The amino-acid sequence of rabbit Cu-Zn superoxide dismutase

The primary structure of Cu-Zn superoxide dismutase from rabbit liver was investigated. The reduced and S-carboxymethylated enzyme was treated with cyanogen bromide, trypsin or Staphylococcus aureus proteinase V8. The resulting peptides were separated by high-performance liquid chromatography and sequenced by automated Edman degradation. With the exception of the N- and C-terminus the complete sequence was established by means of overlapping peptides. The N-terminus is blocked and thus not susceptible to Edman degradation. The amino-acid composition of the tryptic N-terminal peptide corresponds to that of the cytoplasmatic Cu-Zn superoxide dismutases of other mammals investigated. The chromatographic behaviour of these N-terminal peptides on a reversed phase C18 column is also identical, thus suggesting also for the rabbit Cu-Zn superoxide dismutase the N-terminal sequence Ac-Ala-Thr-Lys. The C-terminus was demonstrated to have the sequence -Ile-Ala-Pro by enzymatic degradation with carboxypeptidase Y. The complete amino-acid sequence of the rabbit Cu-Zn superoxide dismutase consists of 152 amino-acids and shows the expected homology to other Cu-Zn enzymes published so far. The aspartate and six histidine residues known to complex the metal ions are conserved at homologous positions. This also applies for the arginine residue near the C-terminus which is supposed to direct the anionic superoxide radical towards the active centre of the enzyme. The amino acid sequence of the rabbit Cu-Zn superoxide dismutase corresponds to those of other mammals in more than 80% of its amino-acid residues. From a total of 152 amino-acid residues the rabbit shares with rat 128, with mouse 130, with horse 127, with pig 126/127, with cattle 130 and with man 131 amino acids in homologous positions. However the Cu-Zn superoxide dismutases of closely related mammals like rats and mice differ in only five amino acid residues of their sequence. A phylogenetic closer relatedness between lagomorphs and rodents than between other orders of mammals, could not be derived from the sequence data given. Rather rodents and lagomorphs are to be considered as two evolutionary independent orders of mammals.     

Primary structure of Cu-Zn superoxide dismutase of Brassica oleracea proves homology with corresponding enzymes of animals, fungi and prokaryotes

The complete amino-acid sequence of Cu-Zn superoxide dismutase from white cabbage (Brassica oleracea) is reported. The polypeptide chain consists of 151 amino acids and has a molecular mass of 15,604 Da. The primary structure of the reduced and S-carboxymethylated protein was determined by automated solid phase sequence analysis of tryptic fragments and peptides obtained by digestion with Staphylococcus aureus proteinase V8. The protein shows a free amino terminus as was found for all non-mammalian Cu-Zn enzymes so far sequenced. Comparison of the amino-acid sequence from the plant Cu-Zn enzyme with those from nine eukaryotic enzymes reveals a high degree of homology (50-64%) among these enzymes. As already described for all the eukaryotic Cu-Zn superoxide dismutases also the plant enzyme shows a low homology (about 28%) with the bacteriocuprein of Photobacterium leiognathi. However, the amino-acid residues involved in metal binding, the half-cystine residues forming the intermolecular disulfide bridge, one of the arginine and some glycine and proline residues are conserved in all eleven Cu-Zn superoxide dismutases. Although the precise role of the 23 completely conserved residues is not yet completely understood, they appear to almost define the minimum structural requirements for optimizing the superoxide dismutation at the catalytic site, since functional differences between the eleven enzymes are not detectable.     

The primary structure of porcine Cu-Zn superoxide dismutase. Evidence for allotypes of superoxide dismutase in pigs

Cu-Zn superoxide dismutase was purified to homogeneity from mixed pig blood and from a single pig. The isolated product had an absorption ratio 280/260 nm of 0.91, a specific activity of 3 000 +/- 200 units (cytochrome c reduction test), and an isoelectric point of 7.5 (chromatofocusing) or 7.25 (isoelectric focusing), respectively. Sequence determination was performed by automated solid-phase Edman degradation of fragments of the reduced S-carboxymethylated proteins obtained by digestion with trypsin or Staphylococcus aureus proteinase V8 or treatment with cyanogen bromide. Acetylation of the N-terminus was confirmed by comparing high performance liquid chromatography retention times of N-terminal peptides with those of authentic samples. Sequencing of the superoxide dismutase of mixed porcine blood revealed heterogeneity (70% Leu; 30% Val at position 29), whereas the sample derived from a single French pig proved to be homogeneous (100% Leu at position 29). The complete sequence of pig superoxide dismutase comprised 152 amino-acid residues, which corresponds to a theoretical molecular mass of 15 800 Da per subunit, and exhibited the expected high homology with those of other mammals. The aspartate and all 7 histidine residues known to complex the metal ions in bovine superoxide dismutase are conserved in the porcine sequence at the homologous positions Asp82 and His45, His47, His62, His70, His79, His119, respectively.     

