An inexpensive system for evaluating the tussive and anti-tussive properties of chemicals in conscious, unrestrained guinea pigs.

INTRODUCTION: Commercial whole-body plethysmography systems used to evaluate the anti-tussive potential of drugs employ sophisticated technology, but these systems may be cost prohibitive for some laboratories. The present study describes an alternative, inexpensive system for evaluating the tussive and anti-tussive potential of drugs in conscious, unrestrained guinea pigs. METHODS: The system is composed of a transparent small animal anesthesia induction box fitted with a microphone, a camera and a pneumotachometer to simultaneously capture audio, video, air flow and air pressure in real time. Data acquisition and analysis was performed using free software for audio and video, and a research pneumotach system for flow and pressure. System suitability testing was performed by exposing conscious, unrestrained guinea pigs to nebulized aqueous solutions of a selective agonist for TRPV1 (citric acid) or a selective agonist for TRPA1 (AITC), with or without pre-treatment with a selective antagonist for TRPV1 (BCTC) or a selective antagonist for TRPA1 (HC-030031). RESULTS: The system easily discerned coughs from other respiratory events like sneezes. System suitability test results are as follows: AITC caused 10.7 (SEM=1.4592) coughs vs. 5.8 (SEM=1.6553) when pre-treated with HC-030031 (P<0.05). Citric acid caused 12.4 (SEM=1.4697) coughs vs. 3.2 (SEM=1.3928) when pre-treated with BCTC (P<0.002). DISCUSSION: We have described in detail an inexpensive system for evaluating the tussive and anti-tussive potential of chemicals in conscious, unrestrained guinea pigs. Suitability testing indicates that the system is comparable to a commercial whole-body plethysmography system for detecting and differentiating between coughs and sneezes. This system may provide some investigators a cost-conscious alternative to more expensive commercial whole-body plethysmography systems.

The catalytic triad in Drosophila alcohol dehydrogenase: pH, temperature and molecular modelling studies.

Drosophila alcohol dehydrogenase belongs to the short chain dehydrogenase/reductase (SDR) family which lack metal ions in their active site. In this family, it appears that the three amino acid residues, Ser138, Tyr151 and Lys155 have a similar function as the catalytic zinc in medium chain dehydrogenases. The present work has been performed in order to obtain information about the function of these residues. To obtain this goal, the pH and temperature dependence of various kinetic coefficients of the alcohol dehydrogenase from Drosophila lebanonensis was studied and three-dimensional models of the ternary enzyme-coenzyme-substrate complexes were created from the X-ray crystal coordinates of the D. lebanonensis ADH complexed with either NAD(+) or the NAD(+)-3-pentanone adduct. The kon velocity for ethanol and the ethanol competitive inhibitor pyrazole increased with pH and was regulated through the ionization of a single group in the binary enzyme-NAD(+) complex, with a DeltaHion value of 74(+/-4) kJ/mol (18(+/-1) kcal/mol). Based on this result and the constructed three-dimensional models of the enzyme, the most likely candidate for this catalytic residue is Ser138. The present kinetic study indicates that the role of Lys155 is to lower the pKa values of both Tyr151 and Ser138 already in the free enzyme. In the binary enzyme-NAD(+) complex, the positive charge of the nicotinamide ring in the coenzyme further lowers the pKa values and generates a strong base in the two negatively charged residues Ser138 and Tyr151. With the OH group of an alcohol close to the Ser138 residue, an alcoholate anion is formed in the ternary enzyme NAD(+) alcohol transition state complex. In the catalytic triad, along with their effect on Ser138, both Lys155 and Tyr151 also appear to bind and orient the oxidized coenzyme.

Metal binding properties of thiols; complexes with horse liver alcohol dehydrogenase.

