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H M Steinman

Publications and source records attributed to H M Steinman.

At least 19 recordsLinked to original sources
PubMed1995

The Bcl-2 oncoprotein functions as a pro-oxidant.

The mechanism by which the bcl-2 oncogene exerts its anti-apoptotic and antioxidant action is unknown. We found that expression of bcl-2 in superoxide dismutase-deficient (SOD-) Escherichia coli resulted in increased transcription of the KatG catalase-peroxidase, a 13-fold increase in KatG activity and a 100-fold increase in resistance to hydrogen peroxide. In addition, mutation rate was increased 3-fold, and katG and oxyR, a transcriptional regulator of katG induction, were required for aerobic survival. These data indicate that Bcl-2 acts as a pro-oxidant in E. coli, i.e. Bcl-2 generates reactive oxygen intermediates. In support of a pro-oxidant mechanism in eukaryotic cells, we found a 73% increase in superoxide dismutase activity in a murine B-cell line overexpressing Bcl-2. Increases in reduced glutathione and in oxyradical damage to DNA, previously observed in other overexpressing cell lines, are additional evidence for a pro-oxidant mechanism. Thus, Bcl-2 does not appear to be an antioxidant. Instead, Bcl-2 appears to influence levels of reactive oxygen intermediates that induce endogenous cellular antioxidants. This activity of Bcl-2 may control entry into apoptosis.

Animals↗
PubMed1995

Function and stationary-phase induction of periplasmic copper-zinc superoxide dismutase and catalase/peroxidase in Caulobacter crescentus.

Although cytosolic superoxide dismutases (SODs) are widely distributed among bacteria, only a small number of species contain a periplasmic SOD. One of these is Caulobacter crescentus, which has a copper-zinc SOD (CuZnSOD) in the periplasm and an iron SOD (FeSOD) in the cytosol. The function of periplasmic CuZnSOD was studied by characterizing a mutant of C. crescentus with an insertionally inactivated CuZnSOD gene. Wild-type and mutant strains showed identical tolerance to intracellular superoxide. However, in response to extracellular superoxide, the presence of periplasmic CuZnSOD increased survival by as much as 20-fold. This is the first demonstration that periplasmic SOD defends against external superoxide of environmental origin. This result has implications for those bacterial pathogens that contain a CuZnSOD. C. crescentus was shown to contain a single catalase/peroxidase which, like Escherichia coli KatG catalase/peroxidase, is present in both the periplasmic and cytoplasmic fractions. The growth stage dependence of C. crescentus catalase/peroxidase and SOD activity was studied. Although FeSOD activity was identical in exponential- and stationary-phase cultures, CuZnSOD was induced nearly 4-fold in stationary phase and the catalase/peroxidase was induced nearly 100-fold. Induction of antioxidant enzymes in the periplasm of C. crescentus appears to be an important attribute of the stationary-phase response and may be a useful tool for studying its regulation.

Catalase↗
PubMed1994

The manganese superoxide dismutase of Escherichia coli K-12 associates with DNA.

Superoxide dismutases (SODs) are vital components in the resistance of aerobic organisms to the toxicity of oxygen. Escherichia coli contains two highly homologous cytoplasmic SODs, a manganese- and an iron-containing enzyme (MnSOD, FeSOD). We previously demonstrated that MnSOD and FeSOD have different physiological functions and that MnSOD is more effective in preventing oxidative damage to DNA. In this report, purified E. coli MnSOD was shown to bind nonspecifically to DNA by electrophoretic mobility shift assay and nitrocellulose-filter binding methodologies. From electrophoretic mobility shift assay, the equilibrium dissociation constants for interaction with a variety of double-stranded and single-stranded oligonucleotides ranged from 1.5 +/- 0.2 to 8.4 +/- 1.3 microM at 20 degrees C. This range of concentrations corresponds to MnSOD concentrations in aerobically grown E. coli. In vivo binding of MnSOD to DNA was supported by colocalization of MnSOD and the E. coli nucleoid in immunoelectron microscopy. Both MnSOD and DNA were inhomogeneously distributed in the cytosol, the concentration of each being higher in the center of the cell and relatively low near the inner membrane. In contrast, there was no evidence for physiologically relevant interaction of FeSOD with DNA. Binding to DNA in vitro was weak, Kd > 40-220 microM, concentrations 7-40 times higher than found in vivo. In addition, the cytoplasmic distribution of FeSOD did not correlate with DNA. FeSOD concentration was higher near the inner membrane and lower in the center of the cytosol. These results demonstrate that E. coli MnSOD associates with DNA in vitro and in vivo. Combined with prior data demonstrating that MnSOD preferentially protects DNA in vivo while an equal enzymatic activity of FeSOD does not (Hopkin, K. A., Papazian, M. A., and Steinman, H. M. (1992) J. Biol. Chem. 267, 24253-24258), our data suggest that E. coli MnSOD acts as a "tethered antioxidant"; association of MnSOD with DNA localizes dismutase activity near a target of oxidative stress and increases protection of DNA from oxidative damage. This model has implications for the therapeutic use of SODs as antioxidants.

