Graciela Berberián

A novel 13 kDa cytoplasmic soluble protein is required for the nucleotide (MgATP) modulation of the Na/Ca exchange in squid nerve fibers

The Na/Ca exchange is a highly regulated transport mechanism in which MgATP, a powerful modulatory intracellular substrate, has important implications for its function. As occurs with some preparations, in squid axons, nucleotide regulation is lost after membrane vesicle isolation. This has been a significant obstacle in the biochemical characterization of the MgATP effect. An important clue in solving this long‐standing puzzle is presented in this work by showing that prolonged intracellular dialysis of squid axons produces a complete run down of the MgATP effect. Here we report that a soluble cytoplasmic factor isolated from fresh squid axoplasm and brain reconstitutes the MgATP stimulation of the Na‐gradient‐dependent 45Ca uptake in squid optic nerve membrane vesicles. Partial purification of this factor uncovers the presence of a novel 13 kDa soluble cytoplasmic protein (SCPr) which, when microinjected in ATP de‐regulated dialyzed squid axons, completely restores the MgATP stimulation of Nao‐dependent Ca efflux. We propose that in the squid preparation this SCPr constitutes the link between the nucleotide and target effector: the Na/Ca exchanger itself, or other plasma membrane structures which may secondarily interact with the exchanger.

DETECTION OF A HIGHLY OUABAIN SENSITIVE ISOFORM OF RAT BRAINSTEM NA,K-ATPASE

The present work provides evidence for the existence in rat brainstem of a form of Na,K-ATPase catalytic subunit that displays a high affinity for ouabain (Kd about 10(-9) M). Its kinetic identification was made out from studies on dose response curves of ouabain inhibition of Na,K-ATPase activity, ouabain inhibition of Na(+)-dependent phosphorylation from ATP and ouabain stabilized phosphoenzyme formation from inorganic phosphate (Pi). In all these studies this isoform comprises around 11 percent of the total Na,K-ATPase enzyme. The PAGE electrophoretic mobility of its phosphoprotein obtained from Pi in the presence of ouabain is lower than that of the alpha-1 form but it cannot be distinguished from that of alpha-2. Whether this highly ouabain sensitive form corresponds to the alpha-3 isoenzyme or represents the translational product of one of the additional genes described for the large catalytic subunit remains at the moment an open question.

The effects of several ligands on the potassium-vanadate interaction in the inhibition of the (Na+ + K+)-ATPase and the Na+, K+ pump

Inhibition by vanadate of the K+-dependent p-nitrophenylphosphatase activity catalyzed by the (Na+ + K+)-ATPase partially purified from pig kidney showed competitive behavior with the substrate, K+ and Mg2+ acted as cofactors in promoting that inhibition. Ligands which inhibited the K+-dependent p-nitrophenyl phosphate hydrolysis (Na+, nucleotide polyphosphates, inorganic phosphate) protected against inhibition by vanadate. The magnitude of that protection was proportional to the inhibition produced in the absence of vanadate. In the presence of only p-nitrophenyl phosphate and Mg2+, or when the protective ligands were tested alone, the activation of p-nitrophenyl phosphate hydrolysis by K+ followed a sigmoid curve in the presence as well in the absence of vanadate. However, the combination of 100 mM NaCl and 3 mM ATP resulted in a biphasic effect of K+ on the p-nitrophenyl phosphate hydrolysis in the presence of vanadate. After an initial rise at low K+ concentration, the p-nitrophenylphosphatase activity declined at high K+ concentrations; this decline became more pronounced as the vanadate concentration was increased. This biphasic response was not seen when a nonphosphorylating ATP analog was combined with Na+ (which favors the nucleotide binding) or with inorganic phosphate (a requirement for K+ - K+ exchange). Experiments with inside-out resealed vesicles from human red cells showed that in the absence of Na+ plus ATP, K+ promoted vanadate inhibition of p-nitrophenylphosphatase activity in a nonbiphasic manner, acting at cytoplasmic sites. On the other hand, in the presence of Na+ plus ATP, the biphasic response of p-nitrophenyl phosphate hydrolysis is due to K+ acting on extracellular sites. In vanadate-poisoned intact red blood cells, the biphasic response of the ouabain-sensitive Rb+ influx as a function of the external Rb+ concentration failed to develop when there was no Na+ in the extracellular media. In addition, in the absence of extracellular Na+, external Rb+ did not influence the magnitude of inhibition. The present findings indicate that external K+ favors vanadate inhibition by displacing Na+ from unspecified extracellular membrane sites.

