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R Bühler

Publications and source records attributed to R Bühler.

At least 37 records · Page 2Linked to original sources
PubMed1991

Double-antibody sandwich enzyme-linked immunosorbent assay for quantitation of endoglucanase I of Trichoderma reesei.

A sensitive and specific enzyme-liked immunosorbent assay for endoglucanase I (EG-I) has been developed. The monoclonal antibody a-EG-I 2, directed against an epitope on the core part of the enzyme, was used to capture the antigen in microtiter plate wells. A second, polyclonal antibody against the enzyme was then used to detect and quantitate the bound antigen. The test was specific for EG-I; neither endoglucanase II nor cellobiohydrolase I or II interfered. As little as 20 pg of EG-I protein could be detected. The coefficients of variation were 3.8% within plates and 6% between plates for a diluted Trichoderma reesei culture supernatant that contained 31 ng of EG-I per ml. Binding of the antigen to the monoclonal antibody was pH dependent and restricted to values between pH 6.5 and 10.5 with a maximum around pH 9. Standard solutions of EG-I were very stable at concentrations as low as 5 ng/ml when prepared in buffer that contained 1% bovine serum albumin and that was stored at -20 degrees C. After 37 weeks the antigenicity was still 97%. With this test it was possible to monitor the production of EG-I in a cellulase-producing strain of T. reesei and to demonstrate the apparent absence of the enzyme in a strain with the eglI gene deleted.

Animals↗
PubMed1991

Perivenous expression of ethanol-inducible cytochrome P450 IIE1 in livers from alcoholics and chronically ethanol-fed rats.

The acinar distribution of ethanol-inducible cytochrome P450 was studied by immunohistochemistry using anti-rat P450 IIE1 serum. In all 17 human liver specimens staining was confined to perivenous hepatocytes. Staining was stronger in livers of alcoholics than in non-alcoholics. A similar selective perivenous staining was observed in rat liver. This pattern was exacerbated by chronic ethanol treatment, with staining appearing especially intense in hepatocytes surrounding the terminal hepatic veins. Our results demonstrate that chronic ethanol intake causes induction of P450 IIE1 in the perivenous region. We propose that this regiospecific expression and induction contributes to the perivenous damage caused by alcohol and several other hepatotoxins known to be metabolized by this enzyme.

Adult↗
PubMed1990

Proteolysis of peripheral nerve myelin in acute experimental allergic neuritis.

Proteolysis of peripheral nerve myelin was studied in rats with experimental allergic neuritis (EAN). In vitro measurements using rat sciatic nerve homogenate and denatured bovine myelin as a substrate showed two myelin specific enzyme activities at pH 3.8 (inhibited by pepstatin) and pH 5.8 (inhibited by PMSF) in the normal rat and newly appearing activities at pH 2.8 (inhibited by pepstatin) and pH 5.0 (not characterized) in the EAN rat. In EAN the proteolytic activity was not restricted to myelin substrate but degraded total sciatic nerve protein as well. Endogenous sciatic nerve protease at pH 5.8 did not significantly change in activity during the course of disease. On the contrary, activity of acid protease at pH 2.8 corresponded well to the disease. Myelin degradation in EAN, therefore, appears to be mainly due to exogenous non-tissue protease.

Acute Disease↗
PubMed1988

Active-site-specific zinc-depleted and reconstituted cobalt(II) human-liver alcohol dehydrogenase. Preparation, characterization and complexation with NADH and trans-4-(N,N-dimethylamino)-cinnamaldehyde.

The active-site zinc atom of the beta 1 beta 1 isozyme of class I alcohol dehydrogenase (EC 1.1.1.1) from human liver was specifically removed by the chelating agent dipicolinic acid. From beta 1 gamma 1 and gamma 1 gamma 1 isozyme the active-site zinc is extracted much more slowly than from beta 1 beta 1 isozyme. Only partially active-site metal-depleted enzyme species were obtained from these isozymes. The active-site-specific reconstituted cobalt(II) derivative of the beta 1 beta 1 isozyme shows spectroscopic properties comparable to those of the active-site-specific reconstituted cobalt(II) horse liver alcohol dehydrogenase. The coenzyme-induced conformational change of the protein leads to a red shift of the d-d band from 648 nm to 673 nm. The chromophoric substrate trans-4-(N,N-dimethylamino)-cinnamaldehyde forms ternary complexes with NADH and the different isozymes, in close analogy to horse liver alcohol dehydrogenase. The differences in the active sites between beta 1 and gamma 1 subunits (threonine-48 instead of serine-48) or between zinc and cobalt(II) are reflected in the visible absorption spectra of the metal-bound chromophoric substrate.

