Dissociation between Ca2+-ATPase and alkaline phosphatase activities in plasma membranes of rat duodenum

The presence of Ca²⁺-ATPase activities with high-affinity sites for Ca²⁺ in brush border as well as basolateral plasma membranes of rat duodenal epithelium has been reported previously (Ghijsen, W.E.J.M. and van Os, C.H. (1979) Nature 279, 802–803). Since both plasma membranes contain alkaline phosphatase (EC 3.1.3.1), which also can be stimulated by Ca²⁺, the substrate specificity of Ca²⁺-induced ATP-hydrolysis has been studied to determine whether or not alkaline phosphatase and Ca²⁺-ATPase are two distinct enzymes. In basolateral fragments, the rate of Ca²⁺-dependent ATP-hydrolysis was greater than that of ADP, AMP and $\text{p-}\text{nitrophenylphosphate}$ at Ca²⁺ concentrations below 25 μM. At 0.2 mM Ca²⁺ the rates of ATP, ADP, AMP and $\text{p-}\text{nitrophenylphosphate}$ hydrolysis were not significantly different. In brush border fragments the rates of ATP, ADP and AMP hydrolysis were identical at low Ca²⁺, but at 0.2 mM Ca²⁺, Ca²⁺-induced hydrolysis of ADP and AMP was greater than either ATP or $\text{p-}\text{nitrophenylphosphate}$. Alkaline phosphatase in brush border and basolateral membranes was inhibited by 75% after addition of 2.5 mM theophylline. Ca²⁺-stimulated ATP hydrolysis at 1 μM Ca²⁺ was not sensitive to theophylline in basolateral fragments while the same activity in brush border fragments was totally inhibited. At 0.2 mM Ca²⁺, Ca²⁺-induced ATP hydrolysis in both basolateral and brush border membranes was sensitive to theophylline. Oligomycin and azide had no effect on Ca²⁺-stimulated ATP hydrolysis, either at low or at high Ca²⁺ concentrations. Chlorpromazine fully inhibited Ca²⁺-stimulated ATP hydrolysis in basolateral fragments at 5 μM Ca²⁺, while it had no effect in brush border fragments. From these results we conclude that, (i) Ca²⁺-ATPase and alkaline phosphatase are two distinct enzymes, (ii) high-affinity Ca²⁺-ATPase is exclusively located in basolateral plasma membranes, (iii) alkaline phosphatase activity, present on both sides of duodenal epithelium, is stimulated slightly by low Ca²⁺ concentrations, but this Ca²⁺-induced activity is inhibited by theophylline and shows no specificity with respect to ATP, ADP or AMP.     

Ca2+ displacement by polymyxin B from sarcolemma isolated by ‘gas dissection’ from cultured neonatal rat myocardial cells

Amphiphilic, cationic Polymyxin B is shown to displace Ca²⁺ from ‘gas dissected’ cardiac sarcolemma in a dose-dependent, saturable fashion. The Ca²⁺ displacement is only partially reversible, 57% and 63%, in the presence of 1 mM or 10 mM Ca²⁺, respectively. Total Ca²⁺ displaced by a non-specific cationic probe, lanthanum (La³⁺), at maximal displacing concentration (1 mM) was $\text{0.172 ± 0.02}\text{nmol/μg}$ membrane protein. At 0.1 mM, Polymyxin B displaced 42% of the total La³⁺-displaceable Ca²⁺ or $\text{0.072 ± 0.01}\text{nmol/μg}$ protein. 5 mM Polymyxin displaced Ca²⁺ in amounts equal to those displaced by 1 mM La³⁺. Pretreatment of the membranes with neuraminidase (removal of sialic acid) and protease leads to a decrease in La³⁺-displaceable Ca²⁺ but to an increase in the fraction displaced by 0.1 mM Polymyxin from 42% to 54%. Phospholipase D (cabbage) treatment significantly increased the La³⁺-displaceable Ca²⁺ to $\text{0.227 ± 0.02}\text{nmol/μg}$ protein ($\text{P < 0.05}$), a gain of 0.055 nmol. All of this phospholipid specific increment in bound Ca²⁺ was displaced by 0.1 mM Polymyxin B. The results suggest that Polymyxin B will be useful as a probe for phospholipid Ca²⁺-binding sites in natural membranes.     

