Kinetic properties of the Ca2+-accumulation system of a rat liver microsomal fraction.

1. By using Ca-EGTA buffers, the Km for Ca2+ uptake into rat liver heavy microsomes (microsomal fraction) was found to be 0.2 microM free Ca2+. 2. In the absence of oxalate, these vesicles accumulate about 20 nmol of Ca2+/mg of protein. Efflux of Ca2+ from the vesicles is much faster at pH 7.6 than at pH 6.8, but does not apparently show saturation kinetics or any stringent requirement for external ions. 3. The steady-state distribution of Ca2+ between the microsomes and the medium in the presence of ATP and the absence of oxalate is dependent on Ca2+ load. When the vesicles are loaded to 50% capacity, the external free Ca2+ concentration is 70 nM. 4. The affinity of heavy microsomes for Ca2+ is such that is seems likely that they has a dominant role in the determination of cytoplasmic free Ca2+ concentrations.     

Ca2+ uptake in bovine adrenocortical microsomes: formation of phosphorylated intermediate of Ca2+ dependent ATPase

Bovine adrenocortical microsomes were prepared and partially purified by discontinuous sucrose density gradient. Light fractions of the microsomes at the interface between 15 and 30% sucrose solution, exhibited ATP dependent Ca2+ uptake. The Ca2+ uptake was dependent on temperature and stimulated by free Ca2+ (the concentration for half maximal activation = 1.0 microM) and Mg2+. The Ca2+ uptake was inhibited by ADP but not affected by 10 mM NaN3 or 0.5 mM ouabain. Calcium release from the microsomes was accelerated by a Ca2+ ionophore, A23187, but not by a Ca2+ antagonist, diltiazem. A microsomal protein with a molecular weight of 100-110 kDa was phosphorylated by [gamma-32P]ATP in the presence of Ca2+, and the Ca2+ dependency was over the same range as the Ca2+ uptake (the concentration for half maximal activation = 3.0 microM). The phosphorylated protein (EP) was stable at acidic pH but labile at alkaline pH and sensitive to hydroxylamine. The rate of EP formation at 0 degrees C in the presence of 1 microM ATP and 10 microM Ca2+ (half time = 0.2 s) was less than that in the sarcoplasmic reticulum (SR) of rabbit skeletal muscle (half time = 0.1 s). The rate of EP decomposition at 0 degrees C after adding EGTA was about 6.7 times slower (rate constant: kd = 4.3 X 10(-3) s-1) than that of SR. It was suggested that adrenocortical microsomes contain a Ca2+ dependent ATPase which function as a Ca2+ pump with similar properties to that of SR.     

Lanthanide ions and skeletal muscle sarcoplasmic reticulm. I. Gadolinium localization by electron microscopy.

The Ca2+-Mg2+-dependent adenosine triphosphatase activity of isolated skeletal muscle sarcoplasmic reticulum was studied in the presence of the lanthanide ion, Gd3+. This ion is a powerful inhibitor, producing half maximal effect at approximately 100 micronM Gd3+. Electron microscopy of the isolated vesicles incubated with 100 micron Gd3+ reveal that electron dense depositis of Gd3+ is taken up within the vesicle's interior. This visualization of Gd3+ is apparently dependent on two factors: (i) the presence of ATP, ADP being ineffective; (ii) sufficient time for most of the ATP to be hydrolysed. Since Gd3+ has about the same ionic radius as Ca2+, and since Ca2+ is normally transported across the sarcoplasmic reticulum membrane and accumulated within the vesicle, it is concluded that the increased charge density of the lanthanide ions is critical to the ion transport mechanism, resulting in their localization at the ATPase site and failure to be transported across the membrane.     

