Stabilization of Na,K-ATPase by ionic interactions

The effect of ions on the thermostability and unfolding of Na,K-ATPase from shark salt gland was studied and compared with that of Na,K-ATPase from pig kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark enzyme inactivates rapidly at 20 degrees C, as does the kidney enzyme at 42 degrees C (but not at 20 degrees C). Increasing ionic strength by addition of 20 mM histidine, or of 1 mM NaCl or KCl, protects both enzymes against this rapid inactivation. As detected by DSC, the shark enzyme undergoes thermal unfolding at lower temperature (Tm approximately 45 degrees C) than does the kidney enzyme (Tm approximately 55 degrees C). Both calorimetric endotherms indicate multi-step unfolding, probably associated with different cooperative domains. Whereas the overall heat of unfolding is similar for the kidney enzyme in either 1 mM or 20 mM histidine, components with high mid-point temperatures are lost from the unfolding transition of the shark enzyme in 1 mM histidine, relative to that in 20 mM histidine. This is attributed to partial unfolding of the enzyme due to a high hydrostatic pressure during centrifugation of DSC samples at low ionic strength, which correlates with inactivation measurements. Addition of 10 mM NaCl to shark enzyme in 1 mM histidine protects against inactivation during centrifugation of the DSC sample, but incubation for 1 h at 20 degrees C prior to addition of NaCl results in loss of components with lower mid-point temperatures within the unfolding transition. Cations at millimolar concentration therefore afford at least two distinct modes of stabilization, likely affecting separate cooperative domains. The different thermal stabilities and denaturation temperatures of the two Na,K-ATPases correlate with the respective physiological temperatures, and may be attributed to the different lipid environments.     

Bulk properties of the lipid bilayer are not essential for the thermal stability of Na,K-ATPase from shark rectal gland or pig kidney

The thermal stability of Na,K-ATPase from pig kidney is markedly greater than that of Na,K-ATPase from shark salt glands. The role of the lipid bilayer is studied by solubilisation of the membrane-bound enzyme in the nonionic detergent octaethyleneglycoldodecylmonoether (C₁₂E₈), addition of excess dioleylphosphatidylcholine (DOPC) or palmitoyloleylphosphatidylcholine (POPC) and reconstitution of membranes by removal of detergent. At 54 °C the reconstituted enzymatically active pig enzyme retains a high thermal stability, and reconstituted shark enzyme retains a low thermal stability, even with a 9-fold excess of DOPC. This result suggests that the origin of the difference in thermal stability is not related to bulk lipid properties of the native membranes.     

Correlation of structural and functional thermal stability of the integral membrane protein Na,K-ATPase

The membrane-bound cation-transporting P-type Na,K-ATPase isolated from pig kidney membranes is much more resistant towards thermal inactivation than the almost identical membrane-bound Na,K-ATPase isolated from shark rectal gland membranes. The loss of enzymatic activity is correlated well with changes in protein structure as determined using synchrotron radiation circular dichroism (SRCD) spectroscopy. The enzymatic activity is lost at a 12°C higher temperature for pig enzyme than for shark enzyme, and the major changes in protein secondary structure also occur at T(m)'s that are ~10-15°C higher for the pig than for the shark enzyme. The temperature optimum for the rate of hydrolysis of ATP is about 42°C for shark and about 57°C for pig, both of which are close to the temperatures for onset of thermal unfolding. These results suggest that the active site region may be amongst the earliest parts of the structure to unfold. Detergent-solubilized Na,K-ATPases from the two sources show the similar differences in thermal stability as the membrane-bound species, but inactivation occurs at a lower temperature for both, and may reflect the stabilizing effect of a bilayer versus a micellar environment.     

Conformational transitions of detergent-solubilized Na,K-ATPase are conveniently monitored by the fluorescent probe 6-carboxy-eosin.

6-carboxy-eosin is introduced as a sensitive, non-covalently bound fluorescent probe for monitoring conformational changes in detergent-solubilized Na,K-ATPase. The dissociation constant for 6-carboxy-eosin is about 0.1 microM in 20 mM NaCl at 6 degrees C (pH 7.0) for Na,K-ATPase solubilized in C12E8. It is shown that the slow conformational change from E2 (in K+) to E1 (in Na+) is 4-fold more rapid in the solubilized state than in the membrane-bound state, both for shark rectal gland and pig kidney Na,K-ATPase. The rate of the E1 to E2 transition is rapid and of the same order of magnitude both for the membrane-bound and the solubilized enzyme. All conformational transitions are considerably slower for pig kidney enzyme than for shark enzyme, both in the membrane-bound and in the solubilized state.     

