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At least 19 recordsLinked to original sources

PubMed1978

Inhibition by ethanol, acetaldehyde and trifluoroethanol of reactions catalysed by yeast and horse liver alcohol dehydrogenases.

1. Produced inhibition by ethanol of the acetaldehyde-NADH reaction, catalysed by the alcohol dehydrogenases from yeast and horse liver, was studied at 25 degrees C and pH 6-9. 2. The results with yeast alcohol dehydrogenase are generally consistent with the preferred-pathway mechanism proposed previously [Dickenson & Dickinson (1975) Biochem. J. 147, 303-311]. The observed hyperbolic inhibition by ethanol of the maximum rate of acetaldehyde reduction confirms the existence of the alternative pathway involving an enzyme-ethanol complex. 3. The maximum rate of acetaldehyde reduction with horse liver alcohol dehydrogenase is also subject to hyperbolic inhibition by ethanol. 4. The measured inhibition constants for ethanol provide some of the information required in the determination of the dissociation constant for ethanol from the active ternary complex. 5. Product inhibition by acetaldehyde of the ethanol-NAD+ reaction with yeast alcohol dehydrogenase was examined briefly. The results are consistent with the proposed mechanism. However, the nature of the inhibition of the maximum rate cannot be determined within the accessible range of experimental conditions. 6. Inhibition of yeast alcohol dehydrogenase by trifluoroethanol was studied at 25 degrees C and pH 6-10. The inhibition was competitive with respect to ethanol in the ethanol-NAD+ reaction. Estimates were made of the dissociation constant for trifluoroethanol from the enzyme-NAD+-trifluoroethanol complex in the range pH6-10.

Acetaldehyde↗

PubMed1977

Circular dichroism of insulin A chain in water: trifluoroethanol mixtures. Use of linear and nonlinear least squares analysis.

Circular dichroic spectra obtained for S-carboxymethylated insulin A chain in water: trifluoroethanol mixtures show that a marked conformational transition occurs as the concentration of trifluoroethanol is increased to (v/v) 83% to give a polypeptide containing about 43% of the residues in the alph-helical conformation. Several proposed methods of analysis, including both linear and two nonlinear lest squares methods, were unable to quantitate the amount of beta-structure present in the polypeptide in 83% trifluoroethanol. Examination of the methods of analysis lead to the conclusion that the current models for far UV CD analysis are not adequare for the data obtained in this study and shows that nonlinear least squares procedures may lead to erroneous conclusions.

Circular Dichroism↗

PubMed1977

Quantitative 250 MHz proton magnetic resonance study of hydrogen-deuterium exchange. Angiotensin II hormone in trifluoroethanol.

Hydrogen-deuterium exchange kinetics of (Asn1-Val5) angiotensin II has been investigated by proton magnetic resonance at 250 MHz in deuterated trifluoroethanol, as an approach to the "in situ" hormone conformation. An interactive program was specially developed to perform the data analysis on a computer similar to those used for spectroscopic data acquisition. Nine exchange sites are evidenced and characterized by their individual kinetic parameters. Three of them are assigned to peptide NH hydrogens, and the six remaining to slowly exchanging side chain protons. At 11 degrees C, more than three peptide hydrogens, sterically hindered or involved in hydrogen bonds, do not exchange. These results corroborate previous circular dichroism and infrared investigations performed in the same solvent, and suggest a family of well-folded conformations, stabilized in trifluoroethanol by internal hydrogen bonds, involving both the backbone and the side chain hydrogens.

Angiotensin II↗

PubMed1976

Sex differences in anaesthetic toxicity: fluroxene and trifluoroethanol in mice.

A sex difference in postanaesthetic mortality after fluroxene anaesthesia was found in Swiss Webster mice. More males succumbed than females. This toxicity was biotransformation-dependent and could be reversed by pretreatment with "opposite" sex hormones. The toxicity of the fluroxene metabolite trifluoroethanol also was more marked in male mice, but was only partially influenced by microsomal enzyme inhibitors or stimulators, or by sex hormones.

Animals↗

PubMed1975

Conformational and spectral analysis of the polypeptide antibiotic N-methylleucine gramicidin S dihydrochloride by nuclear magnetic resonance.

