Smoking and Posttraumatic Stress Disorder Symptomatology in Orofacial Pain.

To explore the impact of interactions between smoking and symptoms of posttraumatic stress disorder (PTSD) on pain intensity, psychological distress, and pain-related functioning in patients with orofacial pain, a retrospective review was conducted of data obtained during evaluations of 610 new patients with a temporomandibular disorder who also reported a history of a traumatic event. Pain-related outcomes included measures of pain intensity, psychological distress, and pain-related functioning. Main effects of smoking status and PTSD symptom severity on pain-related outcomes were evaluated with linear regression analyses. Further analyses tested interactions between smoking status and PTSD symptom severity on pain-related outcomes. PTSD symptom severity and smoking predicted worse pain-related outcomes. Interaction analyses between PTSD symptom severity and smoking status revealed that smoking attenuated the impact of PTSD symptom severity on affective distress, although this effect was not found at high levels of PTSD symptom severity. No other significant interactions were found, but the present results identifying smoking as an ineffective coping mechanism and the likely role of inaccurate outcome expectancies support the importance of smoking cessation efforts in patients with orofacial pain. Smoking is a maladaptive mechanism for coping with pain that carries significant health- and pain-related risks while failing to fulfill smokers' expectations of affect regulation, particularly among persons with orofacial pain who also have high levels of PTSD symptom severity. Addressing smoking cessation is a critical component of comprehensive treatment. Further research is needed to develop more effective ways to help patients with pain and/or PTSD to replace smoking with more effective coping strategies.

On the importance of anandamide structural features for its interactions with DPPC bilayers: effects on PLA2 activity.

The acylethanolamide anandamide (AEA) occurs in a variety of mammalian tissues and, as a result of its action on cannabinoid receptors, exhibits several cannabimimetic activities. Moreover, some of its effects are mediated through interaction with an ion channel-type vanilloid receptor. However, the chemical features of AEA suggest that some of its biological effects could be related to physical interactions with the lipidic part of the membrane. The present work studies the effect of AEA-induced structural modifications of the dipalmitoylphosphatidylcholine (DPPC) bilayer on phospholipase A2 (PLA2) activity, which is strictly dependent on lipid bilayer features. This study, performed by 2-dimethylamino-(6-lauroyl)-naphthalene fluorescence, demonstrates that the effect of AEA on PLA2 activity is concentration-dependent. In fact, at low AEA/DPPC molar ratios (from R = 0.001 to R = 0.04), there is an increase of the enzymatic activity, which is completely inhibited for R = 0.1. X-ray diffraction data indicate that the AEA affects DPPC membrane structural properties in a concentration-dependent manner. Because the biphasic effect of increasing AEA concentrations on PLA2 activity is related to the induced modifications of membrane bilayer structural properties, we suggest that AEA-phospholipid interactions may be important to produce, at least in part, some of the similarly biphasic responses of some physiological activities to increasing concentrations of AEA.

Comparison of three rapid detection systems for type A influenza virus on tracheal swabs of experimentally and naturally infected birds.

The present paper reports of the comparison between three rapid virus detection systems and virus isolation (VI) from pooled tracheal swabs collected from naturally and experimentally infected birds with a low pathogenicity avian influenza virus of the H7N3 subtype. The relative sensitivity, specificity and agreement (K value) were calculated for a commercial antigen capture enzyme immunoassay (AC-EIA) and for two nucleic acid detection tests, a one-step reverse transcriptase-polymerase chain reaction (RT-PCR) and a real-time RT-PCR (RRT-PCR), both targeting the M gene. The results indicate that in experimentally infected turkeys VI was positive from the pooled tracheal swabs collected from day 3 to day 10. One-step RT-PCR was able to detect influenza RNA from samples collected from day 3 to day 12, while RRT-PCR amplified influenza RNA in swabs collected from day 3 to day 15. The AC-EIA test yielded positive results between day 5 and day 10 post-infection. On field samples, the K value between the AC-EIA and VI tests was 0.82. Compared with VI, the relative sensitivity of this test was 88.9% (CI95 = 85.2-92.6) and the relative specificity was 95.7% (CI95 = 93.7-97.7). The K value between the RT-PCR and VI tests was 0.88. Compared with virus isolation, the relative sensitivity of the one-step RT-PCR was 95.6% (CI95 = 93.1-98.0) and the relative specificity was 96.3% (CI95 = 94.4-98.1). The K value between the RRT-PCR and VI tests was 0.92. Compared with virus isolation, the relative sensitivity and specificity of RRT-PCR was 93.3% (CI95 = 90.4-96.3) and 98.4% (CI95 = 97.2-99.6), respectively. Generally speaking, comparison between virus isolation, the AC-EIA test and the two nucleic acid detection methods indicated excellent agreement. Data obtained from both experimental and field study suggest a higher sensitivity of the PCR-based methods compared with the AC-EIA. The economical and practical implications of using one of the rapid tests as an alternative to VI during an avian influenza epidemic are discussed.