Na,K-ATPase structure/function relationships probed by the denaturant urea

Urea interacts with the Na,K-ATPase, leading to reversible as well as irreversible inhibition of the hydrolytic activity. The enzyme purified from shark rectal glands is more sensitive to urea than Na,K-ATPase purified from pig kidney. An immediate and reversible inhibition under steady-state conditions of hydrolytic activity at 37 °C is demonstrated for the three reactions studied: the overall Na,K-ATPase activity, the Na-ATPase activity observed in the absence of K⁺ as well as the K⁺-dependent phosphatase reaction (K-pNPPase) seen in the absence of Na⁺. Half-maximal inhibition is seen with about 1 M urea for shark enzyme and about 2 M urea for pig enzyme. In the presence of substrates there is also an irreversible inhibition in addition to the reversible process, and we show that ATP protects against the irreversible inhibition for both the Na,K-ATPase and Na-ATPase reaction, whereas the substrate paranitrophenylphosphate leads to a slight increase in the rate of irreversible inhibition of the K-pNPPase. The rate of the irreversible inactivation in the absence of substrates is much more rapid for shark enzyme than for pig enzyme. The larger number of potentially urea-sensitive hydrogen bonds in shark enzyme compared to pig enzyme suggests that interference with the extensive hydrogen bonding network might account for the higher urea sensitivity of shark enzyme. The reversible inactivation is interpreted in terms of domain interactions and domain accessibilities using as templates the available crystal structures of Na,K-ATPase. It is suggested that a few interdomain hydrogen bonds are those mainly affected by urea during reversible inactivation.     

Myoglobin with chlorophyllous chromophores: Influence on protein stability

The stabilities of myoglobin, apo-myoglobin, and of two myoglobins with chlorophyllous chromophores (Zn-pheophorbide a and Zn-bacteriopheophorbide a), have been studied by thermal and chemical denaturation. With guanidinium chloride, the stability order is myoglobin > Zn-pheophorbide-myoglobin > Zn-bacteriopheophorbide-myoglobin ∼ apo-myoglobin. The thermal behavior is more complex. The transition temperature of thermal unfolding of the apoprotein (62.4 °C) is increased by Zn-pheophorbide a (83.9 °C) and Zn-bacteriopheophorbide a (82.6 °C) to a similar degree as by the native chromophore, heme (83.5 °C). The recovery with Zn-pheophorbide (92-98%) is even higher than with heme (74-76%), while with Zn-bacteriopheophorbide (40%) it is as low as with the apoprotein (42%). Recovery also depends on the rates of heating, and in particular the time spent at high temperatures. It is concluded that irreversibility of unfolding is related to loss of the chromophores, which are required for proper re-folding.     

Na/K-ATPase assay in the intact guinea pig liver submitted to in situ perfusion

We describe an assay for the enzyme Na/K-ATPase in intact guinea pig livers perfused through the portal vein with modified Hank’s solution. The model uses the measurement of non-radioactive rubidium ion incorporation by liver cells, both in the absence and in the presence of the specific Na/K-ATPase inhibitor ouabain, followed by a rinsing procedure with cold saline. The concentration of Rb⁺ in acid-digested liver lobes was measured by atomic emission spectrometry and Na/K pump activity was calculated by the difference between the incorporation of Rb⁺ in the absence and in the presence of ouabain. The optimal conditions for Rb⁺ incorporation were: perfusion flow rate, 3 ml/min per liver; perfusion time at 37 °C, 60 min; rinsing time with cold saline, 5-10 min; and concentration of ouabain, 3 mM. The calculated ouabain IC₅₀ was 100 μM. The major advantage of this model is the possibility of testing experimental drugs affecting this enzyme in conditions close to those in the intact organ.     

