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Biomedical subjects

W F Beyer

Publications and source records attributed to W F Beyer.

At least 19 recordsLinked to original sources
PubMed1992

The structure of human mitochondrial manganese superoxide dismutase reveals a novel tetrameric interface of two 4-helix bundles.

The 2.2 A resolution crystal structure of recombinant human manganese superoxide dismutase, a homotetrameric enzyme that protects mitochondria against oxygen-mediated free radical damage, has been determined. Within each subunit, both the N-terminal helical hairpin and C-terminal alpha/beta domains contribute ligands to the catalytic manganese site. Two identical 4-helix bundles, symmetrically assembled from the N-terminal helical hairpins, form novel tetrameric interfaces that stabilize the active sites. Structurally altered polymorphic variants with reduced activity, such as tetrameric interface mutant Ile-58 to Thr, may produce not only an early selective advantage, through enhanced cytotoxicity of tumor necrosis factor for virus-infected cells, but also detrimental effects from increased mitochondrial oxidative damage, contributing to degenerative conditions, including diabetes, aging, and Parkinson's and Alzheimer's diseases.

Amino Acid Sequence↗
PubMed1992

Visualization of purified fibronectin-transglutaminase complexes.

It has been reported previously (Turner, P.M., and Lorand, L. (1989) Biochemistry 28, 628-635) that human erythrocyte transglutaminase forms a noncovalent complex with human plasma fibronectin near its collagen-binding domain. In the present study, we show by nondenaturing electrophoresis that guinea pig liver transglutaminase, similarly to the erythrocyte enzyme, forms a complex with human fibronectin. Studies of anisotropic shifts of fluorescein-labeled liver and erythrocyte transglutaminases, upon addition of fibronectin, indicated that both transglutaminases bind to fibronectin with a stoichiometry of about 2:1. Polymerization of fibrinogen by human erythrocyte transglutaminase was inhibited after complex formation with fibronectin. Complexes of fibronectin with either erythrocyte or liver transglutaminase were isolated by glycerol gradient zone sedimentation and examined by rotary shadowing electron microscopy. The globular transglutaminase could be readily identified binding to the thin fibronectin strand. The binding site for transglutaminase was within 5-10 nm of the N terminus of fibronectin, consistent with its proximity to the collagen-binding domain. Under some experimental conditions, the complex of fibronectin with erythrocyte transglutaminase appeared as a ring-shaped structure in which two transglutaminase molecules had probably dimerized. The molecular weight of the erythrocyte transglutaminase was determined by sedimentation equilibrium to be 71,440 +/- 830.

Animals↗
PubMed1991

TolA: a membrane protein involved in colicin uptake contains an extended helical region.

The group A colicins and the DNA of many single-stranded filamentous bacteriophage are able to use combinations of the Tol proteins to gain entrance into or across the membrane of Escherichia coli. The TolA protein is a 421-amino acid residue integral membrane protein composed of three domains. Domain I, consisting of the amino-terminal 47 amino acids, contains a 21-residue hydrophobic segment that anchors the protein in the inner membrane. The remaining 374 amino acids, containing the other two domains, reside in the periplasmic space. Domain III, consisting of the carboxyl-terminal 120 residues, is considered to be the functional domain based on the location of the tolA592 deletion mutation. The internal 262 amino acids comprise domain II, which connects domains I and III together via short regions of polyglycine. It contains a large number of 3- to 5-residue polyalanine stretches, many of which have a repeat of the sequence Lys-Ala-Ala-Ala-(Glu/Asp). Circular dichroism analysis of different portions of TolA show domain II to be predominantly alpha-helical in structure while domain III contains approximately 10% helical structure.

Amino Acid Sequence↗
PubMed1991

In vivo competition between iron and manganese for occupancy of the active site region of the manganese-superoxide dismutase of Escherichia coli.