The 220-MHz proton magnetic resonance spectrum of the cyclic decapeptide antibiotic, mono-N-methylleucine gramicidin S, is reported and all the resonances have been assigned to specific protons of the constituent amino acids. Three methods--temperature dependence and solvent mixture (methanol-trifluoroethanol and dimethyl sulfoxide-trifluoroethanol) dependence of peptide NH proton chemical shifts and proton deuteron exchange--habe been utilized to delineate peptide NH protons. The results of the above methods, coupled with the observed vicinal alpha-CH-NH coupling constants and chemical shifts, indicate that in trifluoroethanol the peptide NH PROTONS OF D-Phe4, D-Phe9, L-Orn2, and L-Val6 are exposed to the sovent, and those of L-Val1, L-Orn7, and L-Leu8 are solvent shielded and intramolecularly hydrogen bonded. In trifluoroethanol, dimethyl sulfoxide, and methanol, the decapeptide has no C2 symmetry, and there are only minor conformational differences in the different solvents. In the proposed conformation in trifluoroethanol, one-half of the decapeptide retained the hydrogen bonding pattern of gramicidin S, i.e. cyclo-(L-Val1 NH--O-C L-Leu8) (a beta turn) and cyclo-(L-Leu8 NH--O-C L-Val1). The second half of the molecule exhibits a different type of stable beta turn involving the ten-atom hydrogen-bonded ring, cyclo-(L-Orn7-NH--O-C D-PHE4).

Computers↗

PubMed1976

Conformational studies of corticotropin1-32 and constitutive peptides by circular dichroism.

Circular dichroism spectra on corticotropin1-32 and its constitutive N-, and C-terminal peptides are determined in water and trifluoroethanol under several conditions in the aromatic and peptide spectral regions. Furthermore, the effects of pH and varied mixtures of water-trifluoroethanol are examined on the corticotropin1-32 molecule. The results show that the N- and C-terminal series have a different behaviour in both aqueous and organic media. Corticotropin and the former peptides display "random" spectra in water, and alpha-helix type spectra in trifluoroethanol, while the latter have "random" spectra in both solvents. In the holopeptide corticotropin, the side chain-side chain effects, as reflected by the titration curves obtained from variations in the aromatic region, support the idea of an helical organization of part of the backbone even in aqueous solution. When going from water to trifluoroethanol corticotropin1-32 undergoes a conformational change which leads to an alpha-helix, following a linear pathway. These results, together with other observations, indicate the possible role of the conformation of corticotropin molecules in their biological life.

Adrenocorticotropic Hormone↗

PubMed1975

Solution behavior, circular dichroism and 22 HMz PMR studies of the bovine myelin basic protein.

Bovine myelin basic protein has been investigated with regard to its solution behavior, circular dichroism and 220 MHz PMR spectral properties. At pH 4.8 gamma/2=0.1 acetate buffer, light scattering yielded a Mr of 17 700 and a virial coefficient of 1.0-10(-4) mol-ml/g2. Above pH 7.0 the protein was found to aggregate to higher mol. wt species. Sedimentation experiments at pH 4.8 yielded s degrees 20,w of 1.27 S at gamma/2=0.1 and 1.46 S at gamma/2=0.35. The diffusion coefficient determined from ultracentrifugal experiments was 7.25-10(-7) cm2/s at gamma/2=0.1 and 0.35. The value of f/f0 from diffusion at pH 4.8 and gamma/2=0.35 was 1.64, corresponding to an axial ratio of 11 to 1. The radius of gyration was calculated as 4.28 nm and the root mean square end to end distance was 10.5 nm. At pH 9.0, gamma/2=0.1, s degrees 20,w was 1.71 S and D degrees 20,w was estimated at 7.4-10(-7) cm2/s. The behavior at pH 9.0 reverted to the behavior at pH 4.8 when the pH was readjusted. The E1%/1cm=5.64 at 276.4 nm and 225 at 196 nm. Titration of the protein with trifluoroethanol elicited three distinct regions of conformation stability having increasing helical content as the mol fraction of trifluoroethanol increased. The results of the present study have permitted some comparison of analogous properties and conformational behavior with the basic membrane protein cytochrome c.

Animals↗

PubMed1978

pH-dependent changes of intrinsic fluorescence of chemically modified liver alcohol dehydrogenases.