Amino-acid sequence of a tetrameric, manganese superoxide dismutase from Thermus thermophilus HB8

The amino-acid sequence of a tetrameric manganese superoxide dismutase from Thermus thermophilus HB8 has been determined. The protein was cleaved with cyanogen bromide (BrCN) into four peptides and their alignment was deduced through the fragment of partial cleavage with BrCN and the peptides were produced by cleavage of the protein with o-iodosobenzoic acid. Most of the peptides were sequenced by solid phase Edman degradation. Some of the peptides were sequenced by the Edman dansyl method after sub-fragmentation by proteinase digestion. The amino-acid sequence consists of 203 residues corresponding to a subunit molecular weight of 23,144.     

Demonstration of Cu-Zn superoxide dismutase in rat liver peroxisomes. Biochemical and immunochemical evidence.

In this study, by using highly purified rat liver peroxisomes, we provide evidence from analytical cell fractionation, Western blot, and immunocytochemical analysis that Cu-Zn superoxide dismutase is present in animal peroxisomes. Treatment with ciprofibrate, a peroxisome proliferator, increased the peroxisomal superoxide dismutase activity by 3-fold with no effect on mitochondrial activity but a marked decrease in cytosolic superoxide dismutase activity, further supporting that besides cytosolic and mitochondrial localization, Cu-Zn superoxide dismutase is present in peroxisomes also. Demonstration of superoxide dismutase in peroxisomes suggests a new role for this organelle in pathophysiological conditions, such as ischemia-reperfusion injury.     

Human recombinant CuZn-superoxide dismutase. Amino acid sequence and location of the disulfide bond

The complete amino acid sequence of recombinant human Cu-Zn superoxide dismutase (CuZnSOD) is presented. The S-carboxymethylated protein was cleaved at lysine residues (with Achromobacter protease I) to provide a set of nine non-overlapping fragments accounting for 90% of the sequence. These fragments were then overlapped and aligned, and the sequence was completed by using peptides generated by cleavage at glutamic acid residues (with S. aureus V8 protease) and at arginine (with clostripain). The recombinant protein contains a single disulfide bond between cysteine residues 57 and 146. The primary sequence of recombinant human CuZnSOD is identical to that predicted by its cDNA sequence.     

Regulation of Mn-SOD activity in the mouse heart: glucose effect

Intraperitoneal injection of glucose was found to cause a dose and time dependent suppression of superoxide dismutase activity in mouse heart. Manganese superoxide dismutase was more sensitive to glucose suppression than Cu-Zn superoxide dismutase. While glucose suppressed the Mn form of the enzyme at the concentration of 1.5 mg/kg, it did not have a significant effect on Cu-Zn superoxide dismutase activity at this concentration. The maximum suppression for both forms of superoxide dismutase activity occurred at 4.5 mg/kg. Glucose also suppressed manganese superoxide dismutase activity in mouse heart for a longer period of time compared to Cu-Zn superoxide dismutase. Glucose suppression also occurred in mouse brain. The glucose suppression effect on manganese superoxide dismutase activity in the heart was partially alleviated by X-irradiation.     

A protein isolated from Brucella abortus is a Cu-Zn superoxide dismutase

Brucella abortus contains a protein that elicits an antigenic response in cattle previously exposed to the organism. The amino acid sequence of the recombinant form of this antigenic protein was determined by gas-phase sequencing of the pyridylethylated protein and its peptides obtained by digestion with cyanogen bromide (CNBr), clostripain, and Staphylococcus aureus V8 protease. The Brucella protein demonstrated 53.6% identity with the Cu-Zn superoxide dismutase (SOD) from Photobacterium leiognathi. Residues essential for metal coordination and enzymatic activity and cysteines required for the formation of the intrasubunit disulfide bridge of Cu-Zn SOD were conserved in the Brucella protein. also exhibited SOD activity that was inhibited by cyanide, which is characteristic of a Cu-Zn SOD. Brucella abortus Cu-Zn SOD is the second prokaryotic Cu-Zn SOD to be sequenced, and the fifth found in prokaryotes. The high degree of conservation between Photobacterium and Brucella Cu-Zn SOD supports the hypothesis of a separately evolved prokaryotic and eukaryotic Cu-Zn SOD gene.     