The metal binding properties of thiols were investigated fluorimetrically and spectrophotometrically using horse liver alcohol dehydrogenase as a model metalloenzyme. The steady-state kinetics revealed that in the presence of the coenzyme the primary interaction of a thiol with the enzyme is by thiolate competing with alcohol for the active zinc site. The experiments with 2-mercaptoethanol and ethanethiol showed that at physiological pH it is enzyme-NAD-thiol complexes which are kinetically important with enzyme-thiol complexes only significant at higher pH. The dissociation constants for the binding of thiols in the ternary enzyme-NAD-thiol complexes showed tighter binding as the pH increases, with dithiols binding more tightly than monothiols. The primary binding to zinc was less dependent on the pKa value of each thiol than on mutual stabilization of zinc bound thiolate by the positive charge on the pyridinium ring of NAD, and by monodentate binding and with some dithiols perhaps bidentate binding. The tighter binding to the enzyme of the thiol when it is more hydrophobic or less polar indicates that the thiols interact not only with the active zinc site but also with a neighboring hydrophobic site. This is important for tight binding thiols which are rigidly held by multivalent binding through being anchored hydrophobically and to positively charged centers like zinc.

Drosophila lebanonensis alcohol dehydrogenase: pH dependence of the kinetic coefficients.

The alcohol dehydrogenase (ADH) from Drosophila lebanonensis shows 82% positional identity to the alcohol dehydrogenases from Drosophila melanogaster. These insect ADHs belong to the short-chain dehydrogenase/reductase family which lack metal ions in their active site. In this family, it appears that the function of zinc in medium chain dehydrogenases has been replaced by three amino acids, Ser138, Tyr151 and Lys155. The present work on D. lebanonensis ADH has been performed in order to obtain information about reaction mechanism, and possible differences in topology and electrostatic properties in the vicinity of the catalytic residues in ADHs from various species of Drosophila. Thus the pH dependence of various kinetic coefficients has been studied. Both in the oxidation of alcohols and in the reduction of aldehydes, the reaction mechanism of D. lebanonensis ADH in the pH 6-10 region was consistent with a compulsory ordered pathway, with the coenzymes as the outer substrates. Over the entire pH region, the rate limiting step for the oxidation of secondary alcohols such as propan-2-ol was the release of the coenzyme product from the enzyme-NADH complex. In the oxidation of ethanol at least two steps were rate limiting, the hydride transfer step and the dissociation of NADH from the binary enzyme-NADH product complex. In the reduction of acetaldehyde, the rate limiting step was the dissociation of NAD+ from the binary enzyme-NAD+ product complex. The pH dependences of the kon velocity curves for the two coenzymes were the opposite of each other, i.e. kon increased for NAD+ and decreased for NADH with increasing pH. The two curves appeared complex and the kon velocity for the two coenzymes seemed to be regulated by several groups in the free enzyme. The kon velocity for ethanol and the ethanol competitive inhibitor pyrazole increased with pH and was regulated through the ionization of a single group in the binary enzyme-NAD+ complex, with a pKa value of 7.1. The kon velocity for acetaldehyde was pH independent and showed that in the enzyme-NADH complex, the pKa value of the catalytic residue must be above 10. The koff velocity of NAD+ appeared to be partly regulated by the catalytic residue, and protonation resulted in an increased dissociation rate. The koff velocity for NADH and the hydride transfer step was pH independent. In D. lebanonensis ADH, the pKa value of the catalytic residue was 0.5 pH units lower than in the ADHS alleloenzyme from D. melanogaster. Thus it can be concluded that while most of the topology of the active site is mainly conserved in these two distantly related enzymes, the microenvironment and electrostatic properties around the catalytic residues differ.

Substrate specificity of sheep liver sorbitol dehydrogenase.

The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo> D-ribo > L-xylo > D-lyxo approximately L-arabino > D-arabino > L-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH > -CH2NH2 > -CH2OCH3 approximately -CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols.

Drosophila melanogaster alcohol dehydrogenase: mechanism of aldehyde oxidation and dismutation.

