An "active enzyme" muscle model

A new model of skeletal muscle contraction is presented from a unified view of muscle physiology, chemical energetics and newly obtained experimental data concerning actomyosin ATPase in vitro.In this model an interaction between actin and myosin, involving two distinct active sites, is considered to be the essential elementary mechanism for muscle contractions. These two sites are located on myosin. One site, forming a myosin-ADP-P, complex, has stored energy derived from ATP splitting before the beginning of a contraction. Another site, forming a myosin-ATP complex, upon interacting with actin, catalyzes ATP hydrolysis, using a fraction of the stored energy. The hydrolysis at the latter site is responsible for tension development, while the stored energy is released to drive the contractile reaction between actin and myosin unidirectionally. (Thus, the two sites act co-operatively and they can be viewed as forming an active enzyme.)There has been a difficulty in explaining the shortening heat production with apparent lack of corresponding chemical change at the early stage of contraction. The active enzyme model accounts for the shortening heat as the irreversible release of the stored energy. The heat production appears to precede its corresponding ATP splitting for “refueling” which occurs after complete exhaustion of the stored energy, while the actomyosin ATP hydrolysis takes place proportionally to the work. At the macroscopic level, the model is compatible with Hill's tension-velocity and heat relation.     

Trinitrophenylation of smooth muscle myosin

The reaction of trinitrobenzenesulfonate with gizzard myosin was studied. The initial phase of the reaction involved two residues and at this level of modification the following was observed: the Mg2+-ATPase of myosin, the actin-activated ATPase of phosphorylated myosin and the phosphorylation kinetics of myosin were not affected. However, trinitrophenylation did induce an activation of the actin-activated ATPase of dephosphorylated myosin and in this respect mimicked the effect of light chain phosphorylation. The Mg2+-dependence of actin-activated ATPase also is altered on trinitrophenylation. These alterations of enzymatic properties could be at least partly explained by the finding that trinitrophenylation favored the 6S conformation of myosin.     

Two components of actin-based retrograde flow in sea urchin coelomocytes

Sea urchin coelomocytes represent an excellent experimental model system for studying retrograde flow. Their extreme flatness allows for excellent microscopic visualization. Their discoid shape provides a radially symmetric geometry, which simplifies analysis of the flow pattern. Finally, the nonmotile nature of the cells allows for the retrograde flow to be analyzed in the absence of cell translocation. In this study we have begun an analysis of the retrograde flow mechanism by characterizing its kinetic and structural properties. The supramolecular organization of actin and myosin II was investigated using light and electron microscopic methods. Light microscopic immunolocalization was performed with anti-actin and anti-sea urchin egg myosin II antibodies, whereas transmission electron microscopy was performed on platinum replicas of critical point-dried and rotary-shadowed cytoskeletons. Coelomocytes contain a dense cortical actin network, which feeds into an extensive array of radial bundles in the interior. These actin bundles terminate in a perinuclear region, which contains a ring of myosin II bipolar minifilaments. Retrograde flow was arrested either by interfering with actin polymerization or by inhibiting myosin II function, but the pathway by which the flow was blocked was different for the two kinds of inhibitory treatments. Inhibition of actin polymerization with cytochalasin D caused the actin cytoskeleton to separate from the cell margin and undergo a finite retrograde retraction. In contrast, inhibition of myosin II function either with the wide-spectrum protein kinase inhibitor staurosporine or the myosin light chain kinase-specific inhibitor KT5926 stopped flow in the cell center, whereas normal retrograde flow continued at the cell periphery. These differential results suggest that the mechanism of retrograde flow has two, spatially segregated components. We propose a "push-pull" mechanism in which actin polymerization drives flow at the cell periphery, whereas myosin II provides the tension on the actin cytoskeleton necessary for flow in the cell interior.     

Production of specific antibodies to contractile proteins and their use in immunofluorescence microscopy. III. Antiobody against human uterine smooth muscle myosin.