A highly sensitive method for determining both Mn- and Cu-Zn superoxide dismutase activities in tissues and blood cells

A highly sensitive method for determining the superoxide dismutase (EC 1.15.1.1) in various tissues and blood cells is described. This method involves inhibition of a cypridina luciferin analog that is chemiluminescence dependent upon O2- generated by hypoxanthine-xanthine oxidase. Manganeous superoxide dismutase, which is sensitive to sodium dodecyl sulfate, was determined and calculated by subtraction of superoxide dismutase activity in tissue extract treated with this detergent (Cu-Zn superoxide dismutase) from that in untreated tissue extract (total superoxide dismutase). Both Mn- and Cu-Zn superoxide dismutase activities were expressed as equivalent nanograms of bovine erythrocyte superoxide dismutase per milliliter. Sensitivity limits of the chemiluminescence methods with 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyraz in-3-one and 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one as cypridina luciferin analogs were 1 ng and 2-3 ng of superoxide dismutase/ml, respectively.     

Some sulfhydryl properties and primary structure of human erythrocyte superoxide dismutase

Human Cu-Zn superoxide dismutase prepared by different methods shows varying properties relevant to its sulfhydryl chemistry. A cysteine residue not found in the analogous bovine enzyme appears to be responsible for its unusual lability. Alkylation of this cysteine results in a marked increase in stability, and this form of the protein may be readily crystallized. The primary structure of the 153 amino acid residues found in the human protein has been determined, and 82% of the residues are identical with those of the bovine enzyme. A significant variation is seen in the portion of those proteins comprising residues 17-36, with eleven changes being noted.     

Interleukin 1 slowly increases lung fibroblast Cu-Zn superoxide dismutase activity levels

Certain pulmonary stress raises rat lung Cu-Zn superoxide dismutase (SOD) activity levels, but cytokines released during stress are reported to exert no regulatory effects on Cu-Zn SOD levels in cultured cells. In contrast, our study found that interleukin 1 (IL-1) can increase Cu-Zn SOD activities in human W138 lung fibroblasts. The difference in results could be explained by differences in experimental conditions. The increases seen here did not occur during the first 24 hr, but Cu-Zn SOD activities more than doubled by 3 days. In addition, little increase occurred unless the medium was changed at 24-hr intervals. On the other hand, some other potential experimental variables showed little or no effects on IL-1-induced increases in Cu-Zn SOD activities. These variables included IL-1 isoform (alpha, beta, or both), IL-1 concentration (0.5, 2, 5, or 7 units IL-1 alpha/ml medium), and the presence or absence of exogenously added copper as CuO or ceruloplasmin. In addition, combining IL-1 with dexamethasone, a synthetic glucocorticoid that enhances some IL-1 actions, produced only additive, not synergistic, increases in Cu-Zn SOD activities. In conclusion, IL-1, in several different experimental protocols, raised lung fibroblast Cu-Zn SOD activity levels, but only after a 1 day lag time. Stress-induced increases in Cu-Zn SOD activity levels in vivo also tend to occur only after lag times.     

The extraction and purification of a peptide from rat insulinoma tissue

A peptide was extracted and purified from rat insulinoma tissue which, although similar, was not identical to normal rat C peptides. The purity of the peptide, called rat insulinoma peptide (RIP), was investigated using polyacrylamide gel electrophoresis, isoelectric focusing and high-performance liquid chromatography. It appears to contain two peptides similar to each other but differing in their isoelectric points. The peptides as assessed by fast atom bombardment mass spectrometry have molecular masses in the region of 1982 Da, given a chain length of approx. 22 amino-acid residues. Evidence obtained using an established rat C peptides radioimmunoassay suggests that RIP shares a common C-terminus with rat C peptides. The antiserum produced to RIP was used to develop a radioimmunoassay using a tracer prepared by iodinating purified tyrosylated RIP.     