Ca2+ transport studied with arsenazo III in Tetrahymena microsomes. Effects of calcium ionophore A23187 and trifluoperazine

Transport of Ca2+ in microsomal membrane vesicles of the Tetrahymena has been investigated using arsenazo III as a Ca2+ indicator. The microsomes previously shown to carry a Mg2+-dependent, Ca2+-stimulated ATPase (Muto, Y. and Nozawa, Y. (1984) Biochim. Biophys. Acta 777, 67-74) accumulated calcium upon addition of ATP and Ca2+ sequestered into microsomal vesicles was rapidly discharged by the Ca2+ ionophore A23187. Kinetic studies indicated that the apparent Km for free Ca2+ and ATP are 0.4 and 59 microM, respectively. The Vmax was about 40 nmol/mg protein per min at 37 degrees C. The calcium accumulated during ATP-dependent uptake was released after depletion of ATP in the incubation medium. Furthermore, addition of trifluoperazine which inhibited both (Ca2+ + Mg2+)-ATPase and ATP-dependent Ca2+ uptake rapidly released the calcium accumulated in the microsomal vesicles. These observations suggest that Tetrahymena microsome contains both abilities to take up and to release calcium and may act as a Ca2+-regulating site in this organism.     

Ca2+-sensitivity of actomyosin ATPase purified from Physarum polycephalum.

A contractile protein closely resembling natural actomyosin (myosin B) of rabbit skeletal muscle was extracted from plasmodia of the slime mold, Physarum polycephalum, by protecting the SH-groups with beta-mercaptoethanol or dithiothreitol. Superprecipitation of the protein induced by Mg2+-ATP at low ionic strength was observed only in the presence of very low concentrations of free Ca2+ ions, and the Mg2+-ATPase [EC 3.6.1.3] reaction was activated 2- to 6-fold by 1 muM of free Ca2+ ions. Crude myosin and actin fractions were separated by centrifuging plasmodium myosin B in the presence of Mg2+-PPi at high ionic strength. The crude myosin showed both EDTA- and Ca2+-activated ATPase activities. The Mg2+-ATPase activity of crude myosin from plasmodia was markedly activated by the addition of pure F-actin from rabbit skeletal muscle. Addition of the F-action-regulatory protein complex prepared from rabbit skeletal muscle as well as the actin fraction of plasmodium caused the same degree of activation as the addition of pure F-actin only in the presence of very low concentrations of Ca2+ ion     

Characterization of Ca2+ transport and enzyme activity in microsomes isolated from guinea-pig stomach smooth muscle

Ca2+ uptake by microsomes prepared from guinea-pig stomach required the presence of both ATP and Mg2+ and was unaffected by NaN3. ATP-dependent Ca2+ uptake increased with increasing free Ca2+ concentration from 0.1 to 5 microM, and further increase in Ca2+ concentration above 5 microM did not enhance the uptake further. Half-saturation occurred at approximately 0.55 microM. The t1/2 values of Ca2+ loss from these vesicles loaded in the presence of oxalate were significantly slower than those in the absence of oxalate. Enzyme activity suggested linkage between Ca2+ uptake and ATPase activity, and most of the azide-sensitive component of ATP hydrolysis was attributable to potent inhibition of ADPase activity.     

Proteolytic activation of the canine cardiac sarcoplasmic reticulum calcium pump

Mild trypsin treatment of canine cardiac microsomes consisting largely of sarcoplasmic reticulum vesicles produced a severalfold activation of oxalate-facilitated calcium uptake. The increase in calcium uptake was associated with an increase in ATP hydrolysis. Proteases other than trypsin were also effective although to a lesser degree. Trypsin produced a shift of the Ca2+ concentration dependency curve for calcium uptake toward lower Ca2+ concentrations, which was almost identical with that produced by phosphorylation of microsomes by cyclic AMP dependent protein kinase when the trypsin and the protein kinase were present at maximally activating concentrations. The Hill numbers (+/- SD) of the Ca2+ dependency after treatment of microsomes with trypsin (1.5 +/- 0.1) or protein kinase (1.7 +/- 0.1) were similar and were not significantly different from those for untreated control microsomes (1.6 +/- 0.1 and 1.8 +/- 0.1, respectively). Autoradiograms of sodium dodecyl sulfate-polyacrylamide electrophoretic gels indicate that 32P incorporation into phospholamban (Mr 27.3K) or its presumed monomeric subunit (Mr 5.5K) was markedly reduced when trypsin-treated microsomes were incubated in the presence of cyclic AMP dependent protein kinase and [gamma-32P]ATP compared to control microsomes incubated similarly but pretreated with trypsin inhibitor inactivated trypsin. The activation of calcium uptake by increasing concentrations of trypsin was paralleled by the reduction of phosphorylation of phospholamban. Trypsin treatment of microsomes previously thiophosphorylated in the presence of cyclic AMP dependent protein kinase and [gamma-35S]thio-ATP did not result in a loss of 35S label from phospholamban, which suggests that phosphorylation of phospholamban protects against trypsin attack.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of ATP-dependent calcium transport activity in mouse pancreatic microsomes

