A possible explanation for effects of Sr2+ on contraction-relaxation cycle in canine stomach

Effects of Sr2+ on smooth muscle activity and Ca2+ movement by plasma membrane (PM) were examined in preparations isolated from canine stomach to determine how Sr2+ modulates the contraction-relaxation cycle in smooth muscle. Sr2+ caused contraction in Ca2+-free solution. In the presence of Sr2+, KCl caused a response similar to that in normal Ringer's solution, this contraction was inhibited by both nitroprusside and verapamil. Sr2+ inhibited Ca2+ uptake by PM in the presence and absence of ATP. Release of Ca2+ from loaded PM vesicles was accelerated by Sr2+. Results suggest that Sr2+ could replace Ca2+ in Ca2+ binding sites in the cell.     

Kinetic properties of the purified Ca2+-translocating ATPase from human erythrocyte plasma membrane

The basic kinetic properties of the solubilized and purified Ca2+-translocating ATPase from human erythrocyte membranes were studied. A complex interaction between the major ligands (i.e., Ca2+, Mg2+, H+, calmodulin and ATP) and the enzyme was found. The apparent affinity of the enzyme for Ca2+ was inversely proportional to the concentration of free Mg2+ and H+, both in the presence or absence of calmodulin. In addition, the apparent affinity of the enzyme for Ca2+ was significantly increased by the presence of calmodulin at high concentrations of MgCl2 (5 mM), while it was hardly affected at low concentrations of MgCl2 (2 mM or less). In addition, the ATPase activity was inhibited by free Mg2+ in the millimolar concentration range. Evidence for a high degree of positive cooperativity for Ca2+ activation of the enzyme (Hill coefficient near to 4) was found in the presence of calmodulin in the slightly alkaline pH range. The degree of cooperativity induced by Ca2+ in the presence of calmodulin was decreased strongly as the pH decreased to acid values (Hill coefficient below 2). In the absence of calmodulin, the Hill coefficient was 2 or slightly below over the whole pH range tested. Two binding affinities of the enzyme for ATP were found. The apparent affinity of the enzyme for calmodulin was around 6 nM and independent of the Mg2+ concentration. The degree of stimulation of the ATPase activity by calmodulin was dependent on the concentrations of both Ca2+ and Mg2+ in the assay system.     

Properties of plasma membrane-induced amylase release from rat parotid secretory granules: effects of Ca2+ and Mg-ATP.

A secretory granular fraction (SG) and a plasma membrane rich fraction (PM) have been isolated from rat parotid gland by differential and Percoll gradient centrifugation. With these two fractions, a cell-free interaction system has been reconstituted to clarify the exocytotic interaction between the secretory granules and plasma membranes, and the conditions of amylase release from SG have been characterized in vitro. The addition of PM into this assay system induced a rapid and transient release of amylase from SG. Some other membranes such as erythrocyte ghosts also mimicked the effect of PM. This release was increased by Ca2+, but was not completely blocked by EGTA. Simultaneous addition of 1 mM ATP with 1 mM MgCl2 (Mg-ATP) in the presence of Ca2+ reduced this release. However, in spite of the existence of Mg-ATP, the stimulation of PM-induced amylase release was caused by Ca2+ in a concentration-dependent manner (10(-7)-10(-3) M). These results suggest that Ca2+ and Mg-ATP should participate as important regulators in the exocytotic interaction between secretory granules and plasma membranes in this system. Furthermore, the differences between our system and intact cells are also discussed.     

The water permeability of Arabidopsis plasma membrane is regulated by divalent cations and pH

Mechanisms that regulate water channels in the plant plasma membrane (PM) were investigated in Arabidopsis suspension cells. Cell hydraulic conductivity was measured with a cell pressure probe and was reduced 4-fold as compared to control values when calcium was added in the pipette and in bathing solution. To assess the significance of these effects in vitro, PM vesicles were isolated by aqueous two-phase partitioning and their water transport properties were characterized by stopped-flow spectrophotometry. Membrane vesicles isolated in standard conditions exhibited reduced water permeability (P(f)) together with a lack of active water channels. In contrast, when prepared in the presence of chelators of divalent cations, PM vesicles showed a 2.3-fold higher P(f) and active water channels. Furthermore, equilibration of purified PM vesicles with divalent cations reduced their P(f ) and water channel activity down to the basal level of membranes isolated in standard conditions. Ca2+ was the most efficient with a half-inhibition of P(f) at 50-100 microM free Ca2+. Water transport in purified PM vesicles was also reversibly blocked by H+, with a half-inhibition of P(f )at pH 7.2-7.5. Thus, both Ca2+ and H+ contribute to a membrane-delimited switch from active to inactive water channels that may allow coupling of water transport to cell signalling and metabolism.     

