ABSTRACT
A product of p-xylene auto-oxidation, p-methylbenzyl hydroperoxide, acts as a very strong reversible inhibitor of the ethanol dehydrogenating activity of horse liver alcohol dehydrogenase. Concentrations of hydroperoxide as low as that of the enzyme active site (about 10(-8) mol.dm-3) in the assay depresses the activity by 50%. Somewhat less potent is benzyl hydroperoxide (derived from toluene) while the (secondary) hydroperoxide derived from ethylbenzene and tert.butyl hydroperoxide and cumyl hydroperoxide do not inhibit HLAD appreciably.
Subject(s)
Alcohol Dehydrogenase/antagonists & inhibitors , Benzyl Compounds/pharmacology , Liver/enzymology , Animals , Horses , In Vitro Techniques , Kinetics , Peroxides/pharmacologyABSTRACT
The strong inhibition of horse liver alcohol dehydrogenase (HLAD) by p-methylbenzyl hydroperoxide (XyHP) is only transient, XyHP behaves also as a pseudo-substrate of the enzyme and in the presence of NAD+, is degraded by HLAD to (as yet unidentified) non-inhibiting products while the NAD+ is converted to a derivative similar to the "NADX", originally observed in an analogous reaction of HLAD with hydrogen peroxide. The apparent KM for XyHP is approximately 10(4) times smaller than that for H2O2. The catalytic constant kcat for HLAD degradation of XyHP is two orders of magnitude less than that for ethanol dehydrogenation. XyHP inhibits both directions of the alcohol-aldehyde interconversion with equal potency. The first step of the inhibition mechanism is a tight binding of XyHP to the binary HLAD-NAD+ complex.
Subject(s)
Alcohol Dehydrogenase/antagonists & inhibitors , Benzyl Compounds/pharmacology , Liver/enzymology , NAD/metabolism , Acetaldehyde/metabolism , Animals , Benzyl Compounds/metabolism , Ethanol/metabolism , Horses , Oxidation-Reduction/drug effectsABSTRACT
The decomposition of p-methylbenzyl hydroperoxide by cytochrome c and other selected heme systems in the absence of reducing agents was investigated. p-Methylbenzaldehyde was identified as the major product. A mechanism for this reaction has been suggested. H2O2 and tertiary cumyl hydroperoxide do not react under these conditions. The ability of organic hydroperoxides to act as oxygen donors in the cytochrome-mediated 7-ethoxyresorufin-O-deethylation was studied. Cumyl and tert.butyl hydroperoxides are able to substitute oxygen in the absence of NADPH while p-methylbenzyl hydroperoxide is not.
Subject(s)
Hemeproteins/chemistry , Peroxides/chemistry , Alcohol Dehydrogenase/antagonists & inhibitors , Animals , Benzene Derivatives/chemistry , Benzyl Compounds/chemistry , Chick Embryo , Chickens , Chromatography, Thin Layer , Cytochrome c Group/metabolism , Eggs/analysis , Methemoglobin/chemistry , NADP/metabolism , Protein Binding , Spectrophotometry, Infrared , Spectrophotometry, UltravioletABSTRACT
Total and specific alcohol dehydrogenase activity has been compared in homogenates of 19 different types of human tissues from different sources. ADH activities were detected in tissues which have not been tested yet, e.g., thyroid gland, adrenal gland, fat tissue, skin tissue, peritoneal membrane, breast tissue, duodenum, and gall bladder. Healthy and pathological human tissue differ in their ADH activity. The percentage of the total activity has been estimated in each tested organ in relation to the total activity of the whole body.
Subject(s)
Alcohol Oxidoreductases/metabolism , Liver/enzymology , Alcohol Dehydrogenase , Humans , Liver Diseases/enzymology , Tissue DistributionABSTRACT
The proposed method for ethanol determination is based on the simultaneous oxidation of ethanol and reduction of nitrosodimethylaniline to a quinonediimine derivative in the presence of NAD and horse liver alcohol dehydrogenase. Quinonediimine formed in this reaction is coupled with salicylamide and the absorbance of the resulting blue indaniline dye is measured. The new method yields identical results when compared with the currently used methods for the determination of blood alcohol (i.e., with the conventional enzymatic method, Widmark's method, and gas chromatography). Its main advantage is high sensitivity, little consumption of both enzyme and coenzyme, and the measurement in the visible range of the spectrum.
Subject(s)
Ethanol/blood , Nitroso Compounds , Spectrophotometry/methods , Alcohol Oxidoreductases/metabolism , Aniline Compounds/analysis , Chromatography, Gas , Coloring Agents , Cyclohexenes , Ethanol/metabolism , Humans , NAD/metabolism , Nitroso Compounds/metabolism , Oxidation-Reduction , Salicylamides , Spectrophotometry, UltravioletSubject(s)
Alcohol Oxidoreductases/blood , Alcoholism/diagnosis , Clinical Enzyme Tests , Disease Susceptibility , Humans , MaleABSTRACT
Alcohol dehydrogenase has been immobilized to the basic copolymer and its several derivatives using various techniques. Enzyme coupling to the supports with amino groups by means of glutaraldehyde was found the most suitable. Activity of alcohol dehydrogenase coupled to these amino supports was comparable to that of the enzyme bound to Sepharose. Thermal and pH stability of alcohol dehydrogenase increased essentially upon immobilization. Kinetic properties of the immobilized enzyme differed from those of free alcohol dehydrogenase, pH optimum shifted to alkaline range, and apparent Michaelis constants for substrates and coenzymes increased. Curvatures observed in Lineweaver-Burk plots for coenzymes suggest an involvement of diffusion effects in the reaction catalyzed by alcohol dehydrogenase linked to these polymers.