A Nerve Cytosolic Factor Is Required for MgATP Stimulation of a Na+ Gradient-Dependent Ca2+ Uptake in Plasma Membrane Vesicles from Squid Optic Nervea

As early as 1979’ and 19822 we collected evidence that in dialyzed axons Ca2t1 was required for CaZt influx through the Nat-Caz+ exchanger. This need for Caz’ binding to nontransport internal sites was unambiguously shown in 1986 by following Na+ efflux through the reversal exchange; we named that effect CaZti regulation, and the intracellular site was called the Ca2 + regulatory site.3 In addition, and in the same paper, we presented data indicating that (i) ATP also stimulated reversal exchange and (ii) there seemed to be a relationship (or interaction) between Ca2+, and ATP stimulation. Later work demonstrated that ATP acted precisely by increasing the apparent affinity of the intracellular regulatory site for calcium ions4 In the work that followed (see Ref. 4 for references), always in dialyzed squid giant axons, we accumulated evidence that consistently suggested the effect of ATP was a consequence of a phosphorylation process that involved the interplay of a coupled kinase(s)-phosphatase(s) system(s). The main features of the ATP stimulation of the Nat-Ca2+ exchanger that led to this conclusion are:

Potassium-p-nitrophenyl phosphate interactions with (Na+ + K+)-ATPase. Their relevance to phosphatase activity.

The K+-dependent p-nitrophenylphosphatase activity catalyzed by purified (Na+ + K+)-ATPase from pig kidney shows substrate inhibition (Ki about 9.5 mM at 2.1 mM Mg2+). Potassium antagonizes and sodium favours this inhibition. In addition , K+ reduces the apparent affinity for substrate activation, whereas p-nitrophenyl phosphate reduces the apparent affinity for K+ activation. In the absence of Mg2+, p-nitrophenyl phosphate, as well as ATP, accelerates the release of Rb+ from the Rb+ occluded unphosphorylated enzyme. With no Mg2+ and with 0.5 mM KCl, trypsin inactivation of (Na+ + K+)-ATPase as a function of time follows a single exponential but is transformed into a double exponential when 1 mM ATP or 5 mM p-nitrophenyl phosphate are also present. In the presence of 3 mM MgCl2, 5 mM p-nitrophenyl phosphate and without KCl the trypsin inactivation pattern is that described for the E1 enzyme form; the addition of 10 mM KCl changes the pattern which, after about 6 min delay, follows a single exponential. These results suggest that (i) the shifting of the enzyme toward the E1 state is the basis for substrate inhibition of the p-nitrophenylphosphatase activity of(Na+ + K+)-ATPase, and (ii) the substrate site during phosphatase activity is distinct from the low-affinity ATP site.

Phosphoarginine regulation of the squid nerve Na+/Ca2+ exchanger: metabolic pathway and exchanger-ligand interactions different from those seen with ATP.