Salts induce structural changes in elongation factor 1alpha from the hyperthermophilic archaeon Sulfolobus solfataricus: a Fourier transform infrared spectroscopic study.

Elongation factor 1alpha from the hyperthermophilic archaeon Sulfolobus solfataricus (SsEF-1alpha) carries the aminoacyl tRNA to the ribosome; it binds GDP or GTP, and it is also endowed with an intrinsic GTPase activity that is triggered in vitro by NaCl at molar concentrations [Masullo, M., De Vendittis, E., and Bocchini, V. (1994) J. Biol. Chem. 269, 20376-20379]. The structural properties of SsEF-1alpha were investigated by Fourier transform infrared spectroscopy. The estimation of the secondary structure of the SsEF-1alpha*GDP complex, made by curve fitting of the amide I' band or by factor analysis of the amide I band, indicated a content of 34-36% alpha-helix, 35-40% beta-sheet, 14-19% turn, and 7% unordered structure. The substitution of the GDP bound with the slowly hydrolyzable GTP analogue Gpp(NH)p induced a slight increase in the alpha-helix and beta-sheet content. On the other hand, the alpha-helix content of the SsEF-1alpha*GDP complex increased upon addition of salts, and the highest effect was produced by 5 M NaCl. The thermal stability of the SsEF-1alpha*GDP complex was significantly reduced when the GDP was replaced with Gpp(NH)p or in the presence of NaBr or NH4Cl, whereas a lower destabilizing effect was provoked by NaCl and KCl. Therefore, the extent of the destabilizing effect of salts depended on the nature of both the cation and the anion. The data suggested that the sodium ion was responsible for the induction of the GTPase activity, whereas the anion modulated the enzymatic activity through destabilization of particular regions of SsEF-1alpha. Finally, the infrared data suggested that, in particular region(s) of the polypeptide chain, the SsEF-1alpha*Gpp(NH)p complex possesses structural conformations which are different from those present in the SsEF-1alpha*GDP complex.

Two-dimensional gel electrophoresis and FTIR spectroscopy reveal both forms of yeast plasma membrane H(+)-ATPase in activated and basal-level enzyme preparations.

Plasma membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae was isolated and purified in its two forms, the activated A-ATPase from glucose-metabolizing cells, and the basal-level B-ATPase from cells with endogenous metabolism only. Using two-dimensional gel electrophoretic analysis, we showed that both enzyme preparations are actually mixtures of the non-active, i.e. non-phosphorylated, and the active, i.e. phosphorylated, forms of the enzyme. Previous deliberations suggesting that the B-ATPase displays some activity which is lower than that of A-ATPase were apparently wrong. It seems that, molecularly speaking, the B-form is actually not active at all, and what activity we measure in our preparation is due to an admixture of the true active form (A-form). Fourier transform infrared spectroscopic study of the secondary structure and particularly thermal denaturation data suggest the possibility that the two enzyme forms interact to form complexes less stable than the single forms. On the whole then, there apparently is a different ratio of the active and inactive forms and/or complexes between the two forms present in all enzyme preparations.

Laurdan fluorescence: a simple method to evaluate sperm plasma membrane alterations.