Three forms of the dimeric manganese superoxide dismutase (MnSOD) were isolated from aerobically grown Escherichia coli which contained 2 Mn, 1 Mn and 1 Fe, or 2 Fe, respectively. These are designated Mn2-MnSOD, Mn,Fe-MnSOD, and Fe2-MnSOD. Substitution of iron in place of manganese, eliminated catalytic activity, decreased the isoelectric point, and increased the native electrophoretic anodic mobility, although circular dichroism, high performance liquid chromatography gel exclusion chromatography, and sedimentation equilibrium revealed no gross changes in conformation. Moreover, replacement of iron by manganese restored enzymatic activity. Fe2-MnSOD and the iron-superoxide (FeSOD) of E. coli exhibit distinct optical absorption spectra. These data indicate that the active site environments of E. coli MnSOD and FeSOD must differ. They also indicate that competition between iron and manganese for nascent MnSOD polypeptide chains occurs in vivo, and copurification of these variably substituted MnSODs can explain the substoichiometric manganese contents and the variable specific activities which have been reported for this enzyme.

Amino Acids↗
PubMed1990

Aseptic osteonecrosis of the humeral trochlea (Hegemann's disease).

A differential diagnosis of diseases affecting the elbow joint in children should also give due consideration to osteonecroses. They are very rare in the context of the humeral trochlea, the case with which we are concerned being one of a total of 15. In the light of this syndrome's relative rarity, we will report on the case of a 7-year-old boy. This is also the first time that a MR image of Hegemann's disease has been shown.

Adolescent↗
PubMed1990

[Functional diagnosis of early arthroses of the extremities].

Patient history and clinical examination are important for the diagnosis of degenerative joint disease. Here the typical statements of a patient suffering from early osteoarthritis are described and, as far as possible, explained. The joint in question must be systematically examined. Furthermore, the neighboring joints and soft-tissue structures, i.e., muscles, tendons etc., should be examined and evaluated with respect to their importance in hindering the functional chain. The neuroreflectory mechanisms involved herein are described.

Humans↗
PubMed1989

Differences between the manganese- and the iron-containing superoxide dismutases of Escherichia coli detected through sedimentation equilibrium, hydrodynamic, and spectroscopic studies.

The genome of Escherichia coli codes for two superoxide dismutases that may contain either iron (FeSOD) or manganese (MnSOD) at the active site. The crystal structures of MnSODs from two bacterial sources (but not E. coli) have been completed, and structural comparisons with the crystal structure of the FeSOD from either E. coli or Pseudomonas ovalis have been made. Despite the low degree (less than 50%) of sequence homology between the E. coli enzymes, the two proteins are suggested to be structurally homologous. Nonetheless, these enzymes exhibit absolute metal cofactor specificity in conferring enzymatic activity to the inactive apoenzyme. This observation is surprising considering the identity of the active site ligands and the similarities in their geometry and surrounding environment. Using analytical ultracentrifugation, we have determined that the solution properties of these two proteins are different. Thus dialysis of FeSOD but not of MnSOD against phosphate buffer in the presence or absence of EDTA caused dissociation of the homodimer. This dissociation appeared to be related to the loss of iron from native FeSOD. Thus, apoFeSOD but not apoMnSOD existed predominantly as a monomer at protein concentrations below 150 micrograms/mL. ApoMnSOD showed no evidence for dissociation under these conditions. Fluorescence data suggest that the tryptophan environments for the two enzymes are also different. The results of these physical measurements lead us to propose that subtle differences, perhaps at the subunit contact faces, exist in the structures of these crystallographically similar proteins.

Apoenzymes↗
PubMed1989

Characterization of a superoxide dismutase mimic prepared from desferrioxamine and MnO2.

The physicochemical properties of a soluble green complex obtained from the reaction of MnO2 and desferrioxamine have been investigated. The superoxide dismutase mimetic activity of this complex has been previously reported [D. Darr et al. (1987) Arch. Biochem. Biophys. 258, 351-355]. Optical spectra, titration experiments, and metal analyses are consistent with the presence of one gram-atom Mn3+ per mole of desferrioxamine. At least one of the hydroxamate groups is oxidized in the green complex. Reaction of Desferal with MnO2 in the presence of ascorbate yields a more active, pink complex. This pink complex is more stable toward EDTA, suggesting that in it all three hydroxamate groups are intact and ligated to the manganese. The physicochemical properties of these complexes were examined and structures are suggested.

Ascorbic Acid↗
PubMed1989

Inactivation of human arginine-143, lysine-143, and isoleucine-143 Cu,Zn superoxide dismutases by hydrogen peroxide: multiple mechanisms for inactivation.