The preparation of highly purified myosin from surgical specimen of human uterine muscle is described. Antibodies were raised in rabbits against this immunogen. In immunodiffusion, they react with uterine and chicken gizzard muscle myosin, no reaction is observed between uterine myosin and the anti-chicken-gizzard- myosin. In immunofluorescence, anti-uterine-myosin stains smooth muscle in the contractile and "modulated" state and non-muscle cells such as fibroblasts, platelets and endothelium of various species. Thus, these antibodies contrast anti-gizzard-myosin, which has previously been shown to be specific for contractile state muscle cells. We therefore conclude that the uterine myosin preparation consists of two immunogens, the one being associated with cell contractility and the other, termed cytoplasmic myosin, with motility and mitosis. The latter is indistinguishable from the myosin present in non-muscle cells and can be absorbed specifically with actomyosin from blood platelets.     

The effect of myosin light chain phosphorylation on the actin-stimulated ATPase activity of myosin minifilaments

It has been shown that in the absence of KCl, the actin-stimulated Mg2+-ATPase activity of rabbit skeletal myosin minifilaments with phosphorylated regulatory lights chains (LC2) exceeds 3-4-fold that of myosin minifilaments with dephosphorylated LC2. Addition of KCl leads to a decrease in the difference between the two ATPase activities. LC2 phosphorylation considerably increases the rate of ATPase reaction and only slightly decreases the affinity of myosin minifilaments for F-actin. It is suggested that the unusual effect of LC2 phosphorylation on the kinetic parameters of the actin-stimulated ATPase reaction of myosin minifilaments can be accounted for by its influence on the interaction between myosin heads which results in the ordered self-assembly of minifilaments.     

Alkaline activation of myosin ATPase: some thermodynamic characteristics]

The increase in temperature leads to a decrease in pKa of the group responsible for the activation of CaATP2- hydrolysis by myosin in the alkaline zone of pH. At 20-25 degrees the pKa value is about 9. The value of ionization heat (deltaHi) calculated from pKa temperature dependence is 7.6+/-+/-0.8 kcal/mol. These values are approximated to the values known for phenol hydroxyl of tyrosine. It has been demonstrated that the acceleration of CaATP2- hydrolysis at alkaline values of pH is accompanied by an increase in the Arrhenius energy of activation (Ea), determined from the temperature dependence of the maximal reaction rate (V). The increase of Ea at alkaline values of pH is apparent and is due to an increase in the concentration of a deprotonized form of the enzyme, having a higher activity. A comparison of activation parameters of the reaction at alkaline and neutral values of pH permits to conclude that the acceleration of CaATP2- hydrolysis at alkaline values of pH is due to the acceleration of the limiting step of the reaction. It has also been found that at alkaline values of pH the power of myosin binding with ADP, a competitive inhibitor and the reaction product, is decreased. It is assumed that the acceleration of ATP hydrolysis at alkaline values of pH is due to accelerated dissociation of the reaction products from the active centre of the enzyme, as a result of ionization of a functional group of myosin, probably of the tyrosine residue.     

The role of microtubules in platelet secretory release

The role of microtubules in platelet aggregation and secretion has been analyzed using platelets permeabilized with digitonin and monoclonal antibodies to alpha (DM1A) and beta (DM1B) subunits of tubulin. Permeabilized platelets were able to undergo aggregation and secretory release. However, threshold doses of agonists capable of eliciting a second wave of aggregation and the platelet release reaction were higher than in control platelets exposed to dimethyl sulfoxide, the solvent for digitonin. Both antibodies to alpha and beta tubulin caused a further increase in the threshold concentration of agonists and inhibited the secretory release of permeabilized platelets, but were ineffective using intact platelets. Neither monoclonal antibody inhibited polymerization or depolymerization of platelet tubulin in vitro. Antibodies to platelet actin and myosin also exhibited an inhibitory activity on platelet aggregation albeit less severe than that observed with the antibodies to alpha and beta tubulin. There was evidence of an interaction between DM1A and DM1B and the antibodies to actin and myosin. The interaction of platelet tubulin and myosin was investigated by two different methods. (1) Coprecipitation of the proteins at low ionic strength at which tubulin by itself did not precipitate and (2) affinity chromatography on columns of immobilized myosin. Tubulin freed of its associated proteins (MAPs) by phosphocellulose chromatography bound to myosin in a molar ratio which approached 2. Platelet actin competed with tubulin for 1 binding site on the myosin molecule. MAPs also reduced the binding stoichiometry of tubulin/myosin. Treatment of microtubule protein with p-chloromercuribenzoate or colchicine did not influence its binding to myosin. DM1A and DM1B inhibited the interaction of tubulin and myosin. This effect could also be demonstrated by reaction of electrophoretic transblots of extracted platelet tubulin with the respective proteins. We interpret these results as evidence for an interference of the two monoclonal antibodies to the tubulin subunits (DM1A and DM1B) with the translocation of microtubule protein from its submembranous site to a more central one during the activation process.     

