铜锌超氧化物歧化酶(SOD)研究进展——从基因到功能

超氧化物歧化酶(SOD,EC 1.15.1.1),己经在多种组织中发现,它能将O2.-催化生成H2O2及O2.迄今为止,已经从哺乳动物体内分离出三种SOD:CuZnSOD(SOD1)、MnSOD(SOD2)TLEC-SOD(胞外超氧化物歧化酶,SOD3),各自具有不同的生化及分子特性.CuZnSOD(SOD1),是一类含有Cu及Zn原子的二聚体,存在于特定细胞的基质内,约占SOD总量的90%.在胞质及周质中,SOD以二聚体形式存在,而在线粒体及质外,则以四聚体形式存在.在保护脑、肺及其它组织的氧化应激中,CuZnSOD被认为起着保护作用.运动神经元肌萎缩侧索硬化症(ALS),据称也与同源二聚体CuZnSOD的错误折叠有关,己经报导,有多个CuZnSOD基因位点突变与ALS有关.本文将从基因的结构、表达、调节及蛋白的结构与功能等方面,对CuZnSOD进行简要论述.     

Molecular cloning and characterization of copper/zinc-superoxide dismutase of Paragonimus westermani

Superoxide dismutases (SODs; EC 1.15.1.1) play important roles in the protection of the parasites against cellular oxygen-mediated killing of the hosts. A copper/zinc-containing SOD (Cu/Zn-SOD) was identified previously from lung fluke, Paragonimus westermani. To expand our understanding of P. westermani SOD, we isolated a complementary DNA encoding a Cu/Zn-SOD, expressed the active enzyme in Escherichia coli, and characterized its biochemical properties. The deduced amino acid (aa) sequence of the gene shared up to 73.7% identities with Cu/Zn-SODs of other helminths and shared well-conserved characteristic motifs and essential aa residues involved in coordinating copper and zinc enzymatic functions. Recombinant Cu/ Zn-SOD exhibited comparable biochemical properties with that of the native enzyme, including pH optima and potassium cyanide-and hydrogen peroxide-sensitive inhibition profiles. The active enzyme consisted of 2 identical subunits covalently linked by disulfide bonds. The enzyme was constitutively expressed throughout various developmental stages of the parasite. The levels increased as P. westermani matured and plateaued in adult stage. Our result suggests the enzyme might play an important role for parasites to survive in the hosts through its superoxide anion-detoxifying function.     

Purification and Properties of Glyoxysomal Cuprozinc Superoxide Dismutase from Watermelon Cotyledons (Citrullus vulgaris Schrad)

A glyoxysomal copper,zinc-containing superoxide dismutase (EC 1.15.1.1) was purified to homogeneity, for the first time, from watermelon cotyledons (Citrullus vulgaris Schrad.). The stepwise purification procedure consisted of acetone precipitation, batch anion-exchange chromatography, anion-exchange Fast Protein Liquid Chromatography and gel-filtration column chromatography. Pure copper,zinc-superoxide dismutase (Cu,Zn-SOD II) had a specific activity of 1211 units per milligram protein and was purified 400-fold, with a yield of 8 micrograms enzyme per gram cotyledon. The glyoxysomal Cu,Zn-SOD had a relative molecular weight of about 33,000 and was composed of two equal subunits of 16,500 Daltons. Metal analysis showed that the enzyme, unlike other Cu,Zn-SODs, contained 1 gram-atom Cu and 1 gram-atom Zn per mole dimer. No iron and manganese were detected. Ultraviolet and visible absorption spectra were reminiscent of other copper,zinc-superoxide dismutases.     

Superoxide dismutase in the housefly,Musca domestica (L.)

Four superoxide dismutase (SOD) (E.C. 1.15.1.1) isozymes were present in whole tissue homogenates of Musca domestica when examined by polyacrylamide gel electrophoresis. One of the isozymes contained manganese, and the other three contained copper and zinc. All were observed in each of the body tagma (head, abdomen, and thorax) and at each developmental stage (egg to adult). The copper- and zinc-containing isozymes purified from newly emerged, adult M. domestica had a relative molecular weight of 34,800 as determined by gel filtration chromatography but consisted of two equal-size subunits of 16,000 as measured by sodium dodecylsulfate polyacrylamide gel electrophoresis. An isoelectric point between 4.8 and 5.1 was measured. Approximately 2 mol each of copper and zinc were present per dimer. The three copper, zinc isozymes were identified as charge variants. The amino acid composition of the enzyme was similar to that of copper, zinc-containing superoxide dismutases from other sources. Purified housefly copper, zinc superoxide dismutase was neither deactivated nor able to protect lactic dehydrogenase against deactivation in the presence of light and rose bengal, a known generator of singlet oxygen. The role of SOD in the phototoxic reaction involving rose bengal is discussed.     

