Role of membrane-associated CA2+ dependent matrix metalloprotease-2 in the oxidant activation of Ca2+ATPase by tertiary butylhydroperoxide

Treatment of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant tert-butylhydroperoxide (t-buOOH) increases Ca²⁺⁺ATPase activity. The smooth muscle plasma membrane possesses a Ca²⁺⁺ dependent protease activity in the gelatin containing zymogram having an apparent molecular mass of 72 kDa. The 72 kDa protease activity was found to be inhibited by EGTA and the tissue inhibitor of metalloprotease-2 (TIMP-2). Since 72 kDa is the molecular mass of MMP-2 and since in our present study the 72 kDa protease in the gelatin containing zymogram is inhibited by matrix metalloprotease inhibitors, EGTA and TIMP-2, it may be suggested that the 72 kDa protease is the MMP-2. In addition to the increasing Ca²⁺⁺ATPase activity, t-buOOH also enhances the activity of the membrane associated Ca²⁺⁺ dependent protease that degrades ¹⁴C-gelatin. The oxidant triggered protease activity and the Ca²⁺⁺ATPase activity were found to be prevented by the antioxidant vitamin E, and also by the Ca²⁺⁺ dependent matrix metalloprotease inhibitors: EGTA and TIMP-2. Adding MMP-2 to the smooth muscle plasma membrane suspension caused an increase in Ca²⁺⁺ATPase activity and pretreatment with TIMP-2 prevents the increase in Ca²⁺⁺ATPase activity. Combined treatment of the smooth muscle plasma membrane with low doses of MMP-2 and t-buOOH augments further the Ca²⁺⁺ATPase activity caused by the respective doses of either t-buOOH or MMP-2. Pretreatment with TIMP-2 prevents the increase in Ca²⁺⁺ATPase activity elicited by the low doses of MMP-2 and/or t-buOOH.     

Matrix metalloprotease 2-mediated activation of Ca(2+)-ATPase by superoxide radical (O2*-) in plasma membrane of bovine pulmonary vascular smooth muscle

The role of the matrix metalloprotease-2 (MMP-2) in regulating Ca(2+)-ATPase activity in bovine pulmonary artery smooth muscle plasma membranes during treatment with the O2*- generating system, hypoxanthine (HPX) plus xanthine oxidase (XO) has been studied. The smooth muscle membranes possess matrix metalloprotease (MMP) activity in gelatin zymogram, having an apparent molecular mass of 72 kDa; the activity is inhibited by the tissue inhibitor of metalloprotease-2 (TIMP-2). Since both protease and MMP-2 have same molecular mass and are inhibited by TIMP-2, it may, therefore, be suggested that the protease is the MMP-2. Treatment of the smooth muscle membrane suspension with the O2*- generating system stimulates MMP-2 activity, as evidenced by an apparent increase in the intensity of the protease activity. O2*- also enhances [14C]-gelatin degradation and Ca(2+)-ATPase activity. The increase in MMP activity, assessed by [14C]-gelatin degradation and Ca(2+)-ATPase activity are inhibited upon pretreatment with superoxide dismutase (SOD). The O2*- triggered MMP and Ca(2+)-ATPase activities in the membrane are found to be inhibited by TIMP-2. The stimulation of the MMP and Ca(2+)-ATPase activities remain unaffected by the inhibitors of serine, thiol and cysteine groups of proteases such as phenylmethylsulfonylfluoride (PMSF), Bowman Birk inhibitor (BBI), chymostatin, N-ethylmaleimide, leupeptin, antipain and pepstatin. Adding pure bovine MMP-2 to the smooth muscle membrane suspension causes an increase in Ca(2+)-ATPase activity, but the pretreatment with TIMP-2 inhibits the increase in the enzyme activity.     