The mechanism of skeletal muscle myosin ATPase. The mechanism of ADP interaction

The mechanism of interaction between ADP and the myosin active center has been studied using a transient kinetic technique. The results show that the interaction of ADP with the myosin active center is a homogeneous process independent of the association state of the active centers; namely, whether ADP interacts with the monomeric myosin subfragment-1, or with the dimeric forms heavy meromyosin and myosin. The kinetics of the interaction conforms to a simple two-step reaction mechanism for ADP binding. The kinetic and thermodynamic constants for this mechanism have been determined. In addition, analysis of the binding isotherm indicates that the two active sites in heavy meromyosin and myosin function as identical and independent sites.     

Immunological cross-reactivity between chicken slow skeletal and ventricular muscle myosin

Antibodies elicited in rabbits against chicken slow skeletal anterior latissimus dorsi and ventricular myosin were analyzed by double immunodiffusion for their ability to react with homologous and heterologous antigen at different stages of immunization (1--12 months). Each anti myosin antiserum formed a single, strong precipitin line with its immunogen after short time of immunization. This reaction was specific for myosin heavy chains as determined by GEDELISA (gel electrophoresis derived enzyme lined immunosorbent assay) test. In rabbits injected with ventricular myosin after long time of immunization a second, fainter precipitin line has generally been observed. The antigenic determinants responsible for this precipitin line have been localized on the light myosin subunits. By comparing the two types of anti myosin antisera with heterologous antigen we have obtained evidence for partial immunological cross-reactivity between slow skeletal and ventricular muscle myosins. In particular, all anti ventricular myosin antisera displayed a marked immunological reactivity with anterior latissimus dorsi myosin whereas most of anti anterior latissimus dorsi myosin antisera showed absence of reciprocity. By means of immunofluorescence and immunoabsorption techniques both common and unique slow skeletal and ventricular antigenic determinants have been demonstrated.     

Contraction and polymerization cooperate to assemble and close actomyosin rings around Xenopus oocyte wounds

Xenopus oocytes assemble an array of F-actin and myosin 2 around plasma membrane wounds. We analyzed this process in living oocytes using confocal time-lapse (four-dimensional) microscopy. Closure of wounds requires assembly and contraction of a classic "contractile ring" composed of F-actin and myosin 2. However, this ring works in concert with a 5-10-microm wide "zone" of localized actin and myosin 2 assembly. The zone forms before the ring and can be uncoupled from the ring by inhibition of cortical flow and contractility. However, contractility and the contractile ring are required for the stability and forward movement of the zone, as revealed by changes in zone dynamics after disruption of contractility and flow, or experimentally induced breakage of the contractile ring. We conclude that wound-induced contractile arrays are provided with their characteristic flexibility, speed, and strength by the combined input of two distinct components: a highly dynamic zone in which myosin 2 and actin preferentially assemble, and a stable contractile actomyosin ring.     

Thermal Inactivation of Myosin A-Adenosine Triphosphatase in the Presence of F-Actin

The influence of the concentration of F-actin on the inactivation of myosin A-ATPase in solution and in suspension has been studied. The reaction departs from typical first-order behavior in that the rate decreases as the reaction proceeds. The extent of this effect varied greatly with the amount of F-actin added and slightly with pH and ionic strength. The interpretation of the experimental results is discussed. A kinetic mechanism which qualitatively accounts for the observed behavior and which suggests the occurrence of two types of actomyosin complexes with respect to susceptibility to denaturation is proposed.

The rate of denaturation of myosin A has been found to decrease greatly on an addition of magnesium and also with a decrease in ionic strength at high (10.3) or low (6.0) pH values.     