Unsaturated diacylglycerol as a possible messenger for the activation of calcium-activated, phospholipid-dependent protein kinase system

A small quantity of unsaturated diacylglycerol (DG) sharply decreased the Ca²⁺ and phospholipid concentrations needed for full activation of a Ca²⁺-activated, phospholipid-dependent multifunctional protein kinase described earlier (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T. and Nishizuka, Y. (1979)$\text{J.}\text{Biol.}\text{Chem.}\text{254}$. 3692–3695). In the presence of unsaturated DG and micromolar order of Ca²⁺, phosphatidylserine (PS) was most relevant with the capacity to activate the enzyme, whereas phosphatidylethanolamine and phosphatidylinositol (PI) were far less effective. Phosphatidylcholine was practically inactive. It is possible, therefore, that unsaturated DG, which may be derived from PI turnover provoked by various extracellular stimulators, acts as a messenger for activating the enzyme, and that Ca²⁺ and various phospholipids such as PI and PS seem to play a role cooperatively in this unique receptor mechanism.     

The role of the sites for ATP of the Ca2+-ATPase from human red cell membranes during Ca2+-phosphatase activity

1. In the presence of ATP, the Ca²⁺ pump of human red cell membranes catalyzes the hydrolysis of $\text{p-}\text{nitrophenyl}$ phosphate. The requirement for ATP of the Ca²⁺-$\text{p-}\text{nitrophenylphosphatase}$ activity was studied in relation to the two classes of site for ATP that are apparent during Ca²⁺ -ATPase activity. 2. (a) The $\text{K}{}_{0.5}$ for ATP as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ extrapolated at 0 mM PNPP is equal to the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of the Ca²⁺ -ATPase. (b) PNPP competes with ATP and its effectiveness is the same regardless the nucleotide acts as the substrate of the Ca²⁺ -ATPase or as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$. 3. PNPP at the high-affinity site does not substitute for ATP as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$. 4. At ATP concentrations that almost saturate the high-affinity site, Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity increases as a function of PNPP along an S-shaped curve, while Ca²⁺ -ATPase activity is partially inhibited along a curve of the same shape and apparent affinity. The fraction of Ca²⁺ -ATPase activity which is inhibited by PNPP is that which results from occupation of the low-affinity site by ATP. 5. Activation of the Ca²⁺ -ATPase by ATP at the low-affinity site is associated with inhibition of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity. Both phenomena take place with the same apparent affinity and along curves of the same shape. 6. Experimental results suggest that: (a) the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity depends on ATP at the high-affinity site; (b) PNPP is hydrolyzed at the low-affinity site; (c) Ca²⁺ -ATPase activity at the high-affinity size persists during Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity.     

Effects of lanthanum on calcium-dependent phenomena in human red cells

Lanthanum (0.25 mM) does not penetrate into fresh or Mg²⁺-depleted cells, whereas it does into ATP-depleted or $\text{ATP + 2,3-diphosphoglycerate-depleted cells}$, into cells containing more than 3 mM calcium, or cells stored for more than 4 weeks in acid/citrate/dextrose solution. In fresh cells loaded with calcium, extracellular lanthanum blocks the active Ca²⁺-efflux completely and inhibits $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ (ATP phosphohydrolase, EC 3.6.1.3) activity to about 50%. In Mg²⁺-depleted cells Ca²⁺-Ca²⁺ exchange is inhibited by lanthanum. Ca²⁺-leak is unaffected by lanthanum up to 0.25 mM concentration; higher lanthanum concentrations reduce leak rate. In NaCl medium Ca²⁺-leak ± S.D. amounts to $\text{0.28 ± 0.08 μ}\text{mol/l}$ of cells per min, whereas in KCl medium to $\text{0.15 ± 0.04 μ}\text{mol/l}$ of cells per min at 2.5 mM [Ca²⁺]_e and 0.25 mM [La³⁺]_e pH 7.1.Lanthanum inhibits Ca²⁺-dependent rapid K⁺ transport in ATP-depleted and propranolol-treated red cells, i.e. whenever intracellular calcium is below a critical level. The inhibition of the rapid K⁺ transport can be attributed to protein-lanthanum interactions on the cell surface, since lanthanum is effectively detached from the membrane lipids by propranolol.Lanthanum at 0.2–0.25 mM concentration has no direct effect on the morphology of red cells. The shape regeneration of Ca²⁺-loaded cells, however, is blocked by lanthanum owing to Ca²⁺-pump inhibition. Using lanthanum the transition in cell shape can be quantitatively correlated to intracellular Ca²⁺ concentrations.     

