The ability of the mitochondrial Ca2+-binding glycoprotein to restore Ca2+ transport in glycoprotein-depleted rat liver mitochondria.

Rat liver mitochondria may be subfractionated in sediment and supernatant fractions by swelling in the presence of EDTA and oxaloacetate. The sediment is largely depleted of the Ca2+-binding glycoprotein and its Ca2+-transporting activity may be as low as 10--20% of the starting value. Both the rate of Ca2+ uptake and the capacity to maintain a high Ca2+ concentration gradient across the membrane are depressed. Addition of an osmotic supernatant to the assay mixture may partially restore the original Ca2+-transporting ability. The active component in the supernatant is the Ca2+-binding glycoprotein. This is shown by the following facts: (a) the effect is enhanced by the addition of the purified glycoprotein to the supernatant; (b) precipitation of the glycoprotein from the supernatant by affinity chromatography-purified antibodies abolishes the stimulatory effect, and (c) in the presence of 130 microM Mg2+, the glycoprotein alone may restore fully the Ca2+-transporting ability of the particles. The maximal velocity is already reached at 0.1 microgram glycoprotein/mg mitochondrial protein.     

Isolation of sarcoplasmic reticulum by zonal centrifugation and purification of Ca2+-pump and Ca2+-binding proteins

A procedure for the isolation of highly purified sarcoplasmic reticulum vesicles from rabbit skeletal muscle has been described using sucrose gradient centrifugation in zonal rotors. The yield of our purest fraction was 300 mg of sarcoplasmic reticulum protein using 1 kg muscle. The sarcoplasmic reticulum vesicles were relatively simple in composition. The Ca²⁺-pump protein accounted for most (approx. two-thirds) of the sarcoplasmic reticulum protein. Two other protein components, a Ca²⁺-binding protein and a M₅₅ protein (approx. 55 000 daltons) each accounted for about 5–10% of the protein. Enrichment in the level of phosphoenzyme by the Ca²⁺-pump protein was regarded as an important index of the purification of sarcoplasmic reticulum vesicles. The sarcoplasmic reticulum vesicles were capable of forming 6.4 nmoles of ³²P-labelled phosphoenzyme per mg protein and had a high capacity of energized Ca²⁺ uptake. The Ca²⁺-dependent formation of phosphoenzyme has been used to estimate the sarcoplasmic reticulum protein content in rabbit skeletal muscle and found to be about 2.5% of the total muscle protein.The Ca²⁺-pump and Ca²⁺-binding proteins were isolated with a purity of 90% or more by treating the purified sarcoplasmic reticulum vesicles with bile acids in the presence of salt. The solubilized Ca²⁺-pump protein reaggregated during dialysis together with phospholipid to form membranous vesicles which were capable of forming approx. 9 nmoles ³²P-labelled phosphoenzyme per mg protein. The Ca²⁺-binding protein was water soluble and contained a high percentage of acidic amino acids (35% of total residues).Ca²⁺ binding by sarcoplasmic reticulum vesicles and by the Ca²⁺-pump and Ca²⁺-binding proteins was studied by equilibrium dialysis. Sarcoplasmic reticulum vesicles and Ca²⁺-pump protein contained nonspecific high-affinity Ca²⁺ binding sites with a capacity of 90–100 and 55–70 nmoles Ca²⁺ per mg protein, respectively. Both of them specifically bound 10–15 nmoles Ca²⁺ per mg protein. The binding constants for nonspecific and specific Ca²⁺ binding by both preparations were approx. 1 μM^?1. The Ca²⁺-binding protein nonspecifically bound 900–1000 nmoles Ca²⁺ per mg protein with a binding constant of about 0.25 μM^?1.     