Multiple mechanisms for accumulation of myosin II filaments at the equator during cytokinesis

Total internal reflection fluorescence microscopy revealed how individual bipolar myosin II filaments accumulate at the equatorial region in dividing Dictyostelium cells. Direct observation of individual filaments in live cells provided us with much convincing information. Myosin II filaments accumulated at the equatorial region by at least two independent mechanisms: (i) cortical flow, which is driven by myosin II motor activities and (ii) de novo association to the equatorial cortex. These two mechanisms were mutually redundant. At the same time, myosin II filaments underwent rapid turnover, repeating their association and dissociation with the actin cortex. Examination of the lifetime of mutant myosin filaments in the cortex revealed that the turnover mainly depended on heavy chain phosphorylation and that myosin motor activity accelerated the turnover. Double mutant myosin II deficient in both motor and phosphorylation still accumulated at the equatorial region, although they displayed no cortical flow and considerably slow turnover. Under this condition, the filaments stayed for a significantly longer time at the equatorial region than at the polar regions, indicating that there are still other mechanisms for myosin II accumulation such as binding partners or stabilizing activity of filaments in the equatorial cortex.     

Analysis of the interaction of rabbit skeletal muscle adenylate deaminase with myosin subfragments. A kinetically regulated system

The interaction of rabbit skeletal muscle adenylate deaminase with myosin fragments (heavy meromyosin and subfragment-2) has been studied by analytical centrifugation, gel chromatography, and stopped flow light scattering. Formation of the complex is highly cooperative with respect to addition of two molecules of adenylate deaminase/molecule of myosin fragment to form a ternary complex. Ternary complex formation is also highly pH-dependent with less complex formed at higher pH values, and the pH dependence is steeper with heavy meromyosin than with subfragment-2. At pH 6.5, the dissociation constant for the heavy meromyosin-deaminase complex is approximately 1.2 X 10(-15) M2. Over the pH range 6.5-7.0, rate constants for the formation and dissociation of both the ternary and binary complexes of adenylate deaminase with heavy meromyosin have been determined. From analysis of the time course of stopped flow light scattering, the association steps are found to be extremely rapid, while the rate constant for dissociation of the first molecule of adenylate deaminase from the ternary complex is quite slow. This rate constant increases as the pH increased, but is sufficiently low that the interacting system does not equilibrate on the time scale of mass transport experiments (sedimentation velocity and gel chromatography), and thus displays apparent "slow" behavior. The kinetic regulatory properties of adenylate deaminase are influenced by heavy meromyosin and subfragment-2, particularly with respect to inhibition by GTP. The association and dissociation of adenylate deaminase and myosin fragments and the resultant changes in kinetic properties of the adenylate deaminase can markedly alter the time course of the enzymatic reaction. The time scale over which this interaction is modulated by changes in pH may have significance in the metabolism of exercising muscle.     

No classical type IIB fibres in dog skeletal muscle

Summary To analyse the fibre type composition of adult dog skeletal muscle, enzyme histochemistry, immunohistochemistry for type I, IIA and IIB myosins, and peptide mapping of myosin heavy chains isolated from typed single fibres were combined. Subdivision of type II fibres into two main classes according to the activity of the m-ATPase after acidic and alkaline preincubation proved to be rather difficult and was only consistently achieved after a very careful adjustment of the systems used. One of these sub-classes of type II fibres stained more strongly for m-ATPase activity after acidic and alkaline preincubation, was oxidative-glycolytic and showed a strong reaction with an anti-type IIA myosin. The other one, however, although unreactive with anti-IIA myosin, was also oxidative-glycolytic, and only showed a faint reaction with an anti-type IIB myosin. Peptide mapping of the myosin heavy chains of typed single fibres revealed two populations of heavy chains among the type II fibre group. Thus, in dog muscle, we are confronted with the presence of two main classes of type II fibres, both oxidative-glycolytic, but differing in the structure of their myosin heavy chains. In contrast to some reports in the literature, no classical type IIB fibres could be detected.     

Myosin II functions in actin-bundle turnover in neuronal growth cones

Retrograde actin flow works in concert with cell adhesion to generate traction forces that are involved in axon guidance in neuronal growth cones. Myosins have been implicated in retrograde flow, but identification of the specific myosin subtype(s) involved has been controversial. Using fluorescent speckle microscopy (FSM) to assess actin dynamics, we report that inhibition of myosin II alone decreases retrograde flow by 51% and the remaining flow can be almost fully accounted for by the 'push' of plus-end actin assembly at the leading edge of the growth cone. Interestingly, actin bundles that are associated with filopodium roots elongated by approximately 83% after inhibition of myosin II. This unexpected result was due to decreased rates of actin-bundle severing near their proximal (minus or pointed) ends which are located in the transition zone of the growth cone. Our study reveals a mechanism for the regulation of actin-bundle length by myosin II that is dependent on actin-bundle severing, and demonstrate that retrograde flow is a steady state that depends on both myosin II contractility and actin-network treadmilling.     