OBJECTIVE: To determine, by a simple fluorescence method, sperm plasma membrane alterations related with changes of lipid bilayer that, together with routine semen analysis, could help to elucidate the causes of the unexplained male infertility problems. DESIGN: Pilot study. SETTING: Andrology laboratory and biochemistry institute, medical school. PATIENT(S): Men whose semen was studied for infertility problems. INTERVENTIONS(S): No therapeutic intervention was performed on patients. MAIN OUTCOME MEASURE(S): Presence of spermatozoa plasma membrane alterations evidenced by evaluation of Laurdan fluorescence Generalized Polarization (GP) and reported as a function of increasing cell concentration, spermatozoa total motility, linear speed, and vitality. RESULT(S): Reporting GP values as a function of increasing sperm cell concentration, it is evident that the samples are distributed in two distinct areas: at >32 x 10(6) cells per milliliter, mean GP value was 0.303 +/- 0.015, whereas for lower sperm cell concentrations, the mean GP was 0.365 +/- 0.026 (P<.001). These data indicate that the spermatozoa plasma membranes are characterized by liquid-crystalline phases with different ordering degree and polarity and that about 50% of samples with normal semen characteristics (> or =20 x 10(6) cells per milliliter) show high GP values. CONCLUSION(S): Laurdan fluorescence can be used as a simple method to evaluate spermatozoa plasma membrane alterations, particularly in a group of infertile men presenting normal semen parameters. In these samples, Laurdan could be used as a simple tool for infertility assessment. In fact, it is known that compositional and physicochemical alterations of bilayer features can be important for the fertilizing ability of spermatozoa because they are necessary for a proper physiological membrane activity.

Specific interaction of cytosolic and mitochondrial glyoxalase II with acidic phospholipids in form of liposomes results in the inhibition of the cytosolic enzyme only.

Kinetics of cytosolic recombinant human glyoxalase II and bovine liver mitochondrial glyoxalase II were studied in the presence of liposomes made of different phospholipids (PLs). Neutral PLs such as egg phosphatidylcholine or dipalmitoylphosphatidylcholine did not affect the enzymatic activity of either enzymatic form. Liposomes made of dioleoyl phosphatidic acid or cardiolipin or phosphatidylserine also did not affect the enzymatic activity of mitochondrial glyoxalase II. Conversely, these negatively charged PLs exerted noncompetitive inhibition on cytosolic glyoxalase II only, dioleoyl phosphatidic acid and bovine brain phosphatidylserine exerting the highest and lowest inhibition, respectively. Binding studies, carried out by using a resonant mirror biosensor, revealed that liposomes made of negatively charged PLs interact specifically with both enzymatic forms of glyoxalase II, whereas interactions were not detected with neutral PLs. Once bound on glyoxalase II, negatively charged liposomes could not be removed by 3 M NaCl, suggesting that interactions between glyoxalase II and negatively charged PLs, besides ionic, may be also hydrophobic. These data suggest a possible role of negatively charged phospholipids in the regulation of level of lactoylglutathione in the cell. The data are also discussed in terms of a possible regulation of reduced glutathione supply to mitochondria.

The esterase from the thermophilic eubacterium Bacillus acidocaldarius: structural-functional relationship and comparison with the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus.

The esterase from the thermophilic eubacterium Bacillus acidocaldarius is a thermophilic and thermostable monomeric protein with a molecular mass of 34 KDa. The enzyme, characterized as a "B-type" carboxylesterase, displays the maximal activity at 65 degrees C. Interestingly, it is also quite active at room temperature, an unusual feature for an enzyme isolated from a thermophilic microorganism. We investigated the effect of temperature on the structural properties of the enzyme, and compared its structural features with those of the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus. In particular, the secondary structure and the thermal stability of the esterase were studied by FT-IR spectroscopy, while information on the conformational dynamics of the enzyme were obtained by frequency-domain fluorometry and anisotropy decays. Our data pointed out that the Bacillus acidocaldarius enzyme possesses a secondary structure rich in alpha-helices as described for the esterase isolated from Archaeoglobus fulgidus. Moreover, infrared spectra indicated a higher accessibility of the solvent ((2)H(2)O) to Bacillus acidocaldarius esterase than to Archaeoglobus fulgidus enzyme suggesting, in turn, a less compact structure of the former enzyme. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the Bacillus acidocaldarius protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics. The data suggested an increase in the protein flexibility on increasing the temperature. Moreover, comparison of Bacillus acidocaldarius esterase with the Archaeoglobus fugidus enzyme fluorescence data indicated a higher flexibility of the former enzyme at all temperatures tested, supporting the infrared data and giving a possible explanation of its unusual relative high activity at low temperatures. Proteins 2000;40:473-481.