Inhibition of Na+/Ca2+ exchanger by peroxynitrite in microsomes of pulmonary smooth muscle: role of matrix metalloproteinase-2

Treatment of bovine pulmonary artery smooth muscle microsomes with peroxynitrite (ONOO-) (100 microM) markedly stimulated matrix metalloproteinase-2 (MMP-2) activity and also enhanced Ca2+ATPase activity and ATP-dependent Ca2+ uptake. Pretreatment of the microsomes with vitamin E (1 mM) and TIMP-2 (50 microg/ml) preserved the increase in MMP-2 activity, Ca2+ATPase activity and also ATP-dependent Ca2+ uptake in the microsomes. In contrast, Na(+)-dependent Ca2+ uptake in the microsomes was inhibited by ONOO- and this was found to be reversed by vitamin E (1 mM) and TIMP-2 (50 microg/ml). However, changes caused by ONOO- in MMP-2 activity, ATP-dependent Ca2+ uptake and Na(+)-dependent Ca2+ uptake were not reversed upon pretreatment of the microsomes with a low concentration of 5 microg/ml of TIMP-2 which, on the contrary, reversed MMP-2 (1 microg/ml)-mediated alteration on these parameters. The inhibition of Na(+)-dependent Ca2+ uptake by ONOO- and MMP-2 overpowered the stimulation of ATP-dependent Ca2+ uptake in the microsomes. Treatment with ONOO- abolished the inhibitory effect of TIMP-2 (5 microg/ml) on MMP-2 (1 microg/ml) causing 14C-gelatin degradation. Overall, the present study suggests that ONOO- inactivated TIMP-2, the ambient inhibitor of MMP-2, leading to activation of the ambient proteinase, MMP-2, and subsequently stimulated Ca2+ATPase activity and ATP-dependent Ca2+ uptake, but inhibited Na(+)-dependent Ca2+ uptake, resulting in a marked decrease in Ca2+ uptake in microsomes of bovine pulmonary artery smooth muscle.     

Role of matrix metalloprotease-2 in oxidant activation of Ca<Superscript>2+</Superscript>ATPase by hydrogen peroxide in pulmonary vascular smooth muscle plasma membrane

Exposure of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant H₂O₂ (1 mM) stimulated Ca²⁺ATPase activity. We sought to determine the role of matrix metalloprotease-2 (MMP-2) in stimulating Ca²⁺ATPase activity by H₂O₂ in the smooth muscle plasma membrane. The smooth muscle membrane possesses a Ca²⁺-dependent protease activity in the gelatin containing zymogram having an apparent molecular mass of 72 kDa. The 72 kDa protease activity was found to be inhibited by EGTA, 1: 10-phenanthroline, a2-macroglobulin and tissue inhibitor of metalloprotease-2 (TIMP-2) indicating that the Ca²⁺-dependent 72 kDa protease is the MMP-2. Western immunoblot studies of the membrane suspension with polyclonal antibodies of MMP-2 and TIMP-2 revealed that MMP-2 and TIMP-2, respectively, are the ambient matrix metalloprotease and the corresponding tissue inhibitor of metalloprotease in the membrane. In addition to increasing the Ca²⁺ATPase activity, H₂O₂ also enhanced the activity of the smooth muscle plasma membrane associated protease activity as evidenced by its ability to degrade¹⁴C-gelatin. The protease activity and the Ca²⁺ATPase activity were prevented by the antioxidant, vitamin E, indicating that the effect produced by H₂O₂ was due to reactive oxidant species(es). Both basal and H₂O₂ stimulated MMP-2 activity and Ca²⁺ATPase activity were inhibited by the general inhibitors of matrix metalloproteases: EGTA, 1: 10-phenanthroline, α₂-macroglobulin and also by TIMP-2 (the specific inhibitor of MMP-2) indicating that H₂O₂ increased MMP-2 activity and that subsequently stimulated Ca²⁺ATPase activity in the plasma membrane. This was further confirmed by the following observations: (i) adding low doses of MMP-2 or H₂O₂ to the smooth muscle membrane suspension caused submaximal increase in Ca²⁺ATPase activity, and pretreatment with TIMP-2 prevents the increase in Ca²⁺ATPase activity; (ii) combined treatment of the membrane with low doses of MMP-2 and H₂O₂ augments further the Ca²⁺ATPase activity caused by the respective low doses of either H₂O₂ or MMP-2; and (iii) pretreatment with TIMP-2 prevents the increase in Ca²⁺ATPase activity in the membrane caused by the combined treatment of MMP-2 and H₂O₂.     

