scholarly journals Phospholipid-protein interactions of the plasma-membrane Ca2+-transporting ATPase. Evidence for a tissue-dependent functional difference

1989 ◽  
Vol 263 (3) ◽  
pp. 687-694 ◽  
Author(s):  
L Missiaen ◽  
L Raeymaekers ◽  
F Wuytack ◽  
M Vrolix ◽  
H de Smedt ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Protein Interactions ◽  
Hill Coefficient ◽  
Functional Difference ◽  
Muscle Enzyme ◽  
Low Concentrations ◽  
Erythrocyte Enzyme ◽  
The Hill ◽  
Stimulation Of

The aim of the present work was to investigate the stimulation of the plasma-membrane Ca2+-transporting ATPase by negatively charged phospholipids. The Ca2+-transporting ATPase was purified from pig stomach smooth muscle and from pig erythrocytes, and was reactivated with phosphatidylcholine (PC) in the presence and absence of negatively charged phospholipids. The substitution of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidic acid (PA) or phosphatidylserine (PS) for PC induced profound changes in the Vmax, the K0.5 and the Hill coefficient of the Ca2+-activation curves for both ATPases. Low concentrations of each of the negatively charged phospholipids increased the Vmax., but high ratios of PIP, PIP2 or PA to PC decreased this parameter. PI, PA and PS increased the Vmax. of the erythrocyte enzyme to a larger extent than that of the smooth-muscle enzyme. This difference was less pronounced for PIP and absent for PIP2. PI (greater than 20% PC substituted), PIP, PIP2, PA and PS all increased the affinity of the two Ca2+-transporting ATPases for Ca2+ in the following order of potency: PIP2 greater than PIP greater than PI approximately PS approximately PA. PI, PA and PS increased the Ca2+ affinity of the smooth-muscle enzyme more than that of the erythrocyte enzyme; this difference was less pronounced for PIP and absent for PIP2. Even in the presence of calmodulin, all of the negatively charged phospholipids were still able to increase the Vmax. of the erythrocyte enzyme, whereas only PIP and PIP2 increased the affinity for Ca2+. The effect of PI at low concentrations (less than 20%) on the erythrocyte enzyme was peculiar in that it caused a decrease in the Ca2+ affinity instead of an increase. This effect was not observed for the smooth-muscle enzyme. All of the negatively charged phospholipids slightly increased the Hill coefficient for Ca2+ of both ATPases, and this effect was additive to that of calmodulin. The stimulation of the erythrocyte enzyme exhibited positive co-operativity towards PI and PIP, whereas that of the smooth-muscle enzyme did not. It is concluded (1) that there is a correlation between the number of negative charges on the phospholipids (PIP2 greater than PIP greater than PA approximately PI approximately PS) and the magnitude of their effect on the Vmax. and the K0.5 for Ca2+, and (2) that the action of the lipids on the smooth-muscle enzyme differs from that on the erythrocyte enzyme, indicating that these two Ca2+-transporting ATPases are not the same.

scholarly journals Role of arginine residues in the stimulation of the smooth-muscle plasma-membrane Ca2+ pump by negatively charged phospholipids

1989 ◽  
Vol 264 (2) ◽  
pp. 609-612 ◽  
Author(s):  
L Missiaen ◽  
L Raeymaekers ◽  
G Droogmans ◽  
F Wuytack ◽  
R Casteels
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Phosphatidic Acid ◽  
Muscle Plasma Membrane ◽  
Low Concentrations ◽  
Important Interaction ◽  
Arginine Residues ◽  
Acidic Phospholipids ◽  
Stimulation Of

Negatively charged phospholipids strongly stimulate the purified plasma membrane Ca2+ pump of erythrocytes [Enyedi, Flura, Sarkadi, Gardos & Carafoli (1987) J. Biol. Chem. 262, 6425-6430] and of smooth muscle [Missiaen, Raeymaekers, Wuytack, Vrolix, De Smedt & Casteels, (1989) Biochem. J. 263, 687-694]. We have investigated the role of arginine residues in the interaction of these acidic phospholipids with the smooth-muscle Ca2+ transport ATPase. The arginine-modifying reagent phenylglyoxal inhiibited the ATPase activity in a time-dependent fashion by decreasing the Vmax. of the Ca2(+)-activation curve. Low concentrations of PtdIns, PtdIns4P, PtdIns(4,5) P2, phosphatidylserine and phosphatidic acid partially prevented this inactivation. This protective effect was however not apparent at higher concentrations of PtdIns4P, PtdIns(4,5) P2 and phosphatidic acid, which may be related to the previously observed inhibition of the enzyme at higher concentrations of these phospholipids. These findings indicate that the functionally important interaction of the acidic lipids with the protein occurs at least partially via arginine residue(s).