Drosophila alcohol dehydrogenase (Adh) catalyses the oxidation of both alcohols and aldehydes. In the latter case, the oxidation is followed by a reduction of the aldehyde, i.e. a dismutation reaction. At high pH, dismutation is accompanied by a small release of NADH, which is not observed at neutral pH. Previously it has been emphasized that kinetic coefficients obtained by measuring the increase in A340, i.e. the release of NADH at high pH is not a direct measure of the aldehyde oxidation reaction and these values cannot be compared with those for alcohol dehydrogenation. In this article we demonstrate that this is not entirely true, and that the coefficients phiB and phiAB, where B is the aldehyde and A is NAD+, are the same for a dismutation reaction and a simple aldehyde dehydrogenase reaction. Thus the substrate specificity of the aldehyde oxidation reaction can be determined by simply measuring the NADH release. The coefficients for oxidation and dehydrogenation reactions (phi0d and phiAd respectively) are complex and involve the constants for the dismutation reaction. However, dead-end inhibitors can be used to determine the quantitative contribution of the kinetic constants for the aldehyde oxidation and reduction pathways to the phi0d and phiAd coefficients. The combination of dead-end and product inhibitors can be used to determine the reaction mechanism for the aldehyde oxidation pathway. Previously, we showed that with Drosophila Adh, the interconversion between alcohols and aldehydes followed a strictly compulsory ordered pathway, although aldehydes and ketones formed binary complexes with the enzyme. This raised the question regarding the reaction mechanism for the oxidation of aldehydes, i.e. whether a random ordered pathway was followed. In the present work, the mechanism for the oxidation of different aldehydes and the accompanying dismutation reaction with the slow alleloenzyme (AdhS) from Drosophila melanogaster has been studied. To obtain reliable results for the liberation of NADH during the initial-rate phase, the reaction was measured with a sensitive recording filter fluorimeter, and the complexes formed with the different dead-end and product inhibitors have been interpreted on the basis of a full dismutation reaction. The results are only consistent with a compulsory ordered reaction mechanism, with the formation of a dead-end binary enzyme-aldehyde complex. Under initial-velocity conditions, the rate of acetate release was calculated to be larger than 2.5 s-1, which is more than ten times that of NADH. The substrate specificity constant (kcat/Km or 1/phiB) with respect to the oxidation of substrates was propan-2-ol>ethanol>acetaldehyde>trimethylacetaldehyde.

Effects of supplemental ascorbic acid on the energy conversion of broiler chicks during heat stress and feed withdrawal.

The objectives of this study were 1) to determine the effects of supplemental ascorbic acid (AA) on the energy conversion of broiler chicks maintained at thermoneutral and potential heat stress temperatures using indirect convective calorimetry; and 2) to determine whether changes in energy conversion are reflected in changes in lipid metabolism. In Experiment 1, 120 2-d-old cockerels, housed in two identical environmental chambers, were maintained under constant light (2.0 +/- 0.2 fc) and recommended thermal conditions (29.6 +/- 0.8 C; 33.4 +/- 8.0% RH) and consumed water and feed ad libitum. Beginning on Day 8 posthatch, one-half of the birds inside each chamber were randomly assigned and received feed supplemented with AA. Beginning on Day 9 posthatch, the temperature inside one chamber was increased to 34 C whereas the other chamber remained thermoneutral. This design resulted in four treatments: 1) thermoneutral (TN: 27.7 +/- 0.8 C; 40.9 +/- 9.4% RH) and 0 mg AA/kg feed (ppm); 2) TN and 150 ppm AA; 3) heat stress (H: 33.8 +/- 0.5 C; 43.3 +/- 7.4% RH) and 0 ppm AA; or 4) H and 150 ppm AA. Also beginning on Day 9 posthatch, birds were randomly assigned to one of three identical, indirect convective calorimeters designed to accommodate TN or H. Oxygen consumption, carbon dioxide production, respiratory quotient, and heat production were evaluated daily for 8 h, through Day 17 posthatch. Following calorimetric measurement, birds were returned to their respective caging unit/chamber for the remainder of the study. Weight gain, feed intake, and gain: feed were also measured over the 9-d study. Heat exposure depressed (P < 0.05) weight gain, feed intake, and gain:feed. Ascorbic acid increased (P < 0.10) weight gain. Oxygen consumption and carbon dioxide and heat production per kilogram0.75 decreased (P < 0.05) with age with no change in the respiratory quotient. Heat exposure lowered (P < 0.001) the respiratory quotient. A temperature by AA interaction was detected in which heat-exposed birds expressed lower (P < 0.10) respiratory quotients when consuming the AA-supplemented diet. In Experiment 2, 18 2-d-old cockerels, housed in an environmental chamber, were maintained under constant light and recommended thermal conditions (29.3 +/- 0.4 C; 41.4 +/- 3.3% RH) and consumed water and feed ad libitum. On Day 9 posthatch, birds were deprived of feed for 24 h with ad libitum access to water supplemented with either 0 or 400 mg AA/L. Blood samples were obtained from each bird before and after feed withdrawal and supplementation. Supplemented birds exhibited elevated (P < 0.01) plasma AA, levels that were not affected by feed deprivation. Feed deprivation increased (P < 0.0001) plasma beta-hydroxybutyrate with no effect of AA, and decreased (P < 0.05) plasma triglycerides in the unsupplemented birds. A feed withdrawal by AA interaction was detected in which plasma triglycerides remained elevated in birds supplemented with AA. These data suggest that supplemental AA influences body energy stores that are used for energy purposes during periods of reduced energy intake.

