Pressure inactivation of tetrameric lactate dehydrogenase homologues of confamilial deep-living fishes

Summary The susceptibility to inactivation by hydrostatic pressure of the tetrameric (Fig. 1) muscletype (M₄) lactate dehydrogenase homologues (LDH, EC 1.1.1.27;l-lactate: NAD⁺ oxidoreductase) from six confamilial macrourid fishes was compared at 4 °C. These marine teleost fishes occur over depths of 260 to 4815 m. The pressures necessary to half-inactivate the LDH homologues are related to the pressures which the enzymes are exposed to in vivo (Table 1); higher hydrostatic pressures are required to inactivate the LDH homologues of the deeper-occurring macrourids. The resistance of the LDH homologues to inactivation by pressure is affected by protein concentration (Fig. 3). After an hour of incubation at pressure, the percent remaining activity approaches an asymptotic value (Fig. 2). The inactivation of the macrourid LDH homologues by pressure was not fully reversible. Assuming that inactivation by pressure was due to dissociation of the native tetramer to monomers, apparent equilibrium constants (K _eq) were calculated. Volume changes (V) were calculated over the range of pressures for which plots inK _eq versus pressure were linear (Fig. 4). The V of dissociation values of the macrourid homologues range from –219 to –439 ml mol^–1 (Table 1). Although the hydrostatic pressures required to inactivate the LDH homologues of the macrourid fishes are greater than those which the enzymes are exposed to in vivo, the pressure-stability of these enzymes may reflect the resistance of these enzymes to pressure-enhanced proteolysis in vivo.     

Ultracentrifugation studies on the dissociation of chemically modified catalases

The dissociation of a series of bovine catalases, in which a proportion of the carboxylic acid groups of glutamic and aspartic acids have been chemically modified by coupling with glycine methyl ester (GME) or ethylenediamine (ED), has been investigated by sedimentation rate and equilibrium methods. Sedimentation equilibrium measurements on GME derivatives have been analysed in terms of a monomer-dimer-trimer- tetramer model. The results show that the association of monomeric (M₁) catalase subunits is consistent with the equilibria 4M₁?2M₂?M₄. The Gibbs energies of association at 284K of the monomeric subunit to dimes (M₂) and tetramers (M₄) were found to be in the range ? 28 to ? 30 kJ mol^?1 and ? 91 to ? 97 kJ mol ^?1, respectively. The Gibbs energy for association of dimer to tetramer is in the range ? 32 to ? 34 kJ mol^?1. Chemical modification of bovine catalase markedly increases its susceptibility to dissociation by sodium n-dodecyl sulphate (SDS) and sedimentation rate measurements suggest that the initial event on addition of SDS is the dissociation of the whole molecule to half-molecules     

Kinetics of reconstitution of porcine muscle lactic dehydrogenase after reversible high pressure dissociation

Porcine muscle lactic dehydrogenase can be reversibly dissociated into monomers at high hydrostatic pressure. The rate of dissociation depends on the conditions of the solvent (Schade et al., 1980, Biochemistry, in press). Maximum yields of reactivation are achieved after dissociation by 20 min incubation in 0.2 M Tris/HCl buffer or 0.2 M KCl at pH 7.6, in the presence of 10 mM dithioerylhritol and 1 mM EDTA, provided that both dissociation and reassociation are performed under anaerobic conditions. At enzyme concentrations of the order of 1 μM reactivation amounts to 9?5%/, the product of reac- tivation being indistinguishable from the enzyme in its initial native state. Based on the long-term stability of the enzyme under the optimum given conditions of reactivation, the kinetics of reconstitution after pressure release were investigated over a wide range of enzyme concentrations (1 nM < c < 1 μM). The weakly sigmoidal kinetics may be described by an irre- versible uni-bimolecular reaction scheme, corresponding to a sequential transconformation-association process. Assuming the protomers to be enzymatically inactive, the kinetic profiles may be fitted by one set of kinetic constants: k_uni = 1.5 × 10^?2 s^?1and k_bi = 7 × 10³ s^?1 M^?1, the association step belonging to either dimer or tetramer formation.     

Formation of dissociated enzyme subunits by chemical treatment during renaturation

When iodoacetate is added to denatured muscle aldolase undergoing renaturation, a major portion of the activity in the resulting enzyme remains in the monomeric form (of about 37,000 M_r). In the absence of iodoacetate, the renatured enzyme exists entirely as the tetramer. Iodoacetate treatment of native aldolase tetramer (M_r = 160,000) does not lead to dissociation. The stabilization of the monomer by iodoacetate treatment is presumably due to modification of a group at the intersubunit region. Active monomers of aldolase could be distinguished from native or renatured aldolase tetramer by gel-filtration and by the sensitivity of the monomer to inactivation in 2.3 m-urea.     