Site-specific mutants of human Cu,Zn superoxide dismutase (Cu,ZnSOD) have been prepared in which the active-site arginine at position 143 (i.e., SODR143) has been replaced by either lysine (SODK143) or isoleucine (SODI143). As reported previously (W.F. Beyer, Jr., et al. (1987) J. Biol. Chem. 262, 11182-11187), SODK143 and SODI143 have 43 and 11%, respectively, of the catalytic activity of SODR143. H2O2, at low concentrations, acts as an affinity reagent for the inactivation of SODR143. At pH 9.0 and 25 degrees C, the process is characterized by a half-saturation constant for H2O2, K50, of 5.1 mM and a maximum pseudo-first-order rate constant for inactivation, Kmax, of 0.53 min-1. At pH 11.5, the corresponding values are 0.63 mM and 1.23 min-1. The active species in the inactivation is likely HO2-, as previously found with yeast and bovine Cu,ZnSODs (see C.L. Borders, Jr., and I. Fridovich (1985) Arch. Biochem. Biophys. 241, 472-476). SODK143 is also inactivated by HO2- by an affinity mechanism, i.e., one where reversible binding of H2O2 (HO2-) is a prerequisite for inactivation. At pH values of 9.0 and 11.5, the kmax values are 0.92 and 1.08 min-1, respectively; however, the corresponding K50 values increase to 42.5 and 15.8 mM, respectively. SODI143 is also inactivated by H2O2, but no evidence for an affinity mechanism was found; instead, a second-order kinetic mechanism was observed. Inactivation of each of the three enzymes is accompanied by the loss of one histidine per subunit. At elevated concentrations of H2O2, a second nonaffinity mechanism of inactivation of both SODR143 and SODK143 was found, in which a second equivalent of H2O2 reacts with the Cu,ZnSOD.HO2- complex to give a competing second-order inactivation. It appears that the positive charge of arginine-143 plays a role in the binding of HO2- at the active site of human Cu,ZnSOD, and that replacement of the arginine by lysine gives an enzyme with a similar affinity mechanism of inactivation, but with a greatly reduced affinity for HO2-. However, replacement with isoleucine causes an entirely different mechanism of inactivation; this raises the possibility that the mechanism of enzyme catalysis of superoxide dismutation by SODI143 is also different.

Arginine↗
PubMed1989

Anaerobic induction of ProMn-superoxide dismutase in Escherichia coli.

Escherichia coli growing anaerobically respond to NO3- plus PQ2+ with a 20-30-fold induction of an inactive form of the manganese-containing superoxide dismutase (MnSOD). Mutants lacking a functional nitrate reductase fail to show this response. This inactive enzyme can be activated by addition of Mn(II) salts to cell extracts in the presence of acidic guanidinium chloride, followed by dialysis against neutral buffer. Direct addition of Mn(II) to cell extracts does not result in activation. However, addition of Mn(II) to purified apo-MnSOD results in partial activation. Inactive, reconstitutable MnSOD is induced 13-fold within 15 min of exposure to NO3- plus PQ2+. Western blot analysis revealed a 15-fold increase in immunoreactive MnSOD under these conditions, suggestive of de novo synthesis of this protein. A strain of E. coli bearing a multicopy plasmid carrying the MnSOD gene (sodA) overproduces inactive MnSOD 19-fold compared to the parent strain under anaerobic conditions. Strains of E. coli with an inactivating insertion in the sodA gene do not induce inactive, reconstitutable MnSOD in response to NO3- plus PQ2+ and lack the immunoreactive MnSOD band. These results, in toto, suggest that the inactive protein synthesized under anaerobic conditions in the presence of NO3- plus PQ2+, acting as an electron sink, is a product of the sodA gene and is devoid of activity due to occupation of the manganese site by another metal.

Anaerobiosis↗
PubMed1989

Biochemical and structural studies of tenascin/hexabrachion proteins.

