State-independent inhibition of the oncogenic Kv10.1 channel by desethylamiodarone, a comparison with amiodarone.

Kv10.1 is a voltage-dependent K channel whose ectopic expression is associated with several human cancers. Additionally, Kv10.1 has structure-function properties which are not yet well understood. We are using drugs of clinical importance in an attempt to gain insight on the relationship between pharmacology and characteristic functional properties of this channel. Herein, we report the interaction of desethylamiodarone (desAd), the active metabolic product of the antiarrhythmic amiodarone with Kv10.1: desAd binds to both closed and open channels, with most inhibition taking place from the open state, with affinity ~ 5 times smaller than that of amiodarone. Current inhibition by desAd and amiodarone is not synergistic. Upon repolarization desAd becomes trapped in Kv10.1 and thereafter dissociates slowly from closed-and-blocked channels. The addition of the Cole-Moore shift plus desAd open-pore-block time courses yields an increasing phase on the steady-state inhibition curve (H∞) at hyperpolarized holding potentials. In contrast to amiodarone, desAd does not inhibit the Kv10.1 Cole-Moore shift, suggesting that a relevant hydrophobic interaction between amiodarone and Kv10.1 participates in the inhibition of the Cole-Moore shift, which is lost with desAd.

Development of new hCaM-Alexa Fluor® biosensors for a wide range of ligands.

Eight new fluorescent biosensors of human calmodulin (hCaM) using Alexa Fluor® 350, 488, 532, and 555 dyes were constructed. These biosensors are thermodynamically stable, functional, and highly sensitive to ligands of the CaM. They resolve the problem of CaM ligands with similar spectroscopic properties to the intrinsic and extrinsic fluorophores of other biosensors previously reported. Additionally, they can be used in studies of protein-protein interaction through Förster resonance energy transfer (FRET). The variation in Tm (range 78.07-81.47 °C; 79.05 to WT) is no larger than two degrees in all cases in regards to CaM WT. The Kds calculated with all biosensors for CPZ and BIMI (a new inhibitor of CaM) are in the range of 0.45-1.86 and 0.69-1.54 µm respectively. All biosensors retain their ability to activate Calcineurin about 70%. Structural models built "in silico" show their possible conformation taking the fluorophores in protein thus we can predict system stability. Finally, these new biosensors represent a biotechnological development applied to an analytical problem, which aims to determine accurately the affinity of inhibitors of CaM without possible interference, to be put forward as possible drugs related to CaM.

New complexes containing the internal alternative NADH dehydrogenase (Ndi1) in mitochondria of Saccharomyces cerevisiae.

Mitochondria of Saccharomyces cerevisiae lack the respiratory complex I, but contain three rotenone-insensitive NADH dehydrogenases distributed on both the external (Nde1 and Nde2) and internal (Ndi1) surfaces of the inner mitochondrial membrane. These enzymes catalyse the transfer of electrons from NADH to ubiquinone without the translocation of protons across the membrane. Due to the high resolution of the Blue Native PAGE (BN-PAGE) technique combined with digitonin solubilization, several bands with NADH dehydrogenase activity were observed on the gel. The use of specific S. cerevisiae single and double mutants of the external alternative elements (ΔNDE1, ΔNDE2, ΔNDE1/ΔNDE2) showed that the high and low molecular weight complexes contained the Ndi1. Some of the Ndi1 associations took place with complexes III and IV, suggesting the formation of respirasome-like structures. Complex II interacted with other proteins to form a high molecular weight supercomplex with a molecular mass around 600 kDa. We also found that the majority of the Ndi1 was in a dimeric form, which is in agreement with the recently reported three-dimensional structure of the protein.

Captopril avoids hypertension, the increase in plasma angiotensin II but increases angiotensin 1-7 and angiotensin II-induced perfusion pressure in isolated kidney in SHR.