Manganese superoxide dismutase from a higher plant

A manganese-containing superoxide dismutase (EC 1.15.1.1) was purified to homogeneity from a higher plant for the first time. The enzyme was isolated fromPisum sativum leaf extracts by thermal fractionation, ammonium sulfate salting out, ion-exchange and gel-filtration column chromatography, and preparative polyacrylamide gel electrophoresis. Pure manganese superoxide dismutase had a specific activity of about 3,000 U mg^-1 and was purified 215-fold, with a yield of 1.2 mg enzyme per kg whole leaf. The manganese superoxide dismutase had a molecular weight of 94,000 and contained one g-atom of Mn per mol of enzyme. No iron and copper were detected. Activity reconstitution experiments with the pure enzyme ruled out the possibility of a manganese loss during the purification procedure. The stability of manganese superoxide dismutase at-20°C, 4°C, 25°C, 50°C, and 60°C was studied, and the enzyme was found more labile at high temperatures than bacterial manganese superoxide dismutases and iron superoxide dismutases from an algal and bacterial origin.Abbreviations NBT nitro blue tetrazolium - SOD superoxide dismutase (EC 1.15.1.1)     

Isolation, Purification, and Subcellular Localization of Isozymes of Superoxide Dismutase from Scots Pine (Pinus sylvestris L.) Needles

Two of four isozymes of superoxide dismutase (SOD) (EC 1.15.1.1) were purified from Scots pine (Pinus sylvestris L.) needles. One form was cytosolic (SOD-1) and the other was associated with chloroplasts (SOD-3). The holoenzyme molecular masses was estimated at approximately 35 kilodaltons by gel filtration. The subunit molecular weight of the dimeric enzymes was estimated to 16.5 kilodaltons (SOD-1) and 20.4 kilodaltons (SOD-3) on sodium dodecyl sulfatepolyacrylamide gels. The NH₂-terminal sequence of the pine enzymes showed similarities to other purified superoxide dismutases located in the corresponding compartment. The cytosolic form revealed two additional amino acids at position 1 and 2 at the NH₂-terminal. Both forms were cyanide- and hydrogenperoxide-sensitive and SOD-3 was found to contain approximately one copper atom per subunit, indicating that they belong to the cupro-zinc SODs. The isoelectric point was 4.9 and 4.5 for SOD-1 and SOD-3, respectively.     

Purification, crystallization and properties of iron-containing superoxide dismutase from Pseudomonas ovalis.

Three electrophoretically distinct superoxide dismutases (EC 1.15.1.1) were observed in the crude extracts from Pseudomonas ovalis. One of these was isolated as an iron-containing superoxide dismutase. It contained 1.4 gatoms of Fe per mol of enzyme, and had a specific activity of 3900 units per mg of protein. A crystallized enzyme contained 1.1 gatoms of Fe per mol of enzyme, and had a specific activity of 3100 units per mg of protein. The results of sedimentation equilibrium and gel filtration indicated a molecular weight of 40,000. S020,W was estimated as 3.18 by sedimentation velocity study. Sodium dodecyl sulfate gel electrophoresis indicated that the enzyme was composed of two subunits, and had a molecular weight of 19,500. Analysis for sulfhydryl groups showed that there were four such groups per mol of enzyme. The spectrum of visible and ultraviolet region, the amino acid composition, the CD spectrum of the enzyme, and the effect of certain compounds on the enzyme, were studied and compared with iron-containing superoxide dismutases isolated from other organisms.     

The hyper-thermostable Fe-superoxide dismutase from the Archaeon Acidianus ambivalens: characterization, recombinant expression, crystallization and effects of metal exchange

