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Biomedical subjects

A F Rega

Publications and source records attributed to A F Rega.

At least 37 records · Page 2Linked to original sources
PubMed1987

Gramicidin S inhibition of the Ca2+-ATPase of human red blood cells.

The cationic amphiphilic polypeptide gramicidin S inhibits the Ca2+-ATPase of human red-cell membranes by lowering the maximum velocity of the high-affinity component and the apparent affinity of the low-affinity component of the velocity-versus-ATP concentration curve of the enzyme. Gramicidin S does not alter the apparent affinity of the Ca2+-ATPase for Ca2+. Calmodulin is not essential for the inhibition, but increases the sensitivity of the enzyme to the inhibitor. The effects of gramicidin S on the Ca2+-ATPase can be reversed with phosphatidylcholine vesicles but not with buffer solutions, suggesting that gramicidin S acts from the lipid phase of the membrane.

Adenosine Triphosphate↗
PubMed1987

Differential effects of compound 48/80 on the ATPase and phosphatase activities of the Ca2+ pump of red cells.

The calmodulin antagonist compound 48/80 inhibits the phosphatase activity of the Ca2+-ATPase lowering its maximum velocity and leaving unaltered its apparent affinity for the substrate regardless on whether phosphatase activity is elicited by Ca2+ plus ATP or by calmodulin. Compound 48/80 has no effect on the Ki for ATP as inhibitor of the phosphatase. These results contrast sharply with the large increase that compound 48/80 induces in the apparent affinity of the regulatory site for the nucleotide of the Ca2+-ATPase and suggest that the active site for phosphatase activity is different from the regulatory site for ATP of the Ca2+-ATPase.

Adenosine Triphosphate↗
PubMed1986

The reaction of Mg2+ with the Ca2+-ATPase from human red cell membranes and its modification by Ca2+.

Media prepared with CDTA and low concentrations of Ca2+, as judged by the lack of Na+-dependent phosphorylation and ATPase activity of (Na+ +K+)-ATPase preparations are free of contaminant Mg2+. In these media, the Ca2+-ATPase from human red cell membranes is phosphorylated by ATP, and a low Ca2+-ATPase activity is present. In the absence of Mg2+ the rate of phosphorylation in the presence of 1 microM Ca2+ is very low but it approaches the rate measured in Mg2+-containing media if the concentration of Ca2+ is increased to 5 mM. The KCa for phosphorylation is 2 microM in the presence and 60 microM in the absence of Mg2+. Results are consistent with the idea that for catalysis of phosphorylation the Ca2+-ATPase needs Ca2+ at the transport site and Mg2+ at an activating site and that Ca2+ replaces Mg2+ at this site. Under conditions in which it increases the rate of phosphorylation, Ca2+ is without effect on the Ca2+-ATPase activity in the absence of Mg2+ suggesting that to stimulate ATP hydrolysis Mg2+ accelerates a reaction other than phosphorylation. Activation of the E1P----E2P reaction by Mg2+ is prevented by Ca2+ after but not before the synthesis of E1P from E1 and ATP, suggesting that Mg2+ stabilizes E1 in a state from which Mg2+ cannot be removed by Ca2+ and that Ca2+ stabilizes E1P in a state insensitive to Mg2+. The response of the Ca2+-ATPase activity to Mg2+ concentration is biphasic, activation with a KMg = 88 microM is followed by inhibition with a Ki = 9.2 mM. Ca2+ at concentration up to 1 mM acts as a dead-end inhibitor of the activation by Mg2+, and Mg2+ at concentrations up to 0.5 mM acts as a dead-end inhibitor of the effects of Ca2+ at the transport site of the Ca2+-ATPase.

Adenosine Triphosphate↗
PubMed1986

The activation of phosphatase activity of the Ca2+-ATPase from human red cell membranes by calmodulin, ATP and partial proteolysis.

Depending on the assay conditions, the ability of the Ca2+-ATPase from intact human red cell membranes to catalyze the hydrolysis of p-nitrophenylphosphate is elicited by either calmodulin or ATP. The response of the phosphatase activity to p-nitrophenylphosphate, ATP, Mg2+ and K+ is the same for the activities elicited by ATP or by calmodulin, suggesting that a single process is responsible for both activities. In media with calmodulin, high-affinity activation is followed by high-affinity inhibition of the phosphatase by Ca2+ so that the activity becomes negligible above 30 microM Ca2+. Under these conditions, addition of ATP leads to a large decrease in the apparent affinity for inhibition by Ca2+. In membranes submitted to partial proteolysis with trypsin, neither calmodulin nor Ca2+ are needed and phosphatase activity is maximal in media without Ca2+. This is the first report of an activity sustained by the Ca2+-ATPase of red cell membranes in the absence of Ca2+. Under these conditions, however, ATP still protects against high-affinity inhibition by Ca2+. These results strongly suggest that during activation by calmodulin, Ca2+ is needed only to form the calmodulin-Ca2+ complex which is the effective cofactor. Protection by ATP of the inhibitory effects of Ca2+ and the induction of phosphatase activity by ATP + Ca2+ suggests that activation of the phosphatase by Ca2+ in media with ATP requires the combination of the cation at sites in the ATPase. Results can be rationalized assuming that E2, the conformer of the Ca2+-ATPase, is endowed with phosphatase activity. Under this assumption, either the calmodulin-Ca2+ complex or partial proteolysis would elicit phosphatase activity by displacing the equilibrium between E1 and E2 towards E2. On the other hand, ATP + Ca2+ would elicit the activity by establishing through a phosphorylation-dephosphorylation cycle a steady-state in which E2 predominates over other conformers of the ATPase.