We investigated captopril effects, an ACE inhibitor, on hypertension development, on Ang II and Ang-(1-7) plasma concentrations, on Ang II-induced contraction in isolated kidneys, and on kidney AT1R from spontaneously hypertensive (SHR) rats. Five weeks-old SHR and Wistar Kyoto (WKY) rats were treated with captopril at 30 mg/kg/day, in drinking water for 2 or 14 weeks. Systolic blood pressure (SBP) was measured, and isolated kidneys were tested for perfusion pressure and AT1R expression; while Ang II and Ang-(1-7) concentrations were determined in plasma. Captopril did not modify SBP in WKY rats and avoided its increase as SHR aged. Plasma Ang-II concentration was ∼4-5 folds higher in SHR rats, and captopril reduced it (P<0.05); while captopril increased Ang-(1-7) by ∼2 fold in all rat groups. Captopril increased Ang II-induced pressor response in kidneys of WKY and SHR rats, phenomenon not observed in kidneys stimulated with phenylephrine, a α1-adrenoceptor agonist. Captopril did not modify AT1R in kidney cortex and medulla among rat strains and ages. Data indicate that captopril increased Ang II-induced kidney perfusion pressure but not AT1R density in kidney of WKY and SHR rats, due to blockade of angiotensin II synthesis; however, ACE inhibitors may have other actions like activating signaling processes that could contribute to their diverse effects.

α(1D)-Adrenoceptor regulates the vasopressor action of α(1A)-adrenoceptor in mesenteric vascular bed of α(1D)-adrenoceptor knockout mice.

1 The pressor action of the α(1A)-adrenoceptor (α(1A)-AR) agonist A61603 (N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl] methanesulfonamide) and the α(1)-ARs agonist phenylephrine and their blockade by selective α(1)-ARs antagonists in the isolated mesenteric vascular bed of wild-type (WT) mice and α(1D)-AR knockout (KO α(1D)-AR) mice were evaluated. 2 The apparent potency of A61603 to increase the perfusion pressure in the mesenteric vascular bed of WT and KO α(1D)-AR mice is 86 and 138 times the affinity of phenylephrine, respectively. 3 A61603 also enhanced the perfusion pressure by ≈1.7 fold in the mesenteric vascular bed of WT mice compared with KO α(1D)-AR mice. 4 Because of its high affinity, low concentrations of the α(1A)-AR selective antagonist RS100329 (5-methyl-3-[3-[4-[2-(2,2,2,-trifluoroethoxy) phenyl]-1-piperazinyl] propyl]-2,4-(1H)-pyrimidinedione) shifted the agonist concentration-response curves to the right in the mesenteric vascular bed of WT and KO α(1D)-AR mice. 5 The α(1D)-AR selective antagonist BMY7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5] decane-7,9-dione) did not modify the A61603 or the phenylephrine-induced pressor effect. 6 The α(1B/D)-ARs alkylating antagonist chloroethylclonidine (CEC) shifted the agonist concentration-response curves to the right and decreased the maximum phenylephrine-induced vascular contraction in KO α(1D)-AR mice when compared to WT mice; however, CEC only slightly modified the contraction induced by A61603. 7 The results indicate that the isolated mesenteric vascular bed of WT and KO α(1D)-AR mice expresses α(1A)-AR, that the pressor action of α(1A)-AR is up-regulated for α(1D)-AR in WT mice and suggest an important role of α(1B)-AR in the vascular pressure evoked by phenylephrine in KO α(1D)-AR mice.

Functional properties of the Ustilago maydis alternative oxidase under oxidative stress conditions.

The mitochondrial respiratory chain of Ustilago maydis contains two terminal oxidases, the cytochrome c oxidase (COX) and the alternative oxidase (AOX). To understand the biochemical events that control AOX activity, we studied the regulation and function of AOX under oxidative stress. The activity of this enzyme was increased by both pyruvate (K(05)=2.6 mM) and purine nucleotides (AMP, K(05)=600 microM) in mitochondria using succinate as respiratory substrate. When U.maydis cells were grown in the presence of antimycin A, the amount of AOX in mitochondria was markedly increased and its selectivity towards AMP and pyruvate changed, suggesting that post-translational events may play a role in the regulation of AOX activity under stress conditions. Addition of antimycin A to isolated mitochondria induced the inactivation of AOX, the formation of lipid peroxides and the loss of glutathione from mitochondria. The two last processes are probably related with the time dependent inactivation of AOX, in agreement with the inhibition of the enzyme by tert-butyl hydroperoxide. Our results suggest that the in vivo operation of AOX in U. maydis depends on the mitochondrial antioxidant machinery, including the glutathione linked systems.

Induction of morphological changes in Ustilago maydis cells by octyl gallate.