In squid nerves the Na+–Ca2+ exchanger is up‐regulated by ATP and phosphoarginine (PA). ATP regulation involves drastic alterations in the Na+i, H+i and Ca2+i interactions with the large intracellular cytoplasmic loop of the exchanger protein. In this work we explored the mechanisms associated with PA regulation in intracellular dialysed squid axons and squid optic nerve membrane vesicles. Dialysed axons were used to measure the four modes of exchange fluxes (Na+o–Ca2+i or forward exchange, Ca2+o–Na+i or reverse exchange, Ca2+o–Ca2+i exchange and Na+o–Na+i exchange) under controlled intra‐ and extracellular conditions. Inside‐out membrane vesicles allowed measurement of the Na+‐gradient‐dependent 45Ca2+ uptake (forward mode) as influenced by ligands and digestion with chymotrypsin from the intracellular side. The results show that, unlike ATP, PA regulation does not affect the H+i, Na+i and Ca2+i interactions with the intracellular ‘regulatory’ loop, but increases the affinity of the intracellular transport sites, preferentially for Ca2+i (about 20‐fold) over Na+i (50%); i.e. PA favours the forward mode over the other exchange modes. Intracellular chymotrypsin digestion removed ATP regulation while leaving modulation by PA unmodified. Western blot analysis suggested that chymotrypsin disrupts the large intracellular loop. Together these results indicate that ATP and PA regulations are associated with different structures inside and outside the exchanger protein. Based on these observations we expanded our previous model for metabolic regulation of the Na+–Ca2+ exchanger by adding to the original ‘ATP region’ a new zone, the ‘PA region’, related to the intracellular transport sites for Na+i and Ca2+i. This new model is able to explain most previous and present results.

Acetyl phosphate can act as a substrate for Na+ transport by (Na++K+)-ATPase

Experiments using liposomes with (Na+ + K+)-ATPase incorporated showed that in the presence of extravesicular Mg2+, acetyl phosphate was able to stimulate Na+ uptake when the liposomes contained Na+ or choline and were K+-free; this acetyl phosphate-dependent Na+ transport was similar to the ATP-dependent transport observed with 0.003 mM or 3 mM ATP. When the intravesicular solution contained K+, there was an ATP-dependent Na+ uptake which was large with 3 mM ATP and small (about the size seen in K+-free liposomes) with 0.003 mM ATP; in this case, although acetyl phosphate produced a slight activation of Na+ transport, the effect was not statistically significant. All ATP and acetyl phosphate-stimulated Na+ transport disappeared in the absence of extravesicular Mg2+ or in the presence of ouabain in the intravesicular solution. These results are consistent with the hypothesis that, at the concentration used, acetyl phosphate can replace ATP in the catalytic but not in the regulatory site of the (Na+ + K+)-ATPase and active Na+ transport system. This suggests that as far as the early stages of the pump cycle are concerned the role of ATP is simply to phosphorylate.

Some properties of the H-ATPase activity present in root plasmalemma of Avena sativa L. Two different enzymes or one enzyme with two ATP sites?

The effects of Mg2+, K+ and ATP on a H-ATPase activity from a native plasmalemma fraction of oat roots were explored at 20 degrees C and pH 6.5. In the presence of 3 mM ATP and no K+, H-ATPase activity vs. [Mg2+] approached a monotonic activation but it became biphasic, with a decline above 3 mM Mg2+, in the presence of 20 mM K+. Mg2+ inhibition occurred also in K-free solutions when [ATP] was lowered to 0.05 mM. Also, an apparent monotonic H-ATPase activation by [K+] at 3.0 mM ATP was transformed in biphasic (inhibition by high [K+]) when [ATP] was reduced to 0.05 mM. The best fits of the ATP stimulation curves of hydrolysis satisfied the sum of two Michaelian functions where that with higher affinity had lower Vmx. Taking into consideration all conditions of activity assay, the high-affinity component (1) had a Km about 11-16 microM and a Vmx around 0.14-0.28 mumol Pi/mg per min whereas that with lower affinity (2) had a Km of 220-540 microM and a Vmx of 0.5-1.0 mumol Pi/mg per min. Km2 was markedly affected by the [K+] and [Mg2+]; at optimal concentrations of these cations (1 mM Mg2+ and 10 mM K+) it had a value of 235 +/- 24 microM which was increased to 540 +/- 35 microM at 20 mM [Mg2+] and 60 mM [K+]. In addition, Vmx1 was reduced to about a half when the concentrations of Mg2+ and K+ were increased to inhibitory levels. These results could be explained by the existence of two different enzymes or one enzyme with two ATP sites. In the second case, we could not tell at this stage if both are catalytic or one is regulatory.