The effects of disulfiram and related compounds on equine hepatic alcohol dehydrogenase.

The reversible inhibition and the irreversible inactivation of equine hepatic alcohol dehydrogenase by disulfiram have been investigated. Disulfiram was found to be a potent competitive reversible inhibitor with KEO,I values at pH 7.0 and 10.0 of 50 microM and 30 microM, respectively. Reversible monodentate binding to the active site zinc is indicated by comparison with related compounds. Disulfiram was also found to chemically modify and inactivate the enzyme in an irreversible reaction, which proceeds via the formation of a reversible enzyme-disulfiram binary complex with a dissociation constant at pH 7.0 of 30 microM. The inactivation reaction has been studied over the pH 6.0 to 10.0 range. The dissociation constants for binding to the enzyme and the apparent first-order rate constants for inactivation have been determined as a function of pH. A pKa of 8.3 for the free enzyme has been assigned to the zinc-water ionization. Similar inhibition and affinity labelling kinetics are exhibited by diethyldithiocarbamate and by 2,2'- and 4,4'-dipyridyl disulphide, which have similar enzyme "on" velocity pKa values of 8.3 and 8.2, respectively. The enzyme is competitively protected from inactivation with disulfiram by 2,2'-dipyridyl, 1,7'-phenanthroline, acetone, and ethanol, all of which combine with the active site zinc to form binary complexes. Acetate gave mixed protection against inactivation due to an additional interaction with the anion binding site of the enzyme. In view of the effect of disulfiram on ethanol metabolism and the polyol pathway, its importance as an aversive drug are considered.

Reversible inhibition of sheep liver sorbitol dehydrogenase by the antidiabetogenic drug 2-hydroxymethyl-4-(4-N,N-dimethylaminosulfonyl-1-piperazino) pyrimidine.

The mechanism of the inhibition of sheep liver sorbitol dehydrogenase by the novel antidiabetogenic drug 2-hydroxymethyl-4-(4-N,N-dimethylaminosulfonyl-1-piperazino) pyrimidine has been investigated by steady-state kinetics over the range pH 5-10. The pyrimidine derivative exhibits mixed inhibition with respect to sorbitol, fructose and coenzyme, due to the formation of enzyme-inhibitor and enzyme-NAD(H)-inhibitor complexes. The formation of each of the binary and ternary complexes is inhibited by protonation and deprotonation of groups which, in the enzyme-inhibitor complex, have pK values of 6.6 and 8.0, respectively.

Reversible inhibition of sheep liver sorbitol dehydrogenase by thiol compounds.