scholarly journals Kinetic studies on the regulation of rabbit liver pyruvate kinase

1973 ◽  
Vol 131 (2) ◽  
pp. 287-301 ◽  
Author(s):  
M. G. Irving ◽  
J. F. Williams
Keyword(s):  
Pyruvate Kinase ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Kinetic Studies ◽  
Rabbit Liver ◽  
Hill Coefficient ◽  
Saturation Curve ◽  
Low Concentrations ◽  
Ammonium Sulphate Fractionation ◽  
The Hill

Two kinetically distinct forms of pyruvate kinase (EC 2.7.1.40) were isolated from rabbit liver by using differential ammonium sulphate fractionation. The L or liver form, which is allosterically activated by fructose 1,6-diphosphate, was partially purified by DEAE-cellulose chromatography to give a maximum specific activity of 20 units/mg. The L form was allosterically activated by K+ and optimum activity was recorded with 30mm-K+, 4mm-MgADP-, with a MgADP-/ADP2- ratio of 50:1, but inhibition occurred with K+ concentrations in excess of 60mm. No inhibition occurred with either ATP or GTP when excess of Mg2+ was added to counteract chelation by these ligands. Alanine (2.5mm) caused 50% inhibition at low concentrations of phosphoenolpyruvate (0.15mm). The homotropic effector, phosphoenolpyruvate, exhibited a complex allosteric pattern (nH+2.5), and negative co-operative interactions were observed in the presence of low concentrations of this substrate. The degree of this co-operative interaction was pH-dependent, with the Hill coefficient increasing from 1.1 to 3.2 as the pH was raised from 6.5 to 8.0. Fructose 1,6-diphosphate interfered with the activation by univalent ions, markedly decreased the apparent Km for phosphoenolpyruvate from 1.2mm to 0.2mm, and transformed the phosphoenolpyruvate saturation curve into a hyperbola. Concentrations of fructose 1,6-diphosphate in excess of 0.5mm inhibited this stimulated reaction. The M or muscle-type form of the enzyme was not activated by fructose 1,6-diphosphate and gave a maximum specific activity of 0.3 unit/mg. A Michaelis–Menten response was obtained when phosphoenolpyruvate was the variable substrate (Km+0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg2+.

Calmodulin stimulation of smooth muscle plasmalemmal vesicle Ca2+ uptake: direct or indirect effect?

1992 ◽  
Vol 263 (2) ◽  
pp. H366-H371
Author(s):  
C. Zhang ◽  
R. J. Paul ◽  
E. G. Kranias
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Current Evidence ◽  
Plasma Membrane Vesicle ◽  
Maximal Rate ◽  
Muscle Contractility ◽  
Smooth Muscle Contractility ◽  
Uptake Rates ◽  
Dependent Protein ◽  
Stimulation Of

Current evidence suggests that Ca(2+)-calmodulin-dependent protein kinase(s) may be involved in the regulation of smooth muscle contractility. The plasmalemmal Ca2+ pump plays an important role in smooth muscle contractility, and this pump is stimulated by calmodulin. However, it is not known whether this is due to direct activation, or calmodulin-dependent protein phosphorylation, or both. We tested these hypotheses using a plasma membrane vesicle preparation from porcine antral smooth muscle. Inclusion of calmodulin in the Ca(2+)-uptake assay decreased the free Ca2+ concentration at which Ca2+ uptake is 50% of the maximal rate (1.5 +/- 0.21 to 0.84 +/- 0.06 microM) of the Ca2+ pump for Ca2+, without changing maximal rate of Ca2+ uptake (Vmax) (8.0 +/- 1.1 to 8.4 +/- 0.7 nmol.min-1.mg-1). In contrast, prephosphorylation in the absence of Ca(2+)-calmodulin increased Ca(2+)-uptake rates at both low (pCa 6.0) and high Ca2+ concentration (pCa 5.0), suggesting an increase in Vmax. Further phosphorylation in the presence of Ca(2+)-calmodulin was not associated with any further increases in the Ca(2+)-uptake rates at pCa 6.0. However, inclusion of calmodulin in the Ca(2+)-uptake assays stimulated the rates (pCa 6.0) of both unphosphorylated and prephosphorylated vesicles to a similar extent. These findings suggest that the stimulation of the smooth muscle plasmalemmal Ca2+ pump by calmodulin is predominantly due to a direct effect via a mechanism distinct from that by plasma membrane phosphorylation.