Adenosine Triphosphate↗
PubMed1985

Two modes of inhibition of the Ca2+ pump in red cells by Ca2+.

Two different and independent modes of inhibition of the Ca2+ pump by Ca2+ can be detected measuring active Ca2+ extrusion from resealed ghosts of human red cells: one requires extracellular and the other requires intracellular Ca2+. Ki for inhibition by extracellular Ca2+ is about 10 mM. Extracellular Mg2+ replaces Ca2+ in inhibiting Ca2+ transport but with an apparent affinity for inhibition about 3-times less than that for Ca2+. Inhibition by external Ca2+ is not affected by Na+ or K+ at both surfaces of the cell membrane, external EGTA, internal Ca2+ or ATP. The apparent affinity for external Ca2+ progressively raises as pH increases. The effects of extracellular Ca2+ and Mg2+ are consistent with the idea that for Ca2+ pumping to proceed, external sites in the pump must be protonated and not occupied by extracellular Ca2+ or Mg2+. Inhibition by intracellular Ca2+ takes place with a Ki of about 1 mM and is independent of external Ca2+. The inhibitory effects of intracellular Ca2+ can be accounted for if Ca2+ and CaATP were competitive inhibitors of the activation of the pump by Mg2+ and MgATP, respectively.

Adenosine Triphosphate↗
PubMed1985

Compound 48/80 and calmodulin modify the interaction of ATP with the (Ca2+ + Mg2+)-ATPase of red cell membranes.

Compound 48/80, an anti-calmodulin agent, reduces the maximum effect of ATP and does not affect the apparent affinity for ATP of the high-affinity site of the Ca2+-ATPase from calmodulin-bound membranes of human red cells. In the same preparation, 48/80 reduces more than 50-times the apparent affinity for ATP of the low-affinity site with little change in the maximum effect of the nucleotide at this site of the Ca2+-ATPase. The effects of compound 48/80 are independent of the concentration of Ca2+ between 30 and 200 microM. The apparent affinity of the low-affinity site of the Ca2+-ATPase for ATP is almost 100-fold less in calmodulin-stripped membranes than in calmodulin-bound membranes. In calmodulin-stripped membranes, exogenous calmodulin increases the apparent affinity for ATP up to the control values. These results indicate that apart from increasing the apparent affinity of the transport site for Ca2+, calmodulin also increases the apparent affinity of the regulatory site of the Ca2+-ATPase for ATP. Since this effect is exerted within the physiological ranges of ATP concentrations, it may participate in the physiological regulation of Ca2+ pumping by calmodulin.

Adenosine Triphosphate↗
PubMed1984

The inhibition of the calcium dependent ATPase from human red cells by erythrosin B.

Erythrosin B, a tetraiodinated derivative of fluorescein, completely inhibits the Ca2+-ATPase activity of human red cell membranes. The effect is exerted by reversible combination to a single class of site(s) of high apparent affinity (Ki = 70 nM). Erythrosin B decreases the maximum velocity and leaves unaltered the apparent affinity of the catalytic site for ATP and, conversely, it decreases the apparent affinity and leaves unaltered the maximum effect of ATP on its regulatory site in the Ca2+-ATPase. These results are consistent with the idea that erythrosin B displaces ATP from the regulatory site without replacing it in its effects.

Adult↗
PubMed1983

The effects of alkali metal ions on active Ca2+ transport in reconstituted ghosts from human red cells.

1. K+, Rb+ or Na+ increase from 30 to 90% the maximum rate of Ca2+ transport from resealed ghosts, leaving unaltered the apparent affinity of the Ca2+ pump for Ca2+. Li+ is ineffective as activator of Ca2+ transport. 2. K+ does not change the temperature dependence of Ca2+ transport. 3. The effects of K+ and Na+ are half-maximal at 4.6 mM and 24 mM, respectively. 4. The site(s) at which K+ or Na+ combine are accessible only from the cytoplasmic surface of the cell membrane. 5. Under conditions in which Na+ activates the Ca2+ pump no Na+ efflux coupled to Ca2+ efflux is apparent.