Complete amino acid sequence of copper-zinc superoxide dismutase from Drosophila melanogaster

The complete amino acid sequence of the Drosophila melanogaster Cu,Zn superoxide dismutase subunit has been determined by automated Edman degradation. Sequence analyses were performed on the intact S-carboxymethylated protein, two fragments derived from CNBr cleavage, and three peptides recovered from mouse submaxillary protease digestion of the reduced and S-carboxymethylated enzyme. The peptides were aligned by characterizing peptides yielded by trypsin and Staphylococcus aureus V8 protease. All the peptides studied were purified exclusively by reverse-phase columns of HPLC and were analyzed with an improved liquid-phase sequencer. A molecular weight of 15,750 (subunit) was calculated from the 151 residues sequenced. The amino acid sequence of the Drosophila superoxide dismutase subunit is compared with that of four other eucaryotes: man, horse, cow, and yeast. Comparison of the five primary structures reveals very different rates of evolution at different times. Copper-zinc superoxide dismutase appears to be a very erratic evolutionary clock. Val-Val-Lys-Ala- Val-Cys-Val-Ile-Asn-Gly-Asp-Ala-Lys-Gly-Thr-Val-Phe-Phe-Glu-Gln- Glu-Ser-Ser-Gly-Thr-Pro-Val-Lys-Val-Ser-Gly-Glu-Val-Cys-Gly-Leu- Ala-Lys-Gly-Leu-His-Gly-Phe-His-Val-His-Glu-Phe-Gly-Asp-Asn-Thr- Asn-Gly-Cys-Met-Ser-Ser-Gly-Pro-His-Phe-Asn-Pro-Tyr-Gly-Lys-Glu- His-Gly-Ala-Pro-Val-Asp-Glu-Asn-Arg-His-Leu-Gly-Asp-Leu-Gly-Asn- Ile-Glu-Ala-Thr-Gly-Asp-Cys-Pro-Thr-Lys-Val-Asn-Ile-Thr-Asp-Ser- Lys-Ile-Thr-Leu-Phe-Gly-Ala-Asp-Ser-Ile-Ile-Gly-Arg-Thr-Val-Val-Val- His-Ala-Asp-Ala-Asp-Asp-Leu-Gly-Gln-Gly-Gly-His-Glu-Leu-Ser-Lys- Ser-Thr-Gly-Asn-Ala-Gly-Ala-Arg-Ile-Gly-Cys-Gly-Val-Ile-Gly-Ile- Ala-Lys.     

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.     

The complete amino-acid sequence of the proteinase inhibitor B from the root of the arrowhead (Sagittaria sagittifolia L.)

After reduction and alkylation of the disulfide bonds of the proteinase inhibitor B from the root of the arrowhead (Sagittaria sagittifolia L.) followed by CNBr cleavage three peptide fragments with 68, 62 and 11 amino-acid residues could be separated on DEAE-Sepharose CL-6B. The peptides or the inhibitor itself were further specifically cleaved either by trypsin or by the mixture of (CH3)2SO/HCl/HBr at the arginyl- and the tryptophyl-peptide bond, respectively. The complete amino-acid sequences of the peptides were determined by manual solid phase DABITC/PITC double coupling micro-method and the primary structure of the arrowhead inhibitor B consisting of 141 amino-acid residues was then elucidated. Twenty pairs of amino-acid residues are repeated in the molecule of this inhibitor, three of these pairs even occur three times. The possible locations of the reactive sites are discussed. On the basis of sequence comparisons between this inhibitor and all other serine proteinase inhibitors the arrowhead inhibitor may belong to a new family.     

A tetrameric iron superoxide dismutase from the eucaryote Tetrahymena pyriformis

An iron-containing superoxide dismutase has been purified from the protozoan Tetrahymena pyriformis. It has a molecular weight of 85,000 and is composed of four subunits of equal size. The tetramer contains 2.5 g atoms of ferric iron. Visible absorption and electron spin resonance spectra closely resemble those of other iron-containing superoxide dismutases. The amino acid sequence of the iron superoxide dismutase was determined. Each subunit is made up of 196 residues, corresponding to a molecular weight of 22,711. Comparison of the primary structure with the known sequences of other iron-containing superoxide dismutases reveals a relatively low degree of identity (33-34%). However, a higher percentage identity is found with mammalian manganese-containing superoxide dismutases (41-42%). The amino acid sequence is discussed in consideration of residues that may distinguish iron from manganese or dimeric from tetrameric superoxide dismutases.     