Functional characterization and oligomerization of a recombinant xyloglucan-specific endo-β-1,4-glucanase (GH12) from Aspergillus niveus

Xyloglucan is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists of a cellulosic backbone (beta-1,4-linked glucosyl residues) that is frequently substituted with side chains. This report describes Aspergillus nidulans strain A773 recombinant secretion of a dimeric xyloglucan-specific endo-β-1,4-glucanohydrolase (XegA) cloned from Aspergillus niveus. The ORF of the A. niveus xegA gene is comprised of 714 nucleotides, and encodes a 238 amino acid protein with a calculated molecular weight of 23.5 kDa and isoelectric point of 4.38. The optimal pH and temperature were 6.0 and 60 °C, respectively. XegA generated a xyloglucan-oligosaccharides (XGOs) pattern similar to that observed for cellulases from family GH12, i.e., demonstrating that its mode of action includes hydrolysis of the glycosidic linkages between glucosyl residues that are not branched with xylose. In contrast to commercial lichenase, mixed linkage beta-glucan (lichenan) was not digested by XegA, indicating that the enzyme did not cleave glucan β-1,3 or β-1,6 bonds. The far-UV CD spectrum of the purified enzyme indicated a protein rich in β-sheet structures as expected for GH12 xyloglucanases. Thermal unfolding studies displayed two transitions with mid-point temperatures of 51.3 °C and 81.3 °C respectively, and dynamic light scattering studies indicated that the first transition involves a change in oligomeric state from a dimeric to a monomeric form. Since the enzyme is a predominantly a monomer at 60 °C, the enzymatic assays demonstrated that XegA is more active in its monomeric state.     

Structure and phase behavior of O-stearoylethanolamine: A combined calorimetric, spectroscopic and X-ray diffraction study

Recent studies show that O-acylethanolamines (OAEs), structural isomers of the putative stress-fighting lipids, namely N-acylethanolamines (NAEs), can be derived from NAEs and are present in biological membranes under physiological conditions. In view of this, we have synthesized O-stearoylethanolamine (OSEA) as a representative OAE and investigated its phase behavior and crystal structure. The thermotropic phase transitions of OSEA dispersed in water and in 150 mM NaCl were characterized using calorimetric, spectroscopic, turbidimetric and X-ray diffraction studies. These studies have revealed that when dispersed in water OSEA undergoes a cooperative phase transition centered at 53.8 °C from an ordered gel phase to a micellar structure whereas in presence of 150 mM NaCl the transition temperature increases to 55.8 °C and most likely the bilayer structure is retained above the phase transition. O-Stearoylethanolamine crystallized in the orthorhombic space group P2₁2₁2₁ with four symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that OSEA molecules adopt a linear structure with all-trans conformation in the acyl chain region. The molecules are organized in a tail-to-tail fashion, similar to the arrangement in a bilayer membrane. These studies are relevant to understanding the role of salt on the phase properties of this new class of lipids.     

NMR characterization of a 264-residue hyperthermostable endo-β-1,3-glucanase

Insight into the hyperthermostable endo-β-1,3-glucanase pfLamA from Pyrococcus furiosus is obtained by using NMR spectroscopy. pfLamA functions optimally at 104 °C and recently the X-ray structure of pfLamA has been obtained at 20 °C, a temperature at which the enzyme is inactive. In this study, near-complete (>99%) NMR assignments are presented of chemical shifts of pfLamA in presence and absence of calcium at 62 °C, a temperature at which the enzyme is biologically active. The protein contains calcium and the effects of calcium on the protein are assessed. Calcium binding results in relatively small chemical shift changes in a region distant from the active site of pfLamA and thus causes only minor conformational modifications. Removal of calcium does not significantly alter the denaturation temperature of pfLamA, implying that calcium does not stabilize the enzyme against global unfolding. The data obtained form the basis for elucidation of the molecular origins involved in conformational stability and biological activity of hyperthermophilic endo-β-1,3-glucanases at extreme temperatures.     

