scholarly journals Differential reactivity of lysine residues of the red blood cell Ca2+ pump involved in the E1-E2 conformational equilibrium

1991 ◽  
Vol 279 (1) ◽  
pp. 121-127 ◽  
Author(s):  
C Donnet ◽  
A J Caride ◽  
H N Fernández ◽  
J P F C Rossi
Keyword(s):  
Atpase Activity ◽  
Blood Cells ◽  
Conformational Equilibrium ◽  
The Other ◽  
Apparent Dissociation Constant ◽  
Apparent Affinity ◽  
High Affinity ◽  
Human Red Blood Cells ◽  
Differential Reactivity ◽  
Lysine Residues

1. Modification of Lys residues of the Ca(2+)-ATPase from human red blood cells with methyl acetimidate (MA) inhibited up to 70% of the Ca(2+)-ATPase activity. Furthermore, calmodulin-activated p-nitrophenyl phosphatase activity was fully inhibited at non-limiting concentrations of MA. 2. Treatment with MA inhibited phosphorylation of the Ca(2+)-ATPase. 3. When the enzyme was treated with 7.2 mM-MA in the presence of 100 microM-Ca2+, Ca(2+)-ATPase activity was decreased by 33%, whereas when the membranes were treated with MA in the presence of 50 microM-VO4(3-), this activity was decreased by only 8%. 4. When membranes were either proteolysed or preincubated with 1 mM-Ca2+, MA quickly inactivated the Ca(2+)-ATPase (k = 1.2 min-1). On the other hand, inactivation of membranes preincubated in the absence of Ca2+ and Mg2+ was slow (k = 0.08 min-1). 5. When the activity was measured in the absence of calmodulin, MA decreased to the same extent the values of KCa (the apparent dissociation constant for Ca2+) and Vmax, but in the presence of calmodulin the treatment decreased Vmax. only. 6. The results are consistent with the idea that MA reacts readily with the Ca(2+)-ATPase when the enzyme is in an E1 conformation, but not an E2 conformation, and that, reciprocally, treatment of the enzyme with MA shifts the enzyme to E1. 7. Provided that Ca2+ is present, ATP, with low apparent affinity (K0.5 = 195 microM), protected against inactivation by MA. However, MA treatment did not change the Km values of either the high-affinity or the low-affinity site for ATP, suggesting that protection results from a shift to a conformation in which the Lys residues are inaccessible to MA.

Ion Transport and the Effects of Moulting in the Freshwater Crayfish Cherax destructor (Decapoda : Parastacidae)

1997 ◽  
Vol 45 (5) ◽  
pp. 539 ◽  
Author(s):  
Samad Zare ◽  
Peter Greenaway
Keyword(s):  
Atpase Activity ◽  
Calcium Flux ◽  
Freshwater Crayfish ◽  
Transport Mechanisms ◽  
Transport Capacity ◽  
Apparent Affinity ◽  
High Affinity ◽  
Cherax Destructor ◽  
Net Uptake ◽  
Moulting Cycle

Sodium and chloride were maintained in approximate equilibrium by intermoult Cherax destructor but net uptake from the water began the day before ecdysis and continued for several days in postmoult. Sodium transport in postmoult was four times as much as that during the intermoult. The apparent affinities of the Na and Cl transport mechanisms, 0.5 ± 0.12 and 0.3 ± 0.04 mmol L-1, respectively, were relatively low compared with other reported values for freshwater crustaceans. Calcium flux was negligible in intermoult animals and net efflux was evident in premoult as calcium resorbed from the skeleton was excreted. Following ecdysis, calcium net influx was high and persisted for many days into postmoult as the new exosketon was calcified. An apparent affinity (Km) of 0.3 ± 0.04 mmol L-1 Ca and a maximal net transport rate (Jmax) of 1.8 ± 0.29 µmol Ca g-1 h-1 were measured in postmoult animals. The gills contained a high-affinity Ca2+ ATPase (K m= 0.040.05 µmol L-1) with a low transport capacity (Jmax = 5.6 nmol mg-1 protein min -1) that did not change during the moulting cycle. It is suggested that postmoult Ca transport was facilitated by Na–Ca exchange driven by elevated branchial Na + K+ -ATPase activity.

scholarly journals Proton-sulfate co-transport: mechanism of H+ and sulfate addition to the chloride transporter of human red blood cells.