Alcohol Dehydrogenase↗
PubMed1985

Structural relationships among class I isozymes of human liver alcohol dehydrogenase.

The alpha subunit of human liver alcohol dehydrogenase has been submitted to structural analysis. Together with earlier work on the beta and gamma subunits, the results allow conclusions on the relationship of all known forms of the class I type of the enzyme. Two segments of the alpha subunit were determined; one was also reinvestigated in the beta and gamma subunits. The results establish 11 residue replacements among class I subunits in the segments analyzed and show that the alpha, beta, and gamma protein chains each are structurally distinct in the active site regions, where replacements affect positions influencing coenzyme binding (position 47; Gly in alpha, Arg in beta and gamma) and substrate specificity (position 48; Thr in alpha and beta, Ser in gamma). Residue 128, previously not detected in beta and gamma subunits, corresponds to a position of another isozyme difference (Arg in beta and gamma, Ser in alpha). The many amino acid replacements in alcohol dehydrogenases even at their active sites illustrate that in judgements of enzyme functions absolute importance of single residues should not be overemphasized. Available data suggest that alpha and gamma are the more dissimilar forms within the family of the three class I subunits that have resulted from two gene duplications. The class distinction of alcohol dehydrogenases previously suggested from enzymatic, electrophoretic, and immunological properties therefore also holds true in relation to their structures.

Alcohol Dehydrogenase↗
PubMed1985

Tumor-derived angiogenesis factors from rat Walker 256 carcinoma: an experimental investigation and review.

Angiogenesis, the process of developing a hemovascular network, is an essential feature of the growth of solid tumors, and is induced by factors secreted by tumor cells. Assay procedures suitable for the investigation of angiogenesis, and for the screening of angiogenesis factors during purification are reviewed; and a number of reports describing the purification of angiogenesis factors, primarily from the rat Walker 256 carcinoma as starting material, are discussed. Work from the authors' laboratory is also presented. Walker 256 cells grown in large-scale culture were the source of a reproducible and homogeneous source of angiogenic material. Factors secreted by these cells were isolated by a series of chromatographic steps. Ion exchange chromatography on carboxymethyl-Sephadex produced two active fractions, one of which was fractionated into several macromolecular species by lectin affinity and hydrophobic adsorption chromatography. The other gave a high mol.wt, active fraction that was resolved into a low mol.wt, active component and a non-angiogenic but possibly carrier molecule with a mol.wt of 140,000. While none of the angiogenic factors were identified chemically, the results demonstrate the existence of both high and low mol.wt tumor-secreted angiogenic substances, confirming the hypothesis for tumor-induced angiogenesis and predicting potential means to interfere with the process of tumor growth.

Amnion↗
PubMed1985

Increased ethanol consumption and blood ethanol levels in rats with portacaval shunts.

In a series of experiments, it was demonstrated that male rats with end-to-side portacaval shunts (PCS) consumed more ethanol and exhibited higher blood ethanol levels than sham-operated control animals in chronic tests with 2% ethanol and water ad libitum. Ethanol intake in the 6 h prior to blood sampling was 2-5 times and blood ethanol 10-50 times higher in PCS than control rats. These effects were not due to the feminization of male rats occurring after a PCS, since female PCS rats exhibited comparable increases of ethanol intake and blood ethanol. In both sexes ethanol elimination rate and alcohol dehydrogenase activity per total liver were lower after PCS than in control rats, explaining the disproportionate increase in blood ethanol relative to ethanol intake. Interestingly, ethanol intake was not abnormal in PCS rats fed a low-protein, low-tryptophan diet (corn) alone or as a supplement to the usual chow diet. Such dietary modulation of ethanol preference in this animal model of chronic liver dysfunction merits further attention.

Alcohol Drinking↗
PubMed1984

Human liver alcohol dehydrogenase. 1. The primary structure of the beta 1 beta 1 isoenzyme.