Biological Transport↗
PubMed1981

Vanadate inhibition of active Ca2+ transport across human red cell membranes.

(1) Vanadate (pentavalent vanadium) inhibits with high affinity (K0.5 = 3 microM) the ATP-dependent Ca2+ efflux in reconstituted ghosts from human red cell. (2) To inhibit Ca2+ efflux vanadate has to have access to the inner surface of the cell membrane (3) The inhibitory effect of vanadate is potentiated by intracellular Mg2+ and by intracellular K+. (4) Ca2+ in the external medium antagonizes the inhibitory effect of vanadate.

Biological Transport, Active↗
PubMed1981

Phosphorylation and dephosphorylation of the Ca2+ pump of human red cells in the presence of monovalent cations.

(1) In the presence of calcium ions, K+ increases the rate and the steady state level of phosphorylation of human red cell membranes by [gamma-32P)ATP. The effect of K+ is mimicked by Rb+, NH4+ and Cs+. Electrophoresis experiments suggest that the phosphorus taken up by the membranes in the presence of K+ is bound to the phosphoenzyme of the Ca2+-ATPase. (2) (Ca2+ + K+)-dependent phosphorylation requires Ca2+ and ATP with the same apparent affinity as the phosphorylation of the Ca2+ pump and the effect of K+ on phosphorylation is exerted with the same apparent affinity as that for the activation of the Ca2+-ATPase by K+. (3) The rate of hydrolysis of phosphoenzyme made in the presence of K+ is higher than that made in its absence and K+ increases the ratio Ca2+-ATPase activity/Ca2+-dependent phosphoenzyme concentration. (4) Results suggest that monovalent cations activate the Ca2+ pump because they increase the level and the turnover of the phosphoenzyme of the Ca2+-ATPase.

Ammonia↗
PubMed1980

Vanadate inhibition of the Ca2+-ATPase from human red cell membranes.

(1) VO3(-) combines with high affinity to the Ca2+-ATPase and fully inhibits Ca2+-ATPase and Ca2+-phosphatase activities. Inhibition is associated with a parallel decrease in the steady-state of the Ca2+-dependent phosphoenzyme. (2) VO3(-) blocks hydrolysis of ATP at the catalytic site. The sites for VO3(-) also exhibit negative interactions in affinity with the regulatory sites for ATP of the Ca2+-ATPase. (3) The sites for VO39-) show positive interaactions in affinity with sites for Mg2+ and K+. This accounts for the dependence on Mg2+ and K+ of the inhibition by VO3(-). Although, with less effectiveness, Na2+ and K+ substitutes for K+ whereas Li+ does not. The apparent affinites for Mg24 and K+ for inhibiton by VO3(-) seem to be less than those for activation of the Ca2+-ATPase. (4) Inhibition by VO3(-) is independent of Ca2+ at concentrations up to 50 microM. Higher concentrations of Ca2+ lead to a progressive release of the inhibitiory effect of VO3(-).

Adenosine Triphosphate↗
PubMed1978

Reversal of the calcium pump in human red cells.

Human red cells containing low ATP and high Pi concentrations were suspended in media with and without 2 mM Ca2+, and the incorporation of (32P)Pi into ATP was measured. There was some incorporation whatever the medium, but in every experiment there was an extra incorporation when the cells were in the Ca2+-containing medium. This extra incorporation was abolished by the ionophore A23187, which collapses the Ca2+ concentration gradient across the membranes, or by LaCl3, which blocks the Ca2+ pump. Starved and phosphate-loaded cells also show an uptake of Ca2+ which is not apparent in fresh cells. Results are consistent with the idea that Ca2+-dependent incorporation of Pi into ATP is catalyzed by the Ca2+ pump using energy derived from the Ca2+ concentration gradient.

Adenosine Triphosphate↗
PubMed1978

Activation of partial reactions of the Ca2+-ATPase from human red cells by Mg2+ and ATP.

(1) At ATP concentrations up to 30 micrometer addition of 0.5 mM MgCl2 in the reaction mixture increases both the rate of formation and the steady-state level of the phosphoenzyme of the Ca2+-ATPase from human red cell membranes. Under these conditions Mg2+ has no effect on the rate of dephosphorylation, which remains slow. (2) In the presence of Mg2+ the rate of dephosphorylation is increased 5 to 10 times by high (1 mM) concentrations of ATP. (3) Provided Mg2+ has reacted with the phosphoenzyme, acceleration of dephosphorylation by ATP takes place in the absence of Mg2+. This suggests that the role of Mg2+ on dephosphorylation is to convert the phosphoenzyme into a form that, after combination with ATP, reacts rapidly with water. (4) The results are consistent with the idea that combination of ATP at a non-catalytic site is needed for rapid dephosphorylation of the Ca2+-ATPase.

Adenosine Triphosphate↗