The formation pathway of i-motif tetramers

The i-motif is a four-stranded structure formed by two intercalated parallel duplexes containing hemiprotonated C•C⁺ pairs. In order to describe the sequence of reactions by which four C-rich strands associate, we measured the formation and dissociation rates of three [TC_n]₄ tetramers (n = 3, 4 and 5), their dissociation constant and the reaction order for tetramer formation by NMR. We find that TC_n association results in the formation of several tetramers differing by the number of intercalated C•C⁺ pairs. The formation rates of the fully and partially intercalated species are comparable but their lifetimes increase strongly with the number of intercalated C•C⁺ pairs, and for this reason the single tetramer detected at equilibrium is that with optimal intercalation. The tetramer half formation times vary as the power −2 of the oligonucleotide concentration indicating that the reaction order for i-motif formation is 3. This observation is inconsistent with a model supposing association of two preformed duplex and suggests that quadruplex formation proceeds via sequential strand association into duplex and triplex intermediate species and that triplex formation is rate limiting.     

Thermodynamics and mechanism of high-pressure deactivation and dissociation of porcine lactic dehydrogenase

Lactic dehydrogenase (LDH) from pig heart and pig skeletal muscle can be reversibly dissociated into monomers at high hydrostatic pressure. The reaction can be quantitatively filled by a reversible consecutive dissociation-unfolding mechanism according to Na = 4M ? 4M* (where N is the native letramer, and M and M* two different conformations of the monomer) (K. Müller, et al., Biophys. Chem. 14 (1981) 101). At P ? 1 kbar, the pressure deactivalion of both isoenzymes (H₄ and M₄) is described by the two-state equilibrium N ? 4M. From the respective equilibrium constant and the temperature and pressure dependence of the change in free energy, the thermodynamic parameters of the dissociation/deactivation may be determined, e.g., for LDH-M₄: Δg_Diss = 110 $\text{kJ}\text{mol}$, ΔSDiss = ?860 J/K per mol, ΔH_Diss = ?124 $\text{kJ}\text{mol}$ (enzyme concentration 10 $\text{μg}\text{ml}$, in Tris-HCl buffer, pH 7.6, I = 0.16 M, 293 K, 0.8 kbar); the dissociation volume is found to be ΔV_Diss = ?420 $\text{ml}\text{mol}$ (0.7 < p < 0.9 kbar). Measurements using 8-anilino-1-naphlhalenesulfonic acid (ANS) as extrinsic fluorophore demonstrate that the occurrence of hydrophobic surface area upon dissociation parallels the decrease in reactivation yield after pressurizarion beyond 1 kbar. Within the range of reversible deactivation (p < 1 kbar) no increase in ANS fluorescence is detectable, thus indicating compensatory effects in the process of subunit dissociation. ²H₂O is found to stabilize the enzyme towards pressure dissociation, in accordance with the involvement of hydrophobic interactions in the subunit contact of both isoenzymes of LDH.     

Activation volumes for the two steps of the conjugate-base mechanism in liquid ammonia

The pressure dependence (10–4000 bar) of the kinetics of the ammoniation of[Co(NH₃)₅X](ClO₄)₂ (X = N₃, Cl) and the isomerization of [Co(NH₃)₅(ONO)](ClO₄)₂ in liquid ammonia is reported. The conjugate-base mechanism is operative for these complexes over the entire pressure range used. Activation and thermodynamic parameters were obtained for each of the two steps of the mechanism for [Co(NH₃)₅(N₃)](ClO₄)₂ at 20 bar. Values for the overall activation volume extrapolated to zero pressure are ΔV³(0) = ?12 (11.35 °C, ONO); ?20 (24.45 °C, N₃) and ?30 (0.50 °C, Cl) cm³ mol^?1. Application of El'yanov and Hamann's empirical relation for the pressure dependence of the ionization of weak acids separates the contributions of the pre-equilibrium (ΔV_CB⁰) and the elimination or isomerization reaction (ΔV₂³) (at zero pressure). The values obtained for [Co(NH₃)₅X](ClO₄)₂ are (givens as X; ΔV_CB⁰ and ΔV₂³ in cm³ mol^?1; T in °C): (ONO; ?16 and ?15; 11.35), (N₃; ?22 and 1;24.45), (Cl; ?22 and 3;0.50). These values fit in the accepted picture of volume effects in cobalt(III) ammine kinetics.     

