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M L Salin

Publications and source records attributed to M L Salin.

At least 37 records · Page 2Linked to original sources
PubMed1993

Purification of a catalase-peroxidase from Halobacterium halobium: characterization of some unique properties of the halophilic enzyme.

A hydroperoxidase purified from the halophilic archaeon Halobacterium halobium exhibited both catalase and peroxidase activities, which were greatly diminished in a low-salt environment. Therefore, the purification was carried out in 2 M NaCl. Purified protein exhibited catalase activity over the narrow pH range of 6.0 to 7.5 and exhibited peroxidase activity between pH 6.5 and 8.0. Peroxidase activity was maximal at NaCl concentrations above 1 M, although catalase activity required 2 M NaCl for optimal function. Catalase activity was greatest at 50 degrees C; at 90 degrees C, the enzymatic activity was 20% greater than at 25 degrees C. Peroxidase activity decreased rapidly above its maximum at 40 degrees C. An activation energy of 2.5 kcal (ca. 10 kJ)/mol was calculated for catalase, and an activation energy of 4.0 kcal (ca. 17 kJ)/mol was calculated for peroxidase. Catalase activity was not inhibited by 3-amino-1,2,4-triazole but was inhibited by KCN and NaN3 (apparent Ki [KiApp] of 50 and 67.5 microM, respectively). Peroxidative activity was inhibited equally by KCN and NaN3 (KiApp for both, approximately 30 microM). The absorption spectrum showed a Soret peak at 404 nm, and there was no apparent reduction by dithionite. A heme content of 1.43 per tetramer was determined. The protein has a pI of 3.8 and an M(r) of 240,000 and consists of four subunits of 60,300 each.

Catalase↗
PubMed1991

Elevation of superoxide dismutase in Halobacterium halobium by heat shock.

Exposure of Halobacterium halobium to 50 degrees C for 2.5 h in an aerobic environment resulted in a greater than twofold increase in the activity of the manganese-containing superoxide dismutase. Nondenaturing polyacrylamide gels stained for enzymatic activity did not reveal any additional isozymes of superoxide dismutase induced by the heat shock. The maximal effect was observed at 50 degrees C, and the elevated levels of activity remained constant during 5 h of recovery at 40 degrees C. The induction of enzymatic activity was sensitive to protein synthesis inhibitors. The results are discussed relative to heat shock and stress-related proteins as well as alterations in metabolism brought about by elevated temperatures.

Base Sequence↗
PubMed1991

Halobacterium halobium Mn-SOD gene: archaebacterial and eubacterial features.

A 1.8 kb PstI fragment from Halobacterium halobium DNA was found to hybridize to synthetic oligonucleotide probes constructed by using the sequence of the N-terminus of a Mn-containing superoxide dismutase purified from H. halobium. The entire insert containing a 600-bp coding sequence for Mn-SOD and its 5' and 3' flanking regions was sequenced. The derived amino acid sequence of the structural gene showed a similarity to other manganic and iron-containing superoxide dismutases in normally conserved regions. Primer extension analysis of the H. halobium Mn-SOD mRNA showed that gene transcription begins 14 bases upstream of the translational start. A Shine-Dalgarno sequence and archaebacterial consensus promoter sequences were observed. Several other promoter and terminator nucleotide sequences homologous to prokaryotic and eukaryotic organisms were found.

Amino Acid Sequence↗
PubMed1991

Chloroplast and mitochondrial mechanisms for protection against oxygen toxicity.

As a consequence of their oxygen rich environment, organelles of photosynthetic tissues are exposed to large fluxes of oxyradicals and reactive oxygen species. Superoxide, hydrogen peroxide, hydroxyl radical and singlet oxygen are all potential by-products of respiratory and photosynthetic systems. Strong reductants found in mitochondria and chloroplasts along with a steady flux of photosynthetically generated oxygen enhance the potential for oxyradical production. Unless neutralized by scavenger substrates or enzymes, these reactive intermediates pose a lethal threat. The presence of superoxide dismutases, catalases, various peroxidases and scavenger substrates are all means of defences available to protect organelles. A balance between oxyradical production and neutralization should exist. Perturbations in generation or in sequestration caused by environmental or nutritional factors might profoundly alter the steady state level of oxyintermediates.

Chloroplasts↗
PubMed1988

Cloning and determination of the nucleotide sequence of the Mn-containing superoxide dismutase gene from Halobacterium halobium.

A group of synthetic 17-mer oligodeoxynucleotides (oligos) was constructed to correspond to a sequence of amino acids situated near the N terminus of the manganese-containing superoxide dismutase (Mn-SOD) purified from the halophilic bacterium, Halobacterium halobium. A cosmid library of a Sau3AI partial digest of halobium DNA, cloned into the BamHI site of pHC79, was probed with the radiolabeled oligos. Cosmid DNA was purified from the clone that showed hybridization at the highest stringency. A 1.8-kb PstI fragment of this DNA which hybridized the probes was subcloned into bacteriophage M13 and transfected into Escherichia coli JM101. The entire insert containing a 600-bp sequence coding for Mn-SOD and its 5'- and 3'-flanking regions was sequenced. The derived amino acid sequence of the structural gene showed a similarity to other manganese and iron-containing SODs in normally conserved regions.