Determination of the amino acid sequence of the beta 1 subunit from the class I (pyrazole-sensitive) human liver alcohol dehydrogenase isoenzyme beta 1 beta 1 revealed a 373-residue structure differing at 48 positions (including a gap) from that of the subunit of the well studied horse liver alcohol dehydrogenase EE isoenzyme. The structure deduced is compatible with known differences in composition, ultraviolet absorbance, electrophoretic mobility and catalytic properties between the horse and human enzymes. All zinc-liganding residues of the horse E subunit are strictly conserved in the human beta 1 subunit, despite an earlier report of a mutation involving Cys-46. This residue therefore remains conserved in all known alcohol dehydrogenase structures. However, the total cysteine content of the beta 1 structure is raised from 14 in the subunit of the horse enzyme to 15 by a Tyr----Cys exchange. Most exchanges are on the surface of the molecule and of a well conserved nature. Substitutions close to the catalytic centre are of interest to explain the altered substrate specificity and different catalytic activity of the beta 1 homodimer. Functionally, a Ser----Thr exchange at position 48 appears to be of special importance, since Thr-48 in beta 1 instead of Ser-48 in the horse enzyme can restrict available space. Four other substitutions also line the active-site pocket, and appear to constitute partly compensated exchanges.

Alcohol Dehydrogenase↗
PubMed1984

Human liver alcohol dehydrogenase. 2. The primary structure of the gamma 1 protein chain.

The primary structure of the gamma 1 subunit of human liver alcohol dehydrogenase isoenzyme gamma 1 gamma 1 was deduced by characterization of 36 tryptic and 2 CNBr peptides. The polypeptide chain is composed of 373 amino acid residues. gamma 1 differs from the beta 1 subunit of human liver alcohol dehydrogenase at 21 positions, and from the E subunit of horse liver alcohol dehydrogenase at 43 positions including a gap at position 128 as in the beta 1 subunit. All zinc-liganding residues from the E subunit of the horse protein and the beta 1 subunit of the human enzyme are conserved, but like beta 1, gamma 1 also has an additional cysteine residue at position 286 (in the positional numbering system of the horse enzyme) due to a Tyr----Cys exchange. Most amino acid exchanges preserve the properties of the residues affected and are largely located on the surface of the molecules, away from the active site and the coenzyme binding region. However, eight positions with charge differences in relation to the E subunit of the horse enzyme are noticed. These result in a net positive charge increase of one in gamma 1 versus E, explaining the electrophoretic mobilities on starch gels. Of functional significance is the conservation of Ser-48 in gamma 1 relative to E. The residue is close to the active site but different (Thr-48) in the beta 1 subunit of the human enzyme. Thus, the closer structural relationship between human gamma 1 and horse E enzyme subunit than between beta 1 and E is also reflected in functionally important residues, explaining a greater similarity between gamma 1 gamma 1 and EE than between beta 1 beta 1 and EE.

Alcohol Dehydrogenase↗
PubMed1984

Differential susceptibility of human alcohol dehydrogenase isoenzymes to anions.

Human liver alcohol dehydrogenase (ADH) isoenzymes beta 1 beta 1, gamma 1 gamma 1 from Caucasian individuals with 'typical' ADH and beta 2 beta 2-Bern from Caucasian individuals with 'atypical' phenotype differed in their susceptibility to anions. At pH 7.0 beta 1 beta 1 and gamma 1 gamma 1 were more active in Tris-HCl buffer than in sodium phosphate buffer but less active in Hepes-NaOH and Mops-NaOH. beta 2 beta 2-Bern showed the same activity in all these buffers. At pH 7.0 and at low concentrations (50-100 mM) chloride activated the ethanol oxidation by beta 1 beta 1 and gamma 1 gamma 1, whereas sulfate showed no effect. At anion concentrations above 100 mM all isoenzymes were inhibited. At pH 10.5 beta 1 beta 1 and gamma 1 gamma 1 were not activated. Measuring the acetaldehyde reduction, no comparable activation by chloride was observed; all three isoenzymes were inhibited, at significantly lower anion concentrations. These anion effects can be correlated with the different primary structures of the isoenzymes around the active site and the coenzyme binding site.

Alcohol Dehydrogenase↗
PubMed1984

Atypical human liver alcohol dehydrogenase: the beta 2-Bern subunit has an amino acid exchange that is identical to the one in the beta 2-Oriental chain.