1982 ◽  
Vol 79 (1) ◽  
pp. 87-113 ◽  
Author(s):  
M A Milanick ◽  
R B Gunn
Keyword(s):  
Blood Cells ◽  
Transport Mechanism ◽  
Molecular Mechanisms ◽  
The Other ◽  
Chloride Flux ◽  
Sulfate Ions ◽  
Human Red Blood Cells ◽  
Anion Transporter ◽  
Mechanism Of Reaction ◽  
Chloride Exchange

Proton and sulfate inhibition of the obligatory chloride-chloride exchange of human erythrocytes was measured at 0 degrees C to determine their mechanism of reaction with the anion transporter. The proton and sulfate that are co-transported by this mechanism at higher temperatures behaved as nontransported inhibitors at 0 degrees C. We analyzed the data in terms of four molecular mechanisms: (1) HSO4- addition to the transporter; (2) ordered addition with the proton first; (3) ordered addition with the sulfate first; (4) random addition to the transporter. The Dixon plots of 1/MCl vs. [SO4] at different proton concentrations were not parallel. Thus protons and sulfate ions were not mutually exclusive inhibitors. The slope of these Dixon plots was independent of pH above 7.0, which indicates that sulfate could bind to the unprotonated carrier and excludes the first two mechanisms. Protons were inhibitors of chloride flux in the absence of sulfate, which indicates that protons could bind to the unloaded carrier and excludes mechanism 3. The KI for sulfate was 4.35 +/0 0.36 mM. The pK for the protonatable group was 5.03 +/- 0.02. The binding of either a proton or sulfate to the carrier decreased the KI of the other by ninefold. The only simple mechanism consistent with the data is a random-ordered mechanism with more transporters loaded with a sulfate than loaded with a proton at the pH and sulfate concentrations of plasma.

On haemolysis by anionic detergents

1957 ◽  
Vol 146 (923) ◽  
pp. 225-241 ◽  
Keyword(s):  
Cell Wall ◽  
Blood Cells ◽  
The Other ◽  
Slow Process ◽  
Ionic Detergents ◽  
Human Red Blood Cells ◽  
Lipoprotein Complex ◽  
Rapid Destruction ◽  
Anionic Detergents ◽  
Kinetics Of

The kinetics of haemolysis of human red blood cells by synthetic anionic detergents has been studied. It is shown that these detergents can destroy red cells by two mechanisms, which normally operate simultaneously, one leading to a rapid destruction of the cells whilst the other is a slow process. The rapid process involves the action of the detergent on free phospholipid in the cell wall. This component is easily removed from the cell membrane, and cells thus treated will not undergo rapid haemolysis. The slow process is identical with that by which most haemolytic agents (saponins, non-ionic detergents and butanol) act, and if it alone can operate yields percentage haemolysistime curves which are sigmoid in shape. This process takes place in several stages and seems to involve the slow breakdown of a lipoprotein complex on the cell surface. If phospholipid is removed from the cell wall, it is slowly replaced from phospholipid bound as lipoprotein or possibly by synthesis from metabolites in the membrane. The equilibria which maintain the structure of the cell wall are discussed.

Peptides from the Plasmodium falciparum STEVOR putative protein bind with high affinity to normal human red blood cells

Peptides ◽  
2005 ◽  
Vol 26 (7) ◽  
pp. 1133-1143 ◽  
Author(s):  
Javier E. García ◽  
Alvaro Puentes ◽  
Hernando Curtidor ◽  
Ricardo Vera ◽  
Luis Rodriguez ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Putative Protein ◽  
High Affinity ◽  
Human Red Blood Cells ◽  
Normal Human