ATP-dependent calcium transport in plasma membrane vesicles from neutrophil leukocytes

Plasma membrane vesicles were prepared from guinea pig peritoneal exudate neutrophils, using nitrogen cavitation to rupture the plasma membrane and differential centrifugation to separate the vesicles. The vesicles were enriched 13.2-fold in (Na+, K+)-ATPase activity and had a cholesterol:protein ratio of 0.15, characteristic of plasma membranes. Contamination of the vesicle preparation with DNA or marker enzyme activities for intracellular organelles was very low. Studies designed to determine vesicle sidedness and integrity indicated that 33% were sealed, inside-out; 41% were sealed, right side-out, and 26% were leaky. The vesicles accumulated 45Ca2+ in a linear fashion for 45 min. The uptake was dependent on the presence of oxalate and MgATP in the incubating medium. Uptake showed a Ka for free Ca2+ of 164 nM and a Vmax of 17.2 nmol/mg . min (based on total protein). GTP, ITP, CTP, UTP, ADP, or AMP supported uptake at rates less than or equal to 11% of ATP. Ca2+ uptake was maximal at pH 7-7.5. Calcium stimulated the hydrolysis of ATP by the vesicles with a Ka for free Ca2+ of 440 nM and Vmax of 17.5 nmol/mg . min (based on total protein). When the Ca2+ uptake rate was based upon those vesicles expected to transport Ca2+ (33% sealed, inside-out vesicles) and Ca2+-stimulated ATPase activity was based upon those vesicles expected to express that activity (26% leaky + 33% sealed, inside-out vesicles), the molar stoichiometry of Ca2+ transported:ATP hydrolyzed was 2.12 +/- 0.12. Calmodulin did not increase either Vmax or Ka for free Ca2+ of the uptake system in the vesicles, even when they were treated previously with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The high affinity of this system for Ca2+, specificity for ATP, physiological pH optimum, and stoichiometry of Ca2+ transported:ATP hydrolyzed suggest that it represents an important mechanism by which neutrophils maintain low levels of cytoplasmic free Ca2+.     

3-O-methylfluorescein phosphate as a fluorescent substrate for plasma membrane Ca2+-ATPase

3-O-methylfluorescein phosphate hydrolysis, catalyzed by purified erythrocyte Ca2+-ATPase in the absence of Ca2+, was slow in the basal state, activated by phosphatidylserine and controlled proteolysis, but not by calmodulin. p-Nitrophenyl phosphate competitively inhibits hydrolysis in the absence of Ca2+, while ATP inhibits it with a complex kinetics showing a high and a low affinity site for ATP. Labeling with fluorescein isothiocyanate impairs the high affinity binding of ATP, but does not appreciably modify the binding of any of the pseudosubstrates. In the presence of calmodulin, an increase in the Ca2+ concentration produces a bell-shaped curve with a maximum at 50 microM Ca2+. At optimal Ca2+ concentration, hydrolysis of 3-O-methylfluorescein phosphate proceeds in the presence of fluorescein isothiocyanate, is competitively inhibited by p-nitrophenyl phosphate and, in contrast to the result observed in the absence of Ca2+, it is activated by calmodulin. In marked contrast with other pseudosubstrates, hydrolysis of 3-O-methylfluorescein phosphate supports Ca2+ transport. This highly specific activity can be used as a continuous fluorescent marker or as a tool to evaluate partial steps from the reaction cycle of plasma membrane Ca2+-ATPases.     

The C-terminal 165 amino acids of the plasma membrane Ca(2+)-ATPase confer Ca2+/calmodulin sensitivity on the Na+,K(+)-ATPase alpha-subunit.