Antibodies↗
PubMed1994

The iron superoxide dismutase of Legionella pneumophila is essential for viability.

Legionella pneumophila, the causative agent of Legionnaires' disease, contains two superoxide dismutases (SODs), a cytoplasmic iron enzyme (FeSOD) and a periplasmic copper-zinc SOD. To study the role of the FeSOD in L. pneumophila, the cloned FeSOD gene (sodB) was inactivated with Tn903dIIlacZ, forming a sodB::lacZ gene fusion. By using this fusion, expression of sodB was shown to be unaffected by a variety of conditions, including several that influence sod expression in Escherichia coli: aeration, oxidants, the redox cycling compound paraquat, manipulation of iron levels in the medium, and the stage of growth. A reproducible twofold decrease in sodB expression was found during growth on agar medium containing charcoal, a potential scavenger of oxyradicals, in comparison with growth on the same medium without charcoal. No induction was seen during growth in human macrophages. Additional copies of sodB+ in trans increased resistance to paraquat. Construction of a sodB mutant was attempted by allelic exchange of the sodB::lacZ fusion with the chromosomal copy of sodB. The mutant could not be isolated, and the allelic exchange was possible only if wild-type sodB was present in trans. These results indicate that the periplasmic copper-zinc SOD cannot replace the FeSOD. The data strongly suggest that sodB is an essential gene and that FeSOD is required for the viability of L. pneumophila. In contrast, Sod- mutants of E. coli and Streptococcus mutans grow aerobically and SOD is not required for viability in these species.

Aconitate Hydratase↗
PubMed1993

Function of periplasmic copper-zinc superoxide dismutase in Caulobacter crescentus.

Caulobacter crescentus is one of a small number of bacterial species that contain a periplasmic copper-zinc superoxide dismutase (CuZnSOD). A C. crescentus mutant, with the CuZnSOD gene interrupted by a promoterless cat gene, was constructed and characterized to analyze CuZnSOD function. Periplasmic SOD does not protect against oxyradical damage in the cytosol or play a major role in maintaining the integrity of the cell envelope. Studies of the effect of sodium citrate on plating efficiency suggest that CuZnSOD protects a periplasmic or membrane function(s) requiring magnesium or calcium.

Cations, Divalent↗
PubMed1992

Functional differences between manganese and iron superoxide dismutases in Escherichia coli K-12.

Superoxide dismutases are enzymes that defend against oxidative stress through decomposition of superoxide radical. Escherichia coli contains two highly homologous superoxide dismutases, one containing manganese (MnSOD) and the other iron (FeSOD). Although E. coli Mn and FeSOD catalyze the dismutation of superoxide with comparable rate constants, it is not known if they are physiologically equivalent in their protection of cellular targets from oxyradical damage. To address this issue, isogenic strains of E. coli containing either Mn or FeSOD encoded on a plasmid and under the control of tac promoter were constructed. SOD specific activity in the Mn and FeSOD strains could be controlled by the concentration of isopropyl beta-thiogalactoside in the medium. The tolerance of these strains to oxidative stress was compared at equal Mn and FeSOD specific activities. Our results indicate that E. coli Mn and FeSOD are not functionally equivalent. The MnSOD is more effective than FeSOD in preventing damage to DNA, while the FeSOD appears to be more effective in protecting a cytoplasmic superoxide-sensitive enzyme. These data are the first demonstration that Mn and FeSOD are adapted to different antioxidant roles in E. coli.

