Phospholipid and calmodulin activation of solubilized calcium-transport ATPase from human erythrocytes: regulation by magnesium

1981 ◽  
Vol 59 (11-12) ◽  
pp. 880-888 ◽  
Author(s):  
Ameera Al-Jobore ◽  
Basil D. Roufogalis
Keyword(s):  
Fatty Acids ◽  
Atpase Activity ◽  
Calcium Transport ◽  
Erythrocyte Membranes ◽  
Transport Atpase ◽  
Partial Reaction ◽  
Low Concentrations ◽  
Human Erythrocyte Membranes ◽  
Solubilized Enzyme ◽  
Triton X

The effect of phospholipids on Triton X-100 solubilized (Ca2+ + Mg2+)-ATPase from human erythrocyte membranes has been examined. The enzyme activity was increased by phosphatidylinositol, phosphatidylserine, and phosphatidic acid at both low (2 μM) and high (65 μM) free Ca2+ concentrations, while phosphatidylcholine had little effect and phosphatidyiethanolamine and cardiolipin inhibited the (Ca2+ + Mg2+)-ATPase activity at all Ca2+ concentrations studied. The diacylglycerol, diolein, inhibited the enzyme at high, but not low, Ca2+ concentrations. Low concentrations of phospholipase A2 (1–2 international units) also activated the solubilized enzyme, at least in part by releasing free fatty acids, as the activation was mimicked by oleic acid (1–2 μmol/mg protein) and was abolished by fatty acid depleted bovine serum albumin. The combined activation by saturating levels of phosphatidylserine and calmodulin was additive at 6.5 mM MgCl2, and probably occurred at distinct sites on a regulatory component of the enzyme. The activation by both effectors was antagonized by MgCl2 from 0.5 to 6.5 mM. Ca2+ inhibition of the enzyme was also antagonized by MgCl2 at similar concentrations. Analysis of various models suggested that phosphatidylserine had two effects on (Ca2+ + Mg2+)-ATPase activity. First, a low Ca2+ affinity form of the enzyme was converted to a high Ca2+ affinity form, which was more sensitive to Ca2+ inhibition. Second, it increased the turnover of the enzyme, probably by enhancing its dephosphorylation, which was mimicked in this study by the Ca2+-dependent p-nitrophenyiphosphatase partial reaction.

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.

scholarly journals Ultrastructure of the intact skeleton of the human erythrocyte membrane.

1986 ◽  
Vol 102 (3) ◽  
pp. 997-1006 ◽  
Author(s):  
B W Shen ◽  
R Josephs ◽  
T L Steck
Keyword(s):  
Human Erythrocyte ◽  
Actin Filaments ◽  
Band 3 ◽  
Carbon Films ◽  
Erythrocyte Membranes ◽  
Accessory Proteins ◽  
Red Cell Membrane ◽  
Human Erythrocyte Membranes ◽  
Low Ionic Strength ◽  
Triton X

Filamentous skeletons were liberated from isolated human erythrocyte membranes in Triton X-100, spread on fenestrated carbon films, negatively stained, and viewed intact and unfixed in the transmission electron microscope. Two forms of the skeleton were examined: (a) basic skeletons, stripped of accessory proteins with 1.5 M NaCl so that they contain predominantly polypeptide bands 1, 2, 4.1, and 5; and (b) unstripped skeletons, which also bore accessory proteins such as ankyrin and band 3 and small plaques of residual lipid. Freshly prepared skeletons were highly condensed. Incubation at low ionic strength and in the presence of dithiothreitol for an hour or more caused an expansion of the skeletons, which greatly increased the visibility of their elements. The expansion may reflect the opening of spectrin from a compact to an elongated disposition. Expanded skeletons appeared to be organized as networks of short actin filaments joined by multiple (5-8) spectrin tetramers. In unstripped preparations, globular masses were observed near the centers of the spectrin filaments, probably corresponding to complexes of ankyrin with band 3 oligomers. Some of these globules linked pairs of spectrin filaments. Skeletons prepared with a minimum of perturbation had thickened actin protofilaments, presumably reflecting the presence of accessory proteins. The length of these actin filaments was highly uniform, averaging 33 +/- 5 nm. This is the length of nonmuscle tropomyosin. Since there is almost enough tropomyosin present to saturate the F-actin, our data support the hypothesis that tropomyosin may determine the length of actin protofilaments in the red cell membrane.

Identification of acrolein from the ozone oxidation of unsaturated fatty acids

1999 ◽  
Vol 18 (11) ◽  
pp. 677-682 ◽  
Author(s):  
R Medina-Navarro ◽  
E Mercado-Pichardo ◽  
O Herńndez-Pérez ◽  
J J Hicks
Keyword(s):  
Fatty Acids ◽  
Unsaturated Fatty Acids ◽  
Biological Systems ◽  
Thiobarbituric Acid ◽  
Environmental Pollutant ◽  
Fused Silica ◽  
Erythrocyte Membranes ◽  
Human Erythrocyte Membranes ◽  
Dependent Form ◽  
By Products