Reversible inhibition of sheep liver sorbitol dehydrogenase by various thiol compounds has been studied. Most species inhibit the enzyme-catalyzed reaction competitively with respect to sorbitol, due to the formation of ternary enzyme-NAD-thiol complexes. The primary interaction of thiol inhibitors with the enzyme active site involves the catalytic zinc atom, and a bidentate mode of binding to the active site metal is indicated for some bifunctional thiols in their ternary complexes. Enzyme-bound thiolate facilitates NAD binding to the enzyme and vice versa, mainly due to mutual electrostatic stabilization. The aromatic thiols 1-thio-1-phenylmethane and 1-thio-2-phenylethane are especially potent inhibitors with an inhibition constant of 0.30 microM at pH 9.9. The inhibitory effect of aliphatic thiols, which is positively correlated with alkyl chain length, parallels that observed previously with the related enzyme horse liver alcohol dehydrogenase and indicates that interaction with an enzymic hydrophobic site is important for inhibitor binding. Several reversible inhibitors afford competitive protection against affinity labelling of the enzyme by 2-bromo-3-(5-imidazolyl) propionic acid due to the formation of binary enzyme-thiol complexes. The present study establishes thionucleosides as a novel class of potent sorbitol dehydrogenase inhibitors. The thionucleosides 6-thioguanosine and 6-thioinosine gave mixed inhibition with respect to sorbitol, due to the formation of enzyme-NAD-inhibitor and enzyme-NADH-inhibitor complexes. In order to enable a correlation of the substrate and inhibitor specificities of the enzyme, the kinetic constants for several sorbitol dehydrogenase substrates were determined. L-threitol and DL-1-phenyl-1,2-ethanediol are good substrates with, at high pH, kinetic constants similar to those of sorbitol. The potent inhibition by dithiothreitol and the aromatic thiols thus parallels the substrate specificity of the enzyme. The sorbitol competitive inhibitor 1-thiosorbitol is also a substrate with, at pH 7.4, a maximum velocity of 0.17 s-1 and a Michaelis constant of 8.6 mM. Dithiothreitol forms a tight ternary complex with the enzyme-NAD complex with a molar absorbance of 16.4 x 10(3) M-1 . cm-1 at 311 nm. A spectrophotometric titration of the enzyme with NAD in the presence of dithiothreitol is described, which enables an accurate determination of the concentration of sorbitol dehydrogenase active sites and confirms the activity assay of the enzyme.

Stereo-selective affinity labelling of sheep liver sorbitol dehydrogenase by chloro-substituted analogues of 2-bromo-3-(5-imidazolyl)propionic acid.

The role of configuration for the affinity labelling of sheep liver sorbitol dehydrogenase by chloro-substituted analogues of 2-bromo-3-(5-imidazolyl)propionate (BrImPpOH) has been studied. A saturation kinetics mechanism applies which includes formation of a reversible complex with the enzyme prior to alkylation of Cys-43. The pseudo first-order inactivation rate-constant, k2, and the dissociation constant for the reversible enzyme-affinity label complex. KEI, were determined at pH 7.4 and 23.5 degrees C. The stereo isomers of each affinity label exhibit different kinetic characteristics but, unlike with horse liver alcohol dehydrogenase, the discrimination between them is not absolute. For the different affinity labels, k2 varies with 2-chloro-3-(5-imidazolyl)methylpropionate (Me-ClImPpOH) > 2-chloro-3-(5-imidazolyl)propionate (ClImPpOH) > 2-chloro-3-(5-imidazolyl)propanol (ClImPOH), consistent with their order of inherent reactivity, and the specificity constant k2/KEI varies with (S)-Me-ClImPpOH > (S)-ClImPpOH > (S)-ClImPpOH > (R)-Me-ClImPpOH > (R)-ClImPpOH. Models of the affinity labels were built into the active site of the predicted subunit structure of the enzyme by using a computer-controlled display system. In each binary complex, the imidazole moiety of the affinity label was liganded to the catalytic zinc atom, and the angle Scys-C alpha-Cl was linear, in accordance with an SN2 mechanism. Both enantiomers of each label could form plausible complexes with the enzyme model, in agreement with the kinetic data. The enantiomeric selectivity, rather than absolute specificity, of the reaction appears due to the anion-binding site in sorbitol dehydrogenase being less developed than in horse liver alcohol dehydrogenase.

Reversible and irreversible inhibition of sheep liver sorbitol dehydrogenase with Cibacron Blue 3GA and Eriochrome Black T.