Asymmetric mutations in the tetrameric R67 dihydrofolate reductase reveal high tolerance to active-site substitutions

Type II R67 dihydrofolate reductase (DHFR) is a bacterial plasmid-encoded enzyme that is intrinsically resistant to the widely-administered antibiotic trimethoprim. R67 DHFR is genetically and structurally unrelated to E. coli chromosomal DHFR and has an unusual architecture, in that four identical protomers form a single symmetrical active site tunnel that allows only one substrate binding/catalytic event at any given time. As a result, substitution of an active-site residue has as many as four distinct consequences on catalysis, constituting an atypical model of enzyme evolution. Although we previously demonstrated that no single residue of the native active site is indispensable for function, library selection here revealed a strong bias toward maintenance of two native protomers per mutated tetramer. A variety of such “half-native” tetramers were shown to procure native-like catalytic activity, with similar K_M values but k_cat values 5- to 33-fold lower, illustrating a high tolerance for active-site substitutions. The selected variants showed a reduced thermal stability (T_m ∼12°C lower), which appears to result from looser association of the protomers, but generally showed a marked increase in resilience to heat denaturation, recovering activity to a significantly greater extent than the variant with no active-site substitutions. Our results suggest that the presence of two native protomers in the R67 DHFR tetramer is sufficient to provide native-like catalytic rate and thus ensure cellular proliferation.     

Evidence for an intermediate in the denaturation and assembly of phosphoglucose isomerase

The denaturation of dimeric rabbit muscle phosphoglucose isomerase in guanidine hydrochloride occurs in two discrete steps consisting of partial unfolding followed by subunit dissociation. In 3.5 to 4.5 m guanidine hydrochloride the enzyme forms a stable denaturation intermediate. Formation of this intermediate abolishes catalytic activity, shifts the protein fluorescence emission maximum from 332 to 345 nm, exposes all of the unavailable sulfhydryl groups, and decreases the s_20,w from 6.8 to 4.6 S. The intermediate dissociates into fully unfolded polypeptide chains with further increases in the concentration of the denaturant. The fluorescence maximum shifts to 352 nm and the s_20,w of the denatured monomer is 1.6 S. From the equilibrium constant for subunit association, 3 × 10⁴M^?1, in 4.7 m guanidine hydrochloride, the apparent free energy of association is estimated to be ?6 kcal mol^?1. Reconstitution of the enzyme protein takes place by the reversal of the steps observed upon denaturation. The denatured monomers refold and associate to reform the dimeric intermediate which then anneals to yield the intact enzyme molecule.     

Electrochemical approach to the mechanism of urea denaturation of horse heart cytochrome c

The effect of urea denaturation on the electroactivity of horse heart cytochrome c has been studied by differential pulse polarography and cyclic voltammetry at a gold electrode; the gold electrode was activated by 4,4'-bipyridine. Essentially, two redox couples with E₀₁ ≈ 0.25 V and E'_oz ≈ ?0.05 V (vs. normal hydrogen electrode) have been detected. The experimental results have been interpreted on the basis of the existence of equilibria between native and denatured electroactive forms; transitory species have been assumed to appear on reduction. The scheme that we have proposed agrees well with the conclusions obtained previously by other authors on conformational changes. Moreover, the advantage of electrochemical techniques in investigating the denaturation process has been underlined.     

Characterization and comparison of arcelin seed protein variants from common bean

Four variants of arcelin, an insecticidal seed storage protein of bean, Phaseolus vulgaris L., were investigated. Each variant (arcelin-1, -2, -3, and -4) was purified, and solubilities and M_rs were determined. For arcelins-1, -2, and -4, the isoelectric points, hemagglutinating activities, immunological cross-reactivities, and N-terminal amino acid sequences were determined. On the basis of native and denatured M_rs, the variants were classified as being composed of dimer protein (arcelin-2), tetramer protein (arcelins-3 and -4), or both dimer and tetramer proteins (arcelin-1). Although the dimer proteins (arcelins-1d and -2) could be distinguished by M_rs and isoelectric points, they were identical for their first 37 N-terminal amino acids and had similar immunological cross-reactions, and bean lines containing these variants had a DNA restriction fragment in common. The tetramer proteins arcelin-1t and arcelin-4 also could be distinguished from each other based on M_rs and isoelectric points; however, they had similar immunological cross-reactions and they were 77 to 93% identical for N-terminal amino acid composition. The similarities among arcelin variants, phytohemagglutinin, and a bean α-amylase inhibitor suggest that they are all encoded by related members of a lectin gene family.     