Electron-dense deposits representing calcium oxalate crystals which result from ATP-dependent calcium uptake have been localized within vesicles of of a heavy microsomal fraction prepared from mouse pancreatic acini. In the absence of either ATP or oxalate, no electron-dense deposits could be observed. By subfractionation of microsomes on discontinuous sucrose gradients, it could be shown that the highest energy-dependent calcium transport activity was associated with the rough endoplasmic reticulum. In rough microsomes, the 45Ca2+-uptake measured was 7 times greater than that of smooth microsomes in the presence of ATP and oxalate and about 3 times greater in he presence of ATP alone. When ribosomes were released from the rough endoplasmic reticulum vesicles by treatment with KCl in the presence of puromycin, the stripped microsomes showed a 40% increase in the specific 45Ca2+-uptake activity measured in he presence of ATP and oxalate and an increase of 80 to 90% in the presence of ATP alone. From these results it can be concluded that the calcium transport activity of microsomes prepared from mouse pancreatic acini is located predominantly in the rough endoplasmic reticulum membrane.     

ATP- and calcium-controlled contraction in a saponin model of Physarum polycephalum

Saponin models of the plasmodial strand of Physarum polycephalum were constructed to study how Ca2+ and ATP regulate the generation of tension. ATP-induced isometric tension in a saponin model increased with an increase in ATP concentration until maximum tension (0.3-1.7 mg) was reached at about 1 mM. The sensitivity of the model to ATP was heightened three to five times in a basic solution containing an ATP-regenerating system, the maximum tension (0.3-0.6 mg) being reached at 0.2 to 0.3 mM ATP. Contraction of the model also depended on the Ca2+ concentration irrespective of the presence or absence of the ATP-regenerating system. The optimal pCa was 7.0, and tension decreased with a Ca2+ concentration above or below this value. These results indicate that the tension generated in the plasmodial strand of Physarum in vivo may be regulated by ATP and/or Ca2+.     

The interaction of lanthanides with isolated sarcoplasmic reticulum vesicles from rabbit skeletal muscle.

The interaction of lanthanides with isolated sarcoplasmic reticulum (SR) vesicles from rabbit skeletal muscle and the effects of lanthanides on 45Ca2+ uptake by the vesicles were studied. 153Gd3+ was taken up by the vesicles in the absence of ATP and oxalate in a time-dependent manner, reaching a maximum total accumulation of 380 nmol 153Gd3+/mg protein after 20 min with 200 microM 153Gd3+. This 153Gd3+ accumulation was not washed out by 1 mM EGTA. The addition of ATP induced the release of 87% of the bound 153Gd3+, leaving behind irreversibly-accumulated 153Gd3+. Pre-incubation of the vesicles with lanthanides in the absence of ATP and oxalate inhibited 45Ca2+ uptake without affecting Ca2+-ATPase activity. The percent inhibition of 45Ca2+ uptake increased with length of pre-incubation of the vesicles with lanthanides, reaching 33% after 20 min of pre-incubation. Increasing the 45Ca2+ concentration or adding ATP or oxalate to the preincubation medium abolished these inhibitory effects on 45Ca2+ uptake.     

