Alteration of Ca2+ fluxes in brain microsomes by K+ and Na+: modulation by sulfated polysaccharides and trifluoperazine

Rat brain microsomes accumulate Ca2+ at the expense of ATP hydrolysis. The rate of transport is not modulated by the monovalent cations K+, Na+, or Li+. Both the Ca2+ uptake and the Ca(2+)-dependent ATPase activity of microsomes are inhibited by the sulfated polysaccharides heparin, fucosylated chondroitin sulfate, and dextran sulfate. Half-maximal inhibition is observed with sulfated polysaccharide concentrations ranging from 0.5 to 8.0 micrograms/ml. The inhibition is antagonized by KCl and NaCl but not by LiCl. As a result, Ca2+ transport by the native vesicles, which in the absence of polysaccharides is not modulated by monovalent cations, becomes highly sensitive to these ions. Trifluoperazine has a dual effect on the Ca2+ pump of brain microsomes. At low concentrations (20-80 microM) it stimulates the rate of Ca2+ influx, and at concentrations > 100 microM if inhibits both the Ca2+ uptake and the ATPase activity. The activation observed at low trifluoperazine concentrations is specific for the brain Ca(2+)-ATPase; for the Ca(2+)-ATPases found in blood platelets and in the sarcoplasmic reticulum of skeletal muscle, trifluoperazine causes only a concentration-dependent inhibition of Ca2+ uptake. Passive Ca2+ efflux from brain microsomes preloaded with Ca2+ is increased by trifluoperazine (50-150 microM), and this effect is potentiated by heparin (10 micrograms/ml), even in the presence of KCl. It is proposed that the Ca(2+)-ATPase isoforms from brain microsomes is modulated differently by polysaccharides and trifluoperazine when compared with skeletal muscle and platelet isoforms.     

Structural replacement of active site monovalent cations by the epsilon-amino group of lysine in the ATPase fragment of bovine Hsc70

We have assessed the ability of the epsilon-amino group of a non-native lysine chain to substitute for a monovalent cation in an enzyme active site. In the bovine Hsc70 ATPase fragment, mutation of cysteine 17 or aspartic acid 206 to lysine potentially allows the replacement of an active site potassium ion with the epsilon-amino nitrogen. We examined the ATP hydrolysis kinetics and crystal structures of isolated mutant ATPase domains. The introduced epsilon-amino nitrogen in the C17K mutant occupies a significantly different position than the potassium ion. The introduced epsilon-amino nitrogen in the D206K mutant occupies a position indistinguishable from that of the potassium in the wild-type structure. Each mutant retains <5% ATPase activity when compared to the wild type under physiological conditions (potassium buffer) although substrate binding is tighter, probably as a consequence of slower release. It is possible to construct a very good structural mimic of bound cation which suffices for substrate binding but not for catalytic activity.     

Functional consequences of a posttransfection mutation in the H2-H3 cytoplasmic loop of the alpha subunit of Na,K-ATPase

During kinetic studies of mutant rat Na,K-ATPases, we identified a spontaneous mutation in the first cytoplasmic loop between transmembrane helices 2 and 3 (H2-H3 loop) which results in a functional enzyme with distinct Na,K-ATPase kinetics. The mutant cDNA contained a single G950 to A substitution, which resulted in the replacement of glutamate at 233 with a lysine (E233K). E233K and alpha1 cDNAs were transfected into HeLa cells and their kinetic behavior was compared. Transport studies carried out under physiological conditions with intact cells indicate that the E233K mutant and alpha1 have similar apparent affinities for cytoplasmic Na+ and extracellular K+. In contrast, distinct kinetic properties are observed when ATPase activity is assayed under conditions (low ATP concentration) in which the K+ deocclusion pathway of the reaction is rate-limiting. At 1 microM ATP K+ inhibits Na+-ATPase of alpha1, but activates Na+-ATPase of E233K. This distinctive behavior of E233K is due to its faster rate of formation of dephosphoenzyme (E1) from K+-occluded enzyme (E2(K)), as well as 6-fold higher affinity for ATP at the low affinity ATP binding site. A lower ratio of Vmax to maximal level of phosphoenzyme indicates that E233K has a lower catalytic turnover than alpha1. These distinct kinetics of E233K suggest a shift in its E1/E2 conformational equilibrium toward E1. Furthermore, the importance of the H2-H3 loop in coupling conformational changes to ATP hydrolysis is underscored by a marked (2 orders of magnitude) reduction in vanadate sensitivity effected by this Glu233 --> Lys mutation.     