Drug Resistance, Microbial↗
PubMed1992

Construction of an Escherichia coli K-12 strain deleted for manganese and iron superoxide dismutase genes and its use in cloning the iron superoxide dismutase gene of Legionella pneumophila.

An Escherichia coli K-12 strain deleted for sodA and sodB (manganese and iron superoxide dismutases) was constructed and characterized by Southern blotting, enzyme assays, and physiological analyses. The sod deletion strain was used to clone the iron superoxide dismutase gene of Legionella pneumophila by complementation to paraquat resistance.

Base Sequence↗
PubMed1990

Crystallographic characterization of a Cu,Zn superoxide dismutase from Photobacterium leiognathi.

Crystals of a copper-zinc superoxide dismutase from Photobacterium leiognathi, a luminescent marine bacterium that is the species-specific symbiont of the ponyfish, have been obtained from 2-methyl-2,4-pentanediol solutions. The space group was determined using screenless small-angle precession photographs, and was confirmed by analyzing area detector diffraction data with the XENGEN programs for indexing and refinement. The crystals are monoclinic, space group C2 (a = 126.4 A, b = 87.0 A, c = 44.4 A, beta = 92.8 A), and have two 32,000 Mr dimers per asymmetric unit. The crystals diffract to at least 2.7 A resolution, are resistant to radiation damage, and are suitable for determination of the structure by X-ray diffraction.

Photobacterium↗
PubMed1990

Copper-zinc superoxide dismutase of Caulobacter crescentus: cloning, sequencing, and mapping of the gene and periplasmic location of the enzyme.

Although widely found in the cytoplasm of eucaryotes, the copper-zinc form of superoxide dismutase (CuZnSOD) has been identified in only a small number of bacterial species. One species is the freshwater bacterium Caulobacter crescentus, which also contains an SOD with iron as the metal cofactor (FeSOD). To investigate the function of this CuZnSOD and its structural relationship to the eucaryotic CuZnSODs, the gene encoding CuZnSOD (sodC) of C. crescentus CB15 was cloned and sequenced. By hybridization to pulsed-field electrophoresis gels, sodC was mapped near cysE in the C. crescentus chromosome. Through analysis of spheroplasts, the two SODs of C. crescentus were shown to be differently localized, CuZnSOD in the periplasm and FeSOD in the cytoplasm. In its natural habitat, C. crescentus is frequently associated with blue-green algae (cyanobacteria). The oxygen evolved by these photosynthetic algae may create an extracellular oxidative stress against which the periplasmic CuZnSOD may defend more effectively than the cytoplasmic FeSOD. Amino acid sequence alignments of C. crescentus CuZnSOD with eucaryotic CuZnSODs and with CuZnSOD of Photobacterium leiognathi (the only other bacterium from which CuZnSOD has been isolated and sequenced) suggest similar supersecondary structures for bacterial and eucaryotic CuZnSODs but reveal four novel substitutions in C. crescentus CuZnSOD: a phenylalanine critical to intrasubunit hydrophobic bonding replaced by alanine, a histidine ligand of zinc replaced by aspartate, and substitutions of two other previously invariant residues that stabilize zinc or both copper and zinc. These amino acid substitutions in C. crescentus CuZnSOD may have implications for its catalysis and stability.

Amino Acid Sequence↗
PubMed1988

Iron superoxide dismutase. Nucleotide sequence of the gene from Escherichia coli K12 and correlations with crystal structures.

The nucleotide sequence of the iron superoxide dismutase gene from Escherichia coli K12 has been determined. Analysis of the DNA sequence and mapping of the mRNA start reveal a unique promoter and a putative rho-independent terminator, and suggest that the Fe dismutase gene constitutes a monocistronic operon. The gene encodes a polypeptide product consisting of 192 amino acid residues with a calculated Mr of 21,111. The published N-terminal amino acid sequence of E. coli B Fe dismutase (Steinman, H. M., and Hill, R. L. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 3725-3729), along with the sequences of seven other peptides reported here, was located in the primary structure deduced from the K12 E. coli gene sequence. A new molecular model for iron dismutase from E. coli, based on the DNA sequence and x-ray data for the E. coli B enzyme at 3.1 A resolution, allows detailed comparison of the structure of the iron enzyme with manganese superoxide dismutase from Thermus thermophilus HB8. The structural similarities are more extensive than indicated by earlier studies and are particularly striking in the vicinity of the metal-ligand cluster, which is surrounded by conserved aromatic residues. The combined structural and sequence information now available for a series of Mn and Fe superoxide dismutases identifies variable regions in these otherwise very similar molecules; the principal variable site occurs in a surface region between the two long helices which dominate the N-terminal domain.