Biochemical evidence for functional heterogeneity of cardiac sarcoplasmic reticulum vesicles

Two subpopulations of cardiac sarcoplasmic reticulum vesicles were resolved functionally, based on their sensitivities to the drug ryanodine. These two subpopulations of sarcoplasmic reticulum vesicles, termed ryanodine-sensitive and ryanodine-insensitive, were separated by preloading crude cardiac microsomes with Ca2+ oxalate in the presence of ATP, followed by sucrose density gradient centrifugation. Ryanodine-insensitive vesicles accumulated most of the Ca2+ oxalate during the preload, and constituted the densest subfraction recovered from the sucrose gradient. These ryanodine-insensitive vesicles exhibited the highest density of Ca2+ pumps, and accounted for 10 to 15% of the total protein in crude cardiac microsomes. Ryanodine-insensitive vesicles continued to transport substantial amounts of Ca2+ after isolation. Ryanodine-sensitive vesicles accumulated negligible Ca2+ during the preload, and were recovered from the lower density regions of the sucrose gradient. On a milligrams of protein basis, these vesicles were present in 7-fold excess over ryanodine-insensitive vesicles. Ryanodine-sensitive vesicles transported low amounts of Ca2+ under normal incubation conditions, but 3 X 10(-4) M ryanodine strikingly increased their Ca2+ uptake 5- to 10-fold. Ca2+ uptake by ryanodine-sensitive vesicles was uniquely regulated by Ca2+ ion concentration. Elevation of the ionized Ca2+ concentration from 2 to 4 microM increased Ca2+ uptake by these vesicles greater than 5-fold, but had no effect on their Ca2+-dependent ATPase activity. These ryanodine- and Ca2+ concentration-dependent effects were apparent for only ryanodine-sensitive vesicles. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed distinct differences in polypeptide staining between ryanodine-sensitive and ryanodine-insensitive vesicles, confirming by an independent method that the two populations of vesicles were different. These data provide the first biochemical evidence for functional and structural heterogeneity of cardiac sarcoplasmic reticulum vesicles.     

Ca2+,Mg2+-ATPase of microsomal membranes from bovine aortic smooth muscle: effects of Sr2+ and Cd2+ on Ca2+ uptake and formation of the phosphorylated intermediate of the Ca2+,Mg2+-ATPase

The effects of various divalent cations on the Ca2+ uptake by microsomes from bovine aortic smooth muscle were studied. High concentrations (1 mM) of Co2+, Zn2+, Mn2+, Fe2+, and Ni2+ inhibited neither the Ca2+ uptake by the microsomes nor the formation of the phosphorylated intermediate (E approximately P) of the Ca2+,Mg2+-ATPase of the microsomes. The cadmium ion, however, inhibited both the Ca2+ uptake and the E approximately P formation by the microsomes. Dixon plot analysis indicated Cd2+ inhibited (Ki = 135 microM) the Ca2+ dependent E approximately P formation in a non-competitive manner. The inhibitory effect of Cd2+ was lessened by cysteine or dithiothreitol. The strontium ion inhibited the Ca2+ uptake competitively, while the E approximately P formation increased on the addition of Sr2+ at low Ca2+ concentrations. At a low Ca2+ concentration (1 microM), Sr2+ was taken up by the aortic microsomes in the presence of 1 mM ATP. It is thus suggested that Sr2+ replaces Ca2+ at the Ca2+ binding site on the ATPase.     

The high affinity (Ca2+-Mg2+)-ATPase in liver plasma membranes is a Ca2+ pump. Reconstitution of the purified enzyme into phospholipid vesicles

The purified (Ca2+-Mg2+)-ATPase from rat liver plasma membranes (Lotersztajn, S., Hanoune, J., and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215) was incorporated into soybean phospholipid vesicles, together with its activator. In the presence of millimolar concentrations of Mg2+, the reconstituted proteoliposomes displayed a rapid, saturable, ATP-dependent Ca2+ uptake. Half-maximal Ca2+ uptake activity was observed at 13 +/- 3 nM free Ca2+, and the apparent Km for ATP was 16 +/- 6 microM. Ca2+ accumulated into proteoliposomes (2.8 +/- 0.2 nmol of Ca2+/mg of protein/90 s) was totally released upon addition of the Ca2+ ionophore A-23187. Ca2+ uptake into vesicles reconstituted with enzyme alone was stimulated 2-2.5-fold by the (Ca2+-Mg2+)-ATPase activator, added exogenously. The (Ca2+-Mg2+)-ATPase activity of the reconstituted vesicles, measured using the same assay conditions as for ATP-dependent Ca2+ uptake activity (e.g. in the presence of millimolar concentrations of Mg2+), was maximally activated by 20 nM free Ca2+, half-maximal activation occurring at 13 nM free Ca2+. The stoichiometry of Ca2+ transport versus ATP hydrolysis approximated 0.3. These results provide a direct demonstration that the high affinity (Ca2+-Mg2+)-ATPase identified in liver plasma membranes is responsible for Ca2+ transport.     