The rate of Ca2+ translocation by sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase measured with intravesicular arsenazo III

Release of Ca²⁺ from the (Ca²⁺ + Mg²⁺)-ATPase into the interior of intact sarcoplasmic reticulum vesicles was measured using arsenazo III, a metallochromic indicator of Ca²⁺. Arsenazo III was placed inside the sarcoplasmic reticulum vesicles by making the vesicles transiently leaky with an osmotic gradient in the presence of arsenazo III. External arsenazo III was then removed by centrifugation. Addition of ATP to the (Ca²⁺ + Mg²⁺)-ATPase in the presence of Ca²⁺ causes the rapid phosphorylation of the enzyme at which time the bound Ca²⁺ becomes inaccessible to external EGTA. The release of Ca²⁺ from the (Ca²⁺ + Mg²⁺)-ATPase to the interior of the vesicle measured with intravesicular arsenazo III was much slower indicating that there is an occluded from the Ca²⁺-binding site which precedes the release of Ca²⁺ into the vesicle. The rate of Ca²⁺ accumulation by sarcoplasmic reticulum vesicles is increased by K⁺ (5–100 mM) and ATP (50–1000 μM) but the initial rate of Ca²⁺ translocation measured after the simultaneous addition of ATP and EGTA to vesicles that were preincubated in Ca²⁺ was not influenced by these concentrations of K⁺ and ATP.     

Regulation of erythrocyte membrane shape by Ca2+

In the presence of 1.0 mM ATP and MgCl₂, the specific viscosity of suspensions of human erythrocyte ghosts decreases 35% in 20 minutes at 22°C. The changes in viscosity are a sensitive index of Mg-ATP dependent shape changes in these membranes. Low concentrations of Ca²⁺ (1 to 5 μM) inhibit Mg-ATP dependent viscosity changes. If ghosts were preincubated with 1 mM Mg-ATP and 20 μM A23187 to produce a maximal decrease in viscosity, addition of 10 μM Ca²⁺ to the preincubated ghosts increased the viscosity to levels observed in ghosts preincubated without ATP. Ca²⁺ (1 to 5 μM) also inhibited Mg²⁺ dependent phosphorylation 30% and stimulated dephosphorylation 25% in ghost membranes. These effects of Ca²⁺ on viscosity and phosphorylation may be due to a membrane bound Ca²⁺ phosphatase activity which dephosphorylates membranes phosphorylated by a Mg²⁺ dependent kinase activity.     

Na+-driven Ca2+ transport in alkalophilic Bacillus

Ca²⁺ transport was studied in membrane vesicles of alkalophilic Bacillus. When Na⁺-loaded membrane vesicles were suspended in KHCO₃/KOH buffer (pH 10) containing Ca²⁺, rapid uptake of Ca²⁺ was observed. The apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for Ca²⁺ measured at pH 10 was about 7 μM, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value shifted to 24 μM when measured at pH 7.4. The efflux of Ca²⁺ was studied with Ca²⁺-loaded vesicles. Ca²⁺ was released when Ca²⁺-loaded vesicles were suspended in medium containing 0.4 M Na⁺.Ca²⁺ was also transported in membrane vesicles driven by an artificial pH gradient and by a membrane potential generated by K⁺-valinomycin in the presence of Na⁺.These results indicate the presence of Ca²⁺/Na⁺ and H⁺/Na⁺ antiporters in the alkalophilic Bacillus A-007.     

Ca2+-ATPase cytochemistry in the spinal cord microvasculature of prenatal rats

Summary Membrane-bound Ca²⁺-ATPase activity was localized cytochemically in the blood vessels of the spinal cord of rat embryos to obtain a better understanding of the membrane activities of vascular cells.The cytochemical method revealed a growth of the parenchymal vasculature. In the parenchyma, reaction product was dense over the entire plasma membrane of voluminous endothelial cells provided with large nuclei and enriched cytoplasmic organelles, suggesting that the endothelial cells may be of a vascular sprout. The parenchymal vessels with a wide lumen were frequently associated with pericytes, and the Ca²⁺-ATPase activity was diminished in intensity on the luminal surface of the flattened endothelial cells. On the other hand, the endothelium of extraparenchymal capillaries exhibited Ca²⁺-ATPase activity primarily on the luminal surface of the plasma membrane. Quercetin, a Ca²⁺-transporting ATPase inhibitor, considerably decreased the abluminal activity in the voluminous endothelial cells with slit-like vascular lumen and the luminal activity of functioning capillary endothelium as well. Thus, a dual activity of Ca²⁺-ATPase, postulating for the activities of Ca²⁺-transporting ATPase and ecto-ATPase, was closely correlated with the maturation processes of the capillary endothelium.     