Limited proteolysis as a tool to study the kinetics of protein folding: Conformational rearrangements in acid-dissociated lactic dehydrogenase as determined by pepsin digestion

Noncovalent aggregation as a side reaction competing with the reconstitution of oligomeric enzymes is enhanced by slow conformational changes within the partially unfolded subunits. This has been shown for lactic dehydrogenase from pig muscle after acid dissociation [G., Zettlmeissl R. Rudolph, and R. Jaenicke (1981)Eur. J. Biochem.121, 169–175]. The present experiments confirm previous spectroscopic evidence (from circular dichroism) applying pepsin digestion and subsequent analysis of the fragments on sodium dodecyl sulfate-polyacrylamide gradient gels. The susceptibility of certain fragmentation sites toward pepsin digestion changes with increasing incubation at acid pH, in accordance with a slow M₁ → M₂ transition of the acid-dissociated monomers. Constant pulses of pepsin at varying times after transferring native enzyme to pH 2.3 yield distinct changes in the fragmentation pattern consisting of undigested monomers (M_r = 35,000) plus 12 fragments ranging from 31,000 to 5000. Short digestion of the M₂ species at low concentrations of pepsin preferentially yields 25,000 and 10,500 fragments (molar ratio pepsin:lactic dehydrogenase = 1:24). The time-dependent decrease of monomers upon incubation in 0.1 m sodium phosphate, pH 2.3, at 20 °C strictly parallels the formation of the two fragments. The quantitative kinetic analysis of the changes in peptide pattern yields a first-order rate constant K₁ = 8 ± 2 × 10^?4 s^?1. The observed increase in proteolytic susceptibility is in the time range of the above mentioned decrease in the far-ultraviolet circular dichroism, and the parallel decrease in the yield of reactivation. The results suggest that during the M₁ → M₂ transition at acid pH a specific interdomain cleavage site is becoming exposed. As taken from the molecular weight of the two main fragments the trp 225-lys 226 peptide bond is the most probable candidate for this cleavage site.     

Studies of Root Function in Zea mays: III. Xylem Sap Composition at Maximum Root Pressure Provides Evidence of Active Transport into the Xylem and a Measurement of the Reflection Coefficient of the Root

The cut ends of excised Zea mays roots were sealed to a pressure transducer and their root pressures recorded. These rose approximately hyperbolically to a maximum value of 4.21 ± 0.34 bar after 30 to 40 minutes. Xylem exudate could not be collected at this pressure since the flow rate was zero. Samples of exudate were collected at lower applied pressures (ΔP), however, and Δπ, the osmotic pressure difference between them and the solution bathing the root, was measured by freezing point depression. A plot of ΔP/Δπ against J_v/Δπ, where J_v is the volume flux, proved to be a straight line whose intercept, equal to σ, the reflection coefficient, was 0.853 ± 0.016. The maximum xylem concentrations of various chemical species were found by a similar extrapolative method and compared with those in the cell sap. This indicated that (a) Ca²⁺, Mg²⁺, NO₃²⁻, SO₄²⁻, and most amino acids move from the cells to the xylem down an electrochemical potential gradient; (b) relative to these ions H⁺, NH₄⁺, glutamine and asparagine are actively transported into the xylem; and (c) H₂PO₄⁻, and K⁺ are actively retained in the symplasm.     

Kinetics of tetramolecular quadruplexes

The melting of tetramolecular DNA or RNA quadruplexes is kinetically irreversible. However, rather than being a hindrance, this kinetic inertia allows us to study association and dissociation processes independently. From a kinetic point of view, the association reaction is fourth order in monomer and the dissociation first order in quadruplex. The association rate constant k_on, expressed in M⁻³·s⁻¹ decreases with increasing temperature, reflecting a negative activation energy (E_on) for the sequences presented here. Association is favored by an increase in monocation concentration. The first-order dissociation process is temperature dependent, with a very positive activation energy E_off, but nearly ionic strength independent. General rules may be drawn up for various DNA and RNA sequence motifs, involving 3–6 consecutive guanines and 0–5 protruding bases. RNA quadruplexes are more stable than their DNA counterparts as a result of both faster association and slower dissociation. In most cases, no dissociation is found for G-tracts of 5 guanines or more in sodium, 4 guanines or more in potassium. The data collected here allow us to predict the amount of time required for 50% (or 90%) quadruplex formation as a function of strand sequence and concentration, temperature and ionic strength.     