Pathways of cytochrome c oxidation by soluble and membrane-bound cytochrome aa3

1980 ◽  
Vol 58 (10) ◽  
pp. 969-977 ◽  
Author(s):  
P. Nicholls ◽  
V. Hildebrandt ◽  
B. C. Hill ◽  
F. Nicholls ◽  
J. M. Wrigglesworth
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
The Other ◽  
Enzyme Preparations ◽  
Apparent Affinity ◽  
High Affinity ◽  
High Affinity Site ◽  
Membrane Bound ◽  
Enzyme Molecules ◽  
Low Ionic Strength

In media of low ionic strength, membraneous cytochrome c oxidase, isolated cytochrome c oxidase, and proteoliposomal cytochrome c oxidase each bind cytochrome c at two sites, one of low affinity (1 μM > Kd′ > 0.2 μM) and readily reversible and the other of high affinity (0.01 μM > Kd) and weakly reversible. When cytochrome c occupies both sites, including the low affinity site, the maximal turnover measured polarographically with ascorbate and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) is independent of TMPD concentration, and lies between 250 and 400 s−1 (30 °C, pH 7.4) for fully activated systems. The apparent affinity of the enzyme for cytochrome c is, however, TMPD dependent. When cytochrome c occupies only the high-affinity site, the maximal turnover is closely dependent upon the concentration of TMPD, which, unlike ascorbate, can reduce bound cytochrome c. As TMPD concentration is increased, the maximal turnover approaches that seen when both sites are occupied. The lower activity of isolated cytochrome aa3 is due to the presence of inactive or inaccessible enzyme molecules. Incorporation of isolated enzyme into phospholipid vesicles restores full activity to all the subsequently accessible cytochrome aa3 molecules. Negatively charged (asolectin) vesicles show a higher cytochrome c affinity at the low-affinity sites than do the other enzyme preparations. A model for the cytochrome c – cytochrome aa3 complexes is put forward in which both sites, when occupied, are fully catalytically competent, but in which occupation of the "tight" site by a catalytically functional cytochrome c molecule is required for overall oxidation of cytochrome c via the "loose" site.

Chloride (or bicarbonate)-dependent copper uptake through the anion exchanger in human red blood cells

1990 ◽  
Vol 259 (4) ◽  
pp. C570-C576 ◽  
Author(s):  
J. O. Alda ◽  
R. Garay
Keyword(s):  
Red Blood Cells ◽  
Dissociation Constant ◽  
Anion Exchanger ◽  
Blood Cells ◽  
Initial Rate ◽  
Disulfonic Acid ◽  
Copper Uptake ◽  
Selective Electrode ◽  
Apparent Dissociation Constant ◽  
Human Red Blood Cells

The initial rate of Cu2+ uptake in human red blood cells was measured by atomic absorption. About 80% of Cu2+ uptake was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) concentrations greater than 5-10 microM. DIDS-sensitive Cu2+ uptake required the presence of external HCO3- or external Cl-. Cl- strongly stimulated Cu2+ uptake following a Michaelis-like function, with apparent dissociation constant (KCl) of 72 +/- 9.4 (SD) mM (n = 6 experiments). HCO3- stimulated DIDS-sensitive Cu2+ uptake following a Michaelis-like function, with apparent dissociation constant (Kbic) of 10 +/- 1.9 (SD) mM (n = 4 experiments). Maximal rates (of Cl(-)- or HCO3(-)-stimulated Cu2+ uptake) were nonadditive. DIDS-sensitive Cu2+ uptake was not modified by physiological concentrations of phosphate or sulfate. Conversely, it was strongly inhibited by physiological concentrations of L-histidine and cysteine (at a Cu2+ concentration of 100 microM, these physiological ligands exhibited KHis and KCys of 50 and 80 microM, respectively). By using a copper-selective electrode, we found that at pH 7-7.4 copper is associated with OH-, particularly in the form of Cu(OH)2 complexes. In conclusion, the anion exchanger is the major transport mechanism for red blood cell Cu2+ uptake. The translocating species can be the monovalent anion complexes of copper with OH-, Cl-, and/or HCO3-.

scholarly journals Correlation between Ca2+ uptake, Ca2+ efflux and phosphoenzyme level in sarcoplasmic-reticulum vesicles