Steady-state and time resolved fluorescence of albumins interacting with N-oleylethanolamine, a component of the endogenous N-acylethanolamines.

The functions of N-acylethanolamines, minor constituents of mammalian cells, are poorly understood. It was suggested that NAEs might have some pharmacological actions and might serve as a cytoprotective response, whether mediated by physical interactions with membranes or enzymes or mediated by activation of cannabinoid receptors. Albumins are identified as the major transport proteins in blood plasma for many compounds including fatty acids, hormones, bilirubin, ions, and many drugs. Moreover, albumin has been used as a model protein in many areas, because of its multifunctional binding properties. Bovine (BSA) and human (HSA) serum albumin are similar in sequence and conformation, but differ for the number of tryptophan residues. This difference can be used to monitor unlike protein domains. Our data suggest that NOEA binds with high affinity to both albumins, modifying their conformational features. In both proteins, NOEA molecules are linked with higher affinity to hydrophobic sites near Trp-214 in HSA or Trp-212 in BSA. Moreover, fluorescence data support the hypothesis of the presence of other NOEA binding sites on BSA, likely affecting Trp-134 environment. The presence of similar binding sites is not measurable on HSA, because it lacks of the second Trp residue.

The thermophilic esterase from Archaeoglobus fulgidus: structure and conformational dynamics at high temperature.

The esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus is a monomeric protein with a molecular weight of about 35.5 kDa. The enzyme is barely active at room temperature, displaying the maximal enzyme activity at about 80 degrees C. We have investigated the effect of the temperature on the protein structure by Fourier-transform infrared spectroscopy. The data show that between 20 degrees C and 60 degrees C a small but significant decrease of the beta-sheet bands occurred, indicating a partial loss of beta-sheets. This finding may be surprising for a thermophilic protein and suggests the presence of a temperature-sensitive beta-sheet. The increase in temperature from 60 degrees C to 98 degrees C induced a decrease of alpha-helix and beta-sheet bands which, however, are still easily detected at 98 degrees C indicating that at this temperature some secondary structure elements of the protein remain intact. The conformational dynamics of the esterase were investigated by frequency-domain fluorometry and anisotropy decays. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics. Remarkably, the tryptophanyl fluorescence emission reveals that the indolic residues remained shielded from the solvent up to 80 degrees C, as shown from the emission spectra and by acrylamide quenching experiments. The relationship between enzyme activity and protein structure is discussed.

Experimental pancreatitis induced by synthetic prooxidant tert-butyl hydroperoxide.

The purpose of this study was to verify whether injection of tert-butyl hydroperoxide (Bu(t)OOH, a well-known prooxidant agent) into the bile-pancreatic duct can induce acute pancreatitis. A rapid blockade of the secretion was observed in the majority of the animals after 3 hours of observation. After 6 hours, the secretion reached a very low level, significantly different compared with controls. In groups of rats injected with Bu(t)OOH, pancreatic weight gain was observed compared with the rats injected with physiologic saline. Histology of pancreata removed 3 hours after injection of Bu(t)OOH showed acinar cell vacuolization, interstitial edema, focal necrosis of pancreatic acini, fat-tissue necrosis, and leukocyte infiltration of the organ. These changes were considerably greater after the 6-hour observation period. Electron-microscopic inspection revealed profound morphologic changes 3 hours after Bu(t)OOH injection. The control rats receiving physiologic saline alone had well-preserved pancreatic tissue structure. In conclusion, injection of the prooxidant agent, tert-butyl hydroperoxide, into common bile-pancreatic duct induces acute necrotizing pancreatitis, which indicates the crucial role of free radical reactions in pathogenesis of this disease.

Effects of fluorescent pseudo-ATP and ATP-metal analogs on secondary structure of Na(+)/K(+)-ATPase.

