scholarly journals Influence of Ca2+ and Mg2+ on the turnover of the phosphomonoester group of phosphatidylinositol 4-phosphate in human erythrocyte membranes

1987 ◽  
Vol 244 (1) ◽  
pp. 183-190 ◽  
Author(s):  
H Hegewald ◽  
E Müller ◽  
R Klinger ◽  
R Wetzker ◽  
H Frunder
Keyword(s):  
Steady State ◽  
External Medium ◽  
Erythrocyte Membranes ◽  
Ionophore A23187 ◽  
Half Maximum ◽  
Maximum Inhibition ◽  
Human Erythrocyte Membranes ◽  
Simultaneous Activation ◽  
Membrane Bound ◽  
Phosphatidylinositol 4

In isolated erythrocyte membranes, increasing the free Mg2+ concentration from 0.5 to 10 mM progressively activates the membrane-bound phosphatidylinositol (PtdIns) kinase and leads to the establishment of a new equilibrium with higher phosphatidylinositol 4-phosphate (PtdIns4P) and lower PtdIns concentrations. The steady-state turnover of the phosphomonoester group of PtdIns4P also increases at high Mg2+ concentrations, indicating a simultaneous activation of PtdIns4P phosphomonoesterase by Mg2+. Half-maximum inhibition of PtdIns kinase occurs at 10 microM free Ca2+ in the presence of physiological free Mg2+ concentrations. Increasing free Mg2+ concentrations overcome Ca2+ inhibition of PtdIns kinase. In the presence of Ca2+, calmodulin activates Ca2+-transporting ATPase 5-fold, but does not alter pool size and radiolabelling of PtdIns4P. In intact erythrocytes, adding EGTA or EGTA plus Mg2+ and the ionophore A23187 to the external medium does not exert significant effects on concentration and radiolabelling of polyphosphoinositides when compared with controls in the presence of 1.4 mM free Ca2+.

Calcium-dependent hydrolyses of polyphosphoinositides in human erythrocyte membranes

1984 ◽  
Vol 62 (6) ◽  
pp. 363-368 ◽  
Author(s):  
R. Blaine Moore ◽  
Stanley H. Appel
Keyword(s):  
Time Course ◽  
Erythrocyte Membranes ◽  
Ionophore A23187 ◽  
Erythrocyte Ghosts ◽  
Calcium Activation ◽  
Activation Curve ◽  
Intact Cells ◽  
Calcium Dependent ◽  
Human Erythrocyte Membranes ◽  
Phosphatidylinositol 4

Incubation of erythrocytes with [32P]phosphate resulted in a linear incorporation of the label into PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate), PtdIns4P (phosphatidylinositol 4-monophosphate), and PA (phosphatidic acid) over a period of 2 h at 37 °C. Exposure of 32P-labelled erythrocyte ghosts to calcium caused a loss of label from PtdIns(4,5)P2 and PtdIns4P, but not PA. The concentration of calcium required for half-maximal hydrolyses of both polyphosphoinositides was about 1 μM. Strontium, at higher concentrations, stimulated the hydrolyses of both polyphosphoinositides but barium, up to 1 mM, had little effect. Intact erythrocytes incubated in the presence of Ca–EGTA buffers and the ionophore A23187 did not show marked losses of [32P]PtdIns(4,5)P2 or [32P]PtdIns4P, but rather exhibited a dramatic increase in the level of [32P]PA. In contrast, cells which had been depleted of their ATP lost significant amounts of [32P]PtdIns(4,5)P2 and [32P]PtdIns4P and had less change in their levels of [32P]PA relative to intact cells. The calcium activation curve and the time course for [32P]PA synthesis in intact cells were similar to the calcium activation curve and the time course for the hydrolyses of [32P]PtdIns(4,5)P2 and [32P]PtdIns4P in ATP-depleted erythrocytes. These results strongly support a link between Ca2+-dependent polyphosphoinositide breakdown and PA synthesis in human erythrocytes.