Amino Acid Sequence↗
PubMed1987

Bacteriocuprein superoxide dismutase of Photobacterium leiognathi. Isolation and sequence of the gene and evidence for a precursor form.

The gene encoding the bacteriocuprein superoxide dismutase from Photobacterium leiognathi, American Type Culture Collection strain 25521, was cloned in a pUC12 vector and sequenced. The nucleotide sequence predicted a 22-residue leader peptide amino-terminal to the known bacteriocuprein sequence. The expected precursor bacteriocuprein was directly identified in the in vitro translation products of the cloned gene by polyacrylamide gel electrophoresis and automated Edman degradation. Enzymatically active bacteriocuprein that lacked the leader peptide was identified in sonic extracts of Escherichia coli hosts containing the cloned gene. A single transcript of 580 nucleotides was observed in blots of total P. leiognathi RNA, and a unique site of transcriptional initiation was identified by primer extension analysis. P. leiognathi bacteriocuprein is the first bacteriocuprein whose gene has been isolated and sequenced and the first copper-zinc superoxide dismutase in which a leader peptide has been found. The presence of a leader peptide suggests that the bacteriocuprein is localized in the membrane or periplasm, in contrast to the eukaryotic copper-zinc superoxide dismutases, which are cytoplasmic enzymes. Such a difference in intracellular location could be important for understanding the presence and function of the uncommon, bacteriocuprein superoxide dismutase in P. leiognathi.

Amino Acid Sequence↗
PubMed1986

Strain variation in bacteriocuprein superoxide dismutase from symbiotic Photobacterium leiognathi.

Photobacterium leiognathi ATCC 25521 (the type strain and light-organ symbiont of ponyfish) is one of the few bacteria that produces a copper-zinc superoxide dismutase, termed bacteriocuprein. We enzymologically and immunologically characterized the bacteriocuprein superoxide dismutases in sonicates from the type strain and nine additional strains of P. leiognathi, each isolated from the light organ of a separate ponyfish specimen, representing seven ponyfish species. The results indicate considerable strain variation. (i) The level of bacteriocuprein enzymatic activity varied greatly among strains from different species of ponyfish. In four of the nine strains, activity was low or undetectable, while in five strains it was comparable to that in the type strain. (ii) The bacteriocuprein in one strain had a specific activity much lower than that of the type strain, and in another strain, no bacteriocuprein activity and no cross-reactive polypeptide were detectable. (iii) A new electrophoretic variant, which migrated slower than that of strains from fish captured in Thailand and Japan, was identified in strains from fish captured in the Philippine Islands. (iv) Enzymological and immunological differences were observed in bacteriocupreins of strains from male and female specimens of the same ponyfish species, for the two species in which specimens of both sexes were examined. These observations raise the possibility that specific variations in the bacteriocupreins of P. leiognathi might be characteristic of the species, geographical source, or sex of the ponyfish host. Thus, the data indicate that the possibility of strain variation should be considered when other species are screened for bacteriocupreins.

Animals↗
PubMed1985

Bacteriocuprein superoxide dismutases in pseudomonads.

Two new instances of the rare bacteriocuprein form of superoxide dismutase have been discovered in Pseudomonas diminuta and P. maltophilia. Each species contains a manganese superoxide dismutase as well. Eight other strains of Pseudomonas and Xanthomonas spp. lacked bacteriocupreins and contained either a manganese or an iron superoxide dismutase. Native molecular weights and isoelectric points were determined for all these bacterial dismutases. A monospecific polyclonal antibody was prepared against the bacteriocuprein from Photobacterium leiognathi; it was not cross-reactive with the bacteriocuprein from either Pseudomonas strain. Bacteriocupreins have previously been identified in only two procaryotes, P. leiognathi and Caulobacter crescentus. The discovery of the Pseudomonas bacteriocupreins reveals a broader distribution, raising the possibility that bacteriocupreins are a continuous line of descent among procaryotes and not isolated evolutionary occurrences, as previous data suggested.