Glycine 699 is pivotal for the motor activity of skeletal muscle myosin

Myosin couples ATP hydrolysis to the translocation of actin filaments to power many forms of cellular motility. A striking feature of the structure of the muscle myosin head domain is a 9-nm long "lever arm" that has been postulated to produce a 5-10-nm power stroke. This motion must be coupled to conformational changes around the actin and nucleotide binding sites. The linkage of these sites to the lever arm has been analyzed by site-directed mutagenesis of a conserved glycine residue (G699) found in a bend joining two helices containing the highly reactive and mobile cysteine residues, SH1 and SH2. Alanine mutagenesis of this glycine (G699A) dramatically alters the motor activity of skeletal muscle myosin, inhibiting the velocity of actin filament movement by > 100-fold. Analysis of the defect in the G699A mutant myosin is consistent with a marked slowing of the transition within the motor domain from a strong binding to a weak binding interaction with actin. This result is interpreted in terms of the role of this residue (G699) as a pivot point for motion of the lever arm. The recombinant myosin used in these experiments has been produced in a unique expression system. A shuttle vector containing a regulated muscle-specific promoter has been developed for the stable expression of recombinant myosin in C2C12 cells. The vector uses the promoter/enhancer region, the first two and the last five exons of an embryonic rat myosin gene, to regulate the expression of an embryonic chicken muscle myosin cDNA. Stable cell lines transfected with this vector express the unique genetically engineered myosin after differentiation into myotubes. The myosin assembles into myofibrils, copurifies with the endogenous myosin, and contains a complement of muscle-specific myosin light chains. The functional activity of the recombinant myosin is readily analyzed with an in vitro motility assay using a species-specific anti-S2 mAb to selectively assay the recombinant protein. This expression system has facilitated manipulation and analysis of the skeletal muscle myosin motor domain and is also amenable to a wide range of structure-function experiments addressing questions unique to the muscle-specific cytoarchitecture and myosin isoforms.     

Reaction of cardiac myosin with a purine disulfide analog of adenosine triphosphate. II. Stoichiometry and subunit location of labeling.

The reaction of bovine cardiac myosin with the site-specific purine disulfide analog of ATP, 6,6'-dithiobis (inosinyl imidodiphosphate), was studied to determine the stoichiometry of labeling and subunit location of the reactive cysteines. The analog inactivates myosin by forming a mixed disulfide between the thiopurine nucleotide and certain key cysteines. The thiopurine nucleotide was displaced quantitatively by 14CN to form the more stable thiocyanato-enzyme derivatives. In cardiac myosin, the reactive cysteines could be categorized into three classes, nonessential, critical, and noncritical. The modification of the critical cysteines (two per myosin) inactivated the EDTA and Ca2+ ATPase activities, the latter to a lesser extent. The nonessential cysteines (two to three per myosin) and the noncritical cysteines (two per myosin), differentiated by their rates of reaction, had no effect on the ATPase activities after modification. Thiocyanato-modified myosin was analyzed by sodium dodecyl sulfate gel electrophoresis to determine the distribution of 14CN in the subunits. The critical cysteines were found on the 21,000-dalton light chain (LC1) and the noncritical cysteines on the heavy chains. More specifically, the critical cysteine modified in cardiac LC1 (determined from the products after cyclization and chain cleavage at the thiocyanatoalanyl residues) was shown to be the thiol residue whose surrounding amino acid sequence is homologous to that of the single cysteine of the skeletal myosin alkali light chains, confirming the likely similar structure and function of these light chains in the two different muscle types.     