scholarly journals Ca2+-dependent and Ca2+-independent degradation of phosphatidylinositol in rabbit vas deferens

1981 ◽  
Vol 194 (1) ◽  
pp. 129-136 ◽  
Author(s):  
K Egawa ◽  
B Sacktor ◽  
T Takenawa
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Vas Deferens ◽  
The Other ◽  
Ionophore A23187 ◽  
K Concentration ◽  
Rabbit Vas Deferens ◽  
Dependent Mechanism ◽  
Stimulation Of

The effects of Ca2+ and acetylcholine on the degradation and synthesis of phosphatidylinositol in rabbit vas deferens was studied in vitro by a pulse–chase technique and by measuring the content of the phospholipid in the tissue. Ca2+-dependent degradation of phosphatidylinositol was found in slices and homogenates prelabelled with myo-[2-3H]inositol. The phosphatidylinositol content of the slices also decreased by a Ca2+-dependent mechanism. On the other hand, removal of intracellular Ca2+ with the ionophore A23187 and EGTA increased the amount of phosphatidylinositol. These results indicate that the intracellular Ca2+ concentration has an important role in regulating the phosphatidylinositol content of the tissue. Increasing the extracellular K+ concentration, which causes an increase in plasma-membrane Ca2+ permeability, did not enhance phosphatidylinositol breakdown nor decrease its tissue content. However, phosphatidylinositol synthesis was clearly inhibited. After stimulation of the smooth muscle with acetylcholine, degradation of phosphatidylinositol was enhanced. Furthermore, the content of phosphatidylinositol in the tissue also decreased. These phenomena were evident even in the absence of Ca2+. The acetylcholine-induced degradation of phosphatidylinositol was blocked by the muscarinic antagonist atropine, but not by the nicotinic antagonist (+)-tubocurarine. The acetylcholine-induced decrease in the phosphatidylinositol content of the tissue led to the compensatory synthesis of phosphatidylinositol. Synthesis was separated from degradation in the same tissue. Compensatory synthesis was inhibited by acetylcholine. The degradation of phosphatidylinositol induced by acetylcholine was not inhibited by 8-bromoguanosine 3′:5′-cyclic monophosphate, indicating that the degradative process was not mediated by an increase in the cyclic nucleotide.

scholarly journals AIF4-induced inhibition of the ATPase activity, the Ca2+-transport activity and the phosphoprotein-intermediate formation of plasma-membrane and endo(sarco)plasmic-reticulum Ca2+-transport ATPases in different tissues. Evidence for a tissue-dependent functional difference

1989 ◽  
Vol 261 (2) ◽  
pp. 655-660 ◽  
Author(s):  
L Missiaen ◽  
F Wuytack ◽  
H De Smedt ◽  
F Amant ◽  
R Casteels
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Intermediate Formation ◽  
Functional Difference ◽  
Transport Activity ◽  
Fast Skeletal Muscle ◽  
Transport Atpase ◽  
Plasmic Reticulum

AIF4- inhibits the (Ca2+ + Mg2+)-ATPase activity of the plasma-membrane and the sarcoplasmic-reticulum Ca2+-transport ATPase [Missiaen, Wuytack, De Smedt, Vrolix & Casteels (1988) Biochem. J. 253, 827-833]. The aim of the present work was to investigate this inhibition further. We now report that AIF4- inhibits not only the (Ca2+ + Mg2+)-ATPase activity, but also the ATP-dependent 45Ca2+ transport, and the formation of the phosphoprotein intermediate by these pumps. Mg2+ potentiated the effect of AIF4-, whereas K+ had no such effect. The plasma-membrane Ca2+-transport ATPase from erythrocytes was 20 times less sensitive to inhibition by AIF4- as compared with the Ca2+-transport ATPase from smooth muscle. The endoplasmic-reticulum Ca2+-transport ATPase from smooth muscle was inhibited to a greater extent than the sarcoplasmic-reticulum Ca2+-transport ATPase of slow and fast skeletal muscle.

