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.

scholarly journals The carbohydrate moiety of band-3 glycoprotein of human erythrocyte membranes

1980 ◽  
Vol 187 (3) ◽  
pp. 677-686 ◽  
Author(s):  
T Tsuji ◽  
T Irimura ◽  
T Osawa
Keyword(s):  
Blood Group ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Band 3 ◽  
Carbohydrate Moiety ◽  
Erythrocyte Membranes ◽  
Gel Chromatography ◽  
Branching Points ◽  
Human Erythrocyte Membranes ◽  
Group A

Band-3 glycoprotein was purified from human blood-group-A erythrocyte membranes by selective solubilization and gel chromatography on Sepharose 6B in the presence of sodium dodecyl sulphate. The purified glycoprotein was subjected to hydrazinolysis in order to release the carbohydrate moiety. The released oligosaccharides were N-acetylated and applied to a column of DEAE-cellulose. Most of the band-3 oligosaccharides obtained were found to be free of sialic acids. When this neutral fraction was subjected to gel chromatography on a column of Sephadex G-50, two broad peaks were observed indicating that the band-3 glycoprotein was heterogeneous in the size of the oligosaccharide moieties. All fractions from gel chromatography were found to contain galactose, mannose, N-acetylglucosamine and fucose. The higher-molecular-weight (mol.wt. 3000-8000) peak consisted of fucose, mannose, galactose, N-acetylglucosamine and N-acetylgalactosamine in a molar proportion of 1.6:3.0:8.4:10.5:0.2. Most of these oligosaccharides were digested with a mixture of beta-galactosidase and beta-N-acetylhexosaminidase after alpha-L-fucosidase treatment to give a small oligosaccharide with the structure alpha Man2-beta Man-beta GlcNAc-GlcNAc. Methylation studies and limited degradation by nitrous acid deamination showed that the oligosaccharides contained the repeating disaccharide Gal beta 1→4GlcNAc beta 1→3, with branching points at C-6 of some of the galactose residues. These results indicate that a major portion of the band-3 oligosaccharide has a common core structure, with heterogeneity in the numbers of the repeating disaccharides, and contains fucose residues both in the peripheral portion and in the core portion. Haemagglutination tests were also carried out to determine the blood-group specificities of the glycoprotein and the results demonstrated the presence of both blood-group-H and I antigenic activities.

scholarly journals Isolation and characterization of a membrane-attack-complex-inhibiting protein present in human serum and other biological fluids

1990 ◽  
Vol 265 (2) ◽  
pp. 471-477 ◽  
Author(s):  
M J Watts ◽  
J R Dankert ◽  
B P Morgan
Keyword(s):  
Cerebrospinal Fluid ◽  
Polyclonal Antibodies ◽  
Biological Fluids ◽  
Polyacrylamide Gel Electrophoresis ◽  
Membrane Attack Complex ◽  
Erythrocyte Membranes ◽  
Dependent Manner ◽  
Isolation And Characterization ◽  
Human Erythrocyte Membranes ◽  
Inhibiting Protein

We have previously reported the isolation of a membrane-attack-complex-inhibiting protein (MIP) from human erythrocyte membranes [Watts, Patel & Morgan (1987) Complement 4, 236] and the production of polyclonal antibodies to this protein. Here we report the identification in plasma, urine, saliva and cerebrospinal fluid of a protein immunochemically identical with the membrane-derived MIP. The protein has been isolated from plasma by immunoaffinity chromatography on an anti-(erythrocyte MIP)-Sepharose column and shown by SDS/polyacrylamide-gel electrophoresis to be of similar molecular mass to the erythrocyte protein (55 kDa non-reduced and 65 kDa under reducing conditions). Monoclonal antibodies have been raised against plasma MIP and used to establish a two-site enzyme-linked immunoadsorbent assay, enabling quantification of MIP in plasma, urine and cerebrospinal fluid. Plasma MIP, though not able to incorporate spontaneously into membranes, was deposited on heterologous and homologous erythrocyte membranes during complement activation in a C8-dependent manner. Depletion of MIP from plasma resulted in enhancement of the lytic capacity of the plasma on heterologous erythrocytes.

Thyroid hormone stimulates release of calmodulin-enhancing activity from human erythrocyte membranes in vitro

1993 ◽  
Vol 84 (2) ◽  
pp. 217-223 ◽  
Author(s):  
William D. Lawrence ◽  
Mark R. Deziel ◽  
Paul J. Davis ◽  
Marion Schoenl ◽  
Faith B. Davis ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Human Erythrocyte ◽  
Erythrocyte Membranes ◽  
Soluble Factor ◽  
Heat Stable ◽  
Enhancing Factor ◽  
Human Erythrocyte Membranes ◽  
Subsequent Addition ◽  
Stable Control

1. Thyroid hormone (L-thyroxine, 10−10mol/l) incubated in vitro with human erythrocyte membranes induced the release of a soluble calmodulin-like material, the 3′:5′-cyclic nucleotide phosphodiesterase-stimulating activity of which was at least six-fold greater than its concentration measured by a specific calmodulin radioimmunoassay. 2. The material had the characteristics of calmodulin in that it stimulated both phosphodiesterase and erythrocyte Ca2+-ATPase activities, cross-reacted with and was neutralized by anti-calmodulin antibody, was adsorbed by phenothiazine-Sepharose and was heat-stable. Control supernatant from the incubation of membranes in the absence of thyroxine contained calmodulin, the bioactivity of which was not enhanced beyond that predicted from radioimmunoassay. Subsequent addition of thyroxine did not increase calmodulin bioactivity. Calmodulin-agarose removed calmodulin-enhancing activity from the supernatant. 3. Thus, the enhancing factor(s) appears to interact directly with calmodulin. These observations indicate that thyroid hormone promotes the release from human erythrocyte membranes of a soluble factor (or factors) which binds to calmodulin and significantly increases its bioactivity. This enhancing activity is similar to that of a calmodulin activator described in a rat model of hypertension (S.-L. Huang et al., J Clin Invest 1988; 82: 276-81).

