scholarly journals Calmodulin affinity chromatography yields a functional purified erythrocyte (Ca2+ + Mg2+)-dependent adenosine triphosphatase

1980 ◽  
Vol 189 (1) ◽  
pp. 81-88 ◽  
Author(s):  
K Gietzen ◽  
M Tejcka ◽  
H U Wolf
Keyword(s):  
Affinity Chromatography ◽  
Adenosine Triphosphatase ◽  
Specific Activity ◽  
Significant Loss ◽  
Erythrocyte Membranes ◽  
Purification Method ◽  
Major Band ◽  
Highly Sensitive ◽  
Human Erythrocyte Membranes ◽  
Triton X

The (Ca2+ + Mg2+)-dependent ATPase of human erythrocyte membranes was solubilized with deoxycholate and purified by calmodulin affinity chromatography to yield a functional enzyme. The method gave an enzyme purified 207-fold as compared with that of the erythrocyte membranes. The molecular weight of the ATPase was in the range 135 000-150 000, as revealed by a single major band after electrophoresis on dodecyl sulphate/polyacrylamide gels. The isolated enzyme was highly sensitive to calmodulin, since the activity was increased about 9-fold. At 37 degrees C and in the presence of calmodulin the purified ATPase had a specific activity of 10.1 mumol/min per mg of protein. Triton X-100 or deoxycholate stimulated the calmodulin-deficient enzyme in a concentration-dependent fashion whereby the calmodulin-sensitivity was lost. The purification method is suitable for studying the lipid-sensitivity of the ATPase, since the lipids can easily be exchanged without a significant loss of activity. A purification procedure described by Niggli, Penniston & Carafoli [(1979) J. Biol. Chem. 254, 9955-9958] resulted in an enzyme that indeed was pure but was lacking a predominant feature, namely the modulation by calmodulin.

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.

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

Influence of associated lipid on the properties of purified bovine erythrocyte acetylcholinesterase

1991 ◽  
Vol 69 (2-3) ◽  
pp. 154-162 ◽  
Author(s):  
Christine P. Nichol ◽  
Basil D. Roufogalis
Keyword(s):  
Phase Separation ◽  
Affinity Chromatography ◽  
Phospholipase C ◽  
Lectin Binding ◽  
Acetylcholinesterase Activity ◽  
Erythrocyte Membranes ◽  
Bovine Erythrocyte ◽  
Hydrophobic Properties ◽  
Native Form ◽  
Triton X

Acetylcholinesterase has been isolated from bovine erythrocyte membranes by affinity chromatography using a m-trimethylammonium ligand. The purified enzyme had hydrophobic properties by the criterion of phase partitioning into Triton X-114. The activity of the hydrophobic enzyme was seen as a slow-moving band in nondenaturing polyacrylamide gels. After treatment with phosphatidylinositol-specific phospholipase C, another form of active enzyme was produced that migrated more rapidly toward the anode in these gels. This form of the enzyme partitioned into the aqueous phase in Triton X-114 phase separation experiments and was therefore hydrophilic. The hydrophobic form bound to concanavalin A in the absence of Triton X-100. As this binding was partially prevented by detergent, but not by α-methyl mannoside, D-glucose, or myo-inositol, it is in part hydrophobic. Erythrocyte cell membranes showed acetylcholinesterase activity present as a major form, which was hydrophobic by Triton X-114 phase separation and in nondenaturing gel electrophoresis moved at the same rate as the purified enzyme. In the membrane the enzyme was more thermostable than when purified in detergent. The hydrophobic enzyme isolated, therefore, represents a native form of the acetylcholinesterase present in the bovine erythrocyte cell membrane, but in isolation its stability becomes dependent on amphiphile concentration. Its hydrophobic properties and lectin binding are attributable to the association with the protein of a lipid with the characteristics of a phosphatidylinositol.Key words: acetylcholinesterase, bovine erythrocytes, phosphatidylinositol-specific phospholipase C, phase separation, affinity chromatography.

scholarly journals Calcium ion-dependent p-nitrophenyl phosphate phosphatase activity and calcium ion-dependent adenosine triphosphatase activity from human erythrocyte membranes

1973 ◽  
Vol 136 (1) ◽  
pp. 185-194 ◽  
Author(s):  
Alcides F. Rega ◽  
Donaldo E. Richards ◽  
Patricio J. Garrahan
Keyword(s):  
Cell Membrane ◽  
Phosphatase Activity ◽  
Human Erythrocyte ◽  
Calcium Ion ◽  
Adenosine Triphosphatase ◽  
Erythrocyte Membranes ◽  
Dependent Atpase ◽  
Nitrophenyl Phosphate ◽  
Inner Surface ◽  
Human Erythrocyte Membranes

In the presence of ATP and of Mg2+, human erythrocyte membranes show a phosphatase activity towards p-nitrophenyl phosphate which is activated by low concentrations of Ca2+. The effect of Ca2+ is strongly enhanced if either K+ or Na+ is also present. Activation of the p-nitrophenyl phosphate phosphatase by Ca2+ reaches a half-maximum at about 8μm-Ca2+ and is apparent only when the ion has access to the inner surface of the cell membrane. Ca2+-dependent phosphatase activity can only be observed if ATP is at the inner surface of the cell membrane, and the presence of ATP seems to be absolutely necessary, since either its removal or its replacement by other nucleoside triphosphates abolishes the activating effect of Ca2+. The properties of the (ATP+Ca2+)-dependent phosphatase are very similar to those of the Ca2+-dependent ATPase (adenosine triphosphatase), also present in erythrocyte membranes, which probably is involved in Ca2+ transport in erythrocytes. The similarities suggest that both activities may be properties of the same molecular system. This view is further supported by the fact that p-nitrophenyl phosphate inhibits to a similar extent Ca2+-dependent ATPase activity and ATP-dependent Ca2+ extrusion from erythrocytes.

scholarly journals The isolation of human-erythrocyte band-3 polypeptide labelled with a photosensitive hydrophobic probe

1980 ◽  
Vol 187 (3) ◽  
pp. 719-725 ◽  
Author(s):  
E Wells ◽  
J B Findlay
Keyword(s):  
Human Erythrocyte ◽  
Band 3 ◽  
Distinct Pattern ◽  
Integral Membrane Proteins ◽  
Erythrocyte Membranes ◽  
Major Band ◽  
Similar Molecular Weight ◽  
Anion Exchange Protein ◽  
Human Erythrocyte Membranes ◽  
Hydrophobic Probe

To investigate the intramembranous domains of the major band-3 polypeptide, human erythrocyte membranes were labelled with 1-azido-4-[125I]iodobenzene. The anion-exchange protein has been isolated by a new procedure that decreases possible contamination by other integral membrane proteins of similar molecular weight. After chemical modification, the protein was subjected to proteolytic cleavage by several proteinases to yield a distinct pattern of peptides, a few of which contained radioactivity.

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