Change in the therapist: the role of patient-induced inspiration

BACKGROUND: The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones, which promote protein folding and participate in many cellular functions. The Hsp70 chaperones are composed of two major domains. The N-terminal ATPase domain binds to and hydrolyzes ATP, whereas the C-terminal domain is required for polypeptide binding. Cooperation of both domains is needed for protein folding. The crystal structure of bovine Hsc70 ATPase domain (bATPase) has been determined and, more recently, the crystal structure of the peptide-binding domain of a related chaperone, DnaK, in complex with peptide substrate has been obtained. The molecular chaperone activity and conformational switch are functionally linked with ATP hydrolysis. A high-resolution structure of the ATPase domain is required to provide an understanding of the mechanism of ATP hydrolysis and how it affects communication between C- and N-terminal domains. RESULTS: The crystal structure of the human Hsp70 ATPase domain (hATPase) has been determined and refined at 1. 84 A, using synchrotron radiation at 120K. Two calcium sites were identified: the first calcium binds within the catalytic pocket, bridging ADP and inorganic phosphate, and the second calcium is tightly coordinated on the protein surface by Glu231, Asp232 and the carbonyl of His227. Overall, the structure of hATPase is similar to bATPase. Differences between them are found in the loops, the sites of amino acid substitution and the calcium-binding sites. Human Hsp70 chaperone is phosphorylated in vitro in the presence of divalent ions, calcium being the most effective. CONCLUSIONS: The structural similarity of hATPase and bATPase and the sequence similarity within the Hsp70 chaperone family suggest a universal mechanism of ATP hydrolysis among all Hsp70 molecular chaperones. Two calcium ions have been found in the hATPase structure. One corresponds to the magnesium site in bATPase and appears to be important for ATP hydrolysis and in vitro phosphorylation. Local changes in protein structure as a result of calcium binding may facilitate phosphorylation. A small, but significant, movement of metal ions and sidechains could position catalytically important threonine residues for phosphorylation. The second calcium site represents a new calcium-binding motif that can play a role in the stabilization of protein structure. We discuss how the information about catalytic events in the active site could be transmitted to the peptide-binding domain.     

Evidence that Ser775 in the alpha subunit of the Na,K-ATPase is a residue in the cation binding pocket

Substitution of alanine for Ser775 in a ouabain-resistant alpha1 sheep isoform causes a 30-fold decrease in apparent affinity for K+ as an activator of the Na,K-ATPase, as well as an increase in apparent affinity for ATP (Arguello, J. M., and Lingrel, J. B (1995) J. Biol. Chem. 270, 22764-22771). This study was carried out to determine whether Ser775 is a direct cation-ligating residue or whether the change in apparent affinity for K+ is secondary to a conformational alteration as evidenced in the change in ATP affinity, with the following results. Kinetics of K+(Rb+) influx into intact cells show that the change is due to a change in K+ interaction at the extracellular surface. The K+ dependence of formation of K+-occluded enzyme (E2(K)) and of the rate of formation of deoccluded enzyme from E2(K) indicate that the Ser775 --> Ala mutation results in a marked increase (>/=30-fold) in rate of release of K+ from E2(K). The high affinity Na+-like competitive antagonist 1,3-dibromo2,4,6-tris-(methylisothiouronium)benzene (Br2TITU), which interacts with the E1 conformation and blocks cytoplasmic cation binding (Hoving, S., Bar-Shimon, M., Tijmes, J. J. , Tal, D. M., and Karlish, S. J. D. (1995) J. Biol. Chem. 270, 29788-29793), inhibits Na+-ATPase of the mutant less than the control enzyme. With intact cells, Br2TITU acts as a competitive inhibitor of extracellular K+ activation of both the mutant and control enzymes. In this case, the mutant was more sensitive to inhibition. With vanadate as a probe of conformation, a difference in conformational equilibrium between the mutant and control enzymes could not be detected under turnover conditions (Na+- ATPase) in the absence of K+. These results indicate that the increase in apparent affinity for ATP effected by the Ser775 --> Ala mutation is secondary to a change in intrinsic cation affinity/selectivity. The large change in affinity for extracellular K+ compared with cytoplasmic Na+ and to Br2TITU binding supports the conclusion that the serine hydroxyl is either part of the K+-gate structure or a direct cation-ligating residue that is shared by at least one Na+ ion, albeit with less consequence on rate constants for Na+ binding or release compared with K+.     