Amino Acid Sequence↗
PubMed1988

Purification of a manganese-containing superoxide dismutase from Halobacterium halobium.

An oxygen-induced superoxide dismutase was purified from the halophilic bacterium, Halobacterium halobium, strain NRL. Due to the high salt requirement for enzyme stability, the purification had to be performed in the presence of 2 M NaCl. The pI of the protein was 4.95. The approximate Mr was 38,500. The subunit size as determined by sodium dodecyl sulfate-electrophoresis was approximately 19,000. Metal analysis showed 1.5 atoms of manganese per dimer, 0.5 atom zinc, and 1.54 atoms copper. The N-terminal sequence of amino acids was determined, and based upon the first 26 amino acids significant homology to other manganese- and iron-containing superoxide dismutases was revealed.

Amino Acid Sequence↗
PubMed1986

A Streptococcus mutans superoxide dismutase that is active with either manganese or iron as a cofactor.

The superoxide dismutase produced by Streptococcus mutans OMZ176 during aerobic growth in a chemically defined medium (modified FMC) that was treated with Chelex 100 (to lower trace metal contamination) and supplemented with high purity manganese was purified (162-fold) by heat treatment, ammonium sulfate precipitation, and chromatofocusing chromatography. The superoxide dismutase produced during aerobic growth in the same medium, but without manganese and supplemented with high purity iron, was similarly purified (220-fold). The molecular masses of each holoenzyme were approximately 43,000 with a subunit mass of 20,700, indicating that the enzymes were dimers of two equally sized subunits. The superoxide dismutase from manganese-grown cells was a manganese enzyme (MnSOD) containing 1.2 atoms of manganese and 0.25 atoms of iron/subunit. The superoxide dismutase from iron-grown cells was an iron enzyme (FeSOD) containing 0.07 atoms of manganese and 0.78 atoms of iron/subunit. The amino acid compositions of the MnSOD and the FeSOD were virtually identical, and their amino-terminal sequences were identical through the first 22 amino acids. Dialysis of the FeSOD with o-phenanthroline and sodium ascorbate generated aposuperoxide dismutase with 94% loss of activity; subsequent dialysis of apoenzyme with either manganese sulfate or ferrous sulfate reconstituted activity (recoveries of 37 and 30%, respectively). Electrophoretic determination of cytoplasmic radioiron distribution indicated that (during aerobic growth) manganese prevented insertion of iron into superoxide dismutase, although the iron levels of at least two other cytoplasmic fractions were not altered by manganese. Therefore, S. mutans used the same aposuperoxide dismutase to form either FeSOD or MnSOD, depending upon which metal was available in the culture medium. Such "cambialistic" enzymes (those capable of making a cofactor substitution) may represent a previously unrecognized family of superoxide dismutases.

Aerobiosis↗
PubMed1985

Purification and characterization of an iron-containing superoxide dismutase from a eucaryote, Ginkgo biloba.

A cyanide-insensitive superoxide dismutase was purified to electrophoretic homogeneity from leaves of the eucaryote, Ginkgo biloba L. A molecular mass of 47,000 was determined for the enzyme, which consisted of two subunits of equal size. The enzyme preparation contained two isoenzymes with isoelectric points of 5.25 and 5.15. Metal analysis after dialysis against EDTA revealed the presence of 1.4 gram atoms of iron per molecule. Approximately 2 gram atoms each of copper and zinc per enzyme molecule were also detected, although removal of copper by other chelators had no effect on enzymatic activity. The purified Ginkgo enzyme exhibited a sensitivity to hydrogen peroxide and insensitivity to cyanide, which is typical of iron-containing superoxide dismutases. Ginkgo iron superoxide dismutase was localized in the stroma of chloroplasts, but was absent from mitochondria. The enzyme from Ginkgo was most similar to iron superoxide dismutases of Nuphar, Brassica, and Escherichia coli when compared on the basis of S delta Q analysis of amino acid composition. Peptide fragments formed by proteolytic digestion of these four enzymes were compared qualitatively; similar-sized fragments which denote possible areas of homology were observed.

Amino Acids↗
PubMed1984

Oxygen toxicity in Streptococcus mutans: manganese, iron, and superoxide dismutase.

When cultured anaerobically in a chemically defined medium that was treated with Chelex-100 to lower its trace metal content, Streptococcus mutans OMZ176 had no apparent requirement for manganese or iron. Manganese or iron was necessary for aerobic cultivation in deep static cultures. During continuous aerobic cultivation in a stirred chemostat, iron did not support the growth rate achieved with manganese. Since the dissolved oxygen level in the chemostat cultures was higher than the final level in the static cultures, manganese may be required for growth at elevated oxygen levels. In medium supplemented with manganese, cells grown anaerobically contained a low level of superoxide dismutase (SOD) activity; aerobic cultivation increased SOD activity at least threefold. In iron-supplemented medium, cells grown anaerobically also had low SOD activity; aerobic incubation resulted in little increase in SOD activity. Polyacrylamide gel electrophoresis of the cell extracts revealed a major band and a minor band of SOD activity in the cells grown with manganese; however, cells grown with iron contained a single band of SOD activity with an Rf value similar to that of the major band found in cells grown with manganese. None of the SOD activity bands were abolished by the inclusion of 2 mM hydrogen peroxide in the SOD activity strain. S. mutans may not produce a separate iron-containing SOD but may insert either iron or manganese into an apo-SOD protein. Alternatively, iron may function in another activity (not SOD) that augments the defense against oxygen toxicity at low SOD levels.