1991 ◽  
Vol 274 (2) ◽  
pp. 427-432 ◽  
Author(s):  
J C Benech ◽  
A Galina ◽  
L de Meis
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Aqueous Medium ◽  
Atp Synthesis ◽  
Dimethyl Sulphoxide ◽  
The Other ◽  
Apparent Affinity ◽  
High Affinity ◽  
Ph Values ◽  
A Value ◽  
Other Hand

Previously [Inesi & de Meis (1989) J. Biol. Chem. 264, 5929-5936] it was shown that dimethyl sulphoxide (Me2SO) increases the amount of Ca2+ accumulated by sarcoplasmic-reticulum vesicles. This effect was related to a decrease in the enzyme affinity for ADP from less than 20 microM to 1 mM. In the present work the apparent affinity of the ADP-sensitive phosphoenzyme for ADP was determined by measuring the rate of ATP synthesis in vesicles previously loaded with Ca2+, at different pH values and in the presence and absence of Me2SO (20%, v/v) and KCl. In all conditions tested, addition of Me2SO never promoted an increase of the apparent Km for ADP to a value higher than 25 microM. ADP inhibits the phosphorylation of the enzyme by Pi. Two components, with Ki values of 80 microM and 8 mM, were detected when vesicles previously loaded with Ca2+ were used. The high-affinity component (Ki 80 microM) was abolished after addition of Me2SO to the medium. Empty vesicles, on the other hand, exhibited only the low-affinity component (Ki 8 mM). During ATP synthesis in a totally aqueous medium, there was a decrease in the phosphoenzyme formed by Pi, after addition of 80-100 microM-ADP to the medium. In the presence of Me2SO this decrease was smaller. The sum of the fractions of phosphoenzyme formed by ATP and by Pi during Ca2+ uptake was higher in the presence of Me2SO than in a totally aqueous medium. Me2SO decreased the passive efflux of Ca2+ measured in the presence of 0.1 mM-Pi and 0.1 mM-MgCl2. In a totally aqueous medium the same decrease was observed when Pi and MgCl2 concentrations were raised to 4 mM. These data suggest that ADP binds to two different enzyme conformations. The binding to one of these conformations (*E) inhibits the phosphorylation of the enzyme by Pi, increases the efflux of Ca2+ and decreases the amount of Ca2+ accumulated by the vesicles.

scholarly journals The Chemical Constitution of Compounds Which Protect Erythrocytes against Freezing Damage

1962 ◽  
Vol 46 (1) ◽  
pp. 167-175 ◽  
Author(s):  
T. Nash
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Room Temperature ◽  
Protective Action ◽  
Freezing Damage ◽  
Basic Character ◽  
High Affinity ◽  
Human Red Blood Cells ◽  
Chemical Constitution ◽  
Neutral Water

Eleven simple neutral water-miscible compounds were tested for protective action against freezing damage to human red blood cells. All the compounds penetrated the cells at room temperature without damage, but only four, N-substituted amides, were active. These results are considered together with previously published work on freezing protection by other low molecuar weight solutes. The affinity of the compounds for water is gauged in two independent ways, and correlates well with protective ability. The chemical constitutional factors responsible for high affinity for water are discussed. It appears that basic character is most important.

scholarly journals The effect of aclarubicin (acl) on human erythrocytes

2011 ◽  
Vol 34 (1) ◽  
pp. 23-29
Author(s):  
Piotr Witczak ◽  
Agnieszka Marczak
Keyword(s):  
Reactive Oxygen Species ◽  
Blood Cells ◽  
Reduced Glutathione ◽  
Human Erythrocytes ◽  
The Other ◽  
Oxygen Species ◽  
Total Glutathione ◽  
Human Red Blood Cells ◽  
20 Nm

The effect of aclarubicin (acl) on human erythrocytesThe present study examines the influence of aclarubicin (ACL) on human red blood cells with a focus on the generation of reactive oxygen species (ROS). A statistically significant increase in ROS levels was observed for all the tested concentrations of aclarubicin (1-20 nM). The depletion of catalase activity and elevated metHb content were also observed. On the other hand, changes in reduced glutathione (GSH) and total glutathione concentrations were not statistically significant. The presented results confirm important role of ROS in ACL cytotoxic activity.

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