The effects of octyl gallate on Ustilago maydis yeast cells were analysed in relation to its capacity to oxidize compounds (pro-oxidant actions). All phenolic compounds tested inhibited the alternative oxidase (AOX). However, only octyl gallate induced a morphological change in yeast cells and collapsed the mitochondrial membrane potential. In contrast to octyl gallate, propyl gallate and nordihydroguaiaretic acid caused only a negligible cell change and the membrane potential was not affected. Our findings show that structurally related phenolic compounds do not necessarily exert similar actions on target cells. Preincubation of U. maydis cells with trolox inhibited the change to pseudohyphal growth produced by octyl gallate. These results suggest that in addition to the inhibitory action of octyl gallate on the AOX, this compound induces a switch from yeast to a mycelium, probably through the formation of lipid peroxides.

Inhibition of cAMP-dependent protein kinase A: a novel cyclo-oxygenase-independent effect of non-steroidal anti-inflammatory drugs in adipocytes.

1. Non-steroidal anti-inflammatory drugs (NSAIDs) [acetylsalicylic acid (ASS), naproxen, nimesulide and piroxicam] decreased adrenaline- or dibutyryl cAMP-stimulated glycerol release in isolated adipocytes. We aimed to determine the mechanism of this NSAIDs action. 2. Non-steroidal anti-inflammatory drugs decreased cAMP-dependent protein kinase A (PKA) activity in rat adipocyte lysates and in a commercial bovine heart PKA holoenzyme. If added before cAMP, NSAIDs impaired PKA activation by the cyclic nucleotide; however, if PKA was first activated by cAMP, NSAIDs were ineffective. NSAIDs were also ineffective against PKA catalytic subunits. 3. Consequently, NSAIDs lowered hormone-sensitive lipase translocation from cytosol to lipid storage droplets in adipocytes lysates, the critical event to promote lipolysis. 4. These results indicate that inhibition of PKA activation explains NSAIDs-induced decrease in adrenaline-stimulated lipolysis. We suggest that reproduction of such inhibition in nociceptive cells might enhance the understanding of the mechanism underlying the analgesic effects of NSAIDs.

Rapid purification and biochemical characterization of glucose kinase from Streptomyces peucetius var. caesius.

Glucose kinase catalyzes the ATP-dependent phosphorylation of glucose. Streptomyces peucetius var. caesius glucose kinase was purified 292-fold to homogeneity. The enzyme has cytosolic localization and is composed of four identical subunits, each of 31 kDa. The purified enzyme easily dissociates into dimers. However, in the presence of 100 mM glucose the enzyme maintains its tetrameric form. Maximum activity was found at 42 degrees C and pH 7.5. Isoelectric focusing of the enzyme showed a pl of 8.4. The N- and C-terminal amino acid sequences were MGLTIGVD and VYFAREPDPIM, respectively. The kinetic mechanism of S. peucetius var. caesius glucose kinase appears to be a rapid equilibrium ordered type, i.e., ordered addition of substrates to the enzyme, where the first substrate is d-glucose. The K(m) values for d-glucose and MgATP(2-) were 1.6 +/- 0.2 and 0.8 +/- 0.1 mM, respectively. Mg(2+) in excess of 10 mM inhibits enzyme activity.

Kinetic characterization of the rotenone-insensitive internal NADH: ubiquinone oxidoreductase of mitochondria from Saccharomyces cerevisiae.

Saccharomyces cerevisiae mitochondria contain an NADH:Q6 oxidoreductase (internal NADH dehydrogenase) encoded by NDI1 gene in chromosome XIII. This enzyme catalyzes the transfer of electrons from NADH to ubiquinone without the translocation of protons across the membrane. From a structural point of view, the mature enzyme has a single subunit of 53 kDa with FAD as the only prosthetic group. Due to the fact that S. cerevisiae cells lack complex I, the expression of this protein is essential for cell growth under respiratory conditions. The results reported in this work show that the internal NADH dehydrogenase follows a ping-pong mechanism, with a Km for NADH of 9.4 microM and a Km for oxidized 2,6-dichorophenolindophenol (DCPIP) of 6.2 microM. NAD+, one of the products of the reaction, did not inhibit the enzyme while the other product, reduced DCPIP, inhibited the enzyme with a Ki of 11.5 microM. Two dead-end inhibitors, AMP and flavone, were used to further characterize the kinetic mechanism of the enzyme. AMP was a linear competitive inhibitor of NADH (Ki = 5.5 mM) and a linear uncompetitive inhibitor of oxidized DCPIP (Ki = 11.5 mM), in agreement with the ping-pong mechanism. On the other hand, flavone was a partial inhibitor displaying a hyperbolic uncompetitive inhibition regarding NADH, and a hyperbolic noncompetitive inhibition with respect to oxidized DCPIP. The apparent intercept inhibition constant (Kii = 5.4 microM) and the slope inhibition constant (Kis = 7.1 microM) were obtained by non linear regression analysis. The results indicate that the ternary complex F-DCPIPox-flavone catalyzes the reduction of DCPIP, although with lower efficiency. The effect of pH on Vmax was studied. The Vmax profile shows two groups with pKa values of 5.3 and 7.2 involved in the catalytic process.