Due to the central role of sorbitol dehydrogenase in diabetic cataract, it is important to examine this enzyme's interaction with different inhibitory compounds such as dyes. The aim of the study was to investigate the binding of Cibacron Blue and Eriochrome Black T to the active site in sorbitol dehydrogenase. These dyes' effect on the enzyme was studied by steady state and affinity labelling kinetics. Both dyes were coenzyme competitive inhibitors with KEI values around 0.5 microM. Essentially the same KEI values were obtained using the dyes as protecting ligands against the affinity label D,L-alpha-Bromo-beta-(5-imidazolyl)-propionic acid. Both dyes were also able to inhibit the enzyme irreversibly through an affinity labelling mechanism, with KEI' values for Cibacron Blue and Eriochrome Black T of 2.2 and 3.1 mM, respectively. Dithiothreitol and NADH were competitive protecting ligands against both dyes. The rate of inactivation was fastest for Cibacron Blue at acid pH values, while the opposite was the case with EBT. Both Cibacron Blue and Eriochrome Black T bind to sorbitol dehydrogenase in two different ways. In both cases the complex formed prior to irreversible inhibition is the weakest. The tighter reversible complexes are suggested to share a common epitope in the coenzyme binding region. Both irreversible complexes involve binding close to the zinc ion at the active site and the sugar binding site. Due to different pH dependences it can be concluded that the affinity labelling mechanism is different for the two dyes and in neither case is the inactivation due to removal of the active site zinc ion.

The effects of disulfiram on equine hepatic alcohol dehydrogenase and its efficiency against alcoholism: vinegar effect.

The effects of disulfiram, its metabolite diethyldithiocarbamate and dithiodipyridine on alcohol metabolism of equine hepatic alcohol dehydrogenase (EC.1.1.1.1.) have been investigated. They were found to form enzyme-NAD(+)-inhibitor complexes which were competitive inhibitors of alcohol metabolism with dissociation constants (KEO,I) at pH 7.0 of 50 microM, 1.3 mM, and 260 microM, respectively. Acetate and vinegar behaved similarly in forming an inhibitory enzyme-NAD(+)-acetate ternary complex competitive with ethanol, with at pH 7.0 essentially identical dissociation constants of 4.0 mM and 3.8 mM, respectively. Disulfiram, diethyldithiocarbamate and dithiodipyridine were also found to exhibit affinity-labelling kinetics with liver alcohol dehydrogenase. The liver enzyme is chemically modified and inactivated in a similar manner by all three reagents via binary enzyme complexes with dissociation constants of 30 microM, 200 microM and 50 microM, respectively. Used as a protector against enzyme inactivation by DL-alpha-bromo-beta-(5-imidazolyl)-propionic acid, disulfiram, diethyl-dithiocarbamate and dithiodipyridine were found to form competitive binary enzyme complexes by binding to the active zinc site with KE,I values of 30 microM, 170 microM and 50 microM, respectively. The disulfiram and acetate binding to zinc results in the formation of binary and ternary complexes which inhibit alcohol metabolism at the enzyme level. Due to many unwanted side-effect), and the easy removal of its anti-drinking effects by drinking vinegar (vinegar effect), disulfiram may still be questioned as an effective drug against alcoholism.

Effect of pH on sheep liver sorbitol dehydrogenase steady-state kinetics.

The variation with pH of the kinetic parameters for sorbitol oxidation and fructose reduction by sheep liver sorbitol dehydrogenase has been studied over the pH 5-10 range. The reaction is compulsory ordered in both directions with the coenzyme as the leading substrate, and the rate-determining step in either direction is the enzyme-coenzyme product dissociation. Throughout the pH range, the lack of a primary kinetic isotope effect on Vm with (2H8) sorbitol confirms that the ternary complexes are not of rate-determining significance under maximum velocity conditions. The association rate constants for NAD and NADH increase and decrease, respectively, towards high pH. NAD binding to the enzyme is dependent upon pK values of 9.2 and 9.6. Whereas the dissociation rate constant for NAD release from the enzyme shows no pronounced variation with pH, NADH release is dependent upon pK values of 7.2 and 7.7. The kinetic constants that characterize the dependence on substrate concentration of the steady-state rate of catalysis vary with pH in accordance with a single pK of 7.1 for sorbitol oxidation and of 7.7 for fructose reduction. These pK values reflect the ionization properties of a catalytically essential group, which is tentatively considered to be either the H2O/OH- ligand binding to the catalytic zinc atom or a histidine residue. Catalysis by sorbitol dehydrogenase, due to the absence of a second ionization contribution, appears not to involve any obligatory step of proton transfer to solution at the ternary complex level. A mechanism for sorbitol dehydrogenase catalysis is proposed.

Effects of supplemental ascorbic acid on the performance of broiler chickens exposed to multiple concurrent stressors.