GrpE-like regulation of the hsc70 chaperone by the anti-apoptotic protein BAG-1

The BAG-1 protein appears to inhibit cell death by binding to Bcl-2, the Raf-1 protein kinase, and certain growth factor receptors, but the mechanism of inhibition remains enigmatic. BAG-1 also interacts with several steroid hormone receptors which require the molecular chaperones Hsc70 and Hsp90 for activation. Here we show that BAG-1 is a regulator of the Hsc70 chaperone. BAG-1 binds to the ATPase domain of Hsc70 and, in cooperation with Hsp40, stimulates Hsc70's steady-state ATP hydrolysis activity approximately 40-fold. Similar to the action of the GrpE protein on bacterial Hsp70, BAG-1 accelerates the release of ADP from Hsc70. Thus, BAG-1 regulates the Hsc70 ATPase in a manner contrary to the Hsc70-interacting protein Hip, which stabilizes the ADP-bound state. Intriguingly, BAG-1 and Hip compete in binding to the ATPase domain of Hsc70. Our results reveal an unexpected diversity in the regulation of Hsc70 and raise the possibility that the observed anti-apoptotic function of BAG-1 may be exerted through a modulation of the chaperone activity of Hsc70 on specific protein folding and maturation pathways.     

Destabilization of peptide binding and interdomain communication by an E543K mutation in the bovine 70-kDa heat shock cognate protein, a molecular chaperone

We have compared 70-kDa heat shock cognate protein (Hsc70) isolated from bovine brain with recombinant wild type protein and mutant E543K protein (previously studied as wild type in our laboratory). Wild type bovine and recombinant protein differ by posttranslational modification of lysine 561 but interact similarly with a short peptide (fluorescein-labeled FYQLALT) and with denatured staphylococcal nuclease-(Delta135-149). Mutation E543K results in 4. 5-fold faster release of peptide and lower stability of complexes with staphylococcal nuclease-(Delta135-149). ATP hydrolysis rates of the wild type proteins are enhanced 6-10-fold by the addition of peptide. The E543K mutant has a peptide-stimulated hydrolytic rate similar to that of wild type protein but a higher unstimulated rate, yielding a mere 2-fold enhancement. All three versions of Hsc70 possess similar ATP-dependent conformational shifts, and all show potassium ion dependence. These data support the following model: (i) in the presence of K+, Mg2+, and ATP, the peptide binding domain inhibits the ATPase; (ii) binding of peptide relieves this inhibition; and (iii) the E543K mutation significantly attenuates the inhibition by the peptide binding domain and destabilizes Hsc70-peptide complexes.     

The ATP-binding cassette (ABC) transporter for maltose/maltodextrins of Salmonella typhimurium. Characterization of the ATPase activity associated with the purified MalK subunit

The ATPase activity associated with the purified MalK subunit of the maltose transport complex of Salmonella typhimurium, a bacterial ATP-Binding Cassette (ABC) transporter (Walter, C., H?ner zu Bentrup, K., and Schneider, E. (1992) J. Biol. Chem. 267, 8863-8869), was characterized in detail. The analysis of the kinetics of ATP hydrolysis yielded a Km value of 70 +/- 4 microM and a Vmax of 1.3 +/- 0.3 mumol/min/mg of protein. Both GTP and CTP also served as substrates. While MalK exhibited nearly the same affinity for GTP as for ATP, the Michaelis constant for CTP as a substrate was much higher. ATP hydrolysis was strongly dependent on the presence of Mg2+ ions. Mn2+ at low concentrations, but neither Ca2+ nor Zn2+ partially substituted for Mg2+. The ATPase activity was optimal at slightly alkaline pH and was stimulated in the presence of both glycerol (7.5%) and dimethyl sulfoxide (Me2SO) (5%). ADP and the non-cleavable substrate analog ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)) were identified as competitive inhibitors. The MalK-ATPase was resistant to specific inhibitors of F-, P-, and V-type ATPases, such as dicyclohexylcarbodiimide, azide, vanadate, or bafilomycin A1. In contrast, micromolar concentrations of the sulfhydryl reagent N-ethylmaleimide strongly inhibited the enzymatic activity. This inhibition was blocked in the presence of ATP. These results suggest that the intrinsic ATPase activity of purified MalK can be clearly distinguished from other ATP-hydrolyzing enzymes, e.g. ion-translocating ATPases.     