scholarly journals The control by Ca2+ of the polyphosphoinositide phosphodiesterase and the Ca2+-pump ATPase in human erythrocytes

1982 ◽  
Vol 202 (1) ◽  
pp. 53-58 ◽  
Author(s):  
C. Peter Downes ◽  
Robert H. Michell
Keyword(s):  
Ionic Strength ◽  
Erythrocyte Membrane ◽  
Erythrocyte Membranes ◽  
Phosphodiesterase 4 ◽  
Maximal Activation ◽  
Human Erythrocyte Membranes ◽  
The Right ◽  
Phosphatidylinositol 4 ◽  
Low Ionic Strength ◽  
Micromolar Range

1. Both the Ca2+-pump ATPase and the polyphosphoinositide phosphodiesterase of the erythrocyte membrane can, when assayed under appropriate conditions, be activated by Ca2+ in the micromolar range. We have therefore compared the mechanisms and affinities for Ca2+ activation of the two enzymes in human erythrocyte membranes, to see whether the polyphosphoinositide phosphodiesterase would be active in normal healthy erythrocytes. 2. At physiological ionic strength and in the presence of calmodulin, the Ca2+-pump ATPase was activated by Ca2+ in a highly co-operative manner, with half-maximal activation occurring at about 0.3μm-Ca2+. At an optimal Ca2+concentration, calmodulin stimulated the Ca2+-sensitive ATPase activity about 10-fold. 3. Ca2+ activated the polyphosphoinositide phosphodiesterase in a non-co-operative manner. The Ca2+ requirements for breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were identical, which supports our previous conclusion that Ca2+ activates a single polyphosphoinositide phosphodiesterase that degrades both lipids with equal facility. Added calmodulin did not affect the activity of the polyphosphoinositide phosphodiesterase. 4. At low ionic strength in the absence of Mg2+, half-maximal activation of the phosphodiesterase was at about 3μm-Ca2+. The presence of 1mm-Mg2+ shifted the Ca2+ activation curve to the right, as did elevation of the ionic strength. When the Ca2+-pump ATPase and the polyphosphoinositide phosphodiesterase were assayed in the same incubations and under conditions of intracellular ionic strength and Mg2+concentration, the ATPase was fully activated at 3μm-Ca2+, whereas no polyphosphoinositide phosphodiesterase activity was detected below 100μm-Ca2+. 5. The Ca2+-pump ATPase of the erythrocyte membrane normally maintains the Ca2+ concentration of healthy erythrocytes below approx. 0.1μm. It therefore seems unlikely that the polyphosphoinositide phosphodiesterase of the erythrocyte membrane ever expresses its activity in a healthy erythrocyte.

scholarly journals The effect of calmodulin on the phosphoprotein intermediate of Mg2+-dependent Ca2+-stimulated adenosine triphosphatase in human erythrocyte membranes

1981 ◽  
Vol 194 (2) ◽  
pp. 481-486 ◽  
Author(s):  
D A Jeffery ◽  
B D Roufogalis ◽  
S Katz
Keyword(s):  
Steady State ◽  
Atpase Activity ◽  
Turnover Rate ◽  
Erythrocyte Membranes ◽  
Steady State Concentration ◽  
Transport Atpase ◽  
Dependent Atpase ◽  
Mgcl2 Concentration ◽  
Human Erythrocyte Membranes ◽  
State Concentration

