Alcides F. Rega

Potassium activated phosphatase from human red blood cells. The mechanism of potassium activation

1. The kinetic behaviour of the K‐activated phosphatase in human red blood cell membranes has been investigated. The concentration of Mg required to give optimal activation is independent of substrate and K concentration, suggesting that Mg combines with the enzyme at a site that is independent of and non‐interacting with the substrate and K sites.

Two classes of site for ATP in the Ca2+-ATPase from human red cell membranes.

(1) The response of the Ca2+-ATPase activity from human red cell membranes to ATP concentrations can be represented by the sum of two Michaelis-like curves: one with a Km of 2.5 micrometer and the other with a Km of 145 micrometer. (2) The maximum Ca2+-ATPase activity elicited by occupation of the site with lower Km represents about 10% of the activity attainable at non-limiting ATP concentrations. (3) 30--50% of the Ca2+-ATPase activity with lower Km remains in the absence of Mg2+ . Mg2+ increases V and the maximum effect of Ca2+, having no effect on the apparent affinities for ATP and Ca2+. (4) The large increase in Ca2+-ATPase activity which results from the occupation of the site with higher Km only takes place when Mg2+ is present. (5) Results are compatible with the idea that the Ca2+-ATPase from human red cell membranes has two classes of site for ATP binding, both of which are occupied when the enzyme catalyzes the hydrolysis of ATP at maximum rate. (6) The properties of the high affinity site suggest that this is the catalytic site of the Ca2+-ATPase. It is proposed that binding of ATP at the low affinity site regulates the turnover of the system.

Calcium ion-dependent phosphorylation of human erythrocyte membranes.

SummaryCalcium ions promote the rapid transfer of the terminal phosphate of ATP to a protein of human erythrocyte membranes. The concentration of Ca2+ for half-maximal effect is 7 μM. At nonlimiting ATP concentrations the level of32P incorporated by the membranes is independent of the presence or absence of Mg2+. The number of phosphorylating sites in a single erythrocyte membrane is about 700. The influence of pH on the rate of hydrolysis of the bound phosphate and its rapid release on exposure to hydroxylamine are both consistent with an acylphosphate bond. The phosphate in the protein undergoes rapid turnover. Enzymatic splitting of the phosphate is stimulated by Mg2+ but not by Ca2+. It is proposed that Mg2+ accelerates the splitting of the phosphate by favoring the conversion of the phosphoprotein from a state of low reactivity to a state of high reactivity towards water. The reactions described probably are intermediate steps in the hydrolysis of ATP catalyzed by the Ca2+-dependent ATPase of human erythrocyte membranes.

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(-).

Potassium-activated phosphatase from human red blood cells

SummaryThe cell membrane K+-activated phosphatase activity was measured in reconstituted ghosts of human red cells having different ionic contents and incubated in solutions of varying ionic composition. When K+-free ghosts are suspended in K+-rich media, full activation of the phosphatase is obtained. Conversely, very little ouabainsensitive activity is detected in K+-rich ghosts suspended in K+-free media. These results, together with the fact that Na+ competitively inhibits the effects of K+ only when present externally, show that the K+ site of the membrane phosphatase is located at the outer surface of the cell membrane. The Mg++ requirements for K+ activation of the membrane phosphatase are fulfilled by internal Mg++. Addition of intracellular Na+ to ATP-containing ghosts raises the apparent affinity of the enzyme for K+, suggesting that the sites where ATP and Na+ produce this effect are located at the inner surface of the cell membrane. The asymmetrical features of the membrane phosphatase are those expected from the proposed role of this enzyme in the Na+−K+-ATPase system.

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.

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.

ATPase and phosphatase activities from human red cell membranes: I. The effects of N-ethylmaleimide.

Summary(i) In human red cell membranes the sensitivity to N-ethylmaleimide of Ca2+-dependent ATPase and phosphatase activities is at least ten times larger than the sensitivity to N-ethylmaleimide of (Na++K+)-ATPase and K+-activated phosphatase activities. All activities are partially protected against N-ethylmaleimide by ATP but not by inorganic phosphate or byp-nitrophenylphosphate. (ii) Protection by ATP of (Na++K+)-ATPase is impeded by either Na+ or K+ whereas only K+ impedes protection by ATP of K+-activated phosphatase. On the other hand, Na+ or K+ slightly protects Ca2+-dependent activities against N-ethylmaleimide, this effect being independent of ATP. (iii) The sensitivity to N-ethylmaleimide of Ca2+-dependent ATPase and phosphatase activities is markedly enhanced by low concentrations of Ca2+. This effect is half-maximal at less than 1 μm Ca2+ and does not require ATP, which suggests that sites with high affinity for Ca2+ exist in the Ca2+-ATPase in the absence of ATP. (iv) Under all conditions tested the response to N-ethylmaleimide of the ATPase and phosphatase activites stimulated by K+ or Na+ in the presence of Ca2+ parallels that of the Ca2+-dependent activities, suggesting that the Ca2+-ATPase system possesses sites at which monovalent cations bind to increase its activity.

The role of the sites for ATP of the Ca2+-ATPase from human red cell membranes during Ca2+-phosphatase activity

Abstract 1. In the presence of ATP, the Ca2+ pump of human red cell membranes catalyzes the hydrolysis of p- nitrophenyl phosphate. The requirement for ATP of the Ca2+- p- nitrophenylphosphatase activity was studied in relation to the two classes of site for ATP that are apparent during Ca2+ -ATPase activity. 2. (a) The K 0.5 for ATP as activator of the Ca2+ - p- nitrophenylphosphatase extrapolated at 0 mM PNPP is equal to the K m of the Ca2+ -ATPase. (b) PNPP competes with ATP and its effectiveness is the same regardless the nucleotide acts as the substrate of the Ca2+ -ATPase or as activator of the Ca2+ - p- nitrophenylphosphatase . 3. PNPP at the high-affinity site does not substitute for ATP as activator of the Ca2+ - p- nitrophenylphosphatase . 4. At ATP concentrations that almost saturate the high-affinity site, Ca2+ - p- nitrophenylphosphatase activity increases as a function of PNPP along an S-shaped curve, while Ca2+ -ATPase activity is partially inhibited along a curve of the same shape and apparent affinity. The fraction of Ca2+ -ATPase activity which is inhibited by PNPP is that which results from occupation of the low-affinity site by ATP. 5. Activation of the Ca2+ -ATPase by ATP at the low-affinity site is associated with inhibition of the Ca2+ - p- nitrophenylphosphatase activity. Both phenomena take place with the same apparent affinity and along curves of the same shape. 6. Experimental results suggest that: (a) the Ca2+ - p- nitrophenylphosphatase activity depends on ATP at the high-affinity site; (b) PNPP is hydrolyzed at the low-affinity site; (c) Ca2+ -ATPase activity at the high-affinity size persists during Ca2+ - p- nitrophenylphosphatase activity.

On the Mechanism of Inhibition of the PMCa2+ -ATPase by Lanthanum

FIGURE 1. Effect of LaCI3 added during phosphorylation. Membranes were preincubated in buffer with 0.2 mM CaClz and 0.5 mM MgClz and phosphorylated 2.5 ms in the same medium with 10 pM (y3?P)ATP plus different concentrations of LaC13. v, was the ratio between the amount of EP and 2.5 ms. (Inset) Effect of LaCI3 on steady-state level of EP. Conditions were identical except that phosphorylation time was 5 seconds.