Horse liver alcohol dehydrogenase specifically carboxymethylated on cysteine-46 (a ligand to the zinc in the active site) or acetimidylated on 25 of the 30 lysine residues per subunit (including residue 228) was studied. The tryptophan fluorescence of these enzymes decreased by 35% as pH was increased, with an apparent pKa of 9.8 +/- 0.2, identical with that of native enzyme. Native enzyme in the presence of 30mM-imidazole, which displaces a water molecule ligated to the zinc, also had a pKa of 9.8. The ionoizable group is thus neither the water molecule nor one of the modified groups. Binding of NAD+ shifted the pKa for the fluorescence transition to 7.6 with native enzyme and to 9.0 with acetimidylated enzyme, but did not shift the pKa of carboxymethylated enzyme. Binding of NAD+ and trifluoroethanol, an unreactive alcohol, gave maximal fluorescence quenching at pH7 with all three enzymes. The acetimidylated enzyme--NAD+--trifluoroethanol complex had an apparent pKa of 5.0, but the pK of the native enzyme complex was experimentally inaccessible. The results are interpreted in terms of coupled equilibria between two different conformational states. On binding of NAD+, the modified enzymes apparently change conformation less readily than does native enzyme, but binding of alcohol can drive the change to completion.

Alcohol Oxidoreductases↗

PubMed1975

Fluroxene toxicity induced by phenobarbital.

Because of reports of fluroxene toxicity in man, the effect of phenobarbital treatment on the toxicity and metabolism of fluroxene was studied in 9 rhesus monkeys. Six monkeys that were exposed to a mean calculated alveolar fluroxene concentration of 5.8% for 4-hr periods up to a total of 16 hr showed no evidence of toxicity. Two animals were sacrificed after a single 4-hr exposure to obtain control measures of fluroxene metabolites in tissues. Four monkeys that had previously survived received exposures to fluroxene and 3 monkeys that had no exposure to fluroxene died during fluroxene anesthesia after treatment with phenobarbital (mean time, 3 hr). Toxicity was manifested by arterial hypotension, pulmonary edema, and arterial hypoxemia. Phenobarbital treatment enhanced production of fluroxene metabolites, including the highly toxic trifluoroethanol. Concentrations of trifluoroethanol in mixed-expired gas, blood, and urine, and of total nonvolatile fluorine in blood, urine, and tissues of animals treated with phenobarbital were 2 to 10 times as in control animals. The results suggest that the rhesus monkey is a valuable model for the study of fluroxene pharmacology and that inclusion of an enzyme-inducing challenge in the evaluation of potential toxicity of other anesthetics seems warranted.

Anesthesia↗

PubMed1978

NMR and enzymatic investigation of the interaction between elastase and sodium trifluoroacetate.

At pH 5.5, sodium trifluoroacetate is a potent competitive inhibitor of porcine elastase (Ki = 2.6 mM) and human leukocyte elastase (Ki = 9.3 mM). For both enzymes the Ki increases strongly with pH. Sodium fluoride is inactive on pancreatic elastase and sodium acetate is a weak inhibitor of this enzyme. Trifluoroethanol inhibits both enzymes but is less active than trifluoroacetate in acidic pH conditions. Bovine trypsin and alpha-chymotrypsin are resistant to the action of sodium trifluoroacetate and trifluoroethanol. The interaction between sodium trifluoroacetate and pancreatic elastase is also demonstrated by 19F NMR spectroscopy. Trifluoroacetyltrialanine is able to displace trifluoroacetate from its complex with pancreatic elastase. In addition, a method using turkey ovomucoid for the active site titration of leukocyte and pancreatic elastase is described.

Animals↗

PubMed1979

Nuclear magnetic resonance studies on a cyclic dodecapeptide analogue of a repeat hexapeptide of tropoelastin: evaluation of secondary structure.

The cyclododecapeptide, (Ala1-Pro2-Gly3-Val4-Gly5-Val6)2, was synthesized and its secondary structure was evaluated from extensive studies in dimethyl sulphoxide, trifluoroethanol and water using NMR methods. A selective decoupling technique in 13C-NMR has been utilized in order to assign the C=O carbon resonances. Temperature dependence of the peptide NH protons and the solvent perturbation of the peptide NH and C=O resonances show the occurrence in all solvents of a beta-turn (a 10-membered H-bond between the Val4 NH and Ala1 C=O) and a gamma-turn, an 11-membered H-bond between the Gly3 NH and the Gly5 C=O; and a possible 14-membered H-bond between the Ala1 NH and the Val4 C=O in dimethyl sulphoxide and trifluoroethanol. These secondary structural features are compared with the linear polyhexapeptide and found the the beta-turn and the gamma-turn are the common conformational features of these peptide systems.