The C-terminal 165 amino acids of the rat brain plasma membrane (PM) Ca(2+)-ATPase II containing the calmodulin binding auto-inhibitory domain was connected to the C-terminus of the ouabain sensitive chicken Na+,K(+)-ATPase alpha 1 subunit. Expression of this chimeric molecule in ouabain resistant mouse L cells was assured by the high-affinity binding of [3H]ouabain. In the presence of Ca2+/calmodulin, this chimeric molecule exhibited ouabain inhibitable Na+,K(+)-ATPase activity; the putative chimeric ATPase activity was absent in the absence of Ca2+/calmodulin and activated by Ca2+/calmodulin in a dose-dependent manner. Furthermore, this chimeric molecule could bind monoclonal IgG 5 specific to the chicken Na+,K(+)-ATPase alpha 1 subunit only in the presence of Ca2+/calmodulin, suggesting that the epitope for IgG 5 in this chimera is masked in the absence of Ca2+/calmodulin and uncovered in their presence. These results propose a direct interaction between the calmodulin binding auto-inhibitory domain of the PM Ca(2+)-ATPase and the specific regions of the Na+,K(+)-ATPase alpha 1 subunit that are structurally homologous to the PM Ca(2+)-ATPase. A comparison of the deduced amino acid sequences revealed several possible regions within the Na+,K(+)-ATPase that might interact with the auto-inhibitory domain of the PM Ca(2+)-ATPase.     

Ca2+ release by inositol trisphosphate from Ca2+-transporting microsomes derived from uterine sarcoplasmic reticulum

Microsomes derived from pregnant uterine sarcoplasmic reticulum, isolated by differential and sucrose density gradient centrifugation, accumulates Ca2+ in the presence of ATP. Inositol trisphosphate caused release of this Ca2+, in a dose dependent manner. 40% of the Ca2+ that can be released by the ionophore A23187 was released by 5 microM inositol trisphosphate. Removal of Mg by EDTA prior to addition of inositol trisphosphate did not change the course of Ca2+ release. These results indicate that by mobilizing intracellular Ca2+, inositol trisphosphate may be the link between hormonal stimuli and smooth muscle contraction.     

Passive calcium influx by plasma membrane vesicles isolated from rat liver

Passive Ca2+ influx independent of ATP addition to the incubation medium, took place in plasma membrane vesicles isolated from rat liver. The rate of Ca2+ influx was found to depend on the concentration of added Ca2+, and on the incubation temperature, and was inhibited by La3+, Hg2+ and by p-chloromercuribenzoate. Influx was not blocked by calcium channel blockers, or affected by a range of uncouplers. Addition of the Ca2+ ionophore A23187 to vesicles that had taken up the ion induced a rapid efflux of Ca2+ especially when EGTA also was added to the incubation medium. A number of divalent cations inhibited Ca2+ influx. The vesicles could be frozen and stored overnight with little loss in activity. The kinetics of Ca2+ influx could be related to that which occurs in the unstimulated perfused rat liver. The data suggest that the plasma membrane vesicle preparation may be useful for further studies on the basal liver cell Ca2+ influx system in vitro.     

Ca2+-stimulated, Mg2+-dependent ATPase activity in neutrophil plasma membrane vesicles. Coupling to Ca2+ transport