Matrix metalloproteinase-2-mediated inhibition of Na+-dependent Ca+ uptake by superoxide radicals (O2*-) in microsomes of pulmonary smooth muscle

Treatment of microsomes (preferably enriched with endoplasmic reticulum) isolated from bovine pulmonary artery smooth muscle tissue with the O2*- -generating system (hypoxanthine (HPX) plus xanthine oxidase (XO)), markedly stimulated matrix metalloproteinase-2 (MMP-2) activity and also enhanced Ca2+ ATPase activity and ATP-dependent Ca2+ uptake. Pretreatment with superoxide dismutase (SOD) and tissue inhibitor of metalloproteinase (TIMP-2) (50 microg ml(-1)), preserved the increase in MMP-2 activity, Ca2+ ATPase activity and also ATP-dependent Ca2+ uptake in the microsomes. In contrast, Na+-dependent Ca2+ uptake in the microsomes was found to be inhibited by the O2*- - generating system. Additionally, O2*- -induced inhibition of Na+-dependent Ca2+ uptake was reversed by SOD and TIMP-2 (50 microg ml(-1)). Electron microscopy revealed that treatment with the O2*- -generating system did not cause any noticeable damage to the microsomes. O2*- -induced changes in MMP-2 activity, ATP-dependent Ca2+ uptake and Na+-dependent Ca2+ uptake, were not reversed upon pretreatment of the microsomes with a low dose (5 microg ml(-1)) of TIMP-2 which, on the contrary, reversed MMP-2 (1 microg ml(-1))-mediated alteration on these parameters. The inhibition of Na+-dependent Ca2+ uptake by O2*- and MMP-2, overpowered the stimulation of ATP-dependent Ca2+ uptake in the microsomes. Treatment of TIMP-2 (5 microg ml(-1)) with the O2*- -generating system abolished the inhibitory effect of TIMP-2 (5 microg ml(-1)) on MMP-2 (1 microg ml(-1)) (measured by (14)C-gelatin degradation). Overall, the present study suggests that O2*- inactivated TIMP-2, the ambient inhibitor of MMP-2, leading to activation of the ambient proteinase, MMP-2, which subsequently stimulated Ca2+ ATPase activity and ATP-dependent Ca2+ uptake, but inhibited Na+-dependent Ca2+ uptake, resulting in a marked decrease in Ca2+ uptake in the smooth muscle microsomes.     

A Mg2+-independent high-affinity Ca2+-stimulated adenosine triphosphatase in the plasma membrane of rat stomach smooth muscle. Subcellular distribution and inhibition by Mg2+

Plasma membrane enriched fraction isolated from the fundus smooth muscle of rat stomach displayed Ca2+-stimulated ATPase activity in the absence of Mg2+. The Ca2+ dependence of such an ATPase activity can be resolved into two hyperbolic components with a high affinity (Km = 0.4 microM) and a low affinity (Km = 0.6 mM) for Ca2+. Distribution of these high-affinity and low-affinity Ca2+-ATPase activities parallels those of several plasma membrane marker enzyme activities but not those of endoplasmic reticulum and mitochondrial membrane marker enzyme activities. Mg2+ also stimulates the ATPase in the absence of Ca2+. Unlike the Mg2+-ATPase and low-affinity Ca2+-ATPase, the plasmalemmal high-affinity Ca2+-ATPase is not sensitive to the inhibitory effect of sodium azide or Triton X-100 treatment. The high-affinity Ca2+-ATPase is noncompetitively inhibited by Mg2+ with respect to Ca2+ stimulation. Such an inhibitory effect of Mg2+ is potentiated by Triton X-100 treatment of the membrane fraction. Calmodulin has little effect on the high-affinity Ca2+-ATPase activity of the plasma membrane enriched fraction with or without EDTA pretreatment. Findings of this novel, Mg2+-independent, high-affinity Ca2+-ATPase activity in the rat stomach smooth muscle plasma membrane are discussed with those of Mg2+-dependent, high-affinity Ca2+-ATPase activities previously reported in other smooth muscle plasma membrane preparations in relation to the plasma membrane Ca2+-pump.     