scholarly journals Structure-activity relationships of retinoids as inhibitors of calmodulin-dependent human erythrocyte Ca2+-ATPase activity and calmodulin binding to membranes

1991 ◽  
Vol 277 (3) ◽  
pp. 603-606 ◽  
Author(s):  
F B Davis ◽  
T J Smith ◽  
P J Davis ◽  
S D Blas
Keyword(s):  
Enzyme Activity ◽  
Retinoic Acid ◽  
Atpase Activity ◽  
Human Erythrocyte ◽  
Enzyme Inhibitors ◽  
Ring Structure ◽  
Erythrocyte Membranes ◽  
Calmodulin Binding ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

All-trans retinoic acid displaces the binding of radiolabelled calmodulin to human erythrocyte membranes, and inhibits the activity of plasma membrane Ca(2+)-stimulated, Mg(2+)-dependent ATPase (Ca(2+)-ATPase; EC 3.6.1.3). This enzyme is dependent upon the action of calmodulin. In this study we explored the structural attributes of the retinoids which confer this ability to inhibit enzyme activity and calmodulin binding. With respect to the fatty acid side-chain, a clear requirement for inhibition is a trans-configuration of the polar end-group. The importance of the ring structure is indicated by the ineffectiveness of polyprenoic acid and a benzene ring retinoid analogue as inhibitors of enzyme activity and calmodulin binding. There was good correlation between the relative potencies of the analogues as enzyme inhibitors and as inhibitors of calmodulin binding. The ability of selected retinoid analogues, at physiological concentrations with respect to all-trans retinoic acid, to inhibit erythrocyte Ca(2+)-ATPase activity and membrane binding of calmodulin underscores the structurally specific effects of these compounds on the interaction of calmodulin with the membrane-bound enzyme.

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 Increase of Na-K-ATPase activity, glutamate, and aspartate uptake in dog erythrocytes associated with hereditary high accumulation of GSH, glutamate, glutamine, and aspartate

Blood ◽  
1983 ◽  
Vol 61 (3) ◽  
pp. 493-499 ◽  
Author(s):  
Y Maede ◽  
M Inaba ◽  
N Taniguchi
Keyword(s):  
Amino Acids ◽  
Atpase Activity ◽  
Convincing Evidence ◽  
Erythrocyte Membranes ◽  
High Potassium ◽  
High Accumulation ◽  
Low Sodium ◽  
Human Erythrocyte Membranes ◽  
High Concentrations ◽  
Glutamine Uptake

Abstract We have found convincing evidence for the presence of Na-K-ATPase and high potassium (K) and low sodium (Na) concentrations in the erythrocytes of some dogs associated with hereditary high concentrations of erythrocyte glutathione and some amino acids, glutamate, glutamine, and aspartate. The Na-K-ATPase activity of the erythrocyte membranes of the dogs was about 3 times that of human erythrocyte membranes, whereas the enzyme activity was not detected in control dogs with a normal level of blood glutathione. The Michaelis constant of the enzyme for ATP (Km ATP) was 6.6 X 10(-3)M in the dogs' erythrocytes and 5.0 X 10(-4)M in the human erythrocytes. The concentration of K in the erythrocytes in the dogs examined was about 11 times that of the controls, whereas the erythrocyte Na concentration in the dogs was about one-third that of the controls. The concentrations of K and Na in the plasma of the dogs were equal to those of the controls. Furthermore, L-3H-glutamate and L-3H-aspartate uptake by those cells with high activity of Na-K-ATPase greatly increased, while L-3H-glutamine uptake was unchanged. It appeared that Na+ and K+ gradients created by Na-K-ATPase across the cell membrane might stimulate glutamate and aspartate uptake by the cells, thus causing the high accumulation of such amino acids in the cells.

scholarly journals Studies on the soluble phosphodiesterases of chicken gizzard smooth muscle

1983 ◽  
Vol 215 (3) ◽  
pp. 627-636 ◽  
Author(s):  
R J Birnbaum ◽  
J F Head
Keyword(s):  
Smooth Muscle ◽  
Deae Cellulose ◽  
Gel Permeation Chromatography ◽  
Hydrolytic Activity ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Dependent Manner ◽  
Chicken Gizzard ◽  
Calmodulin Binding ◽  
Discrete Band

In this study we describe the identification of four soluble forms of cyclic nucleotide phosphodiesterase from chicken gizzard smooth muscle. These isoenzymes were separated from one another by ion-exchange chromatography on DEAE-cellulose and by calmodulin-Sepharose affinity chromatography. Each form migrates as a single discrete band when it is electrophoresed on non-denaturing polyacrylamide gels and stained for phosphodiesterase activity. Each form is also eluted as a single peak on gel-permeation chromatography, giving apparent Mr values of 114 000, 116 000, 122 000 and 59 000. All four enzymes have apparent Km values in the 0-20 microM range, although their relative specificities for cyclic AMP and cyclic GMP differ. Two of the forms bind to calmodulin in a Ca2+-dependent manner; however, only one is activated by calmodulin. The interaction of the second calmodulin-binding form with calmodulin is disrupted by the papaverine derivative verapamil without significantly altering the hydrolytic activity of the enzyme.

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