An experiment was conducted to determine whether ascorbic acid (AA) increases resistance of female Hubbard x Hubbard broiler chicks to multiple concurrent stressors. Stressors imposed from 10 to 17 d posthatch included 2 x 2 x 2 factorial combinations of beak trimming [(B), sham-operated or beak-trimmed and cauterized], coccidiosis [(C), gavage with 0 or 3 x 10(5) sporulated Eimeria tenella oocysts], and heat stress [(H), 28 vs 33 C]. A starter diet was supplemented with AA to provide 0, 150, or 300 ppm (milligrams per kilogram). This resulted in a 2 x 2 x 2 x 3 factorial design with two six-chick replicates of each of the 24 treatment combinations. Data were analyzed using ANOVA and a level of 95% significance. Ascorbic acid increased feed intake and lowered plasma corticosterone and heterophil:lymphocyte ratios. Heat depressed weight gain and feed intake and elevated heterophil:lymphocyte ratios. Heat and AA interacted to improve weight gain and feed intake and lower heterophil:lymphocyte ratios. Coccidiosis depressed weight gain, feed efficiency, and heterophil:lymphocyte ratios. Coccidiosis and AA interacted to increase feed intake and lower plasma corticosterone and heterophil: lymphocyte ratios. Beak trimming increase heterophil:lymphocyte ratios. Beak trimming and AA interacted to increase feed intake and lower heterophil: lymphocyte ratios. Weight gain and feed efficiency decreased whereas heterophil:lymphocyte ratios increased linearly in unsupplemented birds as a function of stressor "order" (the number of stressors imposed simultaneously) indicating an additive effect of systematically increasing the number of stressors. No changes in feed efficiency or heterophil:lymphocyte ratios were detected as a function of stressor order when AA was provided. Ascorbic acid reduced the slope of the regression equation describing the relationship between weight gain and stressor order. It was concluded that AA, particularly at 150 ppm, enhanced performance of broiler chicks exposed to multiple concurrent environmental stressors.

Drosophila melanogaster alcohol dehydrogenase: product-inhibition studies.

The Drosophila melanogaster alleloenzymes AdhS and AdhF have been studied with respect to product inhibition by using the two substrate couples propan-2-ol/acetone and ethanol/acetaldehyde together with the coenzyme couple NAD+/NADH. With both substrate couples the reaction was consistent with an ordered Bi Bi mechanism. The substrates added to the enzyme in a compulsory order, with coenzyme as the leading substrate, to give two interconverting ternary complexes. The second ternary complex broke down with release of products in an obligatory order, with the aldehyde/ketone leaving first. Both the acetaldehyde and acetone products formed binary complexes with the enzyme that affected NAD+ binding. However, only an enzyme-acetone complex seemed to affect NADH binding and hence the reverse reaction. The inhibitory pattern with acetaldehyde as product was also affected by the formation of a ternary enzyme-NAD(+)-acetaldehyde complex, which broke down to acetic acid and NADH. The product-inhibition pattern shown in the present work is different from that published for Drosophila Adh previously and this discrepancy can not be explained by the use of different variants of Drosophila Adh.

The inactivation of sheep liver sorbitol dehydrogenase by pyrophosphate and some analogous metal chelators.

Pyrophosphate and several other metal chelators are shown to inactivate sheep liver sorbitol dehydrogenase. Pyrophosphate, tripolyphosphate, and some bisphosphonates inactivate the enzyme by saturation kinetics involving the formation of a reversible complex. A mechanism for the pyrophosphate-mediated inactivation of sorbitol dehydrogenase is proposed. Steady-state kinetics show that pyrophosphate does not compete with sorbitol for binding to the catalytic zinc atom or with NAD for binding to the anion binding site. The latter is supported by the formation of an E-NAD-pyrophosphate (PPi) complex and by the noncompetitive protection of NADH against inactivation. The rate of enzyme inactivation by pyrophosphate increases with decreasing pH. The pH dependence of the inactivation indicates that a group with a pKa of 6.9 in the free enzyme and in the enzyme-PPi complex is involved. As several zinc-binding reversible inhibitors do not afford protection against pyrophosphate inactivation, the pKa values obtained are considered not to refer to the ionization of the zinc-bound water molecule, but are tentatively suggested to be those of an active site histidine residue. Protection and reactivation by Zn2+ ions indicate that enzyme inactivation results from the loss of the catalytic zinc atom.