The "atypical' human liver alcohol dehydrogenase dimer, homogeneous for beta 2-Bern chains, was isolated from human liver of Caucasian individuals. It is derived from an allelic variant at the ADH2 gene locus and exhibits a considerably higher specific activity and lower pH optimum than its "typical' counterpart (isoenzyme beta 1 beta 1) from the beta 1-chain predominant in Caucasians. Peptides were prepared by trypsin or CNBr cleavage, and were purified by exclusion chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Structural analysis of the peptides showed that beta 2-Bern differs at one position from beta 1. Thus, Arg-47 in beta 1 is substituted by His in beta 2-Bern. This exchange, compatible with a one-base mutation, explains all functional differences by altered interactions with the pyrophosphate moiety of the coenzyme. The difference is also structurally identical to that found for another atypical beta 2-subunit, the beta 2-Oriental type of major Asian occurrence, linking these two atypical forms of human alcohol dehydrogenase.

Alcohol Dehydrogenase↗
PubMed1984

Molecular cloning and characterization of a cDNA for the beta subunit of human alcohol dehydrogenase.

Human alcohol dehydrogenase (ADH) is encoded by at least five genes that fall into three classes. The class I ADH genes encode the three closely related alpha, beta, and gamma polypeptides. Molecular genetic analysis of class I ADH genes has been initiated by isolating a cDNA clone from a human adult liver cDNA library. A synthetic oligonucleotide mixture encoding a portion of the beta subunit of ADH was used as an in situ hybridization probe for the cDNA library. One positively hybridizing clone, pADH12, which contained an 1100-base-pair cDNA insert, was subjected to DNA sequence analysis. The sequence indicated that the cDNA encoded information for the carboxyl-terminal 91 amino acids of a class I ADH and a 3' untranslated region of 593 nucleotides. Comparisons with the carboxyl terminus of the human ADH beta subunit indicated that the cDNA encoded the beta polypeptide. This probe may facilitate genetic studies of various human alcohol-related syndromes, as well as enable basic molecular studies on human ADH gene expression.

Adult↗
PubMed1984

Human alcohol dehydrogenase: structural differences between the beta and gamma subunits suggest parallel duplications in isoenzyme evolution and predominant expression of separate gene descendants in livers of different mammals.

Human alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) occurs in multiple forms, which exhibit distinct electrophoretic mobilities and enzymatic properties. The homogeneous isoenzymes beta 1 beta 1 and gamma 1 gamma 1 were isolated from livers of Caucasians with "typical" ADH phenotype by double ternary complex affinity chromatography and ion exchange chromatography. The differences between the beta 1 and gamma 1 subunits were determined by structural analysis of all tryptic peptides from the carboxymethylated proteins. The human beta 1 and gamma 1 chains differ at 21 of the 373 positions (5.6%). Ten tryptic peptides account for the differences. All residue substitutions are compatible with one-base mutations and result in largely unaltered properties, but five lead to charge differences. Sixteen substitutions are at positions corresponding to the catalytic domain of the well-known horse enzyme; five correspond to the coenzyme-binding domain. Substitutions adjacent to important regions may correlate with differences in coenzyme binding, substrate specificities, and active-site relationships. The residue replacements between the beta 1 and gamma 1 subunits of human ADH are not identical to the known substitutions between ethanol-active (E) and steroid-active (S) subunits of horse ADH. Thus, the duplication leading to human beta 1 and gamma 1 subunits is separate and different from that leading to equine E and S subunits. Both duplications are likely to have occurred after the ancestral separation of human and equine ADH. Of the 21 residues that are different between beta 1/gamma 1, 13 in gamma 1 but only 6 in beta 1 are identical to those of the horse E chain. This suggests a closer relationship between gamma 1 and E, although beta 1 in man and E in the horse are the subunits recovered in highest yield from liver ADH preparations. Consequently, in these two mammalian species, relative activities of genes for an isoenzyme family appear to be different.

Alcohol Dehydrogenase↗
PubMed1984

Biology of disease. Alcoholism and aldehydism: new biomedical concepts.