The secondary structure of Na(+)/K(+)-ATPase after modification of the ATP-binding sites was analyzed. Consistently with recent reports, we found in trypsin-treated Na(+)/K(+)-ATPase additionally to alpha-helix also beta-sheet structures in the transmembrane segments. However, binding of fluorescein 5'-isothiocyanate (FITC), the pseudo-ATP analog, to the ATP-binding site did not affect the secondary structure of undigested Na(+)/K(+)-ATPase. Consequently, fluorescence intensity changes of FITC-labeled Na(+)/K(+)-ATPase commonly used to observe conformational transitions of the enzyme reflect physiological changes of the native structure. The metal complex analogues of ATP, Cr(H(2)O)(4)ATP and Co(NH(3))(4)ATP, on the other hand, affected the secondary structure of Na(+)/K(+)-ATPase. We propose that these changes in the secondary structure are responsible for inhibition of backdoor phosphorylation.

beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: structure and activity in the presence of alcohols.

beta-Glycosidase from the extreme thermophilic archaeon Sulfolobus solfataricus is a tetrameric protein with a molecular mass of 240 kDa, stable in the presence of detergents, and with a maximal activity at temperatures above 95 degrees C. Understanding the structure-activity relationships of the enzyme under different conditions is of fundamental importance for both theoretical and applicative purposes. In this paper we report the effect of methanol, ethanol, 1-propanol, and 1-butanol on the activity of S. solfataricus beta-glycosidase expressed in Escherichia coli. The alcohols stimulated the enzyme activity, with 1-butanol producing its maximum effect at a lower concentration than the other alcohols. The structure of the enzyme was studied in the presence of 1-butanol by circular dichroism, and Fourier-transform infrared and fluorescence spectroscopies. Circular dichroism and steady-state fluorescence measurements revealed that at low temperatures the presence of the alcohol produced no significant changes in the tertiary structure of the enzyme. However, time-resolved fluorescence data showed that the alcohol modifies the protein microenvironment, leading to a more flexible enzyme structure, which is probably responsible for the enhanced enzymatic activity.

Porcine odorant-binding protein: structural stability and ligand affinities measured by fourier-transform infrared spectroscopy and fluorescence spectroscopy.

Infrared spectra show that the binding of the odorants 2-isobuthyl-3-methoxypyrazine (PYR) and 3,7-dimethyl-1-octanol (DMO) stabilises the tertiary structure of porcine OBP-I against thermal denaturation. The fluorescence emission spectrum of the single tryptophan shows a lambdamax at 337 nm, indicating that the residue is not directly exposed to the solvent. Tryptophan does not appear to be involved in the odorant binding process and it is not accessible to the fluorescence quenchers NaI, CsCl and acrylamide. The binding of the fluorescent dye 1-aminoanthracene (1-AMA), a strong ligand, does not modify the tryptophan fluorescence spectrum. In contrast, the lambdamax of 1-AMA bound to OBP-I is shifted from 537 to 481 nm, with a lambdamax intensity increase by a factor of 80. Bound 1-AMA is displaced by odorant molecules in competitive binding assays and can be employed in simple and rapid binding assay, avoiding the use of radioactive ligands. The Scatchard plot shows that 1-AMA binds to OBP-I with a dissociation constant of 1.3 microM and an equimolar stoichiometry.

Steady-state fluorescence and circular dichroism of trout hemoglobins I and IV interacting with tributyltin.

The effect of tributyltin chloride (TBTC) on rainbow trout (Salmo irideus) hemoglobin I (HbI) and hemoglobin IV (HbIV) was characterized by the steady-state fluorescence of intrinsic and extrinsic fluorescent probes. The fluorescence emission spectrum (lambdaex 280 nm) is greatly increased in intensity by the presence of the organotin in both proteins. Circular dichroism spectra in the same samples show a small decrease in theta222, a measure correlated with the percentage of the alpha-helical content. Morever, important changes in near-UV, Soret, and visible regions of CD were induced by TBTC. The correlation of data obtained with trout hemoglobins (HbI and HbIV) with similar measurements on globins suggests that the presence of heme is necessary for the interaction of the organotin compound with the proteins.

Structural-functional relationships in pig heart AMP-deaminase in the presence of ATP, orthophosphate, and phosphatidate bilayers.