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

1985 ◽  
Vol 231 (3) ◽  
pp. 737-742 ◽  
Author(s):  
J Verbist ◽  
F Wuytack ◽  
L Raeymaekers ◽  
R Casteels
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Muscle ◽  
Plasma Membrane ◽  
Atpase Activity ◽  
Membrane Fraction ◽  
Transport Atpase ◽  
Calmodulin Binding ◽  
Dependent Atpase ◽  
Inhibitory Antibodies ◽  
Stimulation Of

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.

Uptake and release of calcium by canine gastric corpus smooth muscle plasma membrane enriched fraction

1983 ◽  
Vol 61 (7) ◽  
pp. 699-704 ◽  
Author(s):  
Yasushi Sakai ◽  
Ashok K. Grover ◽  
Jo-Ann E. T. Fox ◽  
Edwin E. Daniel
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Reaction Time ◽  
Exponential Decay ◽  
Initial Velocity ◽  
Hill Coefficient ◽  
Rapid Loss ◽  
Muscle Plasma Membrane ◽  
Gastric Corpus ◽  
Uptake And Release

Calcium movements across plasma membrane enriched vesicles isolated from canine gastric corpus smooth muscle were investigated. The ATP-dependent Ca2+ uptake increased with time up to 10 min. The uptake for the initial 2-min period was approximately linear with time. The apparent initial velocity of the ATP-dependent Ca2+ uptake increased monotonically with free Ca2+ concentration from 0.1 to 2 μM, and further increases in free Ca2+ concentration did not increase the Ca2+ uptake. The free Ca2+ dependence curve could be described with a Hill coefficient of approximately 1.0 and Km of 0.85 ± 0.01 μM for free Ca2+ concentration. Passive Ca2+ uptake (reaction time = 1 h) also increased with increasing free Ca2+ concentrations from 0.02 to 4.0 mM. Dilution of loaded vesicles in isotonic media containing EGTA led to initial rapid loss (< 1 min) followed by a slower release which showed simple exponential decay. The t1/2 values of the slower Ca2+ loss from these vesicles were 16.1 ± 0.9 min (actively loaded n = 5) and 18.4 ± 0.9 min (passively loaded n = 3), respectively. Dilution in isotonic medium containing both EGTA and A23187 released all the sequestered Ca2+ from these loaded vesicles.

scholarly journals Sodium/calcium exchange in smooth-muscle microsomal fractions

1984 ◽  
Vol 218 (2) ◽  
pp. 421-427 ◽  
Author(s):  
N Morel ◽  
T Godfraind
Keyword(s):  
Smooth Muscle ◽  
Microsomal Fraction ◽  
Rat Aorta ◽  
Maximum Activity ◽  
Hill Coefficient ◽  
Ionophore A23187 ◽  
Half Maximum ◽  
Maximum Inhibition ◽  
Nacl Concentration ◽  
The Hill

The existence of Na+ -dependent Ca2+ transport was investigated in microsomal fractions from the longitudinal smooth muscle of the guinea-pig ileum and from the rat aorta, and its activity was compared with that of the plasmalemmal ATP-dependent Ca2+ pump previously identified in these preparations. The rate of Ca2+ release from plasmalemmal vesicles previously loaded with Ca2+ through the ATP-dependent Ca2+ pump was transiently faster in the presence of 150 mM-NaCl in the medium than in the presence of 150 mM-KCl or -LiCl or 300 mM-sucrose. Na+-loaded vesicles took up Ca2+ when an outwardly directed Na+ gradient was formed across the membrane. The Ca ionophore A23187 induced a rapid release of 85% of the sequestered Ca2+, whereas only 15% was displaced by La3+. Ca2+ accumulated by the Na+-induced Ca2+ transport was released by the addition of NaCl, but not KCl, to the medium. Ca2+ uptake in Na+-loaded vesicles was inhibited in the presence of increasing NaCl concentration in the medium. Half-maximum inhibition was observed with 28 mM-NaCl. Data fitted the Hill equation, with a Hill coefficient (h) of 1.9. Na+-induced Ca2+ uptake was a saturable function of Ca2+ concentration in the medium. Half-maximum activity was obtained with 18 microM-Ca2+ in intestinal-smooth-muscle microsomal fraction and with 50 microM-Ca2+ in aortic microsomal fraction. The results suggest that in these membrane preparations a transmembrane movement of Ca2+ can be driven by a Na+ gradient. However, the Na+-induced Ca2+ transport had a lower capacity, a lower affinity and a slower rate than the ATP-dependent Ca2+ pump.

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