Transport parameters and stoichiometry of active calcium ion extrusion in intact human red cells

Ca²⁺-transport and its energy consumption were studied in intact human red cells loaded with Ca²⁺ by the aid of the ionophore A23187.After the complete elimination of the ionophore the passive Ca²⁺-permeability of the membrane returned to its normal low value, except when the intracellular Ca²⁺-concentration was higher than 3 mM or the ATP level fell below 100 μM. Within these limits the rate of Ca²⁺-extrusion was independent of the cellular ATP content but was greatly enhanced by increasing [Ca²⁺]_i and reached a plateau at about 1 mM intracellular Ca²⁺-concentration. The maximum rate of Ca²⁺-efflux was about 85 μmol/l of cells per min at 37°C, pH 7.4. The activation energy of active Ca²⁺-extrusion was found to be 15 200 cal/mol, and the optimum pH in the suspension was 7.7.Ca²⁺-efflux was not connected with the counter-transport of cations.The Ca²⁺-pump was not affected by ouabain or oligomycin and only partial inhibition could be achieved by the SH-reagents: ethacrynic acid, $\text{N-}\text{ethylmaleimide}$ and $\text{p-}\text{chloromercuribenzoate}$ or with propranolol and ruthenium red. An 80 to 95% inhibition of the active Ca²⁺-extrusion was brought about by 50–250 μM lanthanum, which in the above concentrations caused no aggregation or haemolysis. The inhibition of the Ca²⁺-pump by lanthanum was found to be reversible, the site of inhibition being at the external surface of the cell membrane.To examine the energy consumption of the Ca²⁺-extrusion, ATPase activity was assessed by measuring inorganic phosphate liberation in Ca²⁺-loaded red cells the metabolism of which was inhibited by iodoacetamide + Na⁺-tetrathionate. Ca²⁺-activated ATPase activity connected with the Ca²⁺-pump was distinguished from other Ca²⁺-ATPase by using the non-penetrating inhibitor, lanthanum. The molar ratio of Ca²⁺-transported per ATP split was found to be $\text{2 : 1}$.     

Modulation of lyso-platelet-activating factor:acetyl-CoA acetyltransferase from rat splenic microsomes. The role of calcium ions

The enzyme lyso-platelet-activating factor:acetyl-CoA acetyltransferase (EC 2.3.1.67) was assayed in microsomal fractions from rat spleens. The addition of micromolar Ca²⁺ rapidly enhanced acetyltransferase activity and this activation was reversed by the addition of EGTA in excess of Ca²⁺. The effect of Ca²⁺ was on the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of the enzyme for the substrate acetyl-CoA without showing any significant effect on the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the acetylation reaction. When microsomes were isolated in the presence of 5 mM EGTA, to remove endogenous calmodulin, the same enhancing effect of Ca²⁺ on the acetylation reaction was observed. The addition of exogenous calmodulin to this preparation had no effect on the enzyme activity. Preincubation of spleen microsomes with the calmodulin inhibitor trifluoperazine decreased acetyltransferase in both the presence and the absence of Ca²⁺, indicating an effect of this drug independently of calmodulin. The addition of Mg-ATP to the assay mixture also had no effect on the acetylation reaction. These data suggest that Ca²⁺ modulates acetyltransferase activity from rat spleen microsomes by a mechanism that seems to be independent of calmodulin or protein phosphorylation.     