Ca2+-dependent phosphorylation of rat ovary proteins

A 105,000 × g supernatant fraction from prepubertal rat ovaries was incubated in the presence of [γ-³²P]ATP. Phosphorylated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and identified by autoradiography. Inclusion of Ca²⁺ in the phosphorylation reaction promoted a selective ³²p incorporation into two proteins of M_r = 95,000 and 50,000. Inclusion of chlorpromazine with Ca²⁺ blocked the Ca²⁺-stimulated increase of ³²p incorporation. Our results demonstrate the presence of Ca²⁺-stimulated protein phosphorylation system capable of recognizing endogenous substrate proteins in the prepubertal rat ovary.     

ATP and Ca2+ binding by the Ca2+ pump protein of sarcoplasmic reticulum

The binding of ATP and Ca²⁺ by the Ca²⁺ pump protein of sarcoplasmic reticulum from rabbit skeletal muscle has been studied and correlated with the formation of a phoshorylated intermediate. The Ca²⁺ pump protein has been found to contain one specific ATP and two specific Ca²⁺ binding sites per phosphorylation site. ATP binding is dependent on Mg²⁺ and is severely decreased when a phosphorylated intermediate is formed by the addition of Ca²⁺. In the presence of Mg²⁺ and the absence of Ca²⁺, ATP and ADP bind completely to the membrane. Pre-incubation with N-ethylmaleimide results in inhibition of ATP binding and decrease of Ca²⁺ binding. In the absence of ATP, Ca²⁺ binding is noncooperative at pH 6–7 and negatively cooperative at pH 8. Mg²⁺, Sr²⁺ and La³⁺, in that order, decrease Ca²⁺ binding by the Ca²⁺ pump protein. The affinity of the Ca²⁺ pump protein for both ATP and Ca²⁺ increases when the pH is raised from 6 to 8. At the infection point (pH ≈ 7.3) the binding constants of the Ca²⁺ pump protein-MgATP^2? and Ca²⁺ pump protein-calcium complexes are approx. 0.25 and 0.5 μM^?1, respectively. The unphosphorylated Ca²⁺ pump protein does not contain a Mg²⁺ binding site with an affinity comparable to those of the ATP and Ca²⁺ binding sites.The affinity of the Ca²⁺ pump protein for Ca²⁺ is not appreciably changed by the addition of ATP. The ratio of phosphorylated intermediate formed to bound Ca²⁺ is close to 2 over a 5-fold range of phosphoenzyme concentration. The equilibrium constant for phosphoenzyme formation is less than one at saturating levels of Ca²⁺. The phosphoenzyme is thus a “high-energy” intermediate, whose energy may then be used for the translocation of the two Ca²⁺.A reaction scheme is discussed showing that phosphorylation of sarcoplasmic reticulum proceeds via an enzyme-Ca₂²⁺-MgATP^2? complex. This complex is then converted to a phosphoenzyme intermediate which binds two Ca²⁺ and probably Mg²⁺.     

Reversible shift between two states of Ca2+-ATPase in human erythrocytes mediated by Ca2+ and a membrane-bound activator

The (Ca²⁺ + Mg²⁺)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) from human erythrocytes occurred in two different states, A-state and B-state, depending on the membrane preparation.The A-state showed low maximum activity (V) and the Ca²⁺ activation was characterized by a Hill coefficient, n_H, of about 1 and a Michaelis constant, K_Ca, about 30 μM.The B-state showed high V, a n_H above 1, which indicates positive cooper-activity of Ca²⁺ activation, and a K_Ca of about 1 μM.With varying ATP concentrations, both the A-state and the B-state showed negative cooperativity and slightly different values of K_m.The B-state was shifted to the A-state when the membranes were exposed to low Ca²⁺ concentrations. The shift reached 50% at approx. 0.5 μM Ca²⁺. At the low Ca²⁺ concentrations an activator was released from the membranes.The A-state was shifted to the B-state when the membranes were exposed to Ca²⁺ in the presence of the activator. The shift reached 50% at about 30 μM Ca²⁺. The recovery of high V was time dependent and lasted several minutes. Increasing concentrations of Ca²⁺ and activator accelerated the recovery.It is suggested that the A-state and the B-state correspond to enzyme free of activator and enzyme associated with activator, respectively. Furthermore, the two states may represent a resting and an active state, respectively, of the calcium pump.     