New results of biomedical research in alcoholism show great interindividual, as well as racial, variability with respect to metabolism of alcohol and its first oxidation product, acetaldehyde. Genetic factors play an important part. The enzymes of alcohol and aldehyde metabolism exhibit a genetically determined heterogeneity (isoenzymes and enzyme polymorphisms). This leads to a large variety of individually different enzyme phenotypes. Thus, the hypothesis is put forward that the individual and racial differences in alcohol metabolism are based on the genetically determined variability of the participating enzymes, alcohol dehydrogenase and aldehyde dehydrogenase. Alcohol metabolism and alcohol-induced disturbances of the intermediary metabolism are closely interrelated. Hence, genetic codetermination can also be expected in this regard. As a toxic intermediate of alcohol metabolism, acetaldehyde plays a central role. Three positive ranges of acetaldehyde levels can be defined: (a) the normal range, (b) the "acute aldehyde syndrome" with extremely high levels of acetaldehyde, (c) "chronic aldehydism" with slightly elevated acetaldehyde levels. In Orientals lacking the mitochondrial low Km aldehyde dehydrogenase, acetaldehyde accumulates and produces symptoms of intoxication. This acute aldehyde syndrome is highly aversive and, thus, prevents these individuals from drinking. The effect is similar to the consequences of the inhibition of aldehyde dehydrogenase with disulfiram, a commonly employed drug in the treatment of alcoholics. In alcoholics slightly elevated levels of blood acetaldehyde are observed. There are indications that this chronic aldehydism is not only the consequence of excessive drinking but may reflect a preexisting enzyme pattern that is genetically determined. Therefore, slightly elevated acetaldehyde concentrations could serve as a biologic marker for high risk drinking. Alcohol dehydrogenase is found in many organs and often in specialized cells within a particular tissue. A specific enzyme pattern in these cells could lead to elevated acetaldehyde concentrations, consequently inducing acetaldehyde-related damage. Such organs could represent direct targets even at low blood acetaldehyde levels.

Acetaldehyde↗
PubMed1983

Immunohistochemical localization of alcohol dehydrogenase in human kidney, endocrine organs and brain.

Antibodies against human liver alcohol dehydrogenase (ADH) were produced in rabbits. Peroxidase-labeled protein-A with diaminobenzidine as substrate was used to detect anti-ADH binding in human tissue thin sections. In the kidney, ADH was localized in the epithelia of the tubuli; glomeruli and collecting tubules appeared negative. In prostata and epididymis, the epithelia stained strongly. In the testes, the seminiferous epithelium and the Leydig cells stained higher in the cortex than in the medulla. In the pancreas, the Langerhans islets exhibited particularly high ADH concentrations. In the brain, ADH was localized in neurons of the cerebral cortex, hypothalamus, infundibular stalk of the pituitary, and Purkinje cells of the cerebellum. In summary, ADH could be localized primarily in cells known as targets of ethanol toxicity.

Adrenal Glands↗
PubMed1983

The polymorphisms of alcohol and aldehyde dehydrogenase and their significance for acetaldehyde toxicity.

Evidence is growing that acetaldehyde is responsible for some toxic effects after ethanol intake. Large individual and racial differences in blood and breath acetaldehyde concentrations are observed after alcohol consumption. In many Orientals but few Caucasians extremely high blood acetaldehyde levels occur leading to an acute aldehyde syndrome also observed after treatment with aldehyde dehydrogenase inhibitors. Individuals suffering from the aversive symptoms of that syndrome will be protected from excessive drinking and the related problems. In chronic aldehydism slightly elevated aldehyde concentrations are observed possibly leading to organic injury due to the cytotoxic action of acetaldehyde. Sites exhibiting high alcohol dehydrogenase activity may specifically be affected in alcoholics.

Acetaldehyde↗
PubMed1983

Immunohistochemical localization of alcohol dehydrogenase in the human gastrointestinal tract.

To elucidate the possible role of alcohol dehydrogenase in the generation of damages caused by alcohol abuse, it is important to know the exact localization of this enzyme. Anti-alcohol dehydrogenase antibodies were used to localize the enzyme in human gastrointestinal tissues. Alcohol dehydrogenase was detectable in the mucosa of all parts of the gastrointestinal tract. There is a notable decrease in the amount of alcohol dehydrogenase with increasing distance from the stomach. In the stomach, the reaction for alcohol dehydrogenase was strongest in the mucus-producing cells, moderate in the parietal cells, and weak in the chief cells. Throughout the gut, alcohol dehydrogenase was present in cells exposed to the lumen in amounts comparable to that of the parietal cells. Crypt cells and goblet cells were negative. The results strongly indicate that alcohol dehydrogenase is an intrinsic component of gastrointestinal epithelial cells.

Alcohol Oxidoreductases↗