Relationship between calcium ion transport and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

Calcium uptake by adipocyte endoplasmic reticulum was studied in a rapidly obtained microsomal fraction. The kinetics and ionic requirements of Ca²⁺ transport in this preparation were characterized and compared to those of (Ca²⁺ + Mg²⁺)-ATPase activity. The time course of Ca²⁺ uptake in the presence of 5 mM oxalate was nonlinear, approaching a steady-state level of 10.8–11.5 nmol Ca²⁺/mg protein after 3–4 min of incubation. The rate of Ca²⁺ transport was increased by higher oxalate concentrations with a near linear rate of uptake at 20 mM oxalate. The calculated initial rate of calcium uptake was 18.5 nmol Ca²⁺/mg protein per min. The double reciprocal plot of ATP concentration against transport rate was nonlinear, with apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values of 100 μM and 7 μM for ATP concentration ranges above and below 50 μM, respectively. The apparent $\text{K}\text{m}$ values for Mg²⁺ and Ca²⁺ were 132 μM and 0.36–0.67 μM, respectively. The energy of activation was 23.4 kcal/mol. These kinetic properties were strikingly similar to those of the microsomal ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}$)-ATPase. The presence of potassium was required for maximum Ca²⁺ transport activity. The order of effectiveness of monovalent cations in stimulating both Ca²⁺ transport and ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{-}\text{ATPase}$ activity was $\text{K}{}^{\mathrm{+}}\text{>}\text{Na}{}^{\mathrm{+}}\text{=}\text{NH}{}_4{}^{\mathrm{+}}\text{>}\text{Li}{}^{\mathrm{+}}\text{.}\text{Ca}{}^{\mathrm{2+}}$ transport and ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ activity were both inhibited 10–20% by 6 mM procaine and less than 10% by 10 mM sodium azide. Both processes were completely inhibited by 3 mM dibucaine or 50 μM $\text{p-}\text{chloromercuribenzene}$ sulfonate. The results indicate that Ca²⁺ transport in adipocyte endoplasmic reticulum is mediated by a $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ and suggest an important role for endoplasmic reticulum in control of intracellular Ca²⁺ distribution.     

Influence of monovalent ions on the activity of the (Ca2+ + Mg2+)-ATPase and Ca2+-transport of human red blood cells

In reconstituted human red blood cells a difference was found in (Ca²⁺ + Mg²⁺)-ATPase activity and in Ca²⁺ efflux at 37°C, depending on the side of the membrane at which the monovalent cations K⁺ and Na⁺ were placed. Under the conditions used, (Ca²⁺ + Mg²⁺)-ATPase activity and Ca²⁺ efflux was highest when K⁺ ($\text{35 ± 0.5}\text{mM (± S.E.)}$, mean of four experiments) was at the inside and Na⁺ (130 mM) at the outside of the ghost membrane.     

Calcium uptake by placental plasma membrane vesicles

The Placental plasma membrane vesicles are capable of accumulating up to 190 mM Ca²⁺. This is 24-fold higher than the external Ca²⁺ concentration.This process is dependent on ATP hydrolysis by the placental Ca²⁺-ATPase.The $\text{P}{}_{\mathrm{i}}\text{Ca}$ ratio is dependent on the external Ca²⁺ concentration, and reaches the value of 2 at 10 mM Ca²⁺.Phosphate (5 mM) can double Ca²⁺ uptake when measured in the presence of 5 mM Ca²⁺.Mg²⁺; increased Ca²⁺ uptake only at low Ca²⁺ concentrations, and had no significant effect at 5 mM Ca²⁺.     

Effects of calcium on a parathyroid hormone-sensitive adenylate cyclase inhibitor

Inhibition of parathyroid hormone (PTH)-sensitive adenylate cyclase by {Nle-8, Nle-18, Tyr-34} bPTH-(3–34) amide was studied in thyroparathyroid-ectomized dogs. The inhibitory effect was shown to be markedly enhanced by the addition of calcium ions into the $\text{in}\text{,}\text{vitro}$ assay system. At 0.1 mM Ca²⁺, complete inhibition by the antagonist was obtained. Chelation of exogenous Ca²⁺ by EGTA eliminated the Ca²⁺-induced inhibition. Both the basal and hormone-stimulated activities were decreased in the presence of 0.1 mM Ca²⁺, whereas the addition of EGTA increased both activities. Our results suggest that Ca²⁺ modulates canine renal PTH-sensitive adenylate cyclase and its inhibition by substituted bPTH-(3–34).     