Low concentrations of free Ca2+ stimulated the hydrolysis of ATP by plasma membrane vesicles purified from guinea pig neutrophils and incubated in 100 mM HEPES/triethanolamine, pH 7.25. In the absence of exogenous magnesium, apparent values obtained were 320 nM (EC50 for free Ca2+), 17.7 nmol of Pi/mg X min (Vmax), and 26 microM (Km for total ATP). Studies using trans- 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid as a chelator showed this activity was dependent on 13 microM magnesium, endogenous to the medium plus membranes. Without added Mg2+, Ca2+ stimulated the hydrolysis of several other nucleotides: ATP congruent to GTP congruent to CTP congruent to ITP greater than UTP, but Ca2+-stimulated ATPase was not coupled to uptake of Ca2+, even in the presence of 5 mM oxalate. When 1 mM MgCl2 was added, the vesicles demonstrated oxalate and ATP-dependent calcium uptake at approximately 8 nmol of Ca2+/mg X min (based on total membrane protein). Ca2+ uptake increased to a maximum of approximately 17-20 nmol of Ca2+/mg X min when KCl replaced HEPES/triethanolamine in the buffer. In the presence of both KCl and MgCl2, Ca2+ stimulated the hydrolysis of ATP selectively over other nucleotides. Apparent values obtained for the Ca2+-stimulated ATPase were 440 nM (EC50 for free Ca2+), 17.5 nmol Pi/mg X min (Vmax) and 100 microM (Km for total ATP). Similar values were found for Ca2+ uptake which was coupled efficiently to Ca2+-stimulated ATPase with a molar ratio of 2.1 +/- 0.1. Exogenous calmodulin had no effect on the Vmax or EC50 for free Ca2+ of the Ca2+-stimulated ATPase, either in the presence or absence of added Mg2+, with or without an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid pretreatment of the vesicles. The data demonstrate that calcium stimulates ATP hydrolysis by neutrophil plasma membranes that is coupled optimally to transport of Ca2+ in the presence of concentrations of K+ and Mg2+ that appear to mimic intracellular levels.     

Incorporation of (1-14C)palmitoyl-CoA into phosphatidylcholine by plasma membranes of rat submaxillary glands in vitro.

1. On incubation with the isolated rat submaxillary gland plasma membranes, [1-14C]palmitoyl-CoA was incorporated mainly into phosphatidylcholine and hydrolysed to [1-14C]palmitic acid and CoASH. 2. The addition of lysophosphatidylcholine enhanced the incorporation into phosphatidylcholine and lowered the hydrolysis of palmitoyl-CoA markedly. 3. In the presence of lysophosphatidylcholine, palmitoyl-CoA incorporation into phosphatidylcholine was maximum at 0.1 mM palmitoyl-CoA, 0.5 mM lysophosphatidylcholine and between pH 7.0 and 9.0. 4. The incorporation into phosphatidylcholine was stimulated by Na+, K+ and K-, inhibited by Ca2+ and Mg2+ and unaffected by sodium deoxycholate and ATP. 5. Epinephrine inhibited the incorporation of palmitoyl-CoA into phosphatidylcholine in the presence or absence of ATP, the inhibition being more in the presence of ATP than in its absence. Dibutyryl adenosine 3':5'-monophosphate mimicked the inhibitory effect of epinephrine.     

The plasma membrane calcium pump: regulation by a soluble Ca2+ binding protein

The Ca2+ pump of the plasma membrane of human red blood cells is associated with the activity of a (Ca2+ + Mg2+)-ATPase. Both the ATPase and the pump are stimulated above basal activities by calmodulin, an ubiquitous Ca2+-binding protein. Calmodulin isolated from human red blood cells was shown to be equipotent and equieffective with that isolated from beef brain. Half-maximal activation of ATPase (isolated red blood cell membranes, 37 C) and transport (inside-out red blood cell membrane vesicles, 25 C) were obtained with 2.5 and 4.4 nM calmodulin, respectively. Ca2+ dependence of Ca2+ transport was measured in the absence and in the presence of 50 nM calmodulin. At all Ca2+ concentrations above 2 X 10(-7) M Ca2+, the rate of transport was greater in the presence of calmodulin. The results implicate calmodulin in the regulation of the plasma membrane Ca2+ pump, but the mechanism(s) remain to be elucidated.     

Fe2+ and other divalent metal ions uncouple Ca2+ transport from (Ca2+-Mg2+)-ATPase in rat liver plasma membranes