Regulation of Phosphatidylinositol-4,5-Biphosphate Bound to the Bovine Cardiac Na+/Ca2+ Exchanger

Abstract: Western blot and cross immunoprecipitation analysis with specific antibodies demonstrate that in bovine heart sarcolemmal vesicles phosphatidylinositol‐4,5‐biphosphate (PtdIns‐4,5‐P2) binds strongly to the Na+/Ca2+ exchanger (NCX1). This binding is modulated by ATP, Ca2+, vanadate, exchanger inhibitory peptide (XIP), and PLC‐PtdIns specific in a way resembling the ATP regulation of the exchange fluxes. With 1 μM Ca2+, 3 mM Mg2+, and 0.4 mM vanadate, 1 mM ATP increased about twofold the bound PtdIns‐4,5‐P2, reaching a steady state in 3–5 s at 37°C. With 100 μM Ca2+, ATP had no effect on the PtdIns‐4,5‐P2 bound to NCX1 or on the exchange fluxes. Without vanadate the bound PtdIns‐4,5‐P2 was largely reduced; under this condition ATP failed to increase it and did not stimulate the exchanger. XIP inhibits the exchanger, more noticeable in the absence of ATP. With XIP, ATP does not modify the levels of bound PtdIns‐4,5‐P2; however there is a small but distinct ATP stimulation of the exchanger. Vesicles pretreated with PtdIns‐PLC, showed no de novo, [32P]ATP‐induced, production of PtdIns‐4,5‐P2, but some ATP‐stimulated increase in the bound PtdIns‐4,5‐P2 was detected; however, that increase did not exceed the levels found with vanadate and no ATP. These results indicate that in bovine heart sarcolemmal vesicles, ATP upregulation of NCX1 is related to PtdIns‐4,5‐P2 bound to the exchanger, perhaps over a “threshold” or “unspecific” amount. In addition, vanadate could influence the amount of detected PtdIns‐4,5‐P2 either by inhibiting phosphoinositide‐specific phosphatases and/or by inducing a redistribution of PtdIns‐4,5‐P2 molecules associated with the Na+/Ca2+ exchanger.

Phosphatase activity and potassium transport in liposomes with Na+,K(+)-ATPase incorporated.

We have used liposomes with incorporated pig kidney Na+,K(+)-ATPase to study vanadate sensitive K(+)-K+ exchange and net K+ uptake under conditions of acetyl- and p-nitrophenyl phosphatase activities. The experiments were performed at 20 degrees C. Cytoplasmic phosphate contamination was minimized with a phosphate trapping system based on glycogen, phosphorylase a and glucose-6-phosphate dehydrogenase. In the absence of Mg2+ (no phosphatase activity) 5-10 mM p-nitrophenyl phosphate slightly stimulated K(+)-K+ exchange whereas 5-10 mM acetyl phosphate did not. In the presence of 3 mM MgCl2 (high rate of phosphatase activity) acetyl phosphate did not affect K(+)-K+ exchange whereas p-nitrophenyl phosphate induced a greater stimulation than in the absence of Mg2+; a further addition of 1 mM ADP resulted in a 35-65% inhibition of phosphatase activity with an increase in K(+)-K+ exchange, which sometimes reached the levels seen with 5 mM phosphate and 1 mM ADP. The net K+ uptake in the presence of 3 mM MgCl2 was not affected by acetyl phosphate or p-nitrophenyl phosphate, whereas it was inhibited by 5 mM phosphate (with and without 1 mM ADP). The results of this work suggest that the phosphatase reaction is not by itself associated to K+ translocation. The ADP-dependent stimulation of K(+)-K+ exchange in the presence of phosphatase activity could be explained by the overlapping of one or more step/s of the reversible phosphorylation from phosphate with the phosphatase cycle.