High pressure effects on the activity of glycolytic enzymes

High hydrostatic pressure inhibits growth in most organisms; this may be explained by a deactivation of enzymes involved in essential metabolic pathways. In order to check this hypothesis the enzymic activity of rabbit muscle lactic dehydrogenase and yeast glyceraldehyde-3-phosphate dehydrogenase was investigated in the presence of the coenzyme and excess of substrate at pressures up to 2 kbar.Kinetic analysis of an initial phase of pressure induced activation and of a second phase of reversible deactivation shows that the two enzymes respond to high pressures in different ways leading to a volume of activation of ΔV³(LDH) = 0 ± 1 cm³ mol⁻¹ and ΔV³(GAPDH) = 60 ± 4 cm³ mol⁻¹, respectively. Comparing the lower limits of pressure deaclivation, LDH is found to be more stable towards pressure than GAPDH. At p ≈ 2 kbar total deactivation of both enzymes is observed. A concentration dependent lag of GAPDH reactivation proves dissociation to participate in the process of deactivation, while the effects for LDH are explicable on the basis of reversible denaturation alone.     

The interaction of methylcobalamin with tetracyanoplatinate(II), tetrathiocyanoplatinate(II) and tetrachloroplatinate(II)

The interactions of methylcobalamin (CH₃-B₁₂) with Pt(CN)₄^2?, PtCl₄^2?, and Pt(SCN)₄^2? in aqueous solution were studied by UV-visible and ¹H NMR spectroscopy. Together with earlier results on the mechanism of the Pt(IV)-dependent methyl-transfer reaction from CH₃-B₁₂ to Pt(II), these studies suggest at least three Pt binding sites on CH₃-B₁₂. One site, which is occupied by all three complexes (K₁ = 4 X 10³ M^?1 for Pt(CN)₄^2? and 3 X 10³ M^?1 for PtCl₄^2?), is located on the CoCH₃ side of the corrin macrocycle, and is involved in the methyl-transfer process in the presence of a Pt(IV) complex. An additional site for Pt(SCN)₄^2? is the N-3 of the benzimidazole group, resulting in dissociation of this group from the cobalt. An additional site for Pt(CN)₄^2? has a binding constant of 16 M^1? and ¹H NMR changes indicate perturbation but not dissociation of the benzimidazole group. Only the first interaction is discerned for PtCl₄^2?.     

THE ACTIVATION OF STARFISH EGGS BY ACIDS : II. THE ACTION OF SUBSTITUTED BENZOIC ACIDS AND OF BENZOIC AND SALICYLIC ACIDS AS INFLUENCED BY THEIR SALTS.

1. Comparison of the rates of activation of unfertilized starfish eggs in pure solutions of a variety of parthenogenetically effective organic acids (fatty acids, carbonic acid, benzoic and salicylic acids, chloro- and nitrobenzoic acids) shows that solutions which activate the eggs at the same rate, although widely different in molecular concentration, tend to be closely similar in C_H. The dissociation constants of these acids range from 3.2 x 10^–7 to 1.32 x 10^–3. 2. In the case of each of the fourteen acids showing parthenogenetic action the rate of activation (within the favorable range of concentration) proved nearly proportional to the concentration of acid. The estimated C_H of solutions exhibiting an optimum action with exposures of 10 minutes (at 20°) lay typically between 1.1 x 10^–4 M and 2.1 x 10^–4 M (pH = 3.7–3.96), and in most cases between 1.6 x 10^–4 M and 2.1 x 10^–4 M (pH = 3.7–3.8). Formic acid (C_H = 4.2 x 10^–4 M) and o-chlorobenzoic acid (C_H = 3.5 x 10^–4 M) are exceptions; o-nitrobenzoic acid is ineffective, apparently because of slow penetration. 3. Activation is not dependent on the penetration of H ions into the egg from without, as is shown by the effects following the addition of its Na salt to the solution of the activating acid (acetic, benzoic, salicylic). The rate of activation is increased by such addition, to a degree indicating that the parthenogenetically effective component of the external solution is the undissociated free acid. Apparently the undissociated molecules alone penetrate the egg freely. It is assumed that, having penetrated, they dissociate in the interior of the egg, furnishing there the H ions which effect activation. 4. Attention is drawn to certain parallels between the physiological conditions controlling activation in the starfish egg and in the vertebrate respiratory center.     