Amino Acid Sequence↗

PubMed1978

Fluroxene mutagenicity.

The commercially available volatile anesthetic fluroxene (2,2,2-trifluoroethyl vinyl ether) which contains the stabilizer N-phenyl-1-napthylamine, was tested for mutagenicity using four strains of S. typhimurium, TA1535, TA1537, TA98 and TA100, and one strain of E. coli, WP2. In addition, purified fluroxene; N-phenyl-1-napthylamine; trifluoroethanol, a major metabolite of fluoroxene; and urine from rats anesthetized with fluroxene were tested. Several procedures were utilized including exposure of bacteria to vapor in desiccators and in liquid suspension. Results indicate that fluroxene, but not its stabilizer, was mutagenic to strains TA1535, TA100 and WP2 only in liquid suspension and only in the presence of a rat-liver enzyme system. Trifluoroethanol and urine from fluroxene-treated rat were not mutagenic to any strain of bacteria. These findings indicate that fluroxene is a promutagen which requires preincubation before it is recognized. Further experiments were performed with enzymes prepared from mouse, hamster and human liver. Fluroxene was mutagenic only in the presence of enzymes prepared from Aroclor 1254 pretreated rodents. Since fluroxene was not mutagenic in the presence of enzymes prepared from three human livers, the significance of these findings to man are unclear.

Animals↗

PubMed1978

Hydrogen-deuterium substitution and solvent effects on the nitrogen-15 nuclear magnetic resonance of gramicidin S: evaluation of secondary structure.

Complete assignments of nitrogen-15 resonances of gramicidin S have been made in dimethyl sulfoxide, trifluoroethanol, and in a solvent mixture of dimethyl sulfoxide (50%) and methanol (50%). The assignments are achieved by utilizing the secondary structure of gramicidin S, by comparing the nitrogen-15 spectrum of gramicidin S with that of di-N-methylphenylalanine-gramicidin S and by taking into account the distinguishable value of nitrogen-15 chemical shift for valine in model compounds. Deuterium substitution for labile peptide protons was performed to delineate solvent shielded and deshielded peptide nitrogens and to substantiate further the signal assignments. The solvent titration on going from dimethyl sulfoxide to trifluoroethanol was also performed and shown to have a large deshielding effect on the peptide nitrogen whose corresponding peptide carbonyl, within the peptide moiety, was accessible to the trifluroethanol solvent.

Deuterium↗

PubMed1976

Conformation of gonadotropin releasing hormone.

The conformation of the gonadotropin releasing hormone (Gn-RH), whose primary sequence is pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH2, and of several of its structural analogues has been studied by circular dichroism, optical rotatory dispersion, and fluorescence spectroscopy. The effects of pH, guanidine, and temperature on fluorescence emission have also been examined. Titration data demonstrate that the histidine and tyrosine residues are free of any mutual interactions. The similarity of emission spectra in water and in guanidine hydrochloride solutions precludes significant interactions between the fluorescent groups and other residues. Neither the temperature nor the pH profiles of the emission intensities of either tyrosine or tryptophan reveal any fixed secondary structure in Gn-RH. Both the extent of alkaline quenching and the distance of 10-11 A calculated from Förster energy transfer theory are in accord with a randomly coiled structure with only one residue between tyrosine and tryptophan. Furthermore, the circular dichroism spectrum and optical rotatory dispersion do not exhibit any contributions from peptide bonds in an ordered structure, although there is a perturbation of the peptide absorption region due to overlapping bands from side-chain chromophores. Gn-RH, therefore, appears to behave as a random coil polypeptide in water devoid of any intrachain residue interactions. This nonordered structure in Gn-RH and the lack of any significant differences in the physical-chemical properties of the hormone analogues indicate that a predetermined solution conformation is not required for biological activity. In contrast to its behavior in water, Gn-RH in trifluoroethanol exhibits a conformational transition, with the formation of a beta structure. Differences in conformational changes exhibited by several analogues in trifluoroethanol may be relevant to their relative biological activities at the receptor site.