Isolation of MMP-2 from MMP-2/TIMP-2 complex: characterization of the complex and the free enzyme in pulmonary vascular smooth muscle plasma membrane

We have previously indicated that bovine pulmonary artery smooth muscle plasma membrane possesses a complex of 72-kDa gelatinase and TIMP-2 (MMP-2/TIMP-2 complex) [Mol. Cell. Biochem. 258 (2004) 73]. In this paper, we described isolation of MMP-2 from the MMP-2/TIMP-2 complex, characterizations of the isolated MMP-2 and also the complex. MMP-2/TIMP-2 complex was purified from bovine pulmonary vascular smooth muscle plasma membrane using a combination of purification steps. Heparin-sepharose (100 mM NaCl eluate)-purified preparation contained the MMP-2/TIMP-2 complex. The MMP-2/TIMP-2 complex, which was electrophoresed under reducing condition on the SDS-PAGE and immunobloted with a mixture of polyclonal MMP-2 and TIMP-2 antibodies, revealed two separate immunoreactive bands at their respective electrophoretic migration. Continuous elution electrophoresis of the complex resulted to MMP-2 free of any detectable TIMP-2. The homogeneity of the isolated MMP-2 and the complex was demonstrated by SDS-PAGE under nonreducing condition and also by nondenaturing native-PAGE. The purified TIMP-2 free enzyme electrophoresed as a single band of 72-kDa, which could be activated rapidly and fully by aminophenylmercuric acetate (APMA) with the formation of 62-kDa and 45-kDa active species like native MMP-2 purified from the same source (bovine pulmonary artery smooth muscle). Identical treatment of the MMP-2/TIMP-2 complex with APMA resulted to significantly slower and partial conversion of the active species. Addition of pure TIMP-2 to the TIMP-2 free MMP-2 formed a complex with the progelatinase and prevented the rapid autolytic conversion induced by APMA. Immunoblot study with polyclonal MMP-2 antibody suggested that the isolated 72-kDa gelatinase is the MMP-2. We have also presented additional data indicating that the isolated preparation of 72-kDa gelatinase exhibited properties that are identical with MMP-2 obtained from different sources.     

Inhibitory antibodies to plasmalemmal Ca2+-transporting ATPases. Their use in subcellular localization of (Ca2+ + Mg2+)-dependent ATPase activity in smooth muscle.

Antibodies directed against the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase [(Ca2+ + Mg2+)-dependent ATPase] from pig erythrocytes and from smooth muscle of pig stomach (antral part) were raised in rabbits. Both the IgGs against the erythrocyte (Ca2+ + Mg2+)-ATPase and against the smooth-muscle (Ca2+ + Mg2+)-ATPase inhibited the activity of the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase from smooth muscle. Up to 85% of the total (Ca2+ + Mg2+)-ATPase activity in a preparation of KCl-extracted smooth-muscle membranes was inhibited by these antibodies. The (Ca2+ + Mg2+)-ATPase activity and the Ca2+ uptake in a plasma-membrane-enriched fraction from this smooth muscle were inhibited to the same extent, whereas in an endoplasmic-reticulum-enriched membrane fraction the (Ca2+ + Mg2+)-ATPase activity was inhibited by only 25% and no effect was observed on the oxalate-stimulated Ca2+ uptake. This supports the hypothesis that, in pig stomach smooth muscle, two separate types of Ca2+-transport ATPase exist: a calmodulin-binding ATPase located in the plasma membrane and a calmodulin-independent one present in the endoplasmic reticulum. The antibodies did not affect the stimulation of the (Ca2+ + Mg2+)-ATPase activity by calmodulin.     