By-products of lipoperoxidation reactions may be associated with the genesis or the progression of several diseases as arteriosclerosis, diabetes and cancer, among many others. Acrolein, at first a widely distributed environmental pollutant, is currently known as a compound capable of being generated as a result of metabolic reactions within biological systems, highly toxic and the most electrophilic of the a, b-unsaturated aldehydes formed during lipoperoxidation. In the present study: 1 The separation of acrolein and malondialdehyde was achieved at alkaline pH with the use of high voltage capillary electrophoresis in uncoated fused-silica capillaries. 2 It was demonstrated how the oxidation of fatty acids (arachidonic/linoleic) with ozone generates, in dose-dependent form, acrolein as one of the by-products of the lipoperoxidation process. The oxidation of open human erythrocyte membranes with ozone also generated acrolein. 3 After aldolic condensation, aldol-acrolein derivative has a positive reaction with 2-thiobarbituric acid (TBA) and shows a maximum absorption at 498 nm. This novel characteristic is used in its identification after the separation of the by-products. 4 It is possible to suggest that in the classic reaction of the denominated thiobarbituric acid reactive substances (TBARS), when used as an indicator of the degree of peroxidation in biological systems, a portion of acrolein could be present but dwarfed by the TBAMDA adduct.

scholarly journals A protein activator of Mg2+-dependent, Ca2+-stimulated ATPase in human erythrocyte membranes distinct from calmodulin

1980 ◽  
Vol 187 (2) ◽  
pp. 507-513 ◽  
Author(s):  
Douglas Mauldin ◽  
Basil D. Roufogalis
Keyword(s):  
Atpase Activity ◽  
Sucrose Gradient ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Erythrocyte Membranes ◽  
Dependent Manner ◽  
Calmodulin Binding ◽  
Heat Stable ◽  
Protein Activator ◽  
Human Erythrocyte Membranes

Treatment of extensively washed erythrocyte membranes with 0.1mm-EDTA decreased their Mg2+-dependent, Ca2+-stimulated ATPase [(Mg2++Ca2+)-ATPase] activity. An activator released by this treatment restored the (Mg2++Ca2+)-ATPase to its original value in a Ca2+-dependent manner. This activator was different from calmodulin, as determined by a number of criteria. It was retained on an Amicon XM-100 ultrafiltration membrane (molecular-weight cut-off 100000); it appeared in the void volume of Sephadex G-100 and G-75 columns; it was not retained on a DEAE-cellulose ion-exchange column at ionic strengths similar to those used to retain calmodulin; and it maximally activated (Mg2++Ca2+)-ATPase activity less than calmodulin and at a higher Ca2+ concentration. Like calmodulin, the activator is heat-stable. The activator fraction isolated on a 2.5–15% sucrose gradient in 0.16m-KCl showed a single band of mol.wt. 63000 and no calmodulin on 10%-polyacrylamide/sodium dodecyl sulphate gels. A trace amount of calmodulin was detected in the activator fraction by radioimmunoassay (approx. 10pg/ml of ‘ghosts’), but this amount was insufficient to account for the (Mg2++Ca2+)-ATPase activation. Furthermore, calmodulin-binding protein failed to inhibit (Mg2++Ca2+)-ATPase activity by more than 10–20% in the membrane preparations from which the activator was extracted. It was concluded that erythrocyte membranes contain a (Mg2++Ca2+)-ATPase activator that may attenuate the activation of the Ca2+-transport ATPase by calmodulin.

The inhibitory effects of polyoxyethylene detergents on human erythrocyte acetylcholinesterase and Ca2+ + Mg2+ ATPase

1989 ◽  
Vol 67 (2-3) ◽  
pp. 137-146 ◽  
Author(s):  
R. Blaine Moore ◽  
J. F. Manery ◽  
J. Still ◽  
V. N. Mankad
Keyword(s):  
Human Erythrocyte ◽  
Acetylcholinesterase Activity ◽  
Significant Inhibition ◽  
Erythrocyte Membranes ◽  
Head Group ◽  
Polar Head Group ◽  
Detergent Concentration ◽  
Human Erythrocyte Membranes ◽  
Hydrophobic Tail ◽  
Triton X

The activities of acetylcholinesterase and Ca2+ + Mg2+ ATPase were measured following treatment of human erythrocyte membranes with nonsolubilizing and solubilizing concentrations of Triton X-100. A concentration of 0.1% (v/v) Triton X-100 caused a significant inhibition of both enzymes. The inhibition appears to be caused by perturbations in the membrane induced by Triton X-100 incorporation. No acetylcholinesterase activity and little Ca2+ + Mg2+ ATPase activity were detected in the supernatant at 0.05% Triton X-100 although this same detergent concentration induced changes in the turbidity of the membrane suspension. Also, no inhibition of soluble acetylcholinesterase was observed over the entire detergent concentration range. The inhibition of these enzymes at 0.1% Triton X-100 was present over an eightfold range of membrane protein in the assay indicating an independence of the protein/detergent ratio. The losses in activities of these two enzymes could be prevented by either including phosphatidylserine in the Triton X-100 suspension or using Brij 96 which has the same polyoxyethylene polar head group but an oleyl hydrophobic tail instead of the p-tert-octylphenol group of Triton X-100. The results are discussed in regard to the differential recovery of enzyme activities over the entire detergent concentration range.Key words: Triton X-100, erythrocyte membranes, acetylcholinesterase, Ca2+ + Mg2+ ATPase, polyoxyethylene detergents.

Resistance of Human Erythrocyte Membranes to Triton X-100 and C12E8

2008 ◽  
Vol 227 (1) ◽  
pp. 39-48 ◽  
Author(s):  
Cleyton Crepaldi Domingues ◽  
Annarita Ciana ◽  
Armando Buttafava ◽  
Cesare Balduini ◽  
Eneida de Paula ◽  
...  
Keyword(s):  
Human Erythrocyte ◽  
Erythrocyte Membranes ◽  
Human Erythrocyte Membranes ◽  
Triton X
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