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W Maret

Publications and source records attributed to W Maret.

At least 37 records · Page 2Linked to original sources
PubMed1995

Metallothionein/disulfide interactions, oxidative stress, and the mobilization of cellular zinc.

Glutathione disulfide, the major cellular disulfide, releases zinc from metallothionein (MT) [W. Maret (1994) Oxidative metal release from metallothionein via zinc-thiol/disulfide interchange, Proc. natn. Acad. Sci. U.S.A. 91, 237-241]. Here, the interaction of rabbit liver MT-II with other selected biological disulfides (coenzyme A/glutathione mixed disulfide, coenzyme A disulfide, and cystamine) was investigated by measuring concomitant release of radioactive 65-zinc from MT. These disulfides react more rapidly than glutathione disulfide, thus underscoring the reactivity of zinc sulfur bonds in the clusters of MT and the importance of the MT/disulfide interaction as a chemical mechanism for mobilizing zinc from a thermodynamically stable zinc complex. Two implications of these in vitro findings are discussed. (i) Apparently, in the case of zinc which is redox inert, Nature has availed itself of the redox activity of the cysteine ligand to mobilize the metal, and, presumably to permit redox-control of cellular zinc distribution. The mobilization of zinc from MT suggests a possible function of MT as a physiological zinc donor. (ii) A shift of the glutathione redox balance under conditions of oxidative stress will accelerate metal release from MT. Such a disturbance of metal metabolism has important consequences for the progression of diseases such as Alzheimer's and Parkinson's disease where oxidative stress occurs in affected brain tissue.

Animals↗
PubMed1994

Human liver aldehyde dehydrogenases: new method of purification of the major mitochondrial and cytosolic enzymes and re-evaluation of their kinetic properties.

A new purification procedure, based on dye-adsorption and affinity chromatography, has been developed for the isolation of the two major ALDH isozymes from human liver: ALDH-1 (cytosolic, pI 5.2) and ALDH-2 (mitochondrial, pI 4.9). The procedure affords milligram quantities of ALDH-1 and -2 at 850- and 275-fold purifications, respectively, from 50 g of liver in two days. Kinetic parameters for acetaldehyde oxidation were determined with these purified enzymes, because there is a wide discrepancy in the absolute magnitude of these parameters in the biochemical literature. The Michaelis constants for ALDH-1 and -2, determined from initial velocities (for ALDH-1) and single reaction progress curves (for ALDH-2), are 180 +/- 10 microM and 0.20 +/- 0.02 microM, respectively (pH 7.5 and 9.5, saturating NAD+ in both cases). This three orders of magnitude difference in Km values is much greater than that reported previously in all but one study.

Acetaldehyde↗
PubMed1994

Oxidative metal release from metallothionein via zinc-thiol/disulfide interchange.

Mammalian metallothionein has been postulated to play a pivotal role in cellular zinc distribution. All seven of its metal atoms are bound with high thermodynamic stability in two clusters buried deeply in the molecule. If the protein is to function in metal delivery, there must be a biological mechanism to facilitate metal release. One means to achieve this would be a labilization of the clusters by interaction of metallothionein with an appropriate cellular ligand. To search for such a mediator, we have designed a rapid radiochromatographic method that can detect changes in the zinc content of 65Zn-labeled metallothionein in response to other biomolecules. Using this methodology, we have established that rabbit liver metallothionein 2 interacts with glutathione disulfide with concomitant release of zinc. Under conditions of pseudo-first-order kinetics, the monophasic reaction depends linearly on the concentration of glutathione disulfide in the range from 5 to 30 mM with a second-order rate constant k = 4.9 x 10(-3)s-1.M-1 (pH 8.6; 25 degrees C). Apparently, zinc release does not involve direct access of glutathione disulfide to the inner coordination sphere of the metals. Rather it appears that the solvent-accessible zinc-bound thiolates in two clefts of each domain of metallothionein [Robbins, A. H., McRee, D. E., Williamson, M., Collett, S. A., Xuong, N. H., Furey, W. F., Wang, B. C. & Stout, C. D. (1991) J. Mol. Biol. 221, 1269-1293] participate in a thiol/disulfide interchange with glutathione disulfide. This rate-limiting initial S-thiolation, which occurs with indistinguishable rates in both clusters, then causes the clusters to collapse and release their zinc. Such a mechanism of metal release would link the control of the metal content of metallothionein to the cellular glutathione redox status and raises important questions about the physiological implications of this observation with regard to a role of glutathione in zinc metabolism and in making zinc available for other biomolecules.

Animals↗
PubMed1993

Extrahepatic, differential expression of four classes of human alcohol dehydrogenase.

