Inhibition of Ca-transport ATPase from synaptosomal vesicles by flavonoids

The inhibitory action of the flavonoid quercetin has been examined on the calcium-transport ATPase of synaptosomal vesicles and compared to that of two other flavonoids, morin and rutin. We have found that while quercetin caused a 50% inhibition of calcium transport at a concentration of 15 μM, morin and rutin had similar effects at concentrations of about 200 μM. A similar order of potency was observed also for ATP hydrolysis, though at higher concentrations. Quercetin also strongly inhibited phosphorylation of membrane proteins by ATP in synaptosomal vesicles. Rutin and morin had an almost negligible effect on membrane protein phosphorylation. The order of inhibitory potency of the flavonoids on the Ca²⁺-transport ATPase from synaptosomal vesicles: quercetin > morin > rutin, could be linked to their possible solubility in the membrane lipid phase since: (1) it paralleled their partitioning between a mixture of oil and water; (2) it paralleled their uptake from the reaction mixture by synaptosomal vesicles and phosphatidylcholine liposomes; (3) they had almost equal potency as inhibitors of the water soluble system of histone phosphorylation by protein kinase.     

Inhibition of Ca2+-transport ATPase from synaptosomal vesicles by flavonoids

The inhibitory action of the flavonoid quercetin has been examined on the calcium-transport ATPase of synaptosomal vesicles and compared to that of two other flavonoids, morin and rutin. We have found that while quercetin caused a 50% inhibition of calcium transport at a concentration of 15 microM, morin and rutin had similar effects at concentrations of about 200 microM. A similar order of potency was observed also for ATP hydrolysis, though at higher concentrations. Quercetin also strongly inhibited phosphorylation of membrane proteins by ATP in synaptosomal vesicles. Rutin and morin had an almost negligible effect on membrane protein phosphorylation. The order of inhibitory potency of the flavonoids on the Ca2+-transport ATPase from synaptosomal vesicles: quercetin greater than morin greater than rutin, could be linked to their possible solubility in the membrane lipid phase since: (1) it paralleled their partitioning between a mixture of oil and water; (2) it paralleled their uptake from the reaction mixture by synaptosomal vesicles and phosphatidylcholine liposomes; (3) they had almost equal potency as inhibitors of the water soluble system of histone phosphorylation by protein kinase.     

Inhibition by Sr2+ of specific mitochondrial Ca2+-efflux pathways

The effect of Sr²⁺ on the set point for external Ca²⁺ was studied in rat heart and liver mitochondria with the aid of a Ca²⁺-sensitive electrode. In respiring mitochondria the set point is determined by the rates of Ca²⁺ influx on the Ca²⁺ uniporter and efflux by various mechanisms. We studied the Ca²⁺-Na⁺ exchange pathway in heart mitochondria and the Δψ-modulated efflux pathway in liver mitochondria. Prior accumulation of Sr²⁺ was found to shift the set points towards lower external Ca²⁺ both in heart mitochondria under conditions of Ca²⁺-Na⁺ exchange and in liver mitochondria under conditions that should promote opening of the Δψ-modulated pathway. The effect on the set point was found to be due to inhibition of Ca²⁺ efflux by Sr²⁺ taken up by the mitochondria, while Sr²⁺ efflux was too slow to be measurable.     

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 μM Mg²⁺, the glycoprotein alone may restore fully the Ca²⁺-transporting ability of the particles. The maximal velocity is already reached at 0.1 μg glycoprotein/mg mitochondrial protein.     

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.     

Ca2+- or Mg2+-dependent ATPase in plasma membrane of cultured endothelial cells from bovine carotid artery

ATPase was found in plasma membrane of cultured endothelial cells from bovine carotid artery. The activity of the enzyme solubilized by octaethyleneglycol mono-n-dodecyl ether was enhanced by the addition of Ca²⁺ or Mg²⁺ and was not affected by F-actin and ouabain. V_max was 2.8 and 10.0 μmol P_i/mg protein per h for Ca²⁺- and Mg²⁺-dependent activity, respectively, and the corresponding K_m was 4.8·10^?4 M and 3.2·10^?4 M. Molecular weight of the protein was estimated to be approx. 250 000, as determined by activity-staining electrophoresis with polyacrylamide gels.     