Identification,purification and characterization of matrix metalloproteinase-2 in bovine pulmonary artery smooth muscle plasma membrane

Bovine pulmonary artery smooth muscle tissue possesses matrix metalloproteinase-2 (72 kDa gelatinase: MMP-2; E.C. 3.4.24.24) as revealed by immunoblot studies of its plasma membrane suspension with polyclonal MMP-2 antibody. In this report, we described the purification and partial characterization of MMP-2 in the plasma membrane fraction of the smooth muscle. MMP-2 has been purified from plasma membrane fraction of bovine pulmonary artery smooth muscle to homogeneity using a combination of purification steps. Heparin sepharose purified preparation of 72 kDa progelatinase is composed of two distinct population of zymogens: a 72 kDa progelatinase tightly complexed with TIMP-2 (an ambient tissue inhibitor of metalloprotease in the smooth muscle plasma membrane), and a native 72 kDa progelatinase free of any detectable TIMP-2. The homogeneity of the native 72 kDa progelatinase form is demonstrated by SDS-PAGE under non-reducing condition, non-denaturing native gel electrophoresis. The purified TIMP-2 free proenzyme electrophoresed as a single band of 72 kDa which could be activated by APMA with the formation of 62 and 45 kDa active species. The proenzyme is activated poorly by trypsin but not by plasmin. The purified 72 kDa progelatinase is stable at aqueous solution and does not spontaneously autoactivate. The purified 72 kDa gelatinase exhibited properties that are typical of MMP-2 obtained from other sources. These are: (i) its activity is dependent on the divalent cation, Ca+2, and is inhibited by EDTA, EGTA and 1:1 0-phenanthroline; (ii) it was inhibited by a, macroglobulin but not by the inhibitors of serine, cysteine, thiol, aspartic proteinases and calpains; (iii) it was found to be inhibited by TIMP-2, the specific inhibitor of MMP-2; (iv) like MMP-2, obtained from other sources, its major substrates were found to be collagens (type IV and V) and gelatins (type I, IV and V). Additionally, the purified MMP-2 degrades Dnp-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH (dinitrophenyl labelled peptide), a well known synthetic substrate for the MMP-2.     

Cyclic GMP regulation of the plasma membrane (Ca2+-Mg2+)ATPase in vascular smooth muscle

Plasma membrane (Ca2+-Mg2+)ATPase purified from bovine aortic microsomes by calmodulin affinity chromatography was incorporated into soybean phospholipid liposomes. In the reconstituted proteoliposomes, a protein corresponding to the ATPase was phosphorylated by [gamma-32P]ATP in the presence of cGMP and cGMP-dependent protein kinase. Both the affinity for Ca2+ and the maximum Ca2+ uptake activity by the proteoliposomes were increased by the cGMP-dependent phosphorylation, and there was good parallelism between the Ca2+-uptake rate and the extent of phosphorylation. These results strongly suggest that the Ca2+-transport ATPase of the vascular smooth muscle plasma membrane is regulated through its cGMP-dependent phosphorylation.     

Mg2+- or Ca2+-activated ATPase activities of plasma membranes isolated from vascular smooth muscle

Studies of ATP hydrolysis by various subcellular fractions isolated from rat mesenteric arteries and veins indicate that an apparent ATPase activity, which can be activated by Mg2+ or Ca2+, is primarily associated with the plasma membranes. Although both Mg2+-activated and Ca2+-activated ATPase activities under the optimal condition are substantially lower in venous than in arterial plasma membrane fraction, their dependence on the concentration of Mg2+ and Ca2+ are quite similar in arterial as well as venous plasma membrane fractions. No synergistic effect on ATP hydrolysis was observed in the presence of both Mg2+ and Ca2+. In addition, Mg2+-activated and Ca2+-activated ATPase activities show similar pH dependence, inhibition by deoxycholate, stability toward heat inactivation and substrate specificity. Furthermore, Mg2+-activated and Ca2+-activated ATPase activities were similarly reduced in vascular smooth muscles of spontaneously hypertensive rats. These results suggest that the activation of ATP hydrolysis by Mg2+ or Ca2+ may represent a single enzyme moiety in the plasma membrane of vascular smooth muscle. The possible involvement of such ATPase in the Ca2+ transport function of vascular smooth muscle is discussed.     