Animals↗

PubMed1977

Calorimetric studies of elastin solvation.

The interaction of ox ligamentum nuchae elastin (native, purified and soluble) with four different solvents was investigated by Differential Scanning Calorimetry. The freezable solvent content was determined, at different total solvent contents, from the melting endotherms, and the amount of unfreezable solvent was obtained by extrapolation. For two bifunctional solvents, water and ethylene glycol, the molar ratio "bound solvent/elastin residue" was the same for all the elastin samples investigated, and very close to 1.5. This result is identical to that obtained by other workers for the "water/CO-NH group" of synthetic amorphous polyamides, and it can suggest that the hydration of elastin is interpretable on the basis of a similar model. The model is based on a direct interaction of water with the peptide group, and suggests that 0.5 moles of water are strongly bound, and 1.0 moles are loosely bound to the CO-NH group. The ratio "bound water/elastin residue" is larger for the soluble beta elastin. Different molar ratios are also obtained for monofunctional solvents such as methanol and trifluoroethanol.

Animals↗

PubMed1977

Coenzyme-induced conformational changes and substrate binding in liver alcohol dehydrogenase.

The apoenzyme and holoenzyme structures of liver alcohol dehydrogenase have been determined by X-ray methods to obtain details about coenzyme binding, substrate specificity and the catalytic mechanism. Coenzyme binding induces a conformational change of the protein which partly shields the active site from the solution. The reduced coenzyme binds in an open conformation similar to that of NAD bound to malate dehydrogenase. A hydrogen bond between Thr-178 and the carboxamide group of the coenzyme is essential for proper positioning of the nicotinamide in the active site. Coenzyme analogues in which the carboxamide group is absent or substituted with iodine bind in a different conformation and do not induce the structural change of the protein. Binding of substrate molecules has been studied in crystals obtained from an equilibrium mixture of enzyme, coenzyme and p-bromobenzyl alcohol. The oxygen atom of this substrate as well as that of the inhibitor molecules trifluoroethanol and dimethyl sulphoxide bind directly to the catalytic zinc atom. The substrate-binding region is a deep hydrophobic pocket at the bottom of which the zinc atom mediates electrophilic catalysis of alcohol oxidation.

Alcohol Oxidoreductases↗

PubMed1976

Conformational changes in two neurotoxic proteins from snake venoms.

alpha-Neurotoxin from Naja nigricollis and erabutoxin b from Laticauda semifasciata, two homologous neurotoxic proteins, are studied by circular dichroism, ultraviolet spectroscopy and fluorescence in various water/trifluoroethanol mixtures. The data obtained show that the beta structure of alpha-neurotoxin is conserved in water as well as in the organic solvent. By contrast, erabutoxin b changes from the beta-structure in water to the helix type in trifluoroethanol. The latter induces similarly for both toxins a structural modification around tryptophan 29, a residue common to all neurotoxins known to date. The vicinity of tyrosine 25, another common amino acid, is also altered by the presence of the organic solvent as demonstrated by the sudden increase of reactivity of the phenolic ring towards iodine. The present work affords some evidence for the presence of a particular structure located around the two aromatic residues, which is common to all neurotoxins and able to rearrange independently from the rest of the molecule. Biological importance of this peculiar region is highly probable.

Animals↗

PubMed1978

Conformation of corticotropin. An infrared spectrometry study of hydrogen exchange kinetics.

1H--2H exchange kinetics of the peptide hydrogens in corticotropin have been examined in 2H2O and CF3C2H2O2H solutions by means of infrared absorption measurements. In aqueous solution, around pH 3, the experimental data suggest a partially ordered structure, since in the two corticotropins 1--24 and 1--32 about 6 slowly exchanging peptide protons are numbered. These might belong to the N-terminal part of the molecule. The C-terminal 25--32 octapeptide segment appears to be unordered and slightly destabilizes the overall hormone conformation. For corticotropin1--24 in CF3C2H2O2H, the qualitative interpretation of infrared spectra and the quantitative analysis of exchange data give evidence of a strong stabilization: a predominantly alpha-helical structure is induced by trifluoroethanol.

Adrenocorticotropic Hormone↗