Membrane curvature stress controls the maximal conversion of violaxanthin to zeaxanthin in the violaxanthin cycle—influence of α-tocopherol, cetylethers, linolenic acid, and temperature

Zeaxanthin, an important component in protection against overexcitation in higher plants, is formed from violaxanthin by the enzyme violaxanthin de-epoxidase. We have investigated factors that may control the maximal degree of conversion in the violaxanthin cycle. The conversion of violaxanthin to zeaxanthin in isolated spinach thylakoids was followed at different temperatures and in the presence of lipid packing modifiers. The maximum degree of conversion was found to be 35%, 70% and 80% at 4 °C, 25 °C and 37 °C respectively. In the presence of membrane modifying agents, known to promote non-lamellar structures (H_II), such as linolenic acid the conversion increased, and the maximal level of violaxanthin de-epoxidation obtained was close to 100%. In contrast, substances promoting lamellar phases (L_α), such as α-tocopherol and 8-cetylether (C₁₆EO₈), only 55% and 35% of the violaxanthin was converted at 25 °C, respectively. The results are interpreted in light of the lipid composition of the thylakoid membrane, and we propose a model where a negative curvature elastic stress in the thylakoid lipid bilayer is required for violaxanthin de-epoxidase activity. In this model zeaxanthin with its longer hydrophobic stretch is proposed to promote lamellar arrangements of the membrane. As a result, zeaxanthin relieves the curvature elastic stress, which in turn leads to inactivation of violaxanthin de-epoxidase.     

Oligomerization of the SPP1 scaffolding protein

Viral scaffolding proteins direct polymerization of major capsid protein subunits into icosahedral procapsid structures. The scaffolding protein of bacteriophage SPP1 was engineered with a C-terminal hexahistidine tag (gp11-His₆) and purified. The protein is an α-helical-rich molecule with a very elongated shape as found for internal scaffolding proteins from other phages. It is a 3.3 S tetramer of 93.6 kDa at micromolar concentrations. Intersubunit cross-linking of these tetramers generated preferentially covalently bound dimers, revealing that gp11-His₆ is structurally a dimer of dimers. Incubation at temperatures above 37 °C correlated with a reduction of its α-helical content and a less effective intersubunit cross-linking. Complete loss of secondary structure was observed at temperatures above 60 °C. Refolding of gp11-His₆ thermally denatured at 65 °C led to reacquisition of the protein native ellipticity spectrum but the resulting population of molecules was heterogeneous. Its hydrodynamic behavior was compatible with a mix of 3.3 S elongated tetramers (∼ 90%) and a smaller fraction of 2.4 S dimers (∼ 10%). This population of gp11-His₆ was competent to direct polymerization of the SPP1 major capsid protein gp13 into procapsid-like structures in a newly developed assembly assay in vitro. Although native tetramers were active in assembly, refolded gp11-His₆ showed enhanced binding to gp13 revealing a more active species for interaction with the major capsid protein than native gp11-His₆.     

Ambient temperature-dependent thermoregulatory role of REM sleep

Changes in ambient temperature produce complex effects on sleep–wakefulness. In order to find out the mechanisms involved in temperature-sensitive changes in sleep in rats, their thermal preference, body temperature and sleep were studied before and after the destruction of both peripheral and central warm receptors, by systemic administration of 375 mg/kg capsaicin. Though the pre-treated rats preferred to stay mostly at the ambient temperature of 27 °C, post-treated rats strayed freely into chambers having ambient temperature of 30 °C and 33 °C. Sleep and body temperature of these rats were studied for six hours each, when they were kept at an ambient temperature of 18–36 °C. Total sleep time, especially REM sleep, was maximum at 30 °C in pre-treated rats, but this REM sleep peak at 30 °C disappeared after capsaicin administration. Body temperature increased sharply in post-treated rats, at ambient temperatures above 30 °C. Apart from the ability to defend body temperature at high ambient temperature, avoidance of warm ambient temperature and increase in REM sleep are the behavioral measures which are lost in post-treated rats. Results of this study suggest that the ambient temperature-related increase in REM sleep at 30 °C could be part of the thermoregulatory measures.     

Solubilized (Na+ + K+)-ATPase from shark rectal gland and ox kidney--an inactivation study

The bi-exponential time-course of detergent inactivation at 37 degrees C of C12E8-solubilized (Na+ + K+)-ATPase from shark rectal glands and ox kidney was investigated. The data for shark enzyme, obtained at detergent/protein weight ratios between 2 and 16, are interpreted in terms of a simple model where the membrane bound enzyme is solubilized predominantly as (alpha-beta)2 diprotomers at low detergent concentrations and as alpha-beta protomers at high C12E8 (octaethyleneglycoldodecylmonoether) concentrations. It is observed that the protomers are inactivated 15-fold more rapidly than the diprotomers, and that the rate of inactivation of both oligomers is proportional to the detergent/protein ratio. Inactivation of kidney enzyme was biexponential with a very rapid inactivation of up to 40% of the enzyme activity. The observed rate of inactivation of the slower phase varied with the detergent/protein ratio, but the inactivation pattern for the kidney enzyme could not readily be accommodated within the model for inactivation of the shark enzyme. The rates of inactivation at 37 degrees C were about the same in KCl and NaCl, i.e., in the E2(K) and E1 X Na forms, for both enzymes.     