Charge translocation by the Na+/K+-ATPase investigated on solid supported membranes: cytoplasmic cation binding and release

In the preceding publication (. Biophys. J. 76:000-000) a new technique was described that was able to produce concentration jumps of arbitrary ion species at the surface of a solid supported membrane (SSM). This technique can be used to investigate the kinetics of ion translocating proteins adsorbed to the SSM. Charge translocation of the Na+/K+-ATPase in the presence of ATP was investigated. Here we describe experiments carried out with membrane fragments containing Na+/K+-ATPase from pig kidney and in the absence of ATP. Electrical currents are measured after rapid addition of Na+. We demonstrate that these currents can be explained only by a cation binding process on the cytoplasmic side, most probably to the cytoplasmic cation binding site of the Na+/K+-ATPase. An electrogenic reaction of the protein was observed only with Na+, but not with other monovalent cations (K+, Li+, Rb+, Cs+). Using Na+ activation of the enzyme after preincubation with K+ we also investigated the K+-dependent half-cycle of the Na+/K+-ATPase. A rate constant for K+ translocation in the absence of ATP of 0.2-0.3 s-1 was determined. In addition, these experiments show that K+ deocclusion, and cytoplasmic K+ release are electroneutral.     

Epidemiology of atherosclerotic peripheral arterial occlusive disease in Hong Kong

Incubation of bovine liver mitochondrial rhodanese in dilute, reducing solutions at temperatures ranging between 30 and 45 degreesC conduced to a rapid loss of enzymatic activity. This inactivation was substantially reduced in the presence of millimolar concentrations of alkali metal ions, divalent cations (including Mg2+, Ca2+, and Ba2+) were ineffective. The extent of protection afforded by monovalent cations was highly dependent on their ionic radii, with K+ and Na+ ions being the most effective protective agents. The protection afforded by a number of anions, including thiosulfate, could be totally ascribed to the presence of the accompanying monovalent cation. The overall results indicate that K+ and Na+, at concentrations and temperatures within the physiological range, substantially contribute to the stabilization of the functional structure of rhodanese.     

High selectivity of the i(f) channel to Na+ and K+ in rabbit isolated sinoatrial node cells

The ionic selectivity of the hyperpolarization-activated inward current (i(f)) channel to monovalent cations was investigated in single isolated sinoatrial node cells of the rabbit using the whole-cell patch-clamp technique. With a 140 mM K+ pipette, replacement of 90% external Na+ by Li+ caused a -24.5 mV shift of the fully activated current/voltage I/V curve without a significant decrease of the slope conductance. With a 140 mM Cs+ pipette, the i(f) current decreased almost proportionally to the decrease in external [Na+]o as Li+ was substituted. These responses are practically the same as those observed with N-methyl glucamine (NMG+) substitution, suggesting that the relative permeability of Li+ compared with Na+ for the i(f) channel is as low as that of NMG+. When Cs+ or Rb+ was substituted for internal K+, the fully activated I/V relationship for i(f) showed strong inward rectification with a positive reversal potential, indicating low permeability of the i(f) channel for Cs+ and Rb+. These results show that the i(f) channel is highly selective for Na+ and K+ and will not pass the similar ions Li+ and Rb+. Such a high degree of selectivity is unique and may imply that the structure of the i(f) channel differs greatly from that of other Na+ and K+ conducting channels.     