Stalk segment 5 of the yeast plasma membrane H+-ATPase: mutational evidence for a role in glucose regulation.

In P(2)-type ATPases, a stalk region connects the cytoplasmic part of the molecule, which binds and hydrolyzes ATP, to the membrane-embedded part through which cations are pumped. The present study has used cysteine scanning mutagenesis to examine structure-function relationships within stalk segment 5 (S5) of the yeast plasma-membrane H(+)-ATPase. Of 29 Cys mutants that were made and examined, two (G670C and R682C) were blocked in biogenesis, presumably due to protein misfolding. In addition, one mutant (S681C) had very low ATPase activity, and another (F685C) displayed a 40-fold decrease in sensitivity to orthovanadate, reflecting a shift in equilibrium from the E(2) conformational state toward E(1). By far the most striking group of mutants (F666C, L671C, I674C, A677C, I684C, R687C, and Y689C) were constitutively activated even in the absence of glucose, with rates of ATP hydrolysis and kinetic properties normally seen only in glucose-metabolizing cells. Previous work has suggested that activation of the wild-type H(+)-ATPase results from kinase-mediated phosphorylation in the auto-inhibitory C-terminal region of the 100-kDa polypeptide. The seven residues identified in the present study are located on one face of the S5 alpha-helix, consistent with the idea that mutations along this face serve to release the auto-inhibition.

Modulation of 2-oxoglutarate dehydrogenase complex by inorganic phosphate, Mg(2+), and other effectors.

The interplay of inorganic phosphate (Pi) with other ligands such as Mg(2+), ADP, ATP, and Ca(2+) on the activation of 2-oxoglutarate dehydrogenase complex (2-OGDH) in both isolated enzyme complex and mitochondrial extracts was examined. Pi alone activated the enzyme, following biphasic kinetics with high (K(0.5) = 1.96+/-0.42 mM) and low (K(0.5) = 9.8+/-0.4 mM) affinity components for Pi. The activation by Pi was highly pH-dependent; it increased when the pH raised from 7.1 to 7.6, but it was negligible at pH values below 7.1. Mg-Pi and Mg-ADP, but not Mg-ATP, were more potent activators of 2-OGDH than free Pi and free ADP. ATP inhibited the 2-OGDH activity by chelating the free Mg(2+) and also as a Mg-ATP complex. With or without Mg(2+), ADP, and Pi activated the 2-OGDH by increasing the affinity for 2-OG and the V(m) of the reaction; ATP diminished the V(m), but it increased the affinity for 2-OG in the mitochondrial extract. Pi did not modify the 2-OGDH activation by Ca(2+). The results above mentioned were similar for both preparations, except for hyperbolic kinetics in the isolated enzyme and sigmoidal kinetics in the mitochondrial extracts when 2-oxoglutarate was varied. The data of this study indicated that physiological concentrations of Pi may exert a significant activation of 2-OGDH, which was potentiated by Mg(2+) and high pH, but surpassed by ADP.

Differential response to chloroethylclonidine in blood vessels of normotensive and spontaneously hypertensive rats: role of alpha 1D- and alpha 1A-adrenoceptors in contraction.