Permeability characteristics of erythrocyte ghosts prepared under isoionic conditions by a glycol-induced osmotic lysis

A detailed study has been made of the permeability characteristics of human erythrocyte ghosts prepared under isoionic conditions by a glycol-induced lysis (Billah, M.M., Finean, J.B., Coleman, R. and Michell, R.H. (1976) Biochim. Biophys. Acta 433, 45–54). Impermeability to large molecules such as dextran (average molecular weight 70 000) was restored immediately and spontaneously after each of the 5–7 lyses that were required to remove all of the haemoglobin. Permeabilities to smaller molecules such as MgATP^2?, [³H]inositol and [¹⁴C]choline were initially high but could be greatly reduced by incubation at 37°C for an hour. The extent of such resealing decreased as the number of lyses to which the ghosts had been subjected increased. Both removal of haemoglobin and permeabilities to small molecules were affected significantly by pH, Ca²⁺ concentrations and divalent cation chelators. Maximum resealing was achieved in ghosts prepared in the basic ionic medium (130 mM KCl, 10 mM NaCl, 2 mM MgCl₂, 10 mM $\text{N-2-}\text{hydroxyethylpiperazine}\text{-N′-2-}\text{ethanesulphonic}$ acid (HEPES)) at pH 7.0 (0°C) and with a calcium level around 10^?5 M. Acidic pH facilitated the removal of haemoglobin whilst the presence of divalent cation chelators slowed down its release. Retention of K⁺ by ghosts loaded with K⁺ during the first lysis and subsequently incubated at 37° C was substantial but little K⁺ could be retained within the haemoglobin-free ghosts. Permeability of the ghosts to K⁺ after one lysis was affected by temperature, pH, Ca²⁺ concentrations and by the presence of divalent cation chelators.     

Activation by a calcium-binding protein of guanylate cyclase in Tetrahymena pyriformis.

A Ca²⁺-binding protein (TCBP), which was isolated from $\text{Tetrahymena pyriformis}$, enhanced about 20-fold particulate-bound guanylate cyclase activity in $\text{Tetrahymena}$ cells in the presence of a low concentration of Ca²⁺, while the adenylate cyclase activity was not increased. The enhancement was eliminated by ethylene glycol-bis (β-aminoethyl ether)-N,N′-tetraacetic acid. The enzyme activity was not stimulated by rabbit skeletal muscle troponin-C, the Ca²⁺-binding component of troponin, or other some proteins. In the presence of TCBP, stimulating effect of calcium ion on the enzyme activity was observed within the range of pCa 6.0 to 4.6, and was immediate and reversible.     

State of translocated Ca2+ by sarcoplasmic reticulum inferred from kinetic analysis of calcium oxalate precipitation

Uptake of Ca²⁺ by sarcoplasmic reticulum in the presence of oxalate displays biphasic kinetics. An initial phase of normal uptake is followed by a second phase coincident with precipitation of calcium oxalate inside the vesicles. The precipitation rate induced by actively transported Ca²⁺ is depressed by increasing the added Ca²⁺ concentration. This correlates linearly with the reciprocal of precipitation rate. Therefore, a maximal limit rate could be extrapolated at zero Ca²⁺ ($\text{V}{}_0$). The rate of precipitation, also a function of added amount protein, gives a linear correlation in a double reciprocal plot. Thus, it was possible to estimate the maximal precipitation rate occurring at infinite protein concentration ($\text{V}{}^{\mathrm{\infty }}$). With the combined extrapolated values a maximal expected precipitation rate could be calculated ($\text{V}{}_0{}^{\mathrm{\infty }}$). Kinetics of calcium oxalate precipitation was studied in the absence of calcium uptake and empirical equations relating the rate of precipitation with the added Ca²⁺ were established. Entering $\text{V}{}_0{}^{\mathrm{\infty }}$ in the equations, an internal free Ca²⁺ concentration of approx. 2.5 mM was estimated. Additionally, it is shown that the ionophore X-537A does not supress the Ca²⁺ uptake, if added during the oxalate-dependent phase, albeit the uptake proceeds at a slower rate after the release of approx. 70 nmol Ca²⁺/mg protein. This amount presumably equals the internal free Ca²⁺ not sequestered by oxalate, producing a maximal concentration approx. 14 mM. Taking into account low affinity binding of internal binding sites and the transmembrane Ca²⁺ gradients built up during the uptake of Ca²⁺, values of free Ca²⁺ ranging from 3 to 6 mM, approaching those estimated by the precipitation analysis, could be estimated.     