Reactivation of cell-free models of Physarum plasmodia after myosin reconstitution

Thin-spread glycerol-extracted Physarum plasmodia were treated with N-ethylmaleimide (NEM) to block myosin-ATPase and contractility. After supplementing the models with purified plasmodial myosin, they could be reactivated and contracted upon addition of ATP. Fluorescently labeled actomyosin fibers ruptured during contraction, resulting in beaded or rod-like contraction centers. Glycerol-extracted plasmodia lose their negative Ca++-dependence during extraction. Reconstitution of NEM-treated models with plasmodial myosin partly restored this Ca++-sensitivity. Thus, either myosin or a factor associated with it seems to be involved in the Ca++-dependent regulation of cytoplasmic actomyosin contraction in Physarum. NEM-blocked models reconstituted with skeletal muscle myosin were not reactivated by ATP. The same plasmodia subsequently incubated with plasmodial myosin were able to contract.     

Studies on K+ permeability of rat gastric microsomes

A population of gastric membrane vesicles of high K+ permeability and of lower density than endoplasmic tubulovesicles containing (H+-K+)-ATPase was detected in gastric mucosal microsomes from the rat fasted overnight. The K+-transport activity as measured with 86RbCl uptake had a Km for Rb+ of 0.58 +/- 0.11 mM and a Vmax of 13.7 +/- 1.9 nmol/min X mg of protein. The 86Rb uptake was reduced by 40% upon substituting Cl- with SO2-4 and inhibited noncompetitively by ATP and vanadate with a Ki of 3 and 30 microM, respectively; vanadate also inhibited rat gastric (H+-K+)-ATPase but with a Ki of 0.03 microM. Carbachol or histamine stimulation decreased the population of the K+-permeable light membrane vesicles, at the same time increased K+-transport activity in the heavy, presumably apical membranes of gastric parietal cells, and enabled the heavy microsomes to accumulate H+ ions in the presence of ATP and KCl without valinomycin. The secretagogue-induced shift of K+ permeability was blocked by cimetidine, a H2-receptor antagonist. Four characteristics of the K+ permeability as measured with 86RbCl were common in the resting light and the carbachol-stimulated heavy microsomes; (a) Km for +Rb, (b) anion sensitivity (Cl- greater than SO2-4), (c) potency of various divalent cations (Hg2+, Cu2+, Cd2+, and Zn2+) to inhibit Rb+ uptake, and (d) inhibitory effect of ATP, although the nucleotide sensitivity was latent in the stimulated heavy microsomes. The Vmax for 86RbCl uptake was about 10 times greater in the resting light than the stimulated heavy microsomes. These observations led us to propose that secretagogue stimulation induces the insertion of not only the tubulovesicles containing (H+-K+)-ATPase, but also the light membrane vesicles containing KCl transporter into the heavy apical membranes of gastric parietal cells.     

Magnesium and manganese ions modulate Ca2+ uptake and its energetic coupling in sarcoplasmic reticulum

ATP hydrolysis, either coupled or uncoupled from Ca2+ uptake by sarcoplasmic reticulum (SR), is essentially independent of Mg2+ (millimolar range) up to 50 mM. Conversely, a sharp enhancement of Ca2+ uptake by Mg2+ is observed with a consequent increase of pumping efficiency (Ca2+ per ATP). Therefore, Mg2+ modulates pumping efficiency through the molecular mechanism of the pump itself. Manganese ions also stimulate Ca2+ uptake with an apparent efficiency lower than that of Mg2+. Additionally, Mn2+ competes with Ca2+ for the pump system and is accumulated into SR vesicles. Although the affinity of the pump is about three orders of magnitude higher for Ca2+ than for Mn2+, the capacity of the vesicles for Mn2+ is about three times that commonly observed for Ca2+. It is concluded that Mg2+ (millimolar range) couples ATP hydrolysis to Ca2+ uptake and that active transport of cations (Ca2+ and Mn2+) can proceed without a compensatory countertransport of a divalent cation. Finally, it is suggested that the SR pump operates physically as general cation translocator instead of as a Ca2+-specific pump, as commonly assumed.     