The effects of chloroethylclonidine on alpha(1)-adrenoceptor-mediated contraction in endothelium-denuded caudal arteries and aorta from normotensive Wistar and Wistar Kyoto (WKY), and from spontaneously hypertensive (SHR) rats were evaluated. Chloroethylclonidine elicited concentration-dependent contractions. Maximal contraction was similar in caudal arteries among strains ( approximately 40% of noradrenaline effect). However, chloroethylclonidine elicited a higher contraction in aorta from SHR than from normotensive rats. In Wistar aorta chloroethylclonidine produced the smallest contractile response. In SHR aorta, BMY 7378 and 5-methylurapidil blocked chloroethylclonidine-elicited contraction, while (+)-cyclazocine did not inhibit it; while in caudal arteries, 5-methylurapidil blocked chloroethylclonidine action; the other antagonists had no effect. In chloroethylclonidine-treated aorta noradrenaline elicited biphasic contraction-response curves, indicating a high affinity (pD(2), 8.5 - 7.5) chloroethylclonidine-sensitive component and a low affinity (pD(2), 6.3 - 5.2) chloroethylclonidine-insensitive component. The high affinity component was blocked by chloroethylclonidine; while in caudal arteries noradrenaline elicited monophasic contraction-response curves with pD(2) values (6.5 - 5.7) similar to the low affinity component in aorta. Chloroethylclonidine inhibition of noradrenaline response was greater in aorta than in caudal arteries. Chloroethylclonidine increased the EC(50) values of noradrenaline approximately 1000 fold in aorta and approximately 10 fold in caudal arteries. In SHR aorta BMY 7378 protected alpha(1D)-adrenoceptors and in caudal arteries 5-methylurapidil protected alpha(1A)-adrenoceptors from chloroethylclonidine alkylation, allowing noradrenaline to elicit contraction. These results show marked strain-dependent differences in the ability of chloroethylclonidine to contract aorta; moreover, chloroethylclonidine stimulates alpha(1D)-adrenoceptors in aorta and alpha(1A)-adrenoceptors in caudal arteries. The higher contraction observed in aorta from SHR and WKY suggests an augmented number of alpha(1D)-adrenoceptors in these strains.

An alternative model for the transmembrane segments of the yeast H+-ATPase.

An alternative topological model for the yeast plasma membrane H(+)-ATPase from K. lactis was deduced by joint prediction, using 11 algorithms for the prediction of transmembrane segments complemented with hydrophobic moment analysis. Similarly to the model currently used in the literature, this alternative model contains 10 transmembrane segments, four in the N-half and six in the C-half of the protein. However, the distribution of the membrane-associated segments on the C-half of the enzyme differs in both models. Nine of the 10 transmembrane segments are highly hydrophobic with low hydrophobic moments, and are probably involved in structural roles. The fifth transmembrane segment is, on the other hand, less hydrophobic, with the highest hydrophobic moment, suggesting that this segment might have a dynamic role in the coupling of the hydrolysis of ATP with the translocation of protons across the membrane. The alignment of the Ca(2+)-ATPase, the Na(+)/K(+)-ATPase and the H(+)-ATPase sequences showed that these proteins have the same topology in the N-half, but important differences were found at the C-half of the enzymes. In contrast with the mammalian ATPases, the fifth transmembrane segment in the H(+)-ATPase appears early in the sequence, giving rise to a shorter cytoplasmic central loop. This alternative model will be useful in the designing of site-directed mutagenesis experiments and contains information for the fitting of the amino acid sequence into the transmembrane region of the three-dimensional model of the ATPase.

A novel ATP-diphosphohydrolase from human term placental mitochondria.

This report describes an ATP-diphosphohydrolase activity associated with the inner membrane of human term placental mitochondria. An enriched fraction containing 30 per cent of the total protein and 80 per cent of the total ATP-diphosphohydrolase activity was obtained from submitochondrial particles. ATP-diphosphohydrolase activity was characterized in this fraction. The enzyme had a pH optimum of 8 and catalysed the hydrolysis of triphospho- and diphosphonucleosides other than ATP or ADP. Pyrophosphate was also hydrolysed, but AMP or other monoester phosphates were not. The activity of ATP-diphosphohydrolase was dependent on Mg(2 + ), Ca(2 + )or Mn(2 + )and the enzyme substrate was the cation-nucleotide complex. An excess of free cation produced inhibition.ATP-diphosphohydrolase activity was stimulated at micromolar concentrations of calcium or magnesium in the presence of La-PPi. Negative cooperativity kinetics was observed with all substrates tested. The V(max)ranged from 150 to 300nmol of Pi released/mg/min. The [S](0.5)for nucleotides was 1-10m m and 182m m for PPi. The enzyme was inhibited by orthovanadate, but not by l -phenylalanine, oligomycin, sodium azide, P(1),P(5)-di(adenosine-5')pentaphosphate or sodium fluoride.The experimental evidence showing absence of inhibition by sodium azide and sodium fluoride, hydrolysis of pyrophosphate but not of monoester phosphates, and negative cooperativity suggested that this enzyme was a novel ATP-diphosphohydrolase.