The addition of nanomolar concentrations of free Fe2+, Mn2+, or Co2+ to rat liver plasma membranes resulted in an activation of ATP hydrolysis by these membranes which was not additive with the Ca2+-stimulated ATPase activity coupled to the Ca2+ pump. Detailed analysis showed that, if fact, (i) as for the stimulation of (Ca2+-Mg2+)-ATPase by Ca2+, activation of ATP hydrolysis by Fe2+, Mn3+, or Co2+ followed a cooperative mechanism involving two ions; (ii) two interacting sites for ATP were involved in the activation of both Fe2+- and Ca2+-stimulated ATPase activities; (iii) micromolar concentrations of magnesium caused the same dramatic inhibition of both activities; and (iv) the subcellular distribution of Fe2+-activated ATP hydrolysis activity corresponded to that of plasma membrane markers. This suggests that the (Ca2+-Mg2+)-ATPase might be stimulated not only by Ca2+, but also by Fe2+, Mn2+, or Co2+. However, interaction of (Ca2+-Mg2+)-ATPase with Fe2+, Mn2+, or Co2+ inhibited the Ca2+ pump activity. Furthermore, neither the formation of the phosphorylated intermediate of (Ca2+-Mg2+)-ATPase, nor ATP-dependent (59Fe) uptake could be detected in the presence of Fe2+ concentrations which stimulated ATP hydrolysis. We conclude that: (i) under the influence of certain metal ions, the Ca2+ pump in the liver plasma membrane may be switched to an uncoupled state which displays ATP hydrolysis activity, but does not insure ion transport; (ii) therefore the Ca2+ pump in liver plasma membranes specifically insures Ca2+ transport.     

Identification of a calmodulin-stimulated (Ca2++ Mg2+)-ATPase in a plasma membrane fraction isolated from maize (Zea mays) leaves

Plasma membranes were isolated from light-grown, 14-day-old maize leaves ( Zea mays L . cv. Golden Cross Bantam) using aqueous two-phase partitioning. The plasma membrane (PM) fraction contained < 0.3% of the total chlorophyll, < 0.2% of the mitochondrial marker enzyme activity, minimal contamination by endomembranes and 34% of the total PM.
A calmodulin-stimulated (Ca2++ Mg2+)-ATPase was identified in the PM-enriched fraction. The Ca2++ calmodulin stimulation was dependent on Mg2+, saturated at ca 25 μM total Ca2+, had a pH maximum at 7.2 and was maximally stimulated by 600 n M bovine brain calmodulin. The stimulation was not greatly affected by the anion present and showed a divalent cation specificity of Ca2+ > Sr+2 ± Mn+2 > Co2+± Cu2+ > Ba2+. The napthalenesulfonamide W7, an antagonist of calmodulin action, completely inhibited the calmodulin stimulation at 175 μM , while the less active analogue W5 was ineffective at this concentration. La3+, an inhibitor of PM Ca2+ transport, showed a 50% inhibition of calmodulin-stimulated ATPase activity at ca 200 μM . Taken as a whole, these data demonstrate the presence of a calmodulinstimulated, (Ca2++ Mg2+)-ATPase on the cytoplasmic surface of the plasma membrane of maize leaf cells.     

Changes in affinity for calcium ions with the formation of two kinds of phosphoenzyme in the Ca2+,Mg2+-dependent ATPase of sarcoplasmic reticulum

The amount of Ca2+ bound to the Ca2+,Mg2+-dependent ATPase of deoxycholic acid-treated sarcoplasmic reticulum was measured during ATP hydrolysis by the double-membrane filtration method [Yamaguchi, M. & Tonomura, Y. (1979), J. Biochem. 86, 509--523]. The maximal amount of phosphorylated intermediate (EP) was adopted as the amount of active site of the ATPase. In the absence of ATP, 2 mol of Ca2+ bound cooperatively to 1 mol of active site with high affinity and were removed rapidly by addition of EGTA. AMPPNP did not affect the Ca2+ binding to the ATPase in the presence of MgCl2. Under the conditions where most EP and ADP sensitive at steady state (58 microM Ca2+, 50 microM EGTA, and 20 mM MgCl2 at pH 7.0 and 0 degrees C), bound Ca2+ increased by 0.6--0.7 mol per mol active site upon addition of ATP. The time course of decrease in the amount of bound 45Ca2+ on addition of unlabeled Ca2+ + EGTA was biphasic, and 70% of bound 45Ca2+ was slowly displaced with a rate constant similar to that of EP decomposition. Similar results were obtained for the enzyme treated with N-ethylmaleimide, which inhibits the step of conversion of ADP-sensitive EP to the ADP-insensitive one. Under the conditions where most EP was ADP insensitive at steady state (58 microM Ca2+, 30 microM EGTA, and 20 mM MgCl2 at pH 8.8 and 0 degrees C), the amount of bound Ca2+ increased slightly, then decreased slowly by 1 mol per mol of EP formed after addition of ATP. Under the conditions where about a half of EP was ADP sensitive (58 microM Ca2+, 25 microM EGTA, and 1 mM MgCl2 at pH 8.8 and 0 degrees C), the amount of bound Ca2+ did not change upon addition of ATP. These findings suggest that the Ca2+ bound to the enzyme becomes unremovable by EGTA upon formation of ADP-sensitive EP and is released upon its conversion to ADP-insensitive EP.     