The effect of calmodulin on the formation and decomposition of the Ca2+-dependent phosphoprotein intermediate of the (Mg2+ + Ca2+)-dependent ATPase in erythrocyte membranes was investigated. In the presence of 60 microM-Ca2+ and 25 microM-MgCl2, calmodulin (0.5-1.5 microgram) did not alter the steady-state concentration of the phosphoprotein, but increased its rate of decomposition. Higher calmodulin concentrations significantly decreased the steady-state concentration of phosphoprotein. Calmodulin (0.5-1.7 microgram) increased Ca2+-transport ATPase activity by increasing the turnover rate of its phosphoprotein intermediate. Increasing the MgCl2 concentration from 25 microM to 250 microM increased the (Mg2+ + Ca2+)-dependent ATPase activity, but decreased the concentration of the phosphoprotein intermediate. Similarly to calmodulin, MgCl2 increased the turnover rate of the Ca2+-transport ATPase complex (about 3-fold). At the higher MgCl2 concentration calmodulin did not further affect the decomposition of the phosphoprotein intermediate. It was concluded that both calmodulin and MgCl2 increase the turnover of the Ca2+-pump by enhancing the decomposition of the Ca2+-dependent phosphoprotein intermediate.

Use of Steady-State Laurdan Fluorescence to Detect Changes in Liquid Ordered Phases in Human Erythrocyte Membranes

2006 ◽  
Vol 211 (1) ◽  
pp. 15-25 ◽  
Author(s):  
Rebekah Vest ◽  
Rachel Wallis ◽  
Lauren B. Jensen ◽  
Andrea C. Haws ◽  
Joseph Callister ◽  
...  
Keyword(s):  
Steady State ◽  
Human Erythrocyte ◽  
Erythrocyte Membranes ◽  
Ordered Phases ◽  
Human Erythrocyte Membranes ◽  
Liquid Ordered ◽  
Laurdan Fluorescence

Effects of calcium on the vasopressin-sensitive cAMP metabolism in medullary tubules

1985 ◽  
Vol 249 (6) ◽  
pp. F956-F966 ◽  
Author(s):  
E. Kusano ◽  
N. Murayama ◽  
J. L. Werness ◽  
S. Christensen ◽  
S. Homma ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Rat Kidney ◽  
Detectable Effect ◽  
Ionophore A23187 ◽  
Camp Accumulation ◽  
Half Maximum ◽  
Maximum Inhibition ◽  
Camp Metabolism ◽  
Collecting Tubules ◽  
Stimulation Of

The modulatory effect of Ca on [Arg8]vasopressin-dependent (AVP) cAMP metabolism was studied in medullary collecting tubules (MCT) and medullary ascending limbs (MAL) microdissected from rat kidney. In MCT segments incubated in vitro with AVP, the accumulation of cAMP was enhanced (delta +59%) when Ca was omitted from the incubation medium compared with a medium with 2 mM of ionized calcium (Ca2+). Ionophore A23187 caused a decrease in AVP-stimulated cAMP accumulation in MCT in the presence of 2 mM Ca2+ but not in a Ca2+-free medium. Diltiazem and verapamil enhanced the AVP-stimulated cAMP accumulation in MCT; PTH had no detectable effect. A23187 caused a dose-dependent inhibition of cAMP accumulation stimulated by AVP with forskolin in both MCT and in MAL. However, in MAL the A23187 concentration needed for half-maximum inhibition (6.3 X 10(-6) M) was higher than for MCT (3.9 X 10(-7) M). The maximum inhibition in MAL (-65%) was less than in MCT (-97%). In the presence of 3-isobutyl-1-methylxanthine, AVP-stimulated cAMP accumulation was inhibited by A23187 in MCT (-45%) but not in MAL. Naproxen or ibuprofen did not relieve the inhibitory action of A23187 in MCT. Added Ca2+ inhibited the AVP-stimulated adenylate cyclase in MCT and MAL (half-maximum approximately equal to 5 X 10(-4) M Ca2+) and stimulated cAMP phosphodiesterase (cAMP-PDIE) in both MCT and in MAL (half-maximum approximately equal to 9 X 10(-5) M Ca2+). Incubation of MCT and MAL with A23187 decreased (-50%) the content of ATP. Results suggest that increased influx of extracellular Ca2+ inhibits the AVP-stimulated cAMP accumulation in MCT and to a much lesser degree in MAL. Deceased cAMP accumulation in MCT is probably due to both stimulation of cAMP-PDIE and the inhibition of adenylate cyclase, whereas in MAL it is due to stimulation of cAMP-PDIE. The results suggest that Ca2+ influx exhibits a negative modulatory effect on AVP-dependent cAMP metabolism mainly in MCT.