A comparison of low temperature tolerance traits between closely related aphids from the tropics, temperate zone, and Arctic

The survival of aphids exposed to low temperatures is strongly influenced by their ability to move within and between plants and to survive exposure to potentially lethal low temperatures. Little is known about the physiological and behavioural limitations on aphid movement at low temperatures or how they may relate to lethal temperature thresholds. These questions are addressed here through an analysis of the thermal ecology of three closely related aphid species: Myzus persicae, a ubiquitous temperate zone pest, Myzus polaris, an arctic species, and Myzus ornatus, a sub-tropical species. Lower lethal temperatures (LLT₅₀) of aphids reared at 15 °C were similar for M. persicae and M. polaris (range: −12.7 to −13.9 °C), but significantly higher for M. ornatus (−6.6 °C). The temperature thresholds for activity and chill coma increased with rearing temperature (10, 15, 20, and 25 °C) for all clones. For M. polaris and M. ornatus the slopes of these relationships were approximately parallel; by contrast, for M. persicae the difference in slopes meant that the difference between the temperatures at which aphids cease walking and enter coma increased by approximately 0.5 °C per 1 °C increase in rearing temperature. The data suggest that all three species have the potential to increase population sizes and expand their ranges if low temperature limitation is relaxed.     

Electrogenic sodium-sodium exchange carried out by Na,K-ATPase containing the amino acid substitution Glu779Ala

Na,K-ATPase containing the amino acid substitution glutamate to alanine at position 779 of the alpha subunit (Glu779Ala) supports a high level of Na-ATPase and electrogenic Na+-Na+ exchange activity in the absence of K+. In microsomal preparations of Glu779Ala enzyme, the Na+ concentration for half maximal activation of Na-ATPase activity was 161 +/- 14 mM (n = 3). Furthermore, enzyme activity with 800 mM Na+ was found to be similar in the presence and absence of 20 mM K+. These results showed that Na+, with low affinity, could stimulate enzyme turnover as effectively as K+. To gain further insight into the mechanism of this enzyme activity, HeLa cells expressing Glu779Ala enzyme were voltage clamped with patch electrodes containing 115 mM Na+ during superfusion in K+-free solutions. Electrogenic Na+-Na+ exchange was observed as an ouabain-inhibitable outward current whose amplitude was proportional to extracellular Na+ (Na+(o)) concentration. At all Na+(o) concentrations tested (3-148 mM), exchange current was maximal at negative membrane potentials (V(M)), but decreased as V(M) became more positive. Analyzing this current at each V(M) with a Hill equation showed that Na+-Na+ exchange had a high-affinity, low-capacity component with an apparent Na+(o) affinity at 0 mV (K0(0.5)) of 13.4 +/- 0.6 mM and a low-affinity, high-capacity component with a K0(0.5) of 120 +/- 13 mM (n = 17). Both high- and low-affinity exchange components were V(M) dependent, dissipating 30 +/- 3% and 82 +/- 6% (n = 17) of the membrane dielectric, respectively. The low-affinity, but not the high-affinity exchange component was inhibited with 2 mM free ADP in the patch electrode solution. These results suggest that the high-affinity component of electrogenic Na+-Na+ exchange could be explained by Na+(o) acting as a low-affinity K+ congener; however, the low-affinity component of electrogenic exchange appeared to be due to forward enzyme cycling activated by Na+(o) binding at a Na+-specific site deep in the membrane dielectric. A pseudo six-state model for the Na,K-ATPase was developed to simulate these data and the results of the accompanying paper (Peluffo, R.D., J.M. Argüello, and J.R. Berlin. 2000. J. Gen. Physiol. 116:47-59). This model showed that alterations in the kinetics of extracellular ion-dependent reactions alone could explain the effects of Glu779Ala substitution on the Na,K-ATPase.     