Characterization of the (Ca2+-Mg2+)ATPase purified by calmodulin-affinity chromatography from bovine aortic smooth muscle

A calmodulin inhibitor, trifluoperazine, suppresses ATP-dependent Ca2+ uptake into microsomes prepared from bovine aortic smooth muscle. From this microsomal preparation which we expected to contain calmodulin-dependent Ca2+-transport ATPase [EC 3.6.1.3], we purified (Ca2+-Mg2+)ATPase by calmodulin affinity chromatography. The protein peak eluted by EDTA had calmodulin-dependent (Ca2+-Mg2+)ATPase activity. The major band (135,000 daltons) obtained after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) accounted for about 80% of the total protein eluted. This major band was phosphorylated by [gamma-32P]ATP in a Ca2+-dependent manner. All the 32P incorporated into the major band was released by hydroxylaminolysis. The ATPase reconstituted in soybean phospholipid liposomes showed ATP, calmodulin-dependent Ca2+ uptake. The affinity of the ATPase for Ca2+, Km, was 7 microM and the maximum ATPase activity was 1.4 mumol/mg/min. These values were changed to 0.17 microM and 3.5 mumol/mg/min, respectively by the addition of calmodulin. The activity of the purified (Ca2+-Mg2+)ATPase was inhibited by orthovanadate, and the concentration required for half-maximal inhibition was about 1.8 microM which is close to that of plasma membrane ATPases. Judging from the effect of orthovanadate and the molecular weight, the purified (Ca2+-Mg2+)ATPase was considered to have originated from the plasma membrane not from the sarcoplasmic reticulum.     

A monoclonal antibody to the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle inhibits plasmalemmal (Ca2+ + Mg2+)-dependent ATPase activity.

A monoclonal antibody (2B3) directed against the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle was prepared. This antibody reacts with a 130,000-Mr protein that co-migrates on SDS/polyacrylamide-gel electrophoresis with the calmodulin-binding (Ca2+ + Mg2+)-ATPase purified from smooth muscle by calmodulin affinity chromatography. The antibody causes partial inhibition of the (Ca2+ + Mg2+)-ATPase activity in plasma membranes from pig stomach smooth muscle, in pig erythrocytes and human erythrocytes. It appears to be directed against a specific functionally important site of the plasmalemmal Ca2+-transport ATPase and acts as a competitive inhibitor of ATP binding. Binding of the antibody does not change the Km of the ATPase for Ca2+ and its inhibitory effect is not altered by the presence of calmodulin. No inhibition of (Ca2+ + Mg2+)-ATPase activity or of the oxalate-stimulated Ca2+ uptake was observed in a pig smooth-muscle vesicle preparation enriched in endoplasmic reticulum. These results confirm the existence in smooth muscle of two different types of Ca2+-transport ATPase: a calmodulin-binding (Ca2+ + Mg2+)-ATPase located in the plasma membrane and a second one confined to the endoplasmic reticulum.     

Anion dependence of Ca2+ transport and (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase in rat pancreatic endoplasmic reticulum

