exocytosis during episodes of high secretory activity.     

Polymethoxylated flavones induce Ca(2+)-mediated apoptosis in breast cancer cells

Flavonoids, polyphenolic phytochemicals which include flavones and isoflavones, are present in the common human diet. It has been suggested that these compounds may exert anticancer activity; however, the mechanisms involved remain unknown. We have recently shown (Sergeev, 2004, Biochem Biophys Res Commun 321: 462-467) that isoflavones can activate the novel apoptotic pathway mediated by cellular Ca(2+). Here, we report that polymethoxyflavones (PMFs) derived from sweet orange (Citrus sinensis L.) inhibit growth of human breast cancer cells via Ca(2+)-dependent apoptotic mechanism. The treatment of MCF-7 breast cancer cells with 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-OH-HxMF) and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone (3'-OH-TtMF) induced a sustained increase in concentration of intracellular Ca(2+) ([Ca(2+)](i)) resulting from both depletion of the endoplasmic reticulum Ca(2+) stores and Ca(2+) influx from the extracellular space. This increase in [Ca(2+)](i) was associated with the activation of the Ca(2+)-dependent apoptotic proteases, mu-calpain and caspase-12, as evaluated with the calpain and caspase-12 peptide substrates and antibodies to active (cleaved) forms of the enzymes. Corresponding non-hydroxylated PMFs, 3,5,6,7,8,3',4'-heptamethoxyflavone (HpMF) and 5,6,7,3',4'-pentamethoxyflavone (PtMF), were dramatically less active in inducing Ca(2+)-mediated apoptosis. Our results strongly suggest that the cellular Ca(2+) modulating activity of flavonoids underlies their apoptotic mechanism and that hydroxylation of PMFs is critical for their ability to induce an increase in [Ca(2+)](i) and, thus, activate Ca(2+)-dependent apoptotic proteases.     

Single-channel analysis of the conductance fluctuations induced in lipid bilayer membranes by complement proteins C5b-9

Summary Single-channel analysis of electrical fluctuations induced in planar bilayer membranes by the purified human complement proteins C5b6, C7, C8, and C9 have been analyzed. Reconstitution experiments with lipid bilayer membranes showed that the C5b-9 proteins formed pores only if all proteins were present at one side of the membrane. The complement pores had an average single-channel conductance of 3.1 nS at 0.15m KCl. The histogram of the complement pores suggested a substantial variation of the size of the single channel. The linear relationship between single-channel conductance at fixed ionic strength and the aqueous mobility of the ions in the bulk aqueous phase indicated that the ions move inside the complement pore in a manner similar to the way they move in the aqueous phase. The minimum diameter of the pores as judged from the conductance data is approximately 3 nm. The complement channels showed no apparent voltage control or regulation up to transmembrane potentials of 100 mV. At neutral pH the pore is three to four times more permeable for alkali ions than for chloride, which may be explained by the existence of fixed negatively charged groups in or near the pore. The significance of these observations to current molecular models of the membrane lesion formed by these cytolytic serum proteins is considered.     

Ca(2+)-dependent annexin self-association on membrane surfaces

Annexin self-association was studied with 90 degrees light scattering and resonance energy transfer between fluorescein (donor) and eosin (acceptor) labeled proteins. Synexin (annexin VII), p32 (annexin IV), and p67 (annexin VI) self-associated in a Ca(2+)-dependent manner in solution. However, this activity was quite labile and, especially for p32 and p67, was not consistently observed. When bound to chromaffin granule membranes, the three proteins consistently self-associated and did so at Ca2+ levels (pCa 5.0-4.5) approximately 10-fold lower than required when in solution. Phospholipid vesicles containing phosphatidylserine and phosphatidylethanolamine (1:1 or 1:3) were less effective at supporting annexin polymerization than were those containing phosphatidylserine and phosphatidylcholine (1:0, 1:1, or 1:3). The annexins bound chromaffin granule membranes in a positively cooperative manner under conditions where annexin self-association was observed, and both phenomena were inhibited by trifluoperazine. Ca(2+)-dependent chromaffin granule membrane aggregation, induced by p32 or synexin, was associated with intermembrane annexin polymerization at Ca2+ levels less than pCa 4, but not at higher Ca2+ concentrations, suggesting that annexin self-association may be necessary for membrane contact at low Ca2+ levels but not at higher Ca2+ levels where the protein may bind two membranes as a monomer.     

Antibodies affect the ionic conductance of channels formed by amphotericin B in a lipid bilayer

For the first time poly- and monoclonal antibodies (class IgM) against the polyene antibiotic amphotericin B were obtained affecting the properties of a channel formed by the antibiotic and cholesterol in a lipid bilayer when amphotericin B was added to the solution at one (cis) side of the membrane. In the case of the symmetric distribution of cholesterol in the lipid bilayer, three molecules of monoclonal antibodies bind firmly to the channel at the trans-side of the membrane, thus strongly increasing the mean lifetime of the channel in the open state, and not changing practically the ion conductance of its open state. The antibodies did not alter the properties of these channels when added at the cis-side of the membrane as well as of the channels formed in the lipid bilayer when amphotericin B was added at both membrane sides. The antibodies obtained did not affect the conductance of channels in which amphotericin B and cholesterol were replaced with their analogs levorin and 5 alpha-androstan-3 beta-one, which points to a high specificity of the immunoglobulins isolated. When cholesterol was present only in the cis-monolayer of the lipid bilayer and was absent in the trans-monolayer, the same monoclonal antibodies when added at the trans-side of the membrane blocked the conductance of the channel formed by adding the antibiotic to the solution at the cis-side of the bilayer. The obtained evidence is of interest in elucidating the general features of interaction of antibodies with the ionic channels of cellular and model membranes.     

p-Nitrophenylphosphatase activity catalyzed by plasma membrane (Ca(2+) + Mg(2+)ATPase: correlation with structural changes modulated by glycerol and Ca(2+)

The plasma membrane (Ca²⁺+Mg²⁺)ATPase hydrolyzes pseudo-substrates such as p-nitrophenylphosphate. Except when calmodulin is present, Ca²⁺ ions inhibit the p-nitrophenylphosphatase activity. In this report it is shown that, in the presence of glycerol, Ca²⁺ strongly stimulates phosphatase activity in a dose-dependent manner. The glycerol- and Ca²⁺-induced increase in activity is correlated with modifications in the spectral center of mass (average emission wavenumber) of the intrinsic fluorescence of the enzyme. It is concluded that the synergistic effect of glycerol and Ca²⁺ is related to opposite long-term hydration effects on the substrate binding domain and the Ca²⁺ binding domain.