The secondary structure of pig heart AMP-deaminase (AMP-d) in the absence and in the presence of orthophosphate or dioleoyl phosphatidic acid (DOPA) or ATP was investigated by FT-IR spectroscopy. While the latter substance activates the enzyme, orthophosphate is a well-known negative allosteric effector and DOPA exerts a noncompetitive inhibition on AMP-deaminase. Small changes in the secondary structure of AMP-d were induced by the above mentioned substances. Only DOPA reduced the thermal stability of AMP-d and avoided protein intermolecular interactions suggesting structural-functional relationships in AMP-d in the presence of the above substances and a possible role of phosphatidic acid in the subtle regulation of AMP-d activity by temporary binding of the enzyme to cellular membranes.

Structural analysis of ASCUT-1, a protein component of the cuticle of the parasitic nematode Ascaris lumbricoides.

CUT-1 from the intestinal parasitic nematode Ascaris lumbricoides is a protein component of the insoluble residue of the cuticle, cuticlin. It contains the CUT-1-like domain which is shared by members of a novel family of components of extracellular matrices. The structure and the thermal stability of recombinant CUT-1 from A. lumbricoides (ASCUT-1) were investigated by Fourier-transform infrared (FT-IR) and CD spectroscopy. The data revealed that the secondary structure of the protein at 20 degrees C, both as insoluble inclusion bodies or in soluble form, contains about 50% beta structure, 14% alpha-helix and 25% turns. A tendency of A. lumbricoides CUT-1 to form aggregates was documented by FT-IR spectroscopy which showed also that the addition of SDS disrupts these interactions. Near-ultraviolet CD spectra confirmed these data and suggested that phenylalanine residues are probably involved in intermolecular hydrophobic interactions responsible for the tendency of the protein to aggregate. Near-ultraviolet spectra showed also that part of the cysteine residues forms disulphide bridges responsible for the tertiary architecture of the protein. Finally, FT-IR and CD data revealed that ASCUT-1 is very stable at high temperatures. This stability and the tendency of ASCUT-1 to form aggregates suggest that these properties may be important for a protein which is a component of a particularly resistant extracellular matrix such as the nematode cuticle.

Effect of inhibitor binding to beta subunits of F1ATPase on enzyme thermostability: a kinetic and FT-IR spectroscopic analysis.

FT-IR analysis shows that treatment of F1ATPase with the inhibitors DCCD and Nbf-Cl, in the presence of saturating concentrations of ADP and AMP-PNP and in the absence of Mg2+, does not modify the secondary structure of the enzyme, but significantly modifies its compactness and thermal stability, although to different extents. Nbf-Cl causes a significant increase in stabilisation, in addition to that induced by nucleotides, while DCCD is less effective in this regard. Determination by HPLC of the exchange rate, in the absence of Mg2+, of tightly bound nucleotides of F1ATPase treated with the two inhibitors shows that DCCD does not significantly affect the exchange rate of ADP with AMP-PNP and vice versa in catalytic and non-catalytic tight sites, while Nbf-Cl selectively reduces the enzyme's capacity to exchange ADP bound in the tight catalytic site. It is suggested that the effects of DCCD, unlike those of Nbf-Cl, are closely related to the presence or absence of Mg2+.

Structure of yeast plasma membrane H(+)-ATPase: comparison of activated and basal-level enzyme forms.

Plasma membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae was isolated and purified in its two forms, the activated A-ATPase from glucose-metabolising cells, and the basal-level B-ATPase from cells with endogenous metabolism only. Structure of the two enzyme forms and the effects of beta, gamma-imidoadenosine 5'-triphosphate (AMP-PNP) and of diethylstilbestrol (DES) thereon were analysed by FT-IR spectroscopy. IR spectra revealed the presence of two populations of alpha-helices with different exposure to the solvent in both the A-ATPase and B-ATPase. AMP-PNP did not affect the secondary structure of A-ATPase while DES affected the ratio of the two alpha-helix populations. Thermal denaturation experiments suggested a more stable structure in the B-form than in the A-form. AMP-PNP stabilised the A-ATPase structure while DES destabilised both enzyme forms. IR spectra showed that 60% of the amide hydrogens were exchanged for deuterium in both forms at 20 degrees C. The remaining 40% were exchanged at higher temperatures. The maximum amount of H/D exchange was observed at 50-55 degrees C for both enzyme forms, while in the presence of DES it was observed at lower temperatures. The data do not contradict the possibility that the activation of H(+)-ATPase is due to the C-terminus of the enzyme dissociating from the ATP-binding site which is covered by it in the less active form.