Effect of calmodulin,Ca2+ and Mg2+ on the (Ca2+ + Mg2+)-ATPase of erythrocyte membranes

Calmodulin-depleted isotonic erythrocyte ghosts contain 200 ng residual calmodulin/mg protein which is not removed by extensive washings at pCa²⁺ > 7. Specific activity and Ca²⁺-affinity of the (Ca²⁺ + Mg²⁺)ATPase increase at increasing calmodulin, with K_{0.5 Ca} of 0.38 μM at calmodulin concentrations corresponding to that in erythrocytes. High Ca²⁺ concentrations inhibit the enzyme. Specific activity and Ca²⁺-affinity of the enzyme decrease at increasing Mg²⁺ concentrations. The Ca²⁺ ? Mg²⁺ antagonism is likewise observed at inhibitory Ca²⁺ concentrations.     

Interaction of Na+ and Mg2+ with Ca2+ in pancreatic islets as visualized by chlorotetracycline fluorescence

The fluorescence of microdissected pancreatic islets of ob/ob-mice was studied by microscope photometry after incubation with 10 μM chlorotetracycline. In Krebs-Ringer bicarbonate buffer, excitation at 390 nm yielded peak emission at 530 nm, suggesting that chelated Ca²⁺ was the major source of fluorescence. In support of this interpretation, incubation in Ca²⁺-free buffer markedly decreased the fluorescence, whereas withdrawal of Mg²⁺ increased it. Raising the Mg²⁺ concentration to 15 mM suppressed the fluorescence. In the presence of Ca²⁺, the substitution of choline ions for Na⁺ increased the fluorescence considerably; in the absence of Ca²⁺, however, Na⁺ deficiency had only little effect. Control experiments showed that Na⁺ or choline ions had no effect on the fluorescence of Ca²⁺-chlorotetracycline in 70 or 90% methanol. In 90%, but not in 70%, methanol 15 mM Mg²⁺ slightly quenched the fluorescence from 2.5 mM Ca²⁺ and 10 μM chlorotetracycline. It is suggested that Na⁺, and perhaps Mg²⁺, tends to decrease the amount of membrane-bound Ca²⁺ in the pancreatic islets.     

Effects of adenosine diphosphate on Ca2+ fluxes and Ca2+ accumulation of sarcoplasmic reticulum

Ca²⁺ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37°C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 μM CaCl₂, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca²⁺ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca²⁺ uptake, a second phase of Ca²⁺ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca²⁺ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca²⁺ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca²⁺ influx of sarcoplasmic reticulum near steady state of Ca²⁺ uptake was measured by pulse labeling with ⁴⁵Ca²⁺. The Ca²⁺ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca²⁺ uptake in normal medium, Ca²⁺ influx was balanced by Ca²⁺ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca²⁺ exchange rate at the first plateau of Ca²⁺ uptake was about half of that in normal medium. When the second phase of Ca²⁺ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca²⁺ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 μg/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca²⁺ uptake was also observed. These data suggest that the Ca²⁺ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca²⁺ efflux, which subsequently stimulates Ca²⁺ exchange.     

The Ca2+ permeability of sarcoplasmic reticulum vesicles I. Ca2+ outflow in the non-energized state of the calcium pump

Passive Ca²⁺ permeability of sarcoplasmic reticulum vesicles has been studied after maximal loading with Ca²⁺ (150–200 nmol/mg protein) in the presence of Ca²⁺, MgATP and an ATP generating system of limited capacity. Outflow of accumulated Ca²⁺ in the non-energized state of the system was studied by depletion of the medium of one of the substrates, either MgATP (by complete consumption) or Ca²⁺ (by complexation with EGTA). It was found that Ca²⁺ outflow under these conditions is relatively slow and independent of the medium concentration of Ca²⁺ (5·10^?9–5·10^?5 M) or MgATP (0.7–730 μM). Outflow curves were steep at the beginning of the outflow phase (30–60 nmol/min per mg protein), and outflow proceeded at a much lower rate below 100 nmol Ca²⁺/mg protein. Outflow could be completely inhibited by La³⁺. The Ca²⁺ release curves are not compatible with simple diffusion, and cannot be accounted for by Ca²⁺ binding inside the vesicles. Neither are our observations consistent with permeation mediated via the Ca²⁺ translocation sites involved in active transport. We suggest that non-energized Ca²⁺ outflow may proceed by a process of ion-exchange through negatively charged, water-filled channels in the membrane, the properties of which are altered by a high intravesicular concentration of Ca²⁺.     