Tissue Cholinesterases. A Comparative Study of Their Kinetic Properties

2000 ◽  
Vol 55 (1-2) ◽  
pp. 100-108 ◽  
Author(s):  
Kunjan R. Dave ◽  
Anshu R. Syal ◽  
Surendra S. Katyare
Keyword(s):  
Human Serum ◽  
Erythrocyte Membranes ◽  
Soluble Form ◽  
Kinetic Properties ◽  
Rat Serum ◽  
Human Erythrocyte Membranes ◽  
Substrate Saturation ◽  
Membrane Bound ◽  
The One ◽  
Three Components

Abstract The substrate saturation and temperature-dependent kinetic properties of soluble and membrane-bound forms of acetylcholinestarase (AChE) from brain and butyrylcholinesterase (BChE) from heart and liver were examined. In simultaneous studies these parameters were also measured for AChE in erythrocyte membranes and for BChE in the serum from rat and humans. For both soluble and membrane-bound forms of the enzyme from the three tissues, two components were discernible. In the brain, Km of component I (high affinity) and component II (low affinity) was somewhat higher in membrane-bound form than that of the soluble form components, while the Vmax values were significantly higher by about five fold. In the heart, K m of component II was lower in membrane-bound form than in the soluble form, while Vmax for both the components was about four to six fold higher in the membrane-bound form. In the liver, Vmax was marginally higher for the two components of the membrane-bound enzyme; the Km only of component I was higher by a factor of 2. In the rat erythrocyte membranes three components of AChE were present showing increasing values of Km and Vmax. In contrast, in the human erythrocyte membranes only two components could be detected; the one corresponding to component II of rat erythrocyte membranes was absent. In the rat serum two components of BChE were present while the human serum was found to possess three components. Component I of the human serum was missing in the rat serum. Temperature kinetics studies revealed that the Arrhenius plots were biphasic for most of the systems except for human serum. Membrane binding of the enzyme resulted in decreased energy of activation with shift in phase transition temperature (Tt) to near physiological temperature.

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.

scholarly journals Activation of membrane-bound high-affinity calcium ion-sensitive adenosine triphosphatase of human erythrocytes by bivalent metal ions

1975 ◽  
Vol 147 (2) ◽  
pp. 359-361 ◽  
Author(s):  
H Pfleger ◽  
H U Wolf
Keyword(s):  
Metal Ions ◽  
Calcium Ion ◽  
Metal Ion ◽  
Adenosine Triphosphatase ◽  
Erythrocyte Membranes ◽  
Bivalent Metal ◽  
High Affinity ◽  
Human Erythrocyte Membranes ◽  
Bivalent Metal Ions ◽  
Membrane Bound

The Ca2+-sensitive ATPase (adenosine triphosphatase) of human erythrocyte membranes is activated, not only by Ca2+ ions, but also by a series of other bivalent metal ions including Sr2+, Ba2+, Mn2+, Ni2+, Co2+, Cd2+, Cu2+, Zn2+ and Pb2+. The degree of activation is dependent on the radius of the ion rather than on its nature, in contrast with the dissociation constant of the enzyme--metal ion complex.

scholarly journals Purification and characterization of phosphatidylinositol 4-kinase from human erythrocyte membranes

1991 ◽  
Vol 200 (1) ◽  
pp. 179-185 ◽  
Author(s):  
Reinhard WETZKER ◽  
Reinhard KLINGER ◽  
Justin HSUAN ◽  
Michael J. FRY ◽  
Andrea KAUFFMANN-ZEH ◽  
...  
Keyword(s):  
Human Erythrocyte ◽  
Erythrocyte Membranes ◽  
Purification And Characterization ◽  
Human Erythrocyte Membranes ◽  
Phosphatidylinositol 4
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