Anaerobiosis↗
PubMed1982

Isolation and Characterization of an Iron-Containing Superoxide Dismutase From Water Lily, Nuphar luteum.

A cyanide-insensitive superoxide dismutase (EC 1.15.1.1) was purified from leaves of the water lily Nuphar luteum (L.) Sibth. and Smith Subsp. macrophyllum (Small) Beal. The enzyme had a molecular weight of 46,000 and was composed of two equally sized subunits. Metal analysis showed the protein to contain about 1 gram atom of iron per dimer. The iron-containing superoxide dismutase was sensitive to H(2)O(2) as well as to azide. Antibody to the protein did not cross-react with iron superoxide dismutase isolated from the eucaryote Brassica or with algal extracts.

Journal Article↗
PubMed1981

Porcine superoxide dismutase. Isolation and characterization of a relatively basic cuprozinc enzyme.

A cuprozinc superoxide dismutase has been isolated from pig liver. The enzyme is similar to previously described cuprozinc superoxide dismutases in that it is a dimer of about 32 000 molecular weight consisting of approximately two equally sized subunits, and 2 atoms of copper and two atoms of zinc per molecule. It differs, however, from previously described cuprozinc superoxide dismutases because of its higher isoelectric point; pI 6.8 vs 4.9 for bovine enzyme. The diffusion coefficient for the porcine enzyme was determined to be 7.53 x 10(-7) cm2 s-1, while the equivalent spherical hydrodynamic radius was computed as 28.5 A. The enzyme was observed to undergo self-association with time. Sulfhydryl interaction is postulated to be involved.

Amino Acids↗
PubMed1981

Absence of the iron-containing superoxide dismutase in mitochondria from mustard (Brassica campestris).

Mitochondria were isolated from mature leaves as well as etiolated seedlings of Brassica campestris (mustard), a eukaryote previously shown to possess the iron-containing isoenzyme of superoxide dismutase. On the basis of KCN- and H2O2-sensitivity, and on polyacrylamide-gel analysis, only the cuprozinc and mangano superoxide dismutases were found in mitochondria. The iron-containing enzyme was absent. The mangano enzyme was found in the mitochondrial matrix, whereas the cuprozinc enzyme appeared to be localized in the intermembrane space.

Copper↗
PubMed1981

Chemiluminescence in Wounded Root Tissue : EVIDENCE FOR PEROXIDASE INVOLVEMENT.

Root and stem segments from soybean (Merrill cv. ;Bragg') showed an enhanced chemiluminescence upon mechanical injury. Roots emitted more light than did stems. Light emission was diminished by CN(-) and N(3) (-) but was not affected by rotenone and antimycin A. Catalase quenched chemiluminescence in wounded root segments as did ascorbic acid and hydroquinone. Superoxide dismutase addition resulted in a small diminution in light emission, but mannitol, an OH. scavenger, was without effect. The addition of H(2)O(2) to wounded root segments markedly elevated chemiluminescence in the presence of air as well as under N(2). It is concluded that peroxidases, found abundantly in roots, predominantly contribute to light emission in wounded plant tissue.

Journal Article↗
PubMed1981

Distribution of iron-containing superoxide dismutase in vascular plants.

Superoxide dismutases (EC 1.15.1.1) in vascular plants representing different evolutionary levels were characterized using polyacrylamide gel electrophoresis. The three forms of the enzyme were distinguished from each other based on the following criteria: a) the Cu-Zn enzyme is sensitive to cyanide wherease the Fe and Mn enzymes are not; and b) the Cu-Zn and Fe enzymes are inhibited by H(2)O(2) whereas the Mn enzyme is H(2)O(2)-resistant. Of the 43 plant families investigated, the Fe-containing superoxide dismutase was found in three families: Gingkoaceae, Nymphaceae, and Cruciferae.

Journal Article↗
PubMed1975

Free radicals and inflammation. Protection of phagocytosine leukocytes by superoxide dismutase.

Isolated human polymorphonuclear leukocytes engaged in phagocytosis liberate superoxide radical and hydrogen peroxide into the surrounding medium. These two chemical species react to produce the hydroxyl radical, which attacks the leukocyte and leads to premature death of the cell. The hydroxyl radical may be scavenged by mannitol, or its formation can be prevented by the addition of superoxide dismutase or catalase to the medium, thereby eliminating the premature death of the cells. This phenomenon may partially explain the observed anti-inflammatory activity of superoxide dismutase.

Catalase↗