Inhibition by gossypol of phospholipid-sensitive Ca2+-dependent protein kinase from pig testis

Gossypol, a polyphenolic binaphthalene-dialdehyde extracted from cotton plants which possesses male antifertility action in mammals, is a potent inhibitor of phospholipid-sensitive Ca²⁺-dependent protein kinase from pig testis. Gossypol inhibited Ca²⁺-dependent activity of the enzyme without affecting its basal activity. The IC₅₀ value (concentration causing 50% inhibition) was 31 μM when lysine-rich histone was used as substrate. Kinetic analysis indicated that the compound inhibited the enzyme non-competitively with respect to ATP (K_i = 31 μM) or lysine-rich histone (K_i = 30μM), and competitively with respect to phosphatidylserine (K_i = 2.1 μM). With Ca²⁺, irrespective of the presence or absence of 1,3-diolein, the compound lowered V_max and increased the apparent K_a for Ca²⁺. The compound also inhibited phosphorylation by the enzyme of high-mobility-group 1 protein (one of the endogenous substrate in the testis for the enzyme located in nucleosome), with an IC₅₀ value of 88 μM. These results suggested that a phospholipid-sensitive Ca²⁺-dependent protein phosphorylation system in the testis is involved in the regulation of spermatogenesis.     

Inhibition of synaptosomal enzymes by local anesthetics

The effects of tertiary amine local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and chlorpromazine were investigated for three enzyme activities associated with rat brain synaptosomal membranes, i.e., (Na⁺ + K⁺)-ATPase (ouabain-sensitive), Mg²⁺-ATPase (ouabain-insensitive) and acetylcholinesterase. Approximately the same concentrations of each agent gave 50% inhibition of both ATPase, for example 7.9 and 10 mM tetracaine for Mg²⁺-ATPase and (Na⁺ + K⁺)-ATPase, respectively; these concentrations are 10-fold higher than required for inhibition of mitochondrial F₁-ATPase. The relative inhibitory potency of the several agents was proportional to their octanol/water partition coefficients. Acetylcholinesterase was inhibited by all agents tested, but the ester anesthetics (procaine and tetracaine) were considerably more potent than the others after correction for partition coefficient differences. For tetracaine, 0.18 mM gave 50% inhibition and showed competitive inhibition on a Lineweaver-Burk plot, but for dibucaine a mixed type of inhibition was observed, and 0.63 mM was required for 50% inhibition. Tetracaine evidently binds at the active site, and dibucaine at the peripheral or modulator site, on this enzyme.     

The Ca2+ permeability of sarcoplasmic reticulum vesicles II. Ca2+ efflux in the energized state of the calcium pump

Ca²⁺ efflux from sarcoplasmic reticulum vesicles was studied by measurements of net Ca²⁺ uptake, ⁴⁵Ca²⁺ flux and hydrolysis of energy-rich phosphate. The maximal Ca²⁺ uptake capacity (150–200 nmol/mg protein at pH 6.7, 10 mM MgCl₂ and μ=0.26) was independent of the nature and concentration of the energy-donating substrate (ATP or carbamyl phosphate) and of temperature (15–35°C), suggesting coupling between influx and efflux of Ca²⁺. In the presence of high concentrations of ATP, this efflux of Ca²⁺ was much higher than the passive Ca²⁺ permeation, measured after ATP or Ca²⁺ depletion of the reaction medium. Ca²⁺ efflux was imperceptible at vesicle filling levels below 35–40 nmol Ca²⁺/mg protein, and uncorrelated to the inhibition of the Ca²⁺-ATPase by high intravesicular Ca²⁺ concentrations. Analysis of the data indicated that Ca²⁺ efflux under our conditions probably is associated with one of the Ca²⁺-ATPase partial reactions occurring after dephosphorylation, rather than with a reversal of the Ca²⁺ translocation step in the phosphorylated state of the enzyme. Furthermore, passive Ca²⁺ permeation may be concurrently reduced during the enzymatically active state. It is proposed that both Ca²⁺ efflux and passive Ca²⁺ permeation (Ca²⁺ outflow) proceed via the same channels which are closed (occluded) during part of the Ca²⁺-ATPase reaction cycle.     

Regulation of rat brain (Na+ + K+)-ATPase activity by cyclic AMP

The interaction between the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ and the adenylate cyclase enzyme systems was examined. Cyclic AMP, but not 5′-AMP, cyclic GMP or 5′-GMP, could inhibit the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme present in crude rat brain plasma membranes. On the other hand, the cyclic AMP inhibition could not be observed with purified preparations of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme. Rat brain synaptosomal membranes were prepared and treated with either NaCl or cyclic AMP plus NaCl as described by Corbin, J., Sugden, P., Lincoln, T. and Keely, S. ((1977) J. Biol. Chem. 252, 3854–3861). This resulted in the dissociation and removal of the catalytic subunit of a membrane-bound cyclic AMP-dependent protein kinase. The decrease in cyclic AMP-dependent protein kinase activity was accompanied by an increase in $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity. Exposure of synaptosomal membranes containing the cyclic AMP-dependent protein kinase holoenzyme to a specific cyclic AMP-dependent protein kinase inhibitor resulted in an increase in $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme activity. Synaptosomal membranes lacking the catalytic subunit of the cyclic-AMP-dependent protein kinase did not show this effect. Reconstitution of the solubilized membrane-bound cyclic AMP-dependent protein kinase, in the presence of a neuronal membrane substrate protein for the activated protein kinase, with a purified preparation of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$, resulted in a decrease in overall $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity in the presence of cyclic AMP. Reconstitution of the protein kinase alone or the substrate protein alone, with the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ has no effect on $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity in the absence or presence of cyclic AMP. Preliminary experiments indicate that, when the activated protein kinase and the substrate protein were reconstituted with the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme, there appeared to be a decrease in the Na⁺-dependent phosphorylation of the Na⁺-ATPase enzyme, while the K⁺-dependent dephosphorylation of the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ was unaffected.     

