Patricio J. Garrahan

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 interaction of magnesium ions with the calcium pump of sarcoplasmic reticulum

1. In the presence of Ca2+, ATP phosphorylates the Ca2+ pump of sarcoplasmic reticulum at the same site and to the same extent regardless of whether Mg2+ is added or not to the incubation media, the main effect of added Mg2+ being to increase the rate of phosphorylation. 2. When phosphoenzyme is made in Mg2+-containing media it dephosphorylates about 30-times faster than when it is made in the absence of added Mg2+. Addition of Mg2+ after phosphorylation is uneffective in accelerating the hydrolysis of phosphoenzyme even in solubilized enzyme, suggesting that phosphorylation of the Ca2+ pump results in occlusion of the site at which Mg2+ combines to accelerate the release of phosphate. 3. Occlusion of the site for Mg2+ can be partially reversed by trans-1,2-diaminocyclohexonetetraacetic acid (CDTA). Use was made of this property to demonstrate that for the rapid release of phosphate to occur Mg2+ has to be bound to the enzyme. 4. Results seem to indicate that Mg2+ combines with the Ca2+ pump prior to phosphorylation.

Potassium-activated phosphatase from human red blood cells : The Effects of Adenosine Triphosphate.

SummaryThe behavior of the red cell membrane K+-activated phosphatase is significantly altered by ATP. When ATP is added, the apparent affinity of the enzyme for its substrate and for K+ is lowered, whereas its sensitivity to ouabain is increased. Under these conditions, addition of Na+ raises the apparent affinity of the enzyme for K+ to values well above those found in the absence of Na+ and ATP. The effect of Na+ is blocked by hydroxylamine and oligomycin. Low concentrations of Ca++, Sr++ or Ba++, which have little effect in the absence of ATP, induce large increases in the K+-dependent phosphatase activity in the presence of ATP. This effect is associated with the loss of ouabain sensitivity of the phosphatase. The velocity vs. divalent cation concentration curves of the K+-dependent phosphatase and the (Na++K+)-independent ATPase activities are very similar. The effects of ATP seem to be specific for this nucleotide and are exerted at concentrations similar to those normally found in red cells. They may therefore be relevant to the proposed physiological role of the cell membrane phosphatase.

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.

An unexpected effect of ATP on the ratio between activity and phosphoenzyme level of Na+/K(+)-ATPase in steady state.

According to the Albers-Post model the hydrolysis of ATP catalyzed by the Na+/K(+)-ATPase requires the sequential formation of at least two conformers of a phosphoenzyme (E1P and E2P), followed by the K(+)-stimulated hydrolysis of E2P. In this paper we show that this model is a particular case of a more general class of models in all of which the ratio between ATPase activity (v) and total phosphoenzyme level (EP) in steady state is determined solely by the rate constants of interconversion between phosphoconformers and of dephosphorylation. Since these are thought to be unaffected by ATP, the substrate curves for ATPase activity and EP should be identical in shape so that the ratio v/EP ought to be independent of the concentration of ATP. We tested this prediction by parallel measurements of v and EP as a function of [ATP] in the absence or presence of non-limiting concentrations of K+, Rb+ or NH+4. In the absence of K+ or its congeners, both curves followed Michaelis-Menten kinetics, with almost identical Km values (0.16 microM) so that v/EP remained independent of [ATP]. In the presence of either K+, Rb+ or NH+4, v and EP increased with [ATP] along the sum of two Michaelis-Menten equations. The biphasic response of v is well known but, to the best of our knowledge, our results are the first demonstration that the response of EP to [ATP] is also biphasic. Under these conditions, the ratio v/EP increased with [ATP] from 19.8 to 40.1 s-1 along a hyperbola that was half-maximal at 9.5 microM. To preserve the validity of the current model it seems necessary to assume that ATP acts on the E1P <--> E2P transition and/or on the rate of hydrolysis of E2P. The latter possibility was ruled out. We also found that to fit the Albers-Post model to our data, the rate constant for K+ deocclussion from E2 has to be about 10-times higher than that reported from measurements of partial reactions. The results indicate that the Albers-Post model quantitatively predicts the experimental behavior of the Na(+)-ATPase activity but is unable to do this for the Na+/K(+)-ATPase activity, unless additional and yet unproved hypothesis are included.