The peripheral Ca2+ pump activity of macrophages and neutrophils

Exposure of either alveolar macrophages or blood neutrophils to 0.2 – 1 μM ionophore A23187 in the presence of 0.1 – 1 mM CaCl₂ causes a rapid extracellular release of Ca²⁺, which can be measured by a Ca²⁺-selective electrode. The initial rate at which the cation is extruded from the cells is about 0.1 – 0.2 μg-ions/min/ml of cell water. ATP depletion, but not replacement of extracellular Na⁺ with choline, produces a marked inhibition of Ca²⁺ release from macrophages. When the movements of Ca²⁺ between neutrophils and the incubation medium are followed by an isotopic technique, a transient increase in cell-associated ⁴⁵Ca²⁺ is detected a few seconds after the addition of the ionophore. We suggest that the ionophore A23187 mobilises Ca²⁺ from intracellular stores, with a subsequent cell extrusion of the bivalent cation catalysed by a pump localised at the cell surface. These and other data are consistent with the conclusion that the peripheral Ca²⁺ pump system of macrophages and neutrophils is very similar to the well know Ca²⁺ pump of the red cells with regard to mechanism and capacity.     

Localization of (Ca2+ + Mg2+)-ATPase,Ca2+ pump and other ATPase activities in cardiac sarcolemma

N-Ethylmaleimide was employed as a surface label for sarcolemmal proteins after demonstrating that it does not penetrate to the intracellular space at concentrations below 1·10^?4 M. The sarcolemmal markers, ouabain-sensitive (Na⁺ + K⁺)-ATPase and Na⁺/Ca²⁺-exchange activities, were inhibited in N-ethylmaleimide perfused hearts. Intracellular activities such as creatine phosphokinase, glutamate-oxaloacetate transaminase and the internal phosphatase site of the Na⁺ pump (K⁺-p-nitrophosphatase) were not affected. Almost 20% of the (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ pump were inhibited indicating the localization of a portion of this activity in the sarcolemma. Sarcolemma purified by a recent method (Morcos, N.C. and Drummond, G.I. (1980) Biochim. Biophys. Acta 598, 27–39) from N-ethylmaleimide-perfused hearts showed loss of approx. 85% of its (Ca²⁺ + Mg²⁺-ATPase and Ca²⁺ pump compared to control hearts. (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ pump activities showed two classes of sensitivity to vanadate ion inhibition. The high vanadate affinity class ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for inhibition approx. 1.5 μM) may be localized in the sarcolemma and represented approx. 20% of the total inhibitable activity in agreement with estimates from N-ethylmaleimide studies. Sucrose density fractionation indicated that only a small portion of Mg²⁺-ATPase and Ca²⁺-ATPase may be associated with the sarcolemma. The major portion of these activities seems to be associated with high density particles.     

Ca2+ transport mediated by a synthetic neutral Ca2+-ionophore in biological membranes

The effect of a synthetic neutral ligand on the Ca²⁺ permeability of several biological membranes has been investigated. The ligand had been previously shown to possess Ca²⁺-ionophoric activities in artificial phospholipid membranes. The neutral ionophore is able to transport Ca²⁺ across the membranes of erythrocytes and sarcoplasmic reticulum, when lipophilic anions such as tetraphenylborate or carbonylcyanide $\text{p-}\text{trifluoromethoxyphenylhydrazone}$ (FCCP) are present, presumably to facilitate the diffusion of the charged Ca²⁺-ionophore complex across the hydrophobic core of the membrane.In mitochondria, the neutral ionophore promotes the active transport of Ca²⁺ in response to the negative membrane potential generated by respiration, in the presence of the specific inhibitor of the natural carrier ruthenium red.     