Purification and characterization of a Ca2+- or Mg2+-stimulated ATPase from plasma membrane enriched fractions of Dictyostelium discoideum

Evidence is presented for the presence of both diethylstilbestrol (DES)-sensitive and DES-insensitive Mg2+-ATPase activities in plasma membrane enriched fractions of Dictyostelium discoideum. When removed from the membrane, the DES-sensitive activity is markedly less stable than the DES-insensitive activity, and the two activities display a number of quite distinct properties. The DES-sensitive enzyme has a decided preference for Mg2+ over Ca2+, displays saturation kinetics in response to ATP as substrate (Km = 0.2 mM) and has a narrow pH optimum range. In contrast, the DES-insensitive activity is stimulated equally by Mg2+ or Ca2+, is not saturable by ATP within the mM concentration range and has a much broader pH optimum. The DES-insensitive activity has been purified extensively. The purified enzyme is inhibited by vanadate and fluoride, but is insensitive to N,N'-dicyclohexylcarbodiimide (DCCD), N-ethylmaleimide and thimerosal. In the absence of divalent cations, the enzyme displays a sigmoidal activity curve in response to substrate concentration, which is abolished by addition of either Mg2+ or Ca2+, suggesting a binding site for a divalent cation and a positive cooperative interaction. The enzyme is capable of hydrolyzing other nucleotide triphosphates and ADP, but is without activity on AMP, p-nitrophenyl phosphate and pyrophosphate. The enzyme has an apparent molecular weight of approximately 64,000.     

ATP-dependent Ca2+-uptake by the plasma membrane fraction of the myometrium

The specific activities of Mg2+, Ca2+-ATPase in the plasma membrane fraction of rabbit and cattle myometrium are 8.30 +/- 0.80 and 2.36 +/- 0.48 mkmoles of Pi per mg of protein, respectively. This fraction possesses a higher (in comparison with other subcellular fractions) capacity for ATP-dependent uptake of 45Ca2+ (9.37 +/- 1.66 and 6.86 +/- 0.96 nmoles of 45Ca2+ per mg of protein in 15 min for rabbit and cattle myometrium, respectively); the ratio of ATP-dependent uptake of Ca2+ to adsorbed Ca2+ is also high. Phosphate increases Ca2+ uptake in the presence of ATP and Mg2+. The ionophore A-23187 added to the incubation mixture without ATP and Mg2+ sharply increases Ca2+ binding. An addition of the ionophore at the 15th min of the ATP-dependent Ca2+ uptake causes a complete and rapid release of the accumulated Ca2+. The release of Ca2+ can be also caused by an addition of Na-DS or EGTA to the incubation mixture. This suggests that Ca2+ is accumulated through the plasma membrane inside the closed structures. It was assumed that myometrial sarcolemma plays an essential role in regulation of intracellular Ca2+ concentration in the uterus at rest and that the active Ca2+ efflux from the cells is controlled by the Mg2+, Ca2+-ATPase system.     

Isolation of plasma membrane vesicles from rabbit skeletal muscle and their use in ion transport studies