ABSTRACT
The normal level of human serum alcohol dehydrogenase (EC 1.1.1.1) (ADH) activity which is not measurable by conventional methods was found to be within the range 0.07-0.56 U/1 when measured by a sensitive method based on a coenzyme recycling reaction. In different liver diseases the normal upper limit of serum ADH activity was found to be exceeded up to 70 times. Although ADH activity under pathological conditions usually parallels that of other enzymes, e.g., sorbitol dehydrogenase (EC 1.1.1.14) (SDH) and alanine transaminase (EC 2.6.1.2) (ALT), its relative elevation above the upper normal limit is generally greater, particularly in the early stages of viral hepatitis. Observations on some patients also suggested that very early stages of liver damage, caused by drugs or secondary malignancy, could be detected by increases of serum ADH activity when the activities of some other liver specific enzymes were still within their normal values. A pilot experiment on rats, intoxicated with carbon tetrachloride, showed that serum ADH activity could reflect acute liver parenchymal damage more sensitively than SDH and ALT activity.
Subject(s)
Alcohol Oxidoreductases/blood , Liver Diseases/enzymology , Adolescent , Adult , Aged , Animals , Carbon Tetrachloride Poisoning/enzymology , Enzymes/blood , Female , Hepatitis, Viral, Human/enzymology , Humans , Male , Middle Aged , Rats , Rats, Inbred StrainsABSTRACT
Serum alcohol dehydrogenase (ADH) activity in some individuals contains a fraction that is not inhibited by pyrazole. This might be due to the presence in those sera of pi-ADH, a unique isoenzyme found recently in human liver, and which, according to one hypothesis, could be co-responsible for the development of alcohol dependence.
Subject(s)
Alcohol Oxidoreductases/blood , Isoenzymes/blood , Pyrazoles/pharmacology , Alcohol Oxidoreductases/antagonists & inhibitors , Dose-Response Relationship, Drug , HumansABSTRACT
The interactions of three groups of probes (berberine alkaloids, tricyclic psychopharmaca and acridine derivatives) with isoenzymes of horse liver alcohol dehydrogenase and with rat liver alcohol dehydrogenase have been examined. These compounds inhibit the activity of the EE isoenzyme of horse liver alcohol dehydrogenase but differ in their behaviour towards the steroid-active enzymes (i.e. the ES isoenzyme of horse liver alcohol dehydrognase and alcohol dehydrogenase from rat liver): psychopharmaca inhibit, acridines activate and berberines do not bind. The ligands differ also in their influence on the modification of the EE isoenzyme by iodoacetate. Polarities (expressed as Kosower's Z values) of the respective binding sites on the EE isoenzyme were estimated from optical properties of bound probes. Berberines bind into a very hydrophobic area of the enzyme molecule, the binding site for psychopharmaca is moderately hydrophobic and that for acridines is rather polar. Steric arrangements of the binding sites are also discussed. The data presented confirm the existence of three distinct binding sites for these ligands in the substrate pocket of liver alcohol dehydrogenase.
Subject(s)
Alcohol Oxidoreductases/metabolism , Liver/enzymology , Acridines/pharmacology , Alkaloids/pharmacology , Animals , Binding Sites , Horses , Iodoacetates/pharmacology , Isoenzymes/metabolism , Kinetics , Ligands , Protein Binding , Rats , Spectrometry, Fluorescence , Substrate SpecificitySubject(s)
Alcohol Oxidoreductases/blood , Animals , Horses , Humans , Kinetics , Liver/enzymology , NAD , Pyrazoles , SpectrophotometrySubject(s)
Alcohol Oxidoreductases , Chlorpromazine , Tranquilizing Agents , Alcohol Oxidoreductases/metabolism , Animals , Binding Sites , Binding, Competitive , Chlorpromazine/pharmacology , Dibenzothiepins/pharmacology , Dothiepin/pharmacology , Horses , Hydrogen-Ion Concentration , Kinetics , Protein Binding , Spectrophotometry, Ultraviolet , Tranquilizing Agents/pharmacologySubject(s)
Alcohol Oxidoreductases , Berberine Alkaloids , Liver/enzymology , Adenosine Diphosphate , Binding Sites , Binding, Competitive , Chemical Phenomena , Chemistry , Dioxoles , Ethanol , Fatty Acids , Kinetics , Ligands , Macromolecular Substances , Mathematics , Molecular Conformation , NAD , Phenanthrolines , Pyrazoles , Ribose , Spectrometry, FluorescenceABSTRACT
Ricinine-3,5-(14)C (N-methyl-3-cyano-4-methoxy-2-pyridone) administered to senescent leaves of Ricinus communis L. was translocated to all other tissues of the plant. Developing fruit and especially seeds were found to be labeled the most rapidly. Young growing leaves and other developing tissues of the plant imported ricinine from the senescent leaves much more quickly than mature leaves. Relative intensities of the radioactive ricinine imported and deposited in various tissues indicate a possible functional role of ricinine in the castor bean plant. Data on N-demethyl ricinine presented here may stimulate interest in the possible physiological role of the ricinine to N-demethyl ricinine interconversion.