Electrophoresis, Polyacrylamide Gel↗
PubMed1982

Purification and characterization of gamma-glutamyltransferase from rat pancreas.

gamma-Glutamyltransferase ((5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2.) from rat pancreas has been purified to homogeneity and shown to be a glycoprotein of apparent molecular weight 68000, composed of one heavy and one light subunit, with respective molecular weights 43000 and 25000. At the optimum pH 8.0 the specific activity of the purified enzyme is 630 units/mg protein, with L-gamma-glutamyl-p-nitroanilide as substrate (Km = 0.9 mM) and 20 mM glycylglycine as acceptor. The enzyme is inactivated by the active-site modifying agent and glutamine analogue, 6-diazo-5-oxo-L-norleucine, through a specific and stoichiometric reaction with the light subunit (Ki = 1.2 mM); both the inactivation and the modification of the light subunit are accelerated by maleate and prevented by S-methylglutathione. The enzyme is also inactivated by the fluorescent alkylating agent 5-iodoacetamidofluorescein, by specific and stoichiometric incorporation of the fluorescent moiety into the light subunit, which is likewise prevented by S-methylglutathione, but is unaffected by maleate. Antiserum to rat kidney gamma-glutamyltransferase cross-reacts with the pancreas enzyme in immunodiffusion and inhibits its activity in the p-nitroanilide assay. Despite structural, enzymological and immunological similarities between the pancreas and kidney enzymes, their amino acid compositions are markedly different. The rat pancreas enzyme shows an interesting ontological development, being present in minimal amounts in the fetus, and increasing dramatically on birth and during the following 2 days.

Acyltransferases↗
PubMed1982

Copper-zinc superoxide dismutase from Caulobacter crescentus CB15. A novel bacteriocuprein form of the enzyme.

A bacteriocuprein is a copper- and zinc-containing superoxide dismutase isolated from a bacterium. Until recently, the first and only documented bacteriocuprein was that from the marine bacterium Photobacterium leiognathi, which lives symbiotically with Leiognathid fishes. A new bacteriocuprein has been discovered, purified, and characterized from the free living, non-symbiotic bacterium, Caulobacter crescentus CB15. In its native molecular weight, homodimeric subunit structure, specific activity, and metal content, Caulobacter bacteriocuprein is very similar to the copper-zinc superoxide dismutases isolated from eukaryotes, just as the bacteriocuprein from Photobacterium has been shown to be. However, isolation and compositional analysis of tryptic peptides from Caulobacter bacteriocuprein has suggested that it contains amino acid substitutions at a number of sites which have been strictly conserved among the sequences of the eukaryote copper-zinc dismutases, from yeast to human. Consequently, Caulobacter bacteriocuprein may not be as closely related to the eukaryote enzymes as Photobacterium bacteriocuprein appears to be. Thus, the hypothesis of eukaryote to prokaryote gene transfer, proposed for the origin of the Photobacterium protein, may not be applicable for it. Alternative evolutionary mechanisms may therefore be necessary to explain the presence of the rare bacteriocuprein branch in the family tree of copper-zinc superoxide dismutases.

Amino Acid Sequence↗
PubMed1982

Isolation and biochemical characterization of the tryptic fragments of bovine nasal-cartilage proteoglycan monomer of high buoyant density.

Relatively homogeneous fractions of proteoglycan fragments were prepared from tryptic digests of the 4M-guanidinium chloride extract of bovine nasal cartilage. Glycosaminoglycan-containing fragments were separated from non-proteoglycan contaminants by ion-exchange chromatography and fractionated by equilibrium density-gradient centrifugation under dissociative conditions. The fractions of highest buoyant density were chromatographed on a column of Sepharose 4B, digested with chondroitinase ABC and chromatographed on a column of Sepharose 6B, yielding two distinct fractions: fraction B/6B-4 contained fragments from the chondroitin sulphate-bearing region of the proteoglycan monomer, and fraction B/6B-2 fragments from the keratan sulphate-rich region, most probably including a chondroitin sulphate-bearing monomer segment. By dansyl chloride analysis, fraction B/6B-2 had alanine and leucine as sole and fraction B/6B-4 had isoleucine and leucine as greatly predominant N-terminal amino acids, indicative of the relative homogeneity of these preparations of cartilage proteoglycan monomer fragments.

Amino Acids↗