Calcium transport and Ca2+-ATPase activity in ram spermatozoa plasma membrane vesicles

Plasma membrane vesicles, isolated from ejaculated ram sperm, were found to contain Ca²⁺-activated Mg²⁺-ATPase and Ca²⁺ transport activities. Membrane vesicles that were exposed to oxalate as a Ca²⁺-trapping agent accumulated Ca²⁺ in the presence of Mg²⁺ and ATP. The $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for Ca²⁺ uptake was 33 nmol/mg protein per h, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for Ca²⁺ and ATP were 2.5 μM and 45 μM, respectively. 1 μM of the Ca²⁺ ionophore A23187, added initially, completely inhibited net Ca²⁺ uptake and, if added later, caused the release of Ca²⁺ previously accumulated. A Ca²⁺-activated ATPase was present in the same membrane vesicles which had a $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 1.5 μmol/mg protein per h at free Ca²⁺ concentration of 10 μM. This Ca²⁺-ATPase had $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values of 4.5 μM and 110 μM for Ca²⁺ and ATP, respectively. This kinetic parameter was similar to that observed for uptake of Ca²⁺ by the vesicles. The Ca²⁺-ATPase activity was insensitive to ouabain. Both Ca²⁺ transport and Ca²⁺-ATPase activity were inhibited by the flavonoid quercetin. Thus, ram spermatozoa plasma membranes have both a Ca²⁺ transport activity and a Ca²⁺-stimulated ATPase activity with similar substrate affinities and specificities and similar sensitivity to quercetin.     

Comparative abilities of lanthanide ions La3+ and Tb3+ to substitute for Ca2+ in regulating phospholipid-sensitive Ca2+-dependent protein kinase and myosin light chain kinase

Although lanthanide ions La³⁺ and Tb³⁺ were only slightly able to substitute for Ca²⁺ to activate phospholipid-sensitive Ca²⁺-dependent protein kinase (PL-Ca-PK), they potentiated the ability of a suboptimal concentration of Ca²⁺ to stimulate the enzyme. In comparison, the lanthanides were much more effective Ca²⁺ substitutes for myosin light chain kinase, a calmodulin-sensitive Ca²⁺-dependent protein kinase. Both enzymes, however, were inhibited by high concentrations of lanthanides either in the presence or absence of Ca²⁺. Similar effects of the lanthanides were also noted on phosphorylation of endogeneous substrates in the particulate fraction of rat brain stimulated by either phosphatidylserine/Ca²⁺ or calmodulin/Ca²⁺. The La³⁺- or Tb³⁺-stimulated activity of PL-Ca-PK, as the Ca²⁺-stimulated activity, was inhibited by various agents, such as trifluoperazine, polymyxin B, cobra cytotoxin I, melittin, and spermine.     

On the mechanism by which Mg2+ and adenine nucleotides restore membrane potential in rat liver mitochondria deenergized by Ca2+ and phosphate

The presence of ATP or ADP in the incubation medium prevents the collapse of membrane potential induced by external Ca²⁺ and phosphate. The same adenine nucleotides are unable to restore collapsed membrane potential unless Mg²⁺ are also added. Bongkrekate is also able to prevent the effects of external Ca²⁺ and phosphate and when added after membrane potential has collapsed strongly potentiates the restorative action of ATP or ADP. Atractyloside has an opposite effect.     

A Ca2+-activated ATPase specifically released by Ca2+ shock from Paramecium tetraurelia

Deciliation of Paramecium tetraurelia by a Ca²⁺ shock procedure releases a discrete set of proteins which represent about 1% of the total cell protein. Marker enzymes for cytoplasm (hexokinase), endoplasmic reticulum (glucose-6-phosphatase), peroxisomes (catalase), and lysosomes (acid phosphatase) were not released by this treatment. Among the proteins selectively released is a Ca²⁺-dependent ATPase. This enzyme has a broad substrate specificity which includes GTP, ATP, and UTP, and it can be activated by Ca²⁺, Sr²⁺, or Ba²⁺, but not by Mg²⁺ or by monovalent cations. The crude enzyme has a specific activity of 2–3 μmol/min per mg; the optimal pH for activity is 7.5. ATPase, GTPase, and UTPase all reside in the same protein, which is inhibited by ruthenium red, is irreversibly denatured at 50°C, and which has a sedimentation coefficient of 8–10 S. This enzyme is compared with other surface-derived ATPases of ciliated protozoans, and its possible roles are discussed.