Endocytosis-independent uptake of liposome-encapsulated superoxide dismutase prevents the killing of cultured hepatocytes by tert-butyl hydroperoxide

Liposome-encapsulated (LSOD) or free (FSOD), human recombinant Cu-Zn superoxide dismutase prevented the killing of cultured rat hepatocytes by tert-butyl hydroperoxide (TBHP). A dose of 32 U/ml of LSOD reduced the cell killing by 50%. By contrast, it required 288 U/ml of FSOD to similarly reduce the toxicity of TBHP by 50%. Both LSOD and FSOD increased the cell-associated superoxide dismutase activity of the cultured hepatocytes. Whereas 64 U/ml of LSOD increased cell-associated superoxide dismutase activity fourfold, it required 500 U/ml of FSOD to achieve a similar increase. Furthermore, methylamine, benzyl alcohol, cytochalasin B, oligomycin, and monensin, all inhibitors of endocytosis, prevented the increase in cell-associated superoxide dismutase produced by 500 U/ml of FSOD. These same inhibitors had no effect on the increase in cell-associated superoxide dismutase activity produced by a much lower concentration of LSOD. Thus, liposome-encapsulated superoxide dismutase prevented the cell killing by TBHP more efficiently than free superoxide dismutase because it more efficiently entered the hepatocytes by a mechanism that was independent of the endocytosis responsible for the uptake of FSOD. These data further define the conditions of the toxicity of TBHP. The target hepatocyte must contribute superoxide anions, in addition to the previously shown ferric iron. It is hypothesized that superoxide anions reduce ferric to ferrous iron; the latter then reacts with the hydroperoxide to form tert-butyl alkoxyl radicals. Such radicals are potent oxidizing agents that can initiate the peroxidation of cellular lipids previously shown to lethally injure the hepatocytes.     

The thiol proteinases from the latex of Carica papaya L. III. The primary structure of chymopapain

The amino-acid sequence of chymopapain is presented. It was isolated from the latex of the fruits from the tropical species Carica papaya L. and is, besides papain and papaya proteinase omega, the third thiol proteinase from this source. The primary structure contains 218 amino-acid residues. It was deduced from sequence analysis of the native enzyme and of peptides obtained by tryptic, chymotryptic, peptic, thermolysinolytic and mild acidic hydrolysis. Out of a total of eight cysteine residues, six are involved in the formation of three disulfide bonds, the location of which has been established with the help of peptic and thermolysinolytic peptides and fragments, obtained by mild acidic hydrolysis. Chymopapain shares 126 identical amino-acid residues (58%) with papain and 141 (65%) with papaya proteinase omega, including the three disulfide bridges and the free cysteine in position 25, required for activity. Except some amino-acid residues in the substrate-binding site, all residues involved in the catalytic mechanism are conserved. The homology between papaya proteinases is discussed.     

Amino acid sequence of iron-superoxide dismutase from Pseudomonas ovalis

The amino acid sequence of iron-superoxide dismutase from Pseudomonas ovalis was deduced by the analyses of peptides derived from limited hydrolysis of the aminoethylated or pyridylethylated apoprotein with trypsin, Staphylococcus aureus V8 protease, and dilute acid hydrolysis. The polypeptide chain contains 195 amino acid residues and has a calculated Mr of 21,421. The sequence is highly homologous (65% identity) to the recently published sequence of the iron-superoxide dismutase from Photobacterium leiognathi. It is also homologous to the known sequences of the manganese-superoxide dismutase by sharing 33-53% identical residues. Alignment of the superoxide dismutase sequences and the available structural information from X-ray crystallography suggest that the ligands to the iron in the P. ovalis superoxide dismutase are His-26, His-74, Asp-156 and His-160, which align with the ligands to the manganese in the Thermus thermophilus manganese-superoxide dismutase. The sequence information of the P. ovalis dismutase will facilitate refinement of the X-ray crystallographic data that are now available at 2.9 A resolution.     

A monoclonal antibody against COOH-terminal peptide of human liver manganese superoxide dismutase

Three stable hybridoma cell lines producing monoclonal antibodies specific for human liver manganese superoxide dismutase were established, and one monoclonal antibody, PG 11, was chosen for immunochemical studies. Immunoblotting demonstrated that the monoclonal antibody binds exclusively to the manganese superoxide dismutase. Immunohistochemical studies indicated that the enzyme is localized in the matrix of human liver mitochondria. To localize antibody-binding epitope, synthetic peptides of the NH2-terminal (residues 1-16) and COOH-terminal (residues 182-189, 190-196, and 182-196) parts of the enzyme were synthesized, and then their effects on the binding were studied using an enzyme-linked immunosorbent assay method. All of the above COOH-terminal peptides inhibited the binding whereas the NH2-terminal ones did not, indicating that PG 11 recognizes several peptides of COOH termini of manganese superoxide dismutase. This is the first report of monoclonal antibodies against human manganese superoxide dismutase with a distinct epitope and of the immunocytochemical demonstration of manganese superoxide dismutase.