A method has been developed for the isolation of sealed plasma membrane vesicles from rabbit white skeletal muscle. The final preparation was highly purified as indicated by enrichment of plasma membrane marker enzymes (i.e. ouabain-sensitive (Na+,K+)-ATPase, adenylate cyclase, and acetylcholinesterase). The absence of sarcoplasmic reticulum and mitochondria as contaminants was indicated by the low specific activity of marker enzymes, i.e. Ca2+-ATPase, succinate-cytochrome c reductase, and monoamine oxidase. Thin section and negative staining electron microscopy confirmed the absence of sarcoplasmic reticulum and mitochondrial contamination. The plasma membrane preparation consisted largely of sealed vesicles as observed by electron microscopy and as also demonstrated by latency of enzymic activities, which were unmasked by preincubation with detergent (sodium dodecyl sulfate). Membrane sidedness was estimated from latency of ouabain-sensitive (Na+,K+)-ATPase activity and acetylcholinesterase activity. The latency studies suggest that most of the vesicles are oriented inside out with respect to the orientation of the sarcolemma membrane in the muscle fiber. The inside-out plasma membrane vesicles actively accumulated sodium ions upon addition of ATP. The sodium ions were concentrated greater than 8-fold inside the vesicles and were released upon addition of the ionophore monensin. The sodium ions were taken up in the presence of K+ or NH4+ but not of choline. Uptake was inhibited by low concentrations of vanadate or digitoxin. The Na+ uptake was concomitant with Rb+ efflux. Therefore, the sodium ion transport and the resulting gradients formed appear to have been generated by the ouabain-sensitive (Na+,K+)-ATPase. Batrachotoxin, which opens Na+ channels in excitable tissues, prevents most of the Na+ uptake, suggesting the presence of toxin-activated Na+ channels in these plasma membrane vesicles.     

Calcium regulation by lens plasma membrane vesicles

The role of the plasma membrane in the regulation of lens fiber cell cytosolic Ca2+ concentration has been examined using a vesicular preparation derived from calf lenses. Calcium accumulation by these vesicles was ATP dependent, and was releasable by the ionophore A23187, indicating that calcium was transported into a vesicular space. Calcium accumulation was stimulated by Ca2+ (K1/2 = 0.08 microM Ca2+) potassium (maximally at 50 mM K+), and cAMP-dependent protein kinase; it was inhibited by both vanadate (IC50 = 5 microM) and the calmodulin inhibitor R24571 (IC50 = 5 microM), indicating that this pump was plasma-membrane derived and likely calmodulin dependent. Valinomycin, in the presence of K+, stimulated calcium uptake, suggesting that the calcium pump either countertransports K+, or is regulated in an electrogenic fashion. Inhibition of calcium uptake by selenite and p-chloromercuribenzoate demonstrates the presence of an essential -SH group(s) in this enzyme. Calcium release from calcium-filled lens vesicles was enhanced by Na+, demonstrating that these vesicles also contain a Na:Ca exchange carrier. p-Chloromercuribenzoate and p-chloromercuribenzoate sulfonic acid also promoted calcium release from calcium-filled vesicles, suggesting that this release, like calcium uptake, is in part mediated by a cysteine-containing protein. We conclude that lens fiber cell cytosolic Ca2+ concentration could be regulated by a number of plasma membrane processes. The sensitivity of both calcium uptake and release to -SH reagents has implications in lens cataract formation, where oxidation of lens proteins has been proposed to account for the elevated cytosolic Ca2+ in this condition.     

Adenosine 5''-triphosphate synthesis energized by an artificially imposed membrane potential in membrane vesicles of Escherichia coli.

Adenosine 5'-triphosphate (ATP) synthesis driven by an artificially imposed membrane potential in right-side-out membrane vesicles of Escherichia coli was investigated. Membrane vesicles prepared in the presence of adenosine diphosphate were loaded with K+ by incubation with 0.5 M potassium phosphate. Addition of valinomycin resulted in the synthesis of 0.2 to 0.3 nmol of ATP/mg of membrane protein, whereas no synthesis was observed after addition of nigericin. Addition of K+, dicyclohexylcarbodiimide, carbonylcyanide p-trifluoromethoxyphenylhydrazone, or azide to the assay buffer inhibited ATP synthesis. Adenosine diphosphate and Mg2+ were found to be required. Ca2+, which can replace Mg2+ for the hydrolytic activity of the Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3), could not replace Mg2+ in the synthetic reaction and, in fact, inhibited ATP synthesis even in the presence of Mg2+. Strain NR-70, a mutant lacking the Mg2+-ATPase, was unable to synthesize ATP using an artificially imposed membrane potential. Additionally, the Mg2+-ATPase was found to contain tightly bound ATP.