Effect of temperature on the reversal of the calcium ion pump in sarcoplasmic reticulum.

Sarcoplasmic-reticulum vesicles were actively loaded with Ca2+ in the presence of phosphate, and the ADP-induced Ca2+ efflux and ATP synthesis were measured as a function of temperature. Arrhenius plots show break points for both processes at about 18 and 37 degrees C. Between 18 and 37 degrees C, Ca2+ efflux and ATP synthesis occur with an activation energy of 67.2-71.4 kJ/mol, whereas it is about 189-210 kJ/mol for temperatures below 18 degrees C. Above 37 degrees C, the rates of ADP-induced Ca2+ release and of ATP synthesis sharply decline until the temperature reaches about 42 degrees C. Above this temperature, the Ca2+ efflux increases again even in absence of ADP, although the synthesis of ATP is inhibited, which reflects leakiness of the vesicles. The results show that the transition temperatures for ADP-induced Ca2+ efflux and for ATP synthesis resemble those for active Ca2+ uptake, which indicates that the same coupling mechanism is involved during the inward and outward Ca2+ translocations across the membrane.     

Modification of ATP regulatory function in sarcoplasmic reticulum Ca2(+)-ATPase by hydrophobic molecules

The effects of the three hydrophobic molecules triphenylphosphine, trifluoperazine and 3-nitrophenol on Ca2+ uptake and ATPase activity in sarcoplasmic reticulum vesicles was investigated. When ATP was the substrate, triphenylphosphine (3 microM) increased the amount of Ca2+ accumulated by the vesicles. At high concentrations triphenylphosphine inhibited Ca2+ uptake. This effect varied depending on the ATP concentration and the type of nucleotide used. With ITP there was only inhibition and no activation of Ca2+ uptake by triphenylphosphine. On the other hand, trifluoperazine inhibited Ca2+ accumulation regardless of whether ATP or ITP was used as substrate. When 5 mM oxalate was included in the medium in order to avoid binding of Ca2+ to the low-affinity Ca2(+)-binding sites of the enzyme, both stimulation by triphenylphosphine and inhibition by trifluoperazine were reduced. In leaky vesicles at low Ca2+ concentrations, triphenylphosphine and 3-nitrophenol were competitive inhibitors of ATPase activity at the regulatory site of the enzyme (0.1-1 mM ATP). A striking difference was observed when both the high- and low-affinity Ca2(+)-binding sites were saturated. In this condition, triphenylphosphine and 3-nitrophenol promoted a 3-4-fold increase in the apparent affinity for ATP at its regulatory site.     

Ca2+-binding modulator protein in protozoa and myxomycete.

Ca2+-binding protein with the properties of brain modulator protein of 3,5-cyclic nucleotide phosphodiesterase was identified in Physarum polycephalum plasmodia and in Euglena gracilis and Amoeba proteus cells by urea polyacrylamide gel electrophoresis and activation of cyclic nucleotide phosphodiesterase and of myosin light chain kinase.     

Use of 1-anilino-8-naphthalene-sulfonate as a probe of gastric vesicle transport.

The interaction of 1-anilino-8-naphthalene-sulfonate (ANS) with vesicles derived from hog fundic mucosa was studied in the presence of valinomycin and with the addition of ATP. Evidence was found for two classes of sites, those rapidly accessible to ANS with a KD of 7.5 micronM and those slowly accessible, but rapidly accessed in the presence of valinomycin with a KD of 2.5 micronM. ATP transiently increases the quantum yield of the latter ANS binding sites only in the presence of valinomycin, but does not alter the number of KD of those sites. The time course of this increase correlates with H+ uptake and Rb+ extrusion by those vesicles and H+ carries such as tetrachlorsalicylanilide or nigericin abolish the ATP response. With ATP addition in the presence of SC14N and valinomycin there is transient uptake of SCN-. It is concluded that ANS is acting as a probe of a structural change dependent on a potential and H+ gradient.