Site-directed mutagenesis of mammalian alcohol and sorbitol dehydrogenases map functional differences within the enzyme family.
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Alcohol Dehydrogenase↗
Biomedical subjects
C Karlsson
Publications and source records attributed to C Karlsson.
Pre-adsorption of a cellulose ether onto polymer surfaces: adsorption of adhesins and platelet activation.
The effect of pre-adsorbed cellulose ethers upon the adsorption of plasma proteins at polymer surfaces was measured by an enzyme-linked immunosorbent assay. The resulting adsorption of plasma proteins was correlated with the sticking and activation of platelets at the blood-material interface, measured by scanning electron microscopy and release of beta-thromboglobulin. Retained Ca2+ activity was made possible by hirudin, a specific thrombin inhibitor, from medicinal leeches as an anticoagulant drug, thereby keeping the initial coagulation cascade and calcium-dependent cell-protein interactions intact during the experiments. The results show that pre-adsorption of a hydrophobic cellulose ether with a flocculation temperature < 37 degrees C, on polyurethane and polytetrafluoroethylene, decreased the adsorption of fibrinogen, fibronectin and vitronectin. A corresponding decrease was found in the number of cells sticking to the surface and in the release of beta-thromboglobulin from platelets.
Adsorption↗
Kinetics supramolecular structure and equilibrium properties of fibrinogen adsorption at liquid-solid interfaces.
Adsorption of fibrinogen onto hydrophobic and hydrophilic quartz surfaces was studied by ellipsometry and transmission electron microscopy (TEM) of negatively stained proteins. The initial adsorption at the hydrophobic surface, measured by ellipsometry, can be described by an apparent forward rate constant k1 of 2 x 10(4) M-1 s-1. This constant was time-dependent and is therefore considered as a rate coefficient. The apparent forward rate coefficient of adsorption to a hydrophilic surface was both time-dependent and concentration-dependent, indicating a history-dependent process of adsorption. Plateau levels of adsorption were concentration-dependent and lower at the hydrophilic quartz surface (1.2 pmol/cm2) than at the hydrophobic surface (1.8 pmol/cm2). These surface concentrations correspond to rather tight-packed monolayers of molecules adsorbed end-on. The initial desorption can be described by a first order rate constant (k-1 approximately 10(-4) s-1), down to 80-90% of the initial surface concentration. The dissociation rate then decreased (k-1 approximately 10(-6) s-1) resulting in an apparently stable level of adsorbed protein. Slow changes of the binding strength of adsorbed proteins was seen during 24-72 h adsorption time. Deviations from an ideal equilibrium isotherm were seen both in the time dependence and as concavities in a Scatchard plot, suggesting intermolecular cooperativity. At low bulk concentrations a heterogeneous distribution of fibrinogen molecules was found at the surface below monolayer coverage. The supramolecular structure was characterized by the formation of end-to-end dimers and trimers laying down at the surface. At higher surface concentration adsorbed molecules showed polycrystalline structure with repeated nearest neighbor distances at 16 nm. The distribution of adsorbed fibrinogen molecules indicates that surface-adsorbed fibrinogen may form a two-phase system, containing significant amounts of water. The atypical kinetics and concentration dependence of fibrinogen adsorption may thus be due to properties of a two-dimensional phase separation from a three-dimensional liquid bulk.
Adsorption↗
Sorbitol dehydrogenase: cDNA coding for the rat enzyme. Variations within the alcohol dehydrogenase family independent of quaternary structure and metal content.
Two separate cDNA-clones, together coding for rat sorbitol dehydrogenase, have been isolated from a liver cDNA library in lambda gt11 by screening with oligonucleotide probes. One clone contained a 1020-bp fragment starting at the codon for amino acid residue 104 and ending with a 261-bp 3' non-coding region, the second encompassed the entire 5' region and ended with a 3' truncation corresponding to amino acid residue 315. The coding region consists of 356 amino acid residues, one more than in the human and sheep enzymes. The presence of the extra residue at position 3, a proline, can be explained by a shifted splice point in the mRNA. The primary structure of rat sorbitol dehydrogenase allows triplet comparisons of three distinct rat-ungulate-human enzymes differing in quaternary structure and metal content within the zinc-containing alcohol dehydrogenase family. The variability of sorbitol dehydrogenase (tetramer with one zinc atom/subunit; no activity towards ethanol) is large (18%), exactly like that for the class I alcohol dehydrogenase (dimer with two zinc atoms/subunit; no activity towards sorbitol), differing threefold from that of the class III alcohol dehydrogenase/glutathione-dependent formaldehyde dehydrogenase (dimer with two zinc atoms/subunit; 6% variability) suggesting that the distinct extents of variability within this protein family are independent of substrate specificity, metal content and quaternary structure.