Pressure dependence of activity and stability of dihydrofolate reductases of the deep-sea bacterium Moritella profunda and Escherichia coli

To understand the pressure-adaptation mechanism of deep-sea enzymes, we studied the effects of pressure on the enzyme activity and structural stability of dihydrofolate reductase (DHFR) of the deep-sea bacterium Moritella profunda (mpDHFR) in comparison with those of Escherichia coli (ecDHFR). mpDHFR exhibited optimal enzyme activity at 50 MPa whereas ecDHFR was monotonically inactivated by pressure, suggesting inherent pressure-adaptation mechanisms in mpDHFR. The secondary structure of apo-mpDHFR was stable up to 80 °C, as revealed by circular dichroism spectra. The free energy changes due to pressure and urea unfolding of apo-mpDHFR, determined by fluorescence spectroscopy, were smaller than those of ecDHFR, indicating the unstable structure of mpDHFR against pressure and urea despite the three-dimensional crystal structures of both DHFRs being almost the same. The respective volume changes due to pressure and urea unfolding were − 45 and − 53 ml/mol at 25 °C for mpDHFR, which were smaller (less negative) than the corresponding values of − 77 and − 85 ml/mol for ecDHFR. These volume changes can be ascribed to the difference in internal cavity and surface hydration of each DHFR. From these results, we assume that the native structure of mpDHFR is loosely packed and highly hydrated compared with that of ecDHFR in solution.     

Downhill versus Barrier-Limited Folding of BBL 1: Energetic and Structural Perturbation Effects upon Protonation of a Histidine of Unusually Low pKa

A dispersion of melting temperatures at pH 5.3 for individual residues of the BBL protein domain has been adduced as evidence for barrier-free downhill folding. Other members of the peripheral subunit domain family fold cooperatively at pH 7. To search for possible causes of anomalies in BBL's denaturation behavior, we measured the pH titration of individual residues by heteronuclear NMR. At 298 K, the pK_a of His142 was close to that of free histidine at 6.47 ± 0.04, while that of the more buried His166 was highly perturbed at 5.39 ± 0.02. Protonation of His166 is thus energetically unfavorable and destabilizes the protein by ∼ 1.5 kcal/mol. Changes in C^α secondary shifts at pH 5.3 showed a decrease in helicity of the C-terminus of helix 2, where His166 is located, which was accompanied by a measured decrease of 1.1 ± 0.2 kcal/mol in stability from pH 7 to 5.3. Protonation of His166 perturbs, therefore, the structure of BBL. Only ∼ 1% of the structurally perturbed state will be present at the biologically relevant pH 7.6. Experiments at pH 5.3 report on a near-equal mixture of the two different native states. Further, at this pH, small changes of pH and pK_a induced by changes in temperature will have near-maximal effects on pH-dependent conformational equilibria and on propagation of experimental error. Accordingly, conventional barrier-limited folding predicts some dispersion of measured thermal unfolding curves of individual residues at pH 5.3.     

Mechanism of the rate-determining step of the Na(+),K(+)-ATPase pump cycle

The kinetics of the E(2) --> E(1) conformational change of unphosphorylated Na(+),K(+)-ATPase from rabbit kidney and shark rectal gland were investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24 degrees C). The enzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize initially the E(2) conformation. When rabbit kidney enzyme was mixed with NaCl alone, tris ATP alone or NaCl, and tris ATP simultaneously, a fluorescence decrease was observed. The reciprocal relaxation time, 1/tau, of the fluorescent transient was found to increase with increasing NaCl concentration and reached a saturating value in the presence of 1 mM tris ATP of 54 +/- 3 s(-1) in the case of rabbit kidney enzyme. The experimental behavior could be described by a binding of Na(+) to the enzyme in the E(2) state with a dissociation constant of 31 +/- 7 mM, which induces a subsequent rate-limiting conformational change to the E(1) state. Similar behavior, but with a decreased saturating value of 1/tau, was found when NaCl was replaced by choline chloride. Analogous experiments performed with enzyme from shark rectal gland showed similar effects, but with a significantly lower amplitude of the fluorescence change and a higher saturating value of 1/tau for both the NaCl and choline chloride titrations. The results suggest that Na(+) ions or salt in general play a regulatory role, similar to that of ATP, in enhancing the rate of the rate-limiting E(2) --> E(1) conformational transition by interaction with the E(2) state.