Tenascin is a large, disulfide-bonded glycoprotein of the extracellular matrix. The predominant form of tenascin observed by electron microscopy is a six-armed oligomer, termed a hexabrachion. We have determined the molecular mass of the native human hexabrachion to be 1.9 x 10(6) Da by sedimentation equilibrium analysis and by electrophoresis on non-reducing agarose gels. On reducing polyacrylamide gel electrophoresis (SDS-PAGE), human tenascin showed a single prominent band at 320 kDa and minor bands of 220 and 230 kDa. The molecular weight of the native human hexabrachion is thus consistent with a disulfide-bonded hexamer of the 320 kDa subunits. Upon treatment with neuraminidase, the apparent molecular weights of all human and chicken tenascin subunits on reducing SDS-PAGE were decreased by about 10 kDa. Prolonged incubation with alpha-mannosidase, however, caused no apparent change in the apparent molecular weight of tenascin subunits. Sedimentation in a cesium chloride gradient gave a higher buoyant density for human tenascin than for fibronectin, suggesting that it has a higher degree of glycosylation. The far-UV circular dichroism spectrum indicates a predominance of beta-structure and a lack of collagen-like or alpha-helical structure. When human hexabrachions were reduced and acetylated, the resulting fragments were single arms which sedimented at 6 S in glycerol gradients and migrated at 320 kDa on non-reducing gels. Treatment of tenascin with trypsin and alpha-chymotrypsin also produced large fragments which were fractionated by gradient sedimentation and analyzed by non-reducing SDS-PAGE and electron microscopy. We present a structural model for the assembly of the observed fragments into the elaborate native hexabrachion.

Animals↗
PubMed1989

Chemical modification of iron- and manganese-containing superoxide dismutases from Escherichia coli.

The manganese-containing (MnSOD) and iron-containing (FeSOD) superoxide dismutases from Escherichia coli are extensively (greater than 95%) inactivated by treatment with phenylglyoxal. The relatively high concentrations of phenylglyoxal and high pH required for optimal inactivation suggest that inactivation may be due to modification of an arginine with a "normal" elevated pKa, i.e., one not in an active site cavity where the pKa is likely to be lowered because of lower solvent accessibility and decreased polarity of the local environment. Treatment of either enzyme with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 2-hydroxy-5-nitrobenzyl bromide, m-chloroperoxybenzoate, or tetranitromethane causes no inactivation, while 2,4,6-trinitrobenzenesulfonate, N-acetylimidazole, or diethyl pyrocarbonate cause 55-75% inactivation of each enzyme. Failure of hydroxylamine to reverse inactivation by the latter two suggests that in each instance loss of activity is due to lysine modification. The previously reported inactivation of FeSOD by H2O2 was further investigated, and no evidence was found for an affinity mechanism, i.e., a reversible binding of peroxide that precedes inactivation.

Aldehydes↗
PubMed1988

Does copper (II) ethylenediaminetetraacetate disproportionate superoxide?

W. M. Willingham and J. R. J. Sorenson have recently reported [1988) Biochem. Biophys. Res. Commun. 150, 252-258) that the Cu(II)EDTA complex catalyzes the dismutation of O-2 and thereby call into question several earlier reports. These authors concluded that the widespread use of EDTA to suppress dismutation of O-2 by adventitious Cu(II) and other metals, during assays for superoxide dismutase, is unsound. We have undertaken a clarification of this issue by examining the ability of Cu(II)EDTA to catalyze the dismutation of O-2 and have found this complex to be devoid of this activity at pH 6.0 or at 7.8; whether assayed by the cytochrome c or the nitroblue tetrazolium assays. The probable reasons for the results of Willingham and Sorenson and some peculiarities of the nitroblue tetrazolium assay have been explored.

Cytochrome c Group↗
PubMed1988

An ultrasensitive colorimetric assay for manganese.

An ultrasensitive colorimetric assay for manganese is described. It is based upon the catalysis, by Mn(II), of the photochemical oxidation of o-dianisidine, sensitized by riboflavin. Catalase increases the Mn(II)-catalyzed rate of photosensitized oxidation of dianisidine to the bisazobiphenyl, while superoxide dismutase inhibits the rate. The mechanism appears to involve oxidation of Mn(II) by O2-, followed by oxidation of dianisidine by MnO2+ in equilibrium Mn(III). Cu(II) interferes, but Zn(II), Fe(II), Fe(III), Co(II), and Ni(II) do not. Chelating agents and thiol reductants also interfere. Interference by Cu(II) can be overcome by the addition of cyanide, while interference by organic compounds can be surmounted by wet ashing. This assay provides a linear response to Mn(II) over the range 10-2500 nM. The limit of detection was 5 nM Mn(II).

Catalysis↗