Anion dependence of (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase and its phosphorylated intermediate have been characterized in both "intact" and "broken" vesicles from endoplasmic reticulum of rat pancreatic acinar cells using adenosine 5'-[gamma-32P] triphosphate ([gamma-32P]ATP). In intact vesicles (Ca2+ + K+)-Mg2+-ATPase activity was higher in the presence of Cl- or Br- as compared to NO3-, SCN-, cyclamate-, SO4(2-) or SO3(2-). Incorporation of 32P from [gamma-32P]ATP into the 100-kDa intermediate of this Ca2+ATPase was also higher in the presence of Cl-, Br-, NO3- or SCN- as compared to cyclamate-, SO4(2-) or SO3(2-). When the membrane permeability barrier to anions was abolished by breaking vesicle membrane with the detergent Triton X-100 (0.015%) (Ca2+ + K+)-Mg2+ATPase activity in the presence of weakly permeant anions, such as SO4(2-) and cyclamate-, increased to the level obtained with Cl-. However, 32P incorporation into 100-kDa protein was still higher in the presence of Cl- as compared to cyclamate-, indicating a direct effect of Cl- on the Ca2+ATPase molecule. The anion transport blocker 4,4-diisothiocyanostilbene-2,2-disulfonate (DIDS) inhibited (Ca2+ + K+)-Mg2+ATPase activity to about 10% of the Cl- stimulation level, irrespective of the sort of anions present in both intact and broken vesicles. This indicates a direct effect of DIDS on (Ca2+ + K+)-Mg2+ATPase. K+ ionophore valinomycin influenced (Ca2+ + K+)-Mg2+ATPase activity according to the actual K+ gradient: Ko+ greater than Ki+ caused inhibition, Ko+ less than Ki+ caused stimulation. From these results we conclude that Ca2+ transport into endoplasmic reticulum is coupled to ion movements which must occur to maintain electroneutrality.     

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

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

Ca2+-stimulated, Mg2+-dependent ATPase in bovine thyroid plasma membranes

An isolated plasma membrane fraction from bovine thyroid glands contained a Ca2+-stimulated, Mg2+-dependent adenosine triphosphatase ((Ca2+ + Mg2+)-ATPase) activity which was purified in parallel to (Na+ + K+)-ATPase and adenylate cyclase. The (Ca2+ + Mg2+)-ATPase activity was maximally stimulated by approx. 200 microM added calcium in the presence of approx. 200 microM EGTA (69.7 +/- 5.2 nmol/mg protein per min). In EGTA-washed membranes, the enzyme was stimulated by calmodulin and inhibited by trifluoperazine.     

The rat liver plasma membrane high affinity (Ca2+-Mg2+)-ATPase is not a calcium pump. Comparison with ATP-dependent calcium transporter

The high affinity (Ca2+-Mg2+)-ATPase purified from rat liver plasma membrane (Lin, S.-H., and Fain, J. N. (1984) J. Biol. Chem. 259, 3016-3020) has been further characterized. This enzyme also possesses Mg2+-stimulated ATPase activity with K0.5 of 0.16 microM free Mg2+. However, the Vm of the Mg2+-stimulated activity is only half that of the Ca2+-stimulated ATPase activity. The effects of Ca2+ and Mg2+ on this enzyme are not additive. Both the Ca2+-stimulated ATPase and Mg2+-stimulated ATPase activities have similar affinities for ATP (0.21 mM and 0.13 mM, respectively) and similar substrate specificities (they are able to utilize ATP, GTP, UTP, CTP, ADP, and GDP as substrates); both activities are not inhibited by vanadate, p-chloromercuribenzoate, ouabain, dicyclohexylcarbodiimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, oligomycin, F-, N-ethylmaleimide, La3+, and oxidized glutathione. These properties of the Mg2+- and Ca2+-ATPases indicate that both activities reside on the same protein. A comparison of the properties of this high affinity (Ca2+-Mg2+)-ATPase with those of the liver plasma membrane ATP-dependent Ca2+ transport activity reconstituted into artificial liposomes (Lin, S.-H. (1985) J. Biol. Chem. 260, 7850-7856) suggests that this high affinity (Ca2+-Mg2+)-ATPase is not the biochemical expression of the liver plasma membrane Ca2+ pump. The function of this high affinity (Ca2+-Mg2+)-ATPase remains unknown.     