Alcohol Dehydrogenase↗
Determination of nitecapone and (13C6)nitecapone in human plasma by gas chromatography/mass spectrometry.
A gas chromatographic/mass spectrometric method is developed and validated for simultaneous determination of nitecapone and its 13C6-labelled analogue in human plasma using (2H6,13C6)nitecapone as internal standard. The method involves extraction of the analytes from plasma to ethyl acetate-hexane mixture (20:80) and conversion to bis(trimethylsilyl) ethers prior to determination by gas chromatography/mass spectrometry using selected ion monitoring. The quantification range is 0.5-2000 ng ml-1. Precision ranges from 11.3% (coefficient of variation) at low levels to 2.4% at high levels. Recovery is about 50% in the whole range. The method is applied to a pharmacokinetic study where nitecapone diluted with 14C-labelled nitecapone is given intravenously concomitantly with an oral dose of (13C6)nitecapone.
Carbon Isotopes↗
Avian alcohol dehydrogenase: the chicken liver enzyme. Primary structure, cDNA-cloning, and relationships to other alcohol dehydrogenases.
The major ethanol-active form of chicken liver alcohol dehydrogenase was characterized. The primary structure was determined by peptide analysis and, to a large part, was also deduced by cDNA analysis of a near full-length cDNA clone. The latter was detected by screening of a chicken liver cDNA library with antibodies raised against the purified dehydrogenase. The structure shows that the avian enzyme exhibits characteristics of the complex mammalian alcohol dehydrogenase system, tracing its origin and divergence, and allowing functional correlations. The chicken protein analyzed proves to be a class I alcohol dehydrogenase, with 74% residue identity to gamma chains of the human enzyme, a Km for ethanol of 0.5 mM and a Ki for 4-methyl pyrazole of 2.5 microM. Relationships to the other two classes are non-identical; residue exchanges towards the human classes increase in the order I less than III less than II, and human/chicken differences are less than inter-class differences. Consequently, the origins of the classes are more distant than the avian/mammalian separation. They reflect duplicatory events separated in time, and the lines that lead to present-day classes I and II deviate early. Integrated with the data for the quail enzyme, the structure of the chicken protein shows that within the avian enzymes the degree of variation is comparable to that within the mammalian class I enzymes, which are more variable than the class III forms. The coenzyme-binding and substrate-binding residues of this chicken alcohol dehydrogenase are largely identical to those in the mammalian class I counterparts. However, the subunit-interacting areas are more variable and suggest some relationships of the avian enzyme with both class I and III mammalian forms. One of the residues, Gly260 (mammalian class I numbering system), previously considered characteristic of all alcohol dehydrogenases, is replaced by Gln.
Alcohol Dehydrogenase↗
[When Carina became SHSTF representative it brought the profession closer to the membership. Interview by Jan Thomasson].
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Health Occupations↗
Variability within mammalian sorbitol dehydrogenases. The primary structure of the human liver enzyme.
The primary structure of sorbitol dehydrogenase from human liver has been determined by peptide analysis in order to relate the variability of this enzyme to that of the others within the alcohol dehydrogenase family. The structure obtained reveals 355 residues with an acyl-blocked N-terminus and an unexpected microheterogeneity at position 237 (Gln/Leu). The residue identity between sheep and human liver sorbitol dehydrogenase is 89%. This variability is similar to that of class I alcohol dehydrogenases, but distinctly different from that of class III alcohol dehydrogenases, the structures of which are much more conserved. Consequently, class III alcohol dehydrogenase is thus far unique within this family of dehydrogenases, suggesting a particularly strict requirement for that structure. The variability within sorbitol dehydrogenase involves all segments of the molecule but is largely at surface positions and clusters in one such region, covering positions 214-240, corresponding to a segment of the coenzyme-binding domain. Ligands to the active-site zinc and most residues lining the coenzyme-binding and substrate-binding pockets are conserved. However, provided conformational models are reliable, a charge difference may affect the interactions at the inner part of the substrate pocket, another charge difference may affect the interdomain region, and a size difference the adenine pocket. The primary structure of human liver sorbitol dehydrogenase further shows that the absence of three of the four ligands to a second zinc atom present in alcohol dehydrogenases is a general property of sorbitol dehydrogenase.
Amino Acid Sequence↗
Use of cellulose ethers as peptide antigen carriers in the ELISA.