The role of the myosin ATPase activity in adaptive thermogenesis by skeletal muscle

Resting skeletal muscle is a major contributor to adaptive thermogenesis, i.e., the thermogenesis that changes in response to exposure to cold or to overfeeding. The identification of the "furnace" that is responsible for increased heat generation in resting muscle has been the subject of a number of investigations. A new state of myosin, the super relaxed state (SRX), with a very slow ATP turnover rate has recently been observed in skeletal muscle (Stewart et al. in Proc Natl Acad Sci USA 107:430-435, 2010). Inhibition of the myosin ATPase activity in the SRX was suggested to be caused by binding of the myosin head to the core of the thick filament in a structural motif identified earlier by electron microscopy. To be compatible with the basal metabolic rate observed in vivo for resting muscle, most myosin heads would have to be in the SRX. Modulation of the population of this state, relative to the normal relaxed state, was proposed to be a major contributor to adaptive thermogenesis in resting muscle. Transfer of only 20% of myosin heads from the SRX into the normal relaxed state would cause muscle thermogenesis to double. Phosphorylation of the myosin regulatory light chain was shown to transfer myosin heads from the SRX into the relaxed state, which would increase thermogenesis. In particular, thermogenesis by myosin has been proposed to play a role in the dissipation of calories during overfeeding. Up-regulation of muscle thermogenesis by pharmaceuticals that target the SRX would provide new approaches to the treatment of obesity or high blood sugar levels.     

The effects of calcium and magnesium ions on the adenosine triphosphatase and inosine triphosphatase activities of myosin A

1. The effects of Ca(2+) and Mg(2+) on the enzymic activity of myosin were studied with myosin preparations treated by the ion-exchange resin Chelex-100. A reaction mixture containing 0.05m-potassium chloride was chosen in which the effects of univalent ions such as K(+), Na(+) and Cl(-) do not change significantly with small variations in their concentrations. 2. The relationship between the rate of hydrolysis of ATP or ITP and the concentration of Ca(2+) suggests that a relatively weak binding of Ca(2+) either to myosin or to the substrate nucleotide is responsible for the activation of the enzymic activity. According to the experiments with an ultrafiltration technique, the binding of Ca(2+) to myosin proceeds in at least two steps, the first occurring at one site on every 500000 atomic mass units of myosin with an apparent association constant, K(app.), 1.3x10(6)m(-1), and the second seeming to be so weak that its binding parameters cannot be determined by the method used. The first type of Ca(2+) binding is not observable with N-ethylmaleimide-modified myosin, yet this modified myosin shows activation by Ca(2+) of its adenosine triphosphatase and inosine triphosphatase. 3. The inhibition by Mg(2+) can be related to a binding reaction of Mg(2+) with myosin having K(app.) approximately 10(6)m(-1). Mg(2+) replaces the Ca(2+) bound tightly to myosin. The K(app.) for Mg(2+)-myosin binding calculated by assuming a competition between Ca(2+) and Mg(2+) for the same site is 2.1x10(5)-3.0x10(5)m(-1). When myosin is modified with a thiol reagent (p-mercuribenzoate) at a certain ratio to myosin, the inhibition by Mg(2+) becomes unobservable. 4. The behaviour of the hydrolytic activity of myosin on ATP or ITP in the presence of both Ca(2+) and Mg(2+) is consistent with the explanation that the inhibition by Mg(2+) is due to the tight binding of Mg(2+) to myosin, whereas the activation by Ca(2+) is caused either by a weak binding of Ca(2+) to myosin or by CaATP(2-) or by both.     

Calorimetric studies of the interaction of myosin with ADP.

A calorimetric titration method was used to study ADP binding to native myosin. Data were analyzed by assuming that the myosin molecule has n independent and identical sites for ADP binding. The enthalpy change (deltaH), the binding constant (K), and n were determined. In 0.5 M KCl, 0.01 M MgCl2, and 0.02 M Tris/HCl (pH 7.8), we found: at 0 degrees, deltaH = -57.1 +/- 3.2 kJ-mol-1, log K = 6.42 +/- 0.13, n = 1.49 +/- 0.07; at 12 degrees, deltaH = 73.1 +/- 3.2 kJ-mole-1, log K = 6.08 +/- 0.13, and n = 1.74 +/- 0.07. The average heat capacity change on ADP binding to myosin between 0 and 12 degrees is thus -1.4 +/- 0.4 kJ-mol-1-K-1. Reasonably consistent results were obtained at 25 degrees, suggesting ADP binding to myosin is as strongly exothermic as at lower temperatures, although further interpretation of this result seems unwarranted, mainly because of the instability of myosic at this temperature. The number of protons released on binding of ADP to myosin was determined in separate experiments. The value was 0.19 +/- 0.02 at both 0 and 12 degrees. The reaction of protons with Tris thus contributes about -9.5 kJ-mol-1 to the observed heat on ADP binding.