Inhibition of Hsp70 ATPase activity and protein renaturation by a novel Hsp70-binding protein

A cDNA that codes for an Hsp70-interacting protein (HspBP1) was isolated from a human heart cDNA library using the yeast two-hybrid system. The derived amino acid sequence is unique and therefore represents a new regulator of Hsp70. Northern blots of RNA from human tissues indicate that HspBP1 mRNA has a size of approximately 1.7 kilobase pairs and is present in all tissues analyzed but is most abundant in heart and skeletal muscle. Western blot analysis revealed a protein of approximately 40 kilodaltons detected in cell extracts. The ATPase domain of Hsp70 demonstrated binding to HspBP1. Further experiments showed binding of HspBP1 to Hsp70 and Hsc70 in a total heart extract. HspBP1 (8 microM) inhibited approximately 90% of the Hsp40-activated Hsp70 ATPase activity. HspBP1 prevented ATP binding to Hsp70, and therefore this is the likely mechanism of inhibition. Hsp40-activated ATPase activity is essential for the renaturation activity of Hsp70; therefore, the effects of HspBP1 on renaturation of luciferase in a reticulocyte lysate and a defined system were examined. HspBP1 inhibited renaturation with half-maximal inhibition at 2 microM. These data indicate that we have identified a novel Hsp70-interacting protein that inhibits Hsp70 chaperone activity.     

Kinetic uniformity of the ATP hydrolysis reaction catalyzed by Mg2+-ATPase in smooth muscle cell membrane

The kinetic properties of Mg(2+)-ATPase (EC 3.6.1.3) from myometrium cell plasma membranes have been studied. Under conditions of enzyme saturation with ATP (0.5-1.0 mM) or Mg2+ (1.0-5.0 mM) the initial maximal rates of the Mg(2+)-dependent enzymatic ATP hydrolysis, V0 ATP and V0 Mg, are 27.4 +/- 3.3 and 25.2 +/- 4.1 mumol Pi/hour/mg of protein, respectively. The apparent Michaelis constant, Km, for ATP and of the apparent activation constant, K alpha, for Mg2+ are equal to 28.1 +/- 2.6 and 107.0 +/- 26.0 microM, respectively. The bivalent metal ions used at 1.0 mM suppress the Mg(2+)-dependent hydrolysis of ATP whose efficiency decreases in the following order: Cu2+ > Zn2+ = Ni2+ > Mn2+ > Ca2+ > Co2+. Alkalinization of the incubation medium from pH 6.0 to pH 8.0 stimulates the Mg(2+)-dependent hydrolysis of ATP. It has been found that Mg(2+)-ATPase has the properties of an H(+)-sensitive enzymatic sensor which is characterized by a linear dependence between the initial maximal rate of the reaction, V0, and the pH value. The feasible role of plasma membrane Mg(2+)-ATPase in some reactions responsible for the control of proton and Ca2+ homeostasis in myometrium cells has been investigated.     

Kinetic studies of the effects of K+, Na+ and Li+ on the catalytic activity of human erythrocyte pyridoxal kinase

Kinetic studies were conducted to examine the effects of K+, Na+ and Li+ on human erythrocyte pyridoxal kinase (PK) activity. A dialyzed hemolysate served as the PK source. The substrates used were pyridoxal (PL) and ATP. Determination of the enzymatic activity was based on HPLC separation and fluorimetric detection of PL and pyridoxal 5'-phosphate as semicarbazone derivatives. In comparison to the poor activity of PK assayed without monovalent cation, all tested cations are activators. Among them, K+ is the most effective, improving both PK affinity for the substrates and maximal velocity. Na+ increases maximal velocity and PK affinity for ATP but decreases it for PL. Li+ is a poor activator which seems to modify the enzymatic mechanism from a random to an ordered sequential pattern with ATP bound before PL. Results suggest that K+ and Na+ bind to PK on the same site while Li+ binds on another site. This hypothesis and the mechanism of monovalent cation-PK interaction are compared to other well-known K(+)-activated enzymes.     

Purification and properties of the F1F0 ATPase of Ilyobacter tartaricus, a sodium ion pump