A procedure for surface immobilisation of peptides is described. Insulin, a model for peptide antigens, was covalently coupled to alkyl-hydroxyalkyl-cellulose ethers. The cellulose-insulin conjugate was then adsorbed to the plastic surface of microtitre wells and was used as antigen in an ELISA assay. The adsorbed conjugate was shown to be stable in undiluted plasma or serum whereas adsorbed insulin was removed from the surface by incubation in undiluted serum or plasma. Adsorption of serum albumin or cellulose ether polymers to the microtitre plates followed by incubation with whole blood, showed that adsorbed albumin but not the cellulose ether was exchanged by fibrinogen at the surface. The results indicate that coupling of peptides to alkyl-hydroxyalkyl-cellulose ethers is an efficient means of immobilising peptide antigens to hydrophobic surfaces.
Antigens↗
Indoor radon exposure and active and passive smoking in relation to the occurrence of lung cancer.
Exposure to indoor radon and radon daughters is currently attracting great interest as a possible cause of lung cancer. This concern is supported by several studies, most of them relatively small in numbers or weak in the assessment of exposure. This study encompasses 177 persons with lung cancer and 677 noncancer referents, all deceased and with 30 years or more of residency in the same house in an area with radon-leaking alum shale deposits in the central part of southern Sweden. Exposure categories based on building material, type of house, and ground conditions were created, but measurements of the indoor radon daughter concentration were also made for 142 cases and 264 referents. Active and passive smoking was ascertained through questionnaires sent to the next-of-kin. Overall, the lung cancer risk was approximately twofold with regard to the categories of assumed radon daughter exposure for the rural sector of the population but not for the same categories of the urban sector, possibly because of less precise exposure assessment and influence from other factors. Occasional and passive smokers, as well as passive smokers alone, had a particularly increased risk of lung cancer in association with the increased exposure categories.
Adult↗
Blood-ocular and blood-brain barrier function in streptozocin-induced diabetes in rats.
Edetic acid labeled with chromium 51 was injected intravenously in normal rats and in rats with streptozocin-induced diabetes. One hour after the injection the animals were killed and the concentrations of edetic acid 51Cr in vitreous body, retina, and brain were determined. No significant difference was observed between the two groups for either tissue. In a second series, a mixture of tritiated 1-glucose and aminohippuric acid tagged with carbon 14 was injected instead of edetic acid. A substantial accumulation of aminohippuric acid 14C compared with tritiated 1-glucose was observed in the vitreous body and the brain of diabetic rats in comparison with the control group. It is concluded that untreated streptozocin-induced diabetes in rats for one to two weeks will not cause a generalized increase in the permeability of the blood-ocular or the blood-brain barriers, but organic acids may accumulate in the vitreous body as well as in the brain as a consequence of reduced outward transport through these barriers.
Aminohippuric Acids↗
Choline transport through the blood-retinal and the blood-brain barrier in vivo.
The uptake index method was used to study retinal and cerebral uptake of [14H]labelled choline in rats. In both tissues a saturable uptake was observed. This uptake was significantly inhibited by 10-20 mM unlabelled choline chloride as well as by 10 mM hemicholinium-3. It is concluded that choline passes the blood-retinal barrier by a carrier-mediated transport system similar to that in the blood-brain barrier.
Animals↗
Studies on kidney function in subjects exposed to organic solvents. I. Excretion of albumin and beta-2-microglobulin in the urine.
The urine of 134 subjects, exposed to different organic solvents, and 48 unexposed subjects has been analysed with reference to albumin and beta-2-microglobulin excretion. The exposed subjects excreted significantly larger quantities of albumin than the unexposed. Those exposed to styrene excreted the largest amounts. No significant difference in beta-microglobulin excretion could be demonstrated between the groups.
Adult↗
Evaluation of a new X-ray fluorescent analysis technique for the creation of a Nordic hair database: elemental distributions within the root and the virgin segment of hair fibers.
A new, non-destructive X-ray fluorescence technique for quantitative estimation of elemental content in biological tissues has been developed. Technical and instrumental characteristics of the ITRAX X-ray spectrometer have been evaluated in relation to the properties of biological samples, i.e., human hair fibers. Thus, attenuation variations of the fluorescent X-rays in the hair bulk mass were demonstrated by analysis of sulfur, calcium, and zinc in a virgin part near the root of one hair fiber with elliptical cross section. By rotation of the hair fiber and successive analyses made of the same part of the hair fiber, the results showed that concentrations of elements varied as functions of the diameter of the analyzed hair volume. Other sources of errors are also discussed. The ITRAX instrument allows for precise, fast, non-destructive, simultaneous, quantitative recording of the detected elements and trace elements down to levels of 1 ppm (microg/g). It was used for assessment of normal values of physiologically important elements present in hair in a cohort of normal, healthy Swedish, Caucasian individuals. The database constructed from data retrieved from a conceivably homogeneous ethnic set of individuals represents, to our knowledge, the first of its kind.
Evaluation Studies as Topic↗