An iron-containing superoxide dismutase (SOD; EC 1.15.1.1) of the hyperthermophilic archaeon Acidianus ambivalens (Aa-SOD) has been purified and characterized and the gene has been cloned and sequenced. The SOD from the facultatively aerobic member of the crenarchaeota could be expressed in E. coli. Both, the native as well as the heterologously overproduced protein turned out to have extraordinarily high melting temperatures of 128 degrees C and 124.5 degrees C, respectively. To the best of our knowledge, this is the highest directly measured melting temperature of a native protein. Surprisingly, neither the native nor the recombinant superoxide dismutase displays 100% occupation of the metal coordination sites. Obviously it is not the incorporation of a metal ion that confers the extreme thermostability. Expression of the superoxide dismutase in the presence of different metals such as Fe, Co, Ni, Mn and Cu offered the possibility of studying the hitherto unknown cofactor preference of iron-superoxide dismutase. The recombinant enzyme displayed the highest preference for incorporation of cobalt although iron is used as the natural cofactor. Spectroscopic analysis by EPR, atomic absorption and UVNis spectroscopy as well as activity measurements and differential scanning calorimetry of the metal substituted superoxide dismutases were performed. However, the superoxide dismutase of A. ambivalens is active only with iron but may incorporate other metals equally well in the catalytic center without loss of conformational stability or heat tolerance. The co-form of the enzyme could be crystallized.     

Polymers of Cu/Zn Superoxide Dismutase Produced by Cross-Linking with Glutaraldehyde

Soluble polymers of bovine Cu/Zn superoxide dismutase (EC 1.15.1.1) have been prepared using the homobifunctional cross-linking reagent, glutaraldehyde. A form of the enzyme, a tetramer. with a molecular weight of 64, 000 has been purified by gel filtration. The functional properties of the tctrarner have been investigated. Reconstitution with copper and zinc was required for full activity. After metal reconstitution, the specific activity of the tetramer was shown to be close to 90% that of the native dimerism enzyme.

The serum half-life of the tetramer in rats was found to be increased by a factor of six when compared with native superoxide dismutase. The tissue distribution of the two forms was also found to be direrent with the tetrarner accumulating predominantly in the liver.     

Polymers of Cu/Zn Superoxide Dismutase Produced by Cross-Linking with Glutaraldehyde

Soluble polymers of bovine Cu/Zn superoxide dismutase (EC 1.15.1.1) have been prepared using the homobifunctional cross-linking reagent, glutaraldehyde. A form of the enzyme, a tetramer. with a molecular weight of 64, 000 has been purified by gel filtration. The functional properties of the tctrarner have been investigated. Reconstitution with copper and zinc was required for full activity. After metal reconstitution, the specific activity of the tetramer was shown to be close to 90% that of the native dimerism enzyme.

The serum half-life of the tetramer in rats was found to be increased by a factor of six when compared with native superoxide dismutase. The tissue distribution of the two forms was also found to be direrent with the tetrarner accumulating predominantly in the liver.     

Regulation of superoxide dismutase synthesis in Candida albicans

The synthesis of superoxide dismutase [SOD: EC 1.15.1.1] in response to various cultural conditions was examined in Candida albicans, an opportunistic yeast which causes candidiasis in immunosuppressed patients. SOD plays an important role in protecting cells from the oxidative damage of superoxide radicals. Maximum SOD activity was found after 72 hrs of yeast growth. The optimum pH and temperature for the SOD activity were 7 and 40 °, respectively. The major SOD activity was found in the cytosol fraction and the level of extracellular SOD was very low. The enzyme was stimulated to varying degrees by cholic acid, procaine and tocopherol. On the basis of inhibitor studies and other enzyme properties, the isolated enzyme from C. albicans is identified as copper and zinc superoxide dismutase. This revised version was published online in June 2006 with corrections to the Cover Date.     

Modeling and analysis of soybean (Glycine max. L) Cu/Zn,Mn and Fe superoxide dismutases

Superoxide dismutase (SOD, EC 1.15.1.1) is an important metal-containing antioxidant enzyme that provides the first line of defense against toxic superoxide radicals by catalyzing their dismutation to oxygen and hydrogen peroxide. SOD is classified into four metalloprotein isoforms, namely, Cu/Zn SOD, Mn SOD, Ni SOD and Fe SOD. The structural models of soybean SOD isoforms have not yet been solved. In this study, we describe structural models for soybean Cu/Zn SOD, Mn SOD and Fe SOD and provide insights into the molecular function of this metal-binding enzyme in improving tolerance to oxidative stress in plants.     