Ouabain-insensitive Na+-stimulated ATPase activity of basolateral plasma membranes from guinea-pig kidney cortex cells: II. Effect of Ca2+

The ouabain-insensitive, Mg²⁺-dependent, Na⁺-stimulated ATPase activity present in fresh basolateral plasma membranes from guinea-pig kidney cortex cells (prepared at pH 7.2) can be increased by the addition of micromolar concentrations of Ca²⁺ to the assay medium. The Ca²⁺ involved in this effect seems to be associated with the membranes in two different ways: as a labile component, which can be quickly and easily ‘deactivated’ by reducing the free Ca²⁺ concentration of the assay medium to values lower than 1 μM; and as a stable component, which can be ‘deactivated’ by preincubating the membranes for periods of 3–4 h with 2 mM EDTA or EGTA. Both components are easily activated by micromolar concentrations of Ca²⁺. The $\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ of the system for Na⁺ is the same, 8 mM, whether only the stable component or both components, stable and labile, are working. In other words, the activating effect of Ca²⁺ on the Na⁺-stimulated ATPase is on the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$, and not on the $\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ of the system for Na⁺. The activating effect of Ca²⁺ may be related to some conformational change produced by the interaction of this ion with the membranes, since it can also be obtained by resuspending the membranes at pH 7.8 or by ageing the preparations. Changes in the Ca²⁺ concentration may modulate the ouabain-insensitive, Na⁺-stimulated ATPase activity. This modulation could regulate the magnitude of the extrusion of Na⁺ accompanied by Cl^? and water that these cells show, and to which the Na⁺-ATPase has been associated as being responsible for the energy supply of this mode of Na⁺ extrusion.     

Further characterization of Ca2+-activated phospholipase A2 in cat adrenal cortex

When cat adrenocortical cells were incubated with exogenous phospholipid substrate (autoclaved $\text{E.}$$\text{coli}$) in the presence of corticotropin, there was a Ca²⁺-dependent increase in phospholipid breakdown activity, suggesting that a hormone-stimulated phospholipase is localized to the plasma membrane. Phospholipase activity in a particulate fraction from lysed cells at neutral pH was a function of the Ca²⁺ concentration. The addition of increasing Ca²⁺ concentrations to a subcellular fraction of lysed cells which had been prelabelled with [¹⁴C]arachidonic acid produced graded increases in fatty acid release. A depletion of label from phosphatidylcholine was observed, as well as a marked increase in radioactivity associated with phosphatidylethanolamine. The subcellular fraction of cells prelabelled with [¹⁴C]palmitic acid failed to release fatty acid in response to Ca²⁺, although a loss of label from phosphatidylcholine and a modest gain in label by phosphatidylethanolamine was demonstrable. A Ca²⁺-activated deacylation-reacylation reaction preferentially involving phosphatidylethanolamine was evident in cortical cells prelabelled with archidonic acid; whereas, other Ca²⁺-stimulated lipolytic reactions also appeared to be operative in cells prelabelled with either arachidonic or palmitic acid. The Ca²⁺-dependent mobilization of arachidonic acid from an endogenous phospholipid pool lends additional support to the idea that Ca²⁺-mediated activation of phospholipase A₂ participates in the control of adrenocortical activity. However, since Ca²⁺ also stimulated arachidonic acid liberation from cortical triglycerides, these lipid moieties may also contribute to the observed effects of Ca²⁺ on fatty acid release.     