The amounts of mRNA expressed for different alcohol dehydrogenase (ADH) classes were determined in human tissues by Northern hybridization. ADH classes I, II, and III were expressed in all tissues. The mRNAs were highest for class I ADHs, with particularly strong signals in liver, lung, ileum, colon, and uterus. For class II ADH, such a wide tissue distribution had not been recognized previously. Expression of class III ADH was highest in testis, followed by uterus, colon, and ileum. The amounts of class I and III ADH mRNAs varied significantly, indicating that tissue-specific factors modulate the expression of these enzymes above a basal level. Class V ADH (ADH6) was not detected in any of the tissues, including stomach. This suggests that class V ADH is not identical with human stomach sigma-ADH (class IV). The results support the general proposition that ADHs are not restricted to liver and have functions other than those in ethanol oxidation.

Alcohol Dehydrogenase↗
PubMed1992

Alcohol dehydrogenase genes: restriction fragment length polymorphisms for ADH4 (pi-ADH) and ADH5 (chi-ADH) and construction of haplotypes among different ADH classes.

Of the five human alcohol dehydrogenase (ADH) genes located in the region q21-25 of chromosome 4, genetic markers have been reported previously only for class I enzymes, ADH1-3. Here, new restriction fragment length polymorphisms (RFLPs) are described for the genes of two other classes, ADH4 (pi) and ADH5 (chi or formaldehyde dehydrogenase, FDH). The frequencies and modes of inheritance of these RFLPs were determined with DNA both from unrelated individuals and from families. A polymorphic PstI site is assigned to the fourth intron of the ADH4 gene. Pairwise linkage disequilibrium calculations for these new RFLPs and already known RFLPs at the ADH2 and ADH3 loci establish strong linkage disequilibria between polymorphic MspI and BstXI sites in the ADH5 gene as well as between XbaI and MspI sites in the ADH3 gene. Furthermore, linkage disequilibria were detected between RFLPs of the ADH2 and ADH3 genes as well as between those of the ADH4 and ADH5 genes. The latter disequilibrium implies a hitherto unknown physical proximity of two genes belonging to different ADH classes. The RFLPs were used to construct chromosomal haplotypes that include three ADH classes. Of the 16 possible haplotypes for four RFLP markers used here, 10 were experimentally detected. The potential application of the ADH RFLPs and haplotypes in linkage or association studies of inherited diseases such as familial "alcoholism" is discussed.

Alcohol Dehydrogenase↗
PubMed1991

The pH variation of steady-state kinetic parameters of site-specific Co(2+)-reconstituted liver alcohol dehydrogenase. A mechanistic probe for the assignment of metal-linked ionizations.

To identify ionizations of the active site metal-bound water in horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase; EC 1.1.1.1), the pH, solvent isotope, temperature, and anion dependences of the steady-state kinetic parameters kcat and kcat/KM have been evaluated under initial velocity conditions for the native and the active site-specific Co(2+)-reconstituted enzyme. In the oxidation of benzyl alcohol, a bell-shaped pattern of four prototropic equilibria was observed under conditions of saturating concentrations of NAD+. It is shown that the ionizations governing kcat (pK1 congruent to 6.7, pK2 congruent to 10.6) belong to the ternary enzyme-NAD(+)-alcohol complex, whereas the ionizations governing kcat/KM (pK1' congruent to 7.5, pK2' congruent to 8.9) belong to the binary enzyme-NAD+ complex. The ionizations pK1 and pK1' are not influenced by metal substitution and are ascribed to His-51 on the basis of experimental estimates of their associated enthalpies of ionization. On the other hand, pK2 and pK2' are significantly decreased (delta pKa congruent to 1.0) in the Co(2+)-enzyme and are attributed to the active site metal-bound water molecule. The shape of the pH profiles requires that the metal ion coordinates a neutral water molecule in the ternary enzyme-NAD(+)-alcohol complex under physiological conditions. The possible catalytic role of the water molecule within a pentacoordinate metal ion complex in the active site is discussed.

1-Propanol↗
PubMed1989

Cobalt(II)-substituted class III alcohol and sorbitol dehydrogenases from human liver.