Stabilization of invertase by molecular engineering

Extracellular invertase (EC 3.2.1.26) of Saccharomyces cerevisiae was stabilized against thermal denaturation by intermolecular and intramolecular crosslinking of the surface nucleophilic functional groups with diisocyanate homobifunctional reagents (O?C?N(CH₂)_nN?C?O) of various lengths (n = 4, 6, 8). Crosslinking with 1,4‐diisocyanatobutane (n = 4) proved most effective in enhancing thermostability. Stability was improved dramatically by crosslinking 0.5 mg/mL of protein with 30 μmol/mL of the reagent. Molecular engineering by crosslinking reduced the first‐order thermal denaturation constant at 60°C from 1.567 min^?1 (for the native enzyme) to 0.437 min^?1 (for the stabilized enzyme). Similarly, the best crosslinking treatment increased the activation energy for denaturation from 391 kJ mol^?1 (for the native protein) to 466 kJ mol^?1 (for the stabilized enzyme). Crosslinking was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

The binding of Ni2+ to adenylyl-3',5'-adenosine and to poly(adenylic acid)

Studies of the binding of Ni²⁺ to adenylyl-3',5'-adenosine (ApA) at pH 6-0 by ultraviolet spectrophotometry indicate the formation of a 1:1 complex in the presence of a large excess of metal ion. At 25 °C. and ionic strength μ = 0.5 M, the stability constant of Ni(ApA) is evaluated to be K = 2.6 (±0.6) M^?1. The low stability is taken as evidence that the predominant complex species is one in which the ApA acts as a monodentate ligand, mainly through the adenine group. The rate constants for complex formation and dissociation, k_f = 1430 M^?1 s^?1 and k_b = 665 s^?1 (25°C. μ = 0.5M). determined by the temperature-jump relaxation technique, are consistent with this interpretation. The binding strength of Ni²⁺ to poly(adenylic acid) [poly(A)] has been studied at pH 7.0 using murexide as an indicator of the concentration of free Ni²⁺. Within the concentration range [Ni²⁺ = 1 × 10^?5 × 10^?3 M the data can be represented in the form of a linear Scatchard plot. i.e., the process can be described as the binding of Ni²⁺ to one class of independent binding sites. The number of binding sites per monomer is 0.26, and the stability constant K = 8.2×10³ M^?1 (25°C μ = 0.1 M). In kinetic studies of the reaction of Ni²⁺ with poly(A), two relaxation effects due to complex formation were detected, one with a concentration-independent time constant of about 0.4 ms, the other with a concentration-dependent time constant in the millisecond range. The concentration dependence of the longer relaxation time can be accounted for by a three-step mechanism which consists of a fast second-order association reaction followed by two first-order steps. There is evidence, however, that the overall process is more complicated than expressed by the three-step mechanism.     

Equilibrium and Kinetic Properties of the Interaction between Tetrodotoxin and the Excitable Membrane of the Squid Giant Axon

Squid giant axons were treated with tetrodotoxin (TTX) in concentrations ranging from 1 nM to 25 nM and the resulting decrease in sodium current was followed in time using the voltage clamp technique. The removal of TTX from the bathing solution produced only partial recovery of the sodium current. This suggests that the over-all interaction is more complex than just a reversible reaction. By correcting for the partial irreversibility of the decrease in sodium current, a dissociation constant of 3.31 x 10^-9 M was calculated for the reaction between TTX and the reactive site of the membrane. The data obtained fit a dose-response curve modified to incorporate the correction for partial irreversibility when calculated for a one-to-one stoichiometry. The fit disagreed with that calculated for a reaction between two molecules of TTX with a single membrane-reactive site, but neither supported nor disproved the possibility of a complex formed by two reactive sites with one molecule of TTX. Values of the rate constants for the formation and dissociation of the TTX-membrane complex, k ₁ and k ₂, respectively, were obtained from the kinetic data. The values are: k ₁ = 0.202 x 10⁸ M ^-1, and k ₂ = 0.116 min^-1. The magnitude of the dissociation constant derived from these values is 5.74 x 10^-9 M, which has the same order of magnitude as that obtained from equilibrium measurements. Arrhenius plots of the rate constants gave values for the thermodynamic quantities of activation.