Superoxide,hydrogen peroxide,and the gelling of phloem sap from Cucurbita pepo

The possible involvement of superoxide and hydrogen peroxide in the oxidative gelling of phloem exudate from Cucurbita pepo. was investigated. Neither superoxide dismutase (EC 1.15.1.1) nor catalase (EC 1.11.1.6) inhibited the reaction. Although catalase could not be detected in exudate, both peroxidase (EC. 1.11.1.7) and superoxide dismutase were present in reasonable amounts. Polyacrylamide gel electrophoresis revealed one major and one minor isozyme of superoxide dismutase, both of which were adjudged to contain copper and zinc as their prosthetic metals, on the basis of cyanide inhibition and molecular weight.Abbreviations SOD superoxide dismutase     

Unique features of the sodC-encoded superoxide dismutase from Mycobacterium tuberculosis, a fully functional copper-containing enzyme lacking zinc in the active site

The sodC-encoded Mycobacterium tuberculosis superoxide dismutase (SOD) shows high sequence homology to other members of the copper/zinc-containing SOD family. Its three-dimensional structure is reported here, solved by x-ray crystallography at 1.63-A resolution. Metal analyses of the recombinant protein indicate that the native form of the enzyme lacks the zinc ion, which has a very important structural and functional role in all other known enzymes of this class. The absence of zinc within the active site is due to significant rearrangements in the zinc subloop, including deletion or mutation of the metal ligands His115 and His123. Nonetheless, the enzyme has a catalytic rate close to the diffusion limit; and unlike all other copper/zinc-containing SODs devoid of zinc, the geometry of the copper site is pH-independent. The protein shows a novel dimer interface characterized by a long and rigid loop, which confers structural stability to the enzyme. As the survival of bacterial pathogens within their host critically depends on their ability to recruit zinc in highly competitive environments, we propose that the observed structural rearrangements are required to build up a zinc-independent but fully active and stable copper-containing SOD.     

Characterization of the iron superoxide dismutase gene of Azotobacter vinelandii: sodB may be essential for viability

Azotobacter vinelandii contains two superoxide dismutases (SODs), a cytoplasmic iron-containing enzyme (FeSOD), and a periplasmic copper/zinc-containing enzyme (CuZnSOD). In this study, the FeSOD was found to be constitutive, while the activity of CuZnSOD increased as the culture entered the stationary phase. Total SOD (units/mg protein) in stationary phase cells grown under nitrogen-fixing conditions was not significantly different from those grown under non-nitrogen-fixing conditions. The gene encoding FeSOD (sodB) was isolated from an A. vinelandii cosmid library. A 1-kb fragment containing the coding region and 400 base pairs of upstream sequence was cloned and sequenced. The nucleotide sequence and the deduced amino acid sequence had a high degree of homology with other bacterial FeSODs, particularly with P. aeruginosa. Attempts to construct a sodB mutant by recombination of a sodB::kan insertion mutation into the multicopy chromosome of A. vinelandii were unsuccessful even in the presence of SOD mimics or nutritional supplements. These results suggest that FeSOD may be essential for the growth and survival of A. vinelandii, and that the periplasmic CuZnSOD cannot replace the function of FeSOD.     

The primary structure of iron-superoxide dismutase from Photobacterium leiognathi

The complete amino acid sequence of iron-superoxide dismutase from Photobacterium leiognathi was determined. The sequence was deduced following characterization of the peptides obtained from tryptic, chymotryptic, and Staphylococcus aureus V-8 protease digests of the apoprotein. The amino acid sequence listed below is made up of 193 residues. It is the first complete sequence to be determined for an iron-superoxide dismutase. The iron-superoxide dismutase shows the same order of homology with the manganese-superoxide dismutases as these enzymes show among themselves. No homology was observed with the copper/zinc-containing class of superoxide dismutases. Ala-Phe-Glu-Leu-Pro-Ala-Leu-Pro-Phe-Ala-Met-Asn-Ala-Leu-Glu-Pro-His-Ile- Ser-Gln-Glu-Thr-Leu-Glu-Tyr-His-Tyr-Gly-Lys-His-His-Asn-Thr-Tyr-Val-Val- Lys-Leu-Asn-Gly-Leu-Val-Glu-Gly-Thr-Glu-Leu-Ala-Glu-Lys-Ser-Leu-Glu-Glu- Ile-Ile-Lys-Thr-Ser-Thr-Gly-Gly-Val-Phe-Asn-Asn-Ala-Ala-Gln-Val-Trp-Asn- His-Thr-Phe-Tyr-Trp-Asn-Cys-Leu-Ala-Pro-Asn-Ala-Gly-Gly-Glu-Pro-Thr-Gly- Glu-Val-Ala-Ala-Ala-Ile-Glu-Lys-Ala-Phe-Gly-Ser-Phe-Ala-Glu-Phe-Lys-Ala- Lys-Phe-Thr-Asp-Ser-Ala-Ile-Asn-Asn-Phe-Gly-Ser-Ser-Trp-Thr-Trp-Leu-Val- Lys-Asn-Ala-Asn-Gly-Ser-Leu-Ala-Ile-Val-Asn-Thr-Ser-Asn-Ala-Gly-Cys-Pro- Ile-Thr-Glu-Glu-Gly-Val-Thr-Pro-Leu-Leu-Thr-Val-Asp-Leu-Trp-Glu-His-Ala- Tyr-Tyr-Ile-Asp-Tyr-Arg-Asn-Leu-Arg-Pro-Ser-Tyr-Met-Asp-Gly-Phe-Trp-Ala- Leu-Val-Asn-Trp-Asp-Phe-Val-Ser-Lys-Asn-Leu-Ala-Ala.     