Effect of spermine on activity of purified Ca2+, Mg2+-ATPase from plasma membranes of myometrium cells

Effect of endogenous polyamine spermine, a relaxant of smooth muscle, on the activity of myometrium cell plasma membrane Ca2+, Mg(2+)-ATPase was studied. It was observed a tendency to activation of enzyme at the spermine concentrations 0.1-0.5 mM, the increase of the polyamine concentrations up to 10 mM inhibited. ATPase by 80% (I50 = 5.5 +/- 0.3 mM). Spermine inhibited enzyme decreasing its turnover rate and affinity for Ca2+. The ATPase affinity for Mg2+ increased in the presence of spermine. It was revealed, that the inhibitory effect of spermine is changed by the stimulatory effect under the increase of Ca2+ concentration (up to 2.6 microM), that correlates with the relaxing effect of this polyamine on the smooth muscle.     

2,5-Di(tert-butyl)-1,4-benzohydroquinone--a novel inhibitor of liver microsomal Ca2+ sequestration

Treatment of rat liver microsomes with 2,5-di(tert-butyl)-1,4-benzohydroquinone caused a dose-related inhibition (Ki congruent to 1 microM) of ATP-dependent Ca2+ sequestration. This was paralleled by a similar impairment of the microsomal Ca2+-stimulated ATPase activity. In contrast, the hydroquinose failed to induce Ca2+ release from Ca2+-loaded liver mitochondria (supplied with ATP), and inhibited neither the mitochondrial F1F0-ATPase nor the Ca2+-stimulated ATPase activity of the hepatic plasma membrane fraction. The inhibition of microsomal Ca2+ sequestration was not associated with any apparent alteration of membrane permeability or loss of other microsomal enzyme activities or modification of microsomal protein thiols. These findings suggest that 2,5-di(tert-butyl)-1,4-benzohydroquinone is a potent and selective inhibitor of liver microsomal Ca2+ sequestration which may be a useful tool in studies of Ca2+ fluxes in intact cells and tissues.     

Activation of erythrocyte Ca2+-plus-Mg2+-stimulated adenosine triphosphatase by protein kinase (cyclic AMP-dependent) inhibitor. Comparison with calmodulin

Purified protein kinase (cyclic AMP-dependent) inhibitor (PKI) from bovine heart stimulated Ca(2+)+Mg(2+)-stimulated ATPase activity in human erythrocytes, the stimulation being maximal at 2mug/0.6ml. By contrast, PKI from rabbit skeletal muscle had no effect. Bovine heart PKI stimulated Ca(2+)+Mg(2+)-stimulated ATPase by increasing the Ca(2+)-sensitivity of the enzyme. This contrasted with the stimulation by calmodulin, which increased the maximum velocity of the Ca(2+)+Mg(2+)-dependent ATPase in addition to its effect on the Ca(2+)-sensitivity. Both membrane-bound and Triton X-100-solubilized Ca(2+)+Mg(2+)-stimulated ATPase activities were stimulated by PKI, indicating that the stimulation did not require an intact membrane structure. At low Ca(2+) concentration the stimulation by PKI and saturating concentrations of calmodulin were additive, suggesting that the two effectors acted by distinct mechanisms. Although 5mum-cyclic AMP inhibited Ca(2+)+Mg(2+)-stimulated ATPase activity by about 20% when measured at low ATP concentrations, probably by stimulation of phosphorylation by an endogenous protein kinase, the stimulation by PKI (about 100%) was not solely due to its antagonism of the protein kinase. This interpretation was supported by a number of observations. First, modification of arginine residues of bovine heart PKI abolished its inhibition of cyclic AMP-dependent protein kinase, but had no effect on the stimulation of Ca(2+)+Mg(2+)-stimulated ATPase. Secondly, trifluoperazine (20mum) antagonized the stimulation of Ca(2+)+Mg(2+)-dependent ATPase by PKI, similarly to its antagonism of calmodulin stimulation, but it did not affect the inhibition of protein kinase by PKI. We conclude that different mechanisms are involved in the inhibition of protein kinase and the stimulation of Ca(2+)+Mg(2+)-stimulated ATPase by PKI.