Inhibition and activation studies on sheep liver sorbitol dehydrogenase.

Reversible inhibition and activation, as well as protection against affinity labelling with DL-2-bromo-3-(5-imidazolyl)propionic acid, of sheep liver sorbitol dehydrogenase have been studied. The results presented are discussed in terms of enzyme active-site properties and may have potential applications for drug design. Kinetics with mainly sorbitol competitive inhibitors reveals that aliphatic thiols are generally the most potent inhibitors of enzyme activity. Inhibition and inactivation by heterocyclics parallel that seen previously with sorbitol dehydrogenase from other sources as well as with alcohol dehydrogenase from yeast. However, there are significant differences in relation to the structurally similar horse liver alcohol dehydrogenase, as the catalytic zinc of sorbitol dehydrogenase is more easily removed by chelating molecules. Several aldose reductase inhibitors are shown to also inhibit sorbitol dehydrogenase, but at concentrations unlikely to be reached clinically. Enzyme activation has been observed with various compounds, in particular halo-alcohols and detergents. Several inhibitors provide competitive protection against enzyme inactivation by DL-2-bromo-3-(5-imidazolyl)propionic acid. This enables the dissociation constants for binary enzyme-inhibitor complexes to be determined. NADH protects noncompetitively against inactivation. The presence of some binary and ternary enzyme-NADH complexes is indicated from fluorescence emission spectra, as a shift in the fluorescence maximum and intensity is observed due to their formation.

Inhibition and inactivation of horse liver alcohol dehydrogenase with the imidazobenzodiazepine Ro 15-4513.

The imidazobenzodiazepine ethyl 8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine -3-carboxylate (Ro 15-4513) is important as a potential "drink and drive" drug due to effects on receptors in brain neurones, resulting in alcohol intoxication-antagonistic properties. Because of the molecule's importance its effect on alcohol metabolism in liver has been investigated. Ro 15-4513 was found to be, like its parent compound the 8-fluoro analogue flumazenil, a reversible alcohol competitive inhibitor of horse liver alcohol dehydrogenase (EC 1.1.1.1) with a dissociation constant of 345 microM at pH 7.0. Due to its azido group Ro 15-4513 was developed as a potential photoaffinity-labeling reagent for benzodiazepine receptors. Used with horse liver alcohol dehydrogenase, the enzyme is chemically modified and inactivated in a Michaelis-Menten type reaction via a reversible enzyme-Ro 15-4513 complex with a dissociation constant of 8.6 mM at pH 7.0. The inactivation reaction has been studied over the pH 6.0-10.0 range. The dissociation constants for the binding of Ro 15-4513 to the enzyme and the first-order rate constants for inactivation have been determined as a function of pH. These give pKa values of 7.2 and 8.8 for the free enzyme, the latter being assigned to the zinc-water ionization. The enzyme is protected from inactivation in a competitive manner by flumazenil and by many heterocyclic and thiol compounds which combine with the active-site zinc. Flumazenil has a similar binding affinity as Ro 15-4513 with an enzyme-flumazenil dissociation constant of 6.0 mM at pH 7.0. Ro 15-4513 may also have potential as a photoaffinity-labeling reagent for other metallo enzymes. Whether the effects of Ro 15-4513 on alcohol-metabolizing enzymes are also of clinical significance remains to be determined.

Drosophila alcohol dehydrogenase: stereoselective hydrogen transfer from ethanol.

Drosophila alcohol dehydrogenase shows a broad substrate specificity, with secondary alcohols being better substrates than primary alcohols. This specificity indicates that the active site contains two hydrophobic interaction sites and hence, a primary alcohol should be able to bind in two productive modes. This was tested by studying the activity of the enzyme with ethanol, [2H6]-ethanol and 1S-[2H1]-ethanol. An identical primary kinetic isotope effect of 2.5 for the two deuterated ethanols showed that deuterium was transferred from the enantiomeric 1S-[2H1]-ethanol to the coenzyme. Thus, ethanol interacts with only one hydrophobic region of the active site.