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⁺.     

Trifluoperazine inhibition of calmodulin-sensitive Ca2+-ATPase and calmodulin insensitive (Na+ + K+)- and Mg2+-ATPase activities of human and rat red blood cells

Trifluoperazine dihydrochloride-induced inhibition of calmodulin-activated Ca²⁺-ATPase and calmodulin-insensitive (Na⁺ + K⁺)- and Mg²⁺-ATPase activities of rat and human red cell lysates and their isolated membranes was studied. Trifluoperazine inhibited both calmodulin-sensitive and calmodulin-insensitive ATPase activities in these systems. The concentration of trifluoperazine required to produce 50% inhibition of calmodulin-sensitive Ca²⁺-ATPase was found to be slightly lower than that required to produce the same level of inhibition of other ATPase activities. Drug concentrations which inhibited calmodulin-sensitive ATPase completely, produced significant reduction in calmodulin-insensitive ATPases as well. The data presented in this report suggest that trifluoperazine is slightly selective towards calmodulin-sensitive Ca²⁺-ATPase but that it is also capable of inhibiting calmodulin-insensitive (Na⁺ + K⁺)-ATPase and Mg²⁺-ATPase activities of red cells at relatively low concentrations. Thus the action of the drug is not due entirely to its interaction with calmodulin-mediated processes, and trifluoperazine cannot be assumed to be a selective inhibitor of calmodulin interactions under all circumstances.     

Evidence for a Ca2+-transport deficiency in patients with cystic fibrosis

Inside-out vesicles (10) were prepared from red cells obtained from cystic fibrosis (CF) patients and approximately aged-matched controls. Ca²⁺-uptake activity was found to be significantly reduced in the preparations derived from CF patients. This reduction was apparent at all time points studied (10 sec-120 min) and at all free calcium concentrations used (10–150 μM). Reciprocal plots of the data revealed that the K_diss for calcium of the calcium pump, was similar in control and CF samples but that the V_Ca²⁺ was significantly reduced (P<0.001) in the CF preparations. Calmodulin prepared from red cell hemolysates of controls was found to stimulate Ca²⁺-transport activity to a similar extent in both CF and control samples.     

Stimulation by calmodulin of Ca2+ uptake and (Ca2+-Mg2+) ATPase activity in membrane fractions from ox neurohypophyses

Calmodulin stimulated ⁴⁵Ca²⁺ uptake into a plasma membrane enriched fraction from ox neurohypophysial nerve endings and into a microsome fraction. The ⁴⁵Ca²⁺ uptake and the (Ca²⁺-Mg²⁺) ATPase activity in the plasma membrane fraction exhibited similar pCa and calmodulin sensitivities, suggesting that the enzyme activity is the biochemical expression of a high affinity Ca²⁺ pump. Calmodulin thus seems to play a role in regulation of the intracellular free Ca²⁺ concentration in the neurohypophysis.     

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.     

Inhibition of oxidative phosphorylation by a Ca2+-induced diminution of the adenine nucleotide translocator

The mechanism through which internal Ca²⁺ inhibits oxidative phosphorylation of rat heart mitochondria has been explored. In parallel to a Ca²⁺-induced diminution of the activity of the adenine nucleotide translocator, an efflux of internal adenine nucleotides is observed. The efflux of adenine nucleotides depends on the amount of Ca²⁺ accumulated by the mitochondria and on the time that Ca²⁺ remains in the mitochondria; this efflux is atractyloside insensitive. These results suggest that internal Ca²⁺, by inducing a lowering of the internal concentration of adenine nucleotides, diminishes the rate of exchange of adenine nucleotides via the translocase, and in consequence of oxidative phosphorylation. Under conditions in which the Ca²⁺-induced release of adenine nucleotides takes place, no gross changes of the permeability properties of the membrane are observed. As revealed by studies with arsenate, respiratory activity and the function of the ATPase in the direction of ATP synthesis are not affected by internal Ca²⁺.