The catalytic zinc atoms in class III (chi) alcohol dehydrogenase (ADH) and sorbitol dehydrogenase (SDH) from human liver have been specifically removed and replaced by cobalt(II) with a new ultrafiltration technique. The electronic absorption spectrum of class III cobalt ADH (epsiolon 638 = 870 M-1 cm-1) is nearly identical with those of active site substituted horse EE and human class I (beta 1 beta 1) cobalt ADH. Thus, the coordination environment of the catalytic metal is strictly conserved in these enzymes. However, significant differences are noted when the spectra of class III ADH-coenzyme complexes are compared to the corresponding spectra of the horse enzyme. The spectrum of class III ADH.NADH is split into three bands, centered at 680, 638, and 562 nm. The class III ADH.NAD+ species resembles the alkaline form of the corresponding horse enzyme complex but without exhibiting the pH dependence of the latter. These spectral changes underscore the role of the coenzymes in differentially fine tuning the catalytic metal for its particular function in each ADH. The noncatalytic zinc of class III ADH exchanges with cobalt at pH 7.0. While 9 residues out of 15 in the loop surrounding the noncatalytic zinc of class III ADH differ from those of the class I ADH, the electronic absorption spectra of cobalt in the noncatalytic metal site of class III ADH establish that the coordination environment of this site is conserved as well. The spectrum of cobalt SDH differs significantly from those of cobalt ADHs.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Dehydrogenase↗
PubMed1989

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↗
PubMed1989

Fe(II)-substituted horse liver alcohol dehydrogenase, a model for non-heme iron enzymes. Various states of iron-dioxygen interaction investigated by Mössbauer and EPR spectroscopy.

The catalytic Zn(II) ion of horse liver alcohol dehydrogenase (EE isozyme) was replaced by Fe(II), providing a novel iron protein with the unusual coordination of Fe(II) to two cysteines residues, one histine residue and water. The electronic structure of iron in this system was characterized by Mössbauer spectroscopy at various temperatures as well as applied magnetic fields and analysed in terms of the spin Hamiltonian formalism. The novelty we found is an unusually weak spin coupling (/J/ less than 0.1 cm-1) of a paramagnet (S = 1) with iron (S = 2). From EPR and biochemical studies we conclude that the corresponding chemical species is triplet oxygen (O2). The quantitative determination of the coupling energy was possible utilizing the competition between Zeemann interaction and spin coupling at weak magnetic fields and low temperature. Oxidation experiments followed by Mössbauer spectroscopy showed that the spin-coupled system is an outer-sphere Fe(II) . (O2)aq complex occurring as an intermediate during a Fe(II)-catalyzed dioxygen activation. We observed two additional Fe(II) species after treatment with O2 and dithionite. The spin Hamiltonian parameters of iron in the coupled system are presented. The results are compared with those of iron in other non-heme iron proteins.

Alcohol Dehydrogenase↗
PubMed1988

Purification and characterization of human liver sorbitol dehydrogenase.

Sorbitol dehydrogenase from human liver was purified to homogeneity by affinity chromatography on immobilized triazine dyes, conventional cation-exchange chromatography, and high-performance liquid chromatography. The major form is a tetrameric, NAD-specific enzyme containing one zinc atom per subunit. Human liver sorbitol dehydrogenase oxidizes neither ethanol nor other primary alcohols. It catalyzes the oxidation of a secondary alcohol group of polyol substrates such as sorbitol, xylitol, or L-threitol. However, the substrate specificity of human liver sorbitol dehydrogenase is broader than that of the liver enzymes of other sources. The present report describes the stereospecific oxidation of (2R,3R)-2,3-butanediol, indicating a more general function of sorbitol dehydrogenase in the metabolism of secondary alcohols. Thus, the enzyme complements the substrate specificities covered by the three classes of human liver alcohol dehydrogenase.

Amino Acids↗
PubMed1988

Retinal is not formed in vitro by enzymatic central cleavage of beta-carotene.

Rat intestinal mucosa was prepared and incubated with beta-carotene by the procedure of Goodman and Olson [Goodman, DeW. S., & Olson, J.A. (1969) Methods Enzymol. 15, 462-475] to determine beta-carotene cleavage activity. A new detection system for the reaction products of the described enzyme beta-carotene 15,15'-dioxygenase (EC 1.13.11.21) employs solvent extraction of retinoids and carotenoids followed by high-performance liquid chromatography separation and photometric detection of the pigments. It has not detected any newly formed retinal or other retinoids in the intestinal protein preparations from normal or vitamin A deficient rats. The latter were chosen as a possible source of more active enzyme preparations. With corresponding blank samples subjected to identical conditions of incubation but without added protein, small amounts of beta-apocarotenals could be detected. They were previously reported as cleavage products of beta-carotene [Ganguly, J., & Sastry, P.S. (1985) World Rev. Nutr. Diet. 45, 198-220] but are clearly not formed as a result of an enzymatic reaction. The failure to detect in vitro enzymatic central or random cleavage of the beta-carotene molecule in extracts of rat intestinal mucosa emphasizes the need to reevaluate the existing theory of conversion of beta-carotene into vitamin A.

Animals↗