Characterization of a Manganese Superoxide Dismutase from the Higher Plant Pisum sativum

A manganese-containing superoxide dismutase (EC 1.15.1.1) was fully characterized from leaves of the higher plant Pisum sativum L., var. Lincoln. The amino acid composition determined for the enzyme was compared with that of a wide spectrum of superoxide dismutases and found to have a highest degree of homology with the mitochondrial manganese superoxide dismutases from rat liver and yeast. The enzyme showed an apparent pH optimum of 8.6 and at 25°C had a maximum stability at alkaline pH values. By kinetic competition experiments, the rate constant for the disproportionation of superoxide radicals by pea leaf manganese superoxide dismutase was found to be 1.61 × 10⁹ molar⁻¹·second⁻¹ at pH 7.8 and 25°C. The enzyme was not sensitive to NaCN or to H₂O₂, but was inhibited by N₃⁻. The sulfhydryl reagent p-hydroxymercuribenzoate at 1 mm concentration produced a nearly complete inhibition of the manganese superoxide dismutase activity. The metal chelators o-phenanthroline, EDTA, and diethyldithiocarbamate all inhibited activity slightly in decreasing order of intensity. A comparative study between this higher plant manganese superoxide dismutase and other dismutases from different origins is presented.     

Characterization of an iron-containing superoxide dismutase from a higher plant, Citrus limonum

An iron-containing superoxide dismutase (SOD, EC 1.15.1.1) was fully characterized from leaves of the higher plant Citrus limonum R. cv. Verna. This enzyme is the first iron-containing SOD to be characterized in the plant family Rutaceae . The purified Fe-SOD has a molecular mass of about 47 kDa and is composed of two non-covalently joined equal subunits. The amino acid composition determined for the enzyme was compared with that of a wide range of SODs and had highest degree of homology with the Fe-SODs from Brassica campestris and Nuphar luteum . The enzyme was more labile at high temperatures than some eucaryotic and procaryotic Fe-SODs. It showed a maximum stability at pH 7.8. The sensitivity of the enzyme to cyanide, hydrogen peroxide and o -phenanthroline was similar to those reported for other Fe-SODs. but the lemon enzyme was comparatively resistant to H₂O₂. By kinetic competition experiments, the rate constant for the disproportionation of superoxide radicals by lemon Fe-SOD was found to be 1.9 × 10⁹ M ⁻¹ s⁻¹ at pH 7.8 and 25°C. A comparative study between the molecular properties of this higher plant Fe-SOD and SODs from different origins is presented.     

Purification and Properties of Manganese Superoxide Dismutase from Norway Spruce (Picea abies L. Karst)

A manganese-containing superoxide dismutase (SOD; EC 1.15.1.1 [EC] )was purified to electrophoretic homogeneity from seeds of Norwayspruce (Picea abies L.). The apparent molecular mass of thepurified enzyme was 86 kDa, as determined by gel filtration.The subunit molecular mass, estimated by SDS-polyacrylamidegel electrophoresis, was 22 kDa both in the presence and inthe absence of 2-mercaptoethanol. Thus, the native enzyme isa homotetramer with subunits that were not linked by disulfidebonds. The isoelectric point of this Mn-SOD was 5.5. The specificactivity of the Mn-SOD was strongly pH-dependent and was 400units per nmol SOD at pH 7.8 and 30 units per nmol SOD at pH10.4. The first 25 amino acid residues in the amino terminalregion of spruce Mn-SOD exhibited a high degree of sequencehomology to those of Mn-SODs from other organisms. In Mn-deficientneedles the activity of Mn-SOD was only half of that in non-deficientneedles, whereas the activity of CuZn-SOD was doubled. (Received May 20, 1994; Accepted October 31, 1994)