The ATPase of Ilyobacter tartaricus was solubilized from the bacterial membranes and purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed the usual subunit pattern of a bacterial F1F0 ATPase. The polypeptides with apparent molecular masses of 56, 52, 35, 16.5, and 6.5 kDa were identified as the alpha, beta, gamma, epsilon, and c subunits, respectively, by N-terminal protein sequencing and comparison with the sequences of the corresponding subunits from the Na(+)-translocating ATPase of Propionigenium modestum. Two overlapping sequences were obtained for the polypeptides moving with an apparent molecular mass of 22 kDa (tentatively assigned as b and delta subunits). No sequence could be determined for the putative a subunit (apparent molecular mass, 25 kDa). The c subunits formed a strong aggregate with the apparent molecular mass of 50 kDa which required treatment with trichloroacetic acid for dissociation. The ATPase was inhibited by dicyclohexyl carbodiimide, and Na+ ions protected the enzyme from this inhibition. The ATPase was specifically activated by Na+ or Li+ ions, markedly at high pH. After reconstitution into proteoliposomes, the enzyme catalyzed the ATP-dependent transport of Na+, Li+, or Hi+. Proton transport was specifically inhibited by Na+ or Li+ ions, indicating a competition between these alkali ions and protons for binding and translocation across the membrane. These experiments characterize the I. tartaricus ATPase as a new member of the family of FS-ATPases, which use Na+ as the physiological coupling ion for ATP synthesis.     

Dephosphorylation kinetics of pig kidney Na+,K+-ATPase

The kinetics of K+-stimulated dephosphorylation of the Na+,K+-ATPase were investigated at pH 7.4, 24 degrees C, and an ATP concentration of 1.0 mM via the stopped-flow technique using the fluorescent label RH421. Two different mixing procedures were used: (a) premixing with ATP to allow phosphorylation to go to completion, followed by mixing with KCl; and (b) simultaneous mixing with ATP and KCl. Using mixing procedure (a), the dephosphorylation rate constant of enzyme complexed with K+ ions could be determined directly to be 190 s-1).     

BAG-1 modulates the chaperone activity of Hsp70/Hsc70

The 70 kDa heat shock family of molecular chaperones is essential to a variety of cellular processes, yet it is unclear how these proteins are regulated in vivo. We present evidence that the protein BAG-1 is a potential modulator of the molecular chaperones, Hsp70 and Hsc70. BAG-1 binds to the ATPase domain of Hsp70 and Hsc70, without requirement for their carboxy-terminal peptide-binding domain, and can be co-immunoprecipitated with Hsp/Hsc70 from cell lysates. Purified BAG-1 and Hsp/Hsc70 efficiently form heteromeric complexes in vitro. BAG-1 inhibits Hsp/Hsc70-mediated in vitro refolding of an unfolded protein substrate, whereas BAG-1 mutants that fail to bind Hsp/Hsc70 do not affect chaperone activity. The binding of BAG-1 to one of its known cellular targets, Bcl-2, in cell lysates was found to be dependent on ATP, consistent with the possible involvement of Hsp/Hsc70 in complex formation. Overexpression of BAG-1 also protected certain cell lines from heat shock-induced cell death. The identification of Hsp/Hsc70 as a partner protein for BAG-1 may explain the diverse interactions observed between BAG-1 and several other proteins, including Raf-1, steroid hormone receptors and certain tyrosine kinase growth factor receptors. The inhibitory effects of BAG-1 on Hsp/Hsc70 chaperone activity suggest that BAG-1 represents a novel type of chaperone regulatory proteins and thus suggest a link between cell signaling, cell death and the stress response.     

Can we use erythrocytes for the study of the activity of the ubiquitous Na+/H+ exchanger (NHE-1) in essential hypertension?