Trehalose-mediated protection of the plasma membrane H+-ATPase from Kluyveromyces lactis during freeze-drying and rehydration.

During freeze-drying and rehydration, the activity of the H+-ATPase from the plasma membrane of Kluyveromyces lactis was preserved by increasing concentrations of carbohydrates. When the H+-ATPase was freeze-dried in the absence of carbohydrates the activity was lost. The protective efficiency of carbohydrates was as follows: trehalose > maltose > sucrose > glucose > galactose. Each carbohydrate exhibited the maximal protection at a concentration of 20 mg carbohydrate per milligram of protein or above. No structural changes of the rehydrated H+-ATPase were detected by intrinsic fluorescence measurements. Trehalose, at 20 mg/mg protein, protected the enzyme activity completely during freeze-drying and rehydration. Rehydration temperature was critical; at 20 degrees C or below, activity was fully retained, while at 30, 40, or 50 degrees C activity decreased in proportion with temperature. The trehalose-protected freeze-dried H+-ATPase was stored at different temperatures for up to 60 days. Storage at 4 degrees C resulted in retention of most of the enzymatic activity, while storage at 20 or 30 degrees C resulted in loss of activity. The protection of the H+-ATPase by trehalose suggests that this carbohydrate might protect other membrane enzymes from inactivation during handling.

Inactivation of the Kluyveromyces lactis H+-ATPase by dicyclohexylcarbodiimide: binding stoichiometry and effect of nucleophiles.

Dicyclohexylcarbodiimide (DCCD) inactivated the plasma membrane H+-ATPase (EC 3.6.1.35) from Kluyveromyces lactis, with a second-order rate constant of 420 M(-1) min(-1). The inhibition kinetics was apparently complex, due to degradation of DCCD with time. Neither Mg2+ nor Mg-ADP affected the inactivation of the ATPase by DCCD. In contrast, vanadate, a transition state analog of phosphate, partially protected the enzyme with a Kd of 14 microM, indicating a coupling between the DCCD-reactive site and the vanadate-binding site. The incubation of H+-ATPase with 14C-DCCD showed that the incorporation of 1.2 mol of DCCD/mol ATPase leads to complete inactivation. The hydrophobic carbodiimide reacted with the protonated form of the carboxylic group, which displayed a pKa of 7.4, strongly suggesting that the residue is in the hydrophobic environment of the membrane. Benzylamine increased the rate of inactivation by DCCD. In this case, full inactivation of the enzyme was associated with the incorporation of 2.4 mol of DCCD/mol of enzyme, indicating the opening of new reactive sites, resulting from a conformational change induced by benzylamine.

Reactive cysteines of the yeast plasma-membrane H+-ATPase (PMA1). Mapping the sites of inactivation by N-ethylmaleimide.

We have taken advantage of cysteine mutants described previously (Petrov, V. V., and Slayman, C. W. (1995) J. Biol. Chem. 270, 28535-28540) to map the sites at which N-ethylmaleimide (NEM) reacts with the plasma-membrane H+ATPase (PMA)1 of Saccharomyces cerevisiae. When membrane vesicles containing the ATPase were incubated with NEM, six of nine mutants with single cysteine substitutions showed sensitivity similar to the wild-type enzyme. By contrast, C221A and C532A were inactivated more slowly than the wild-type control, and the C221, 532A double mutant was completely resistant, indicating that Cys-221 and Cys-532 are NEM-reactive residues. In the presence of 10 mM MgADP, the wild-type ATPase was partially protected against NEM; parallel experiments with the C221A and C532A mutants showed that the protection occurred at Cys-532, located in or near the nucleotide-binding site. Unexpectedly, the inactivation of the C409A ATPase was approximately 4-fold more rapid than in the case of the wild-type enzyme. Experiments with double mutants made it clear that this resulted from an acidic shift in pKa and a consequent acceleration of the reaction rate at Cys-532. One simple interpretation is that substitution of Cys-409 leads to a local conformational change within the central hydrophilic domain. Consistent with this idea, the reaction of fluorescein 5'-isothiocyanate at Lys-474 was also stimulated approximately 3. 5-fold by the C409A mutation. Taken together, the results of this study provide new information about the reactivity of individual Cys residues within the ATPase and pave the way to tag specific sites for structural and functional studies of the enzyme.