The significance of physiological [Ca2+] and [Mg2+] for in vitro experiments on synaptic transmission

Since 1932 $\text{in}$$\text{vitro}$ physiological and pharmacological studies on neuromuscular and other types of synaptic transmission have been carried out usually in Krebs' of similar balanced electrolyte solutions. It has been disregarded, however, that although the total calcium [Ca_t] (2.5 mM) and [Mg_t] (1.2 mM), are about the same in human plasma and in Krebs' solution, the physiologically important [Ca²⁺] and [Mg²⁺], primarily because of binding to plasma proteins, are much lower in plasma (1.1 and 0.6 mM) than in Krebs' solution (2.0 and 1.1 mM). We observed that in a modified Krebs' solution in which the [Ca_t] and [Mg_t] are 1.4 and 0.9 mM respectively and the [Ca²⁺] and [Mg²⁺] are about the same as in human plasma, the Ca²⁺ dependent volley output of acetylcholine is less and the inhibition of the electrically induced isometric twitch tension of the rat phrenic nerve - hemidiaphragm preparation by nondepolarizing neuromuscular blocking agents and certain antibiotics is greater than in conventional Krebs' solution, in which the [Ca²⁺] and [Mg²⁺] are higher than $\text{in}$$\text{vivo}$. Similarly, during electrical field stimulation of the guinea-pig myenteric plexus - longitudinal muscle preparation volley output of acetylcholine is lower and the inhibition of the isometric contraction of the muscle by normophine is greater in modified than in conventional Krebs' solution. It is suggested that for greater relevance to $\text{in}$$\text{vivo}$ conditions the [Ca²⁺] and [Mg²⁺] of balanced electrolyte solutions used in in vitro experiments on synaptic transmission should be the same as in human plasma or in the plasma of the species of the experimental animal.     

Interactions of La3+ with phosphatidylserine vesicles Binding,phase transition,leakage, 31P-NMR and fusion

The interaction of La³⁺ with phosphatidylserine vesicles is studied by differential scanning calorimetry, ¹⁴⁰La binding, ³¹P-NMR chemical shifts and relaxation rates, carboxyfluorescein and [¹⁴C]sucrose release, X-ray diffraction and freeze-fracture electron microscopy. In the presence of La³⁺ concentrations above 1 mM and an incubation temperature of 38°C, i.e., at the phase transition temperature of the complex La/phosphatidylserine, the binding ratio of La/lipid exceeds a $\text{1}\text{3}$ ratio, reaching saturation at a $\text{1}\text{2}$ ratio. Analysis, employing a modified Gouy-Chapman equation, indicates a significant increase in the intrinsic binding constant of La/phosphatidylserine when the La³⁺ concentration exceeds the threshold concentration for leakage. The analysis illustrates that at the molecular level the binding of La³⁺ can be comparable to or even weaker than that of Ca²⁺, but that even when present at smaller concentrations La³⁺ competes with and partially displaces Ca²⁺ from membranes or other negatively charged surfaces. The results suggest that the sequence La³⁺>Ca²⁺>Mg²⁺ reflects both the binding strength of these cations to phosphatidylserine as well as their ability to induce leakage, enhancement of ³¹P spin-lattice relaxation rates, fusion and other structural changes. The leakage, fusion, and other structural changes are more pronounced at the phase transition temperature of the La/lipid complex.     

Ca2+-binding properties of a unique ATPase inhibitor protein isolated from mitochondria of bovine heart and rat skeletal muscle

Previous studies showed that Ca²⁺ induced monomer to active dimer interconversion of a mitochondrial ATPase inhibitor protein from bovine heart or rat skeletal muscle (Yamada, E.W., Huzel, N.J. and Dickison, J.C. (1981) J. Biol. Chem. $\text{256}$, 10203–10207). Initial equilibrium dialysis measurements of Ca²⁺ binding showed that this unique protein possesses three binding sites of high affinity with a maximum of one mol of Ca²⁺ bound/mol of protein monomer. Magnesium (1 mM) did not affect the first association constant but increased the second and third by about 1.2 and 1.5 fold, respectively. That the apparent association constants varied with concentration of protein monomer was in agreement with the self-associating nature of the protein. Scatchard plots at three concentrations of protein intersected at a molar ratio of about 0.5 ($\text{Ca}{}^{\mathrm{2+}}\text{monomer}$). K_a1 and K_a2 values of 4.2 μM and 12.1 μM, respectively, were estimated by extrapolation of apparent constants to infinite dilution of protein. K_a3 (51.3 μM) was estimated by extrapolation of double reciprocal plots of apparent constants versus protein concentration to infinite levels of protein. A model for Ca²⁺ binding by this self-associating protein is described. Trifluoperazine had no effect on the activity of the inhibitor protein from either tissue.