Both Na+/Li+ countertransport and electrochemical proton gradient (delta mu(H+))-induced Na+ and H+ fluxes are increased in erythrocytes from patients with essential hypertension. It was assumed that these abnormalities are related to ubiquitous (housekeeping) forms of the Na+/H+ exchanger (NHE-1). To examine this hypothesis, we compared kinetic and regulatory properties of erythrocyte Na+/Li+ countertransport and delta mu(H+)-induced Na+ and H+ fluxes with data obtained for cloned isoforms of the Na+/H+ exchanger. In human erythrocytes, Na+/Li+ countertransport exhibited a hyperbolic dependence on [Na+]0 with a K0.5 of approximately 30 to 40 mmol/L. The activity of this carrier was increased by two-fold in the fraction of erythrocytes enriched with the old cells, was inhibited by 0.1 mmol/L phloretin, and was insensitive to both 1 mmol/L amiloride and ATP depletion. In contrast, delta mu(H+)-induced 22Na influx was exponentially increased at [Na+]0 > 60 mmol/L, was insensitive to phloretin, was partly decreased by both 1 mmol/L amiloride and ATP depletion, and was the same in total erythrocytes and in the old cells. The values of Na+/Li+ countertransport and delta mu(H+)-induced Na+ influx in erythrocytes from different species were not correlating and their ratio in human, rat, and rabbit erythrocytes was 10:1:170 and 1:5:1 for Na+/ Li+ countertransport and delta mu(H+)-induced Na+ influx, respectively. In contrast to the majority of nonepithelial cells and cells transfected with an ubiquitous isoform of Na+/H+ exchanger, both delta mu(H+)-induced Na+ influx and Na+/Li+ countertransport in human erythrocytes were completely insensitive to ethylisopropyl amiloride (20 micromol/L) and cell shrinkage. Thus, our data strongly suggest that human erythrocyte Na+/Li+ countertransport and delta mu(H+)-induced Na+/H+ exchange are mediated by the distinct transporters. Moreover, because the properties of these erythrocyte transporters and NHE-1 are different, it complicates the use of erythrocytes for the identification of the mechanism for activating the ubiquitous form of Na+/H+ exchanger in primary hypertension.     

Kinetics of Na(+)-dependent conformational changes of rabbit kidney Na+,K(+)-ATPase

The kinetics of Na(+)-dependent partial reactions of the Na+,K(+)-ATPase from rabbit kidney were investigated via the stopped-flow technique, using the fluorescent labels N-(4-sulfobutyl)-4-(4-(p-(dipentylamino)phenyl)butadienyl)py ridinium inner salt (RH421) and 5-iodoacetamidofluorescein (5-IAF). When covalently labeled 5-IAF enzyme is mixed with ATP, the two labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau 1 approximately 155 s-1 for 5-IAF-labeled enzyme and 1/tau 1 approximately 200 s-1 for native enzyme (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3-->E2P(Na+)3 + ADP). The smaller amplitude slow phase, 1/tau 2 = 30-45 s-1, is attributed to the relaxation of the dephosphorylation/rephosphorylation equilibrium in the absence of K+ ions (E2P<==>E2). The Na+ concentration dependence of 1/tau 1 showed half-saturation at a Na+ concentration of 6-8 mM, with positive cooperatively involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high-affinity ATP-binding site determined from the ATP concentration dependence of 1/tau 1 was 8.0 (+/- 0.7) microM. It was found that P3-1-(2-nitrophenyl)ethyl ATP, tripropylammonium salt (NPE-caged ATP), at concentrations in the hundreds of micromolar range, significantly decreases the value of 1/tau 1, observed. This, as well as the biexponential nature of the kinetic traces, can account for previously reported discrepancies in the rates of the reactions investigated.     

The hydroxyl of threonine 13 of the bovine 70-kDa heat shock cognate protein is essential for transducing the ATP-induced conformational change

The mechanism by which ATP binding transduces a conformational change in 70-kDa heat shock proteins that results in release of bound peptides remains obscure. Wei and Hendershot demonstrated that mutating Thr37 of hamster BiP to glycine impeded the ATP-induced conformational change, as monitored by proteolysis [(1995) J. Biol. Chem. 270, 26670-26676]. We have mutated the equivalent resitude of the bovine heat shock cognate protein (Hsc70), Thr13, to serine, valine, and glycine. Solution small-angle X-ray scattering experiments on a 60-kDa fragment of Hsc70 show that ATP binding induces a conformational change in the T13S mutant but not the T13V or T13G mutants. The kinetics of ATP-induced tryptophan fluorescence intensity changes in the 60-kDa proteins is biphasic for the T13S mutant but monophasic for T13V or T13G, consistent with a conformational change following initial ATP binding in the T13S mutant but not the other two. Crystallographic structures of the ATPase fragments of the T13S and T13G mutants at 1.7 A resolution show that the mutations do not disrupt the ATP binding site and that the serine hydroxyl mimics the threonine hydroxyl in the wild-type structure. We conclude that the hydroxyl of Thr13 is essential for coupling ATP binding to a conformational change in Hsc70. Molecular modeling suggests this may result from the threonine hydroxyl hydrogen-bonding to a gamma-phosphate oxygen of ATP, thereby inducing a structural shift within the ATPase domain that couples to its interactions with the peptide binding domain.