BIOCHEMICAL

MEDICINE

AND

METABOLIC

BIOLOGY

‘%,406-415

(1991)

Human Manganese Superoxide Dismutase Is Readily Detectable by a Copper Blotting Technique STEPHEN G. KALER,’ Human

Genetics

Branch,

RICHARD J. MARAIA,

AND WILLIAM

National Institute of Child Health and Human Institutes of Health, Bethesda, Maryland 20892

A. GAHL

Development,

National

Received July 26, 1991 Manganese-superoxide dismutase (MnSOD) in human fibroblast and liver lysates was found to bind copper avidly under the conditions of a copper blotting technique which also detects known copper-binding proteins. Competition studies suggest that the copper-binding site of the molecule under these conditions is distinct from its manganese-binding site. Copper blotting provides a sensitive way to detect MnSOD in human tissues, and may be generally applicable to studies of copper-binding by biological molecules. 8 1~1 Academic Press, Inc.

The superoxide dismutases (SODS) constitute an important family of metalloenzymes which protect cells against the toxic effects of superoxide anions generated during normal aerobic metabolism (1). Two general classes of SODS are recognized: those which contain copper and zinc, and those which contain manganese or iron. In addition to their metal content, these classes of SODS may be distinguished from each other by amino acid sequence, subcellular distribution, and chromosomal localization (2,3). The manganese-containing form of SOD (MnSOD) is a mitochondrial matrix enzyme which has also been found in the cytosol of human liver extracts (4). MnSOD belongs to an evolutionary class of SODS in which some forms (prokaryotic) contain iron instead of manganese (1) but are thought to utilize the same amino acid residues (one aspartate and three histidines) as ligands for either metal (5,6). Studies on the MnSOD of Escherichiu coli have shown that reconstitution of the apoenzyme by manganese could be prevented by a number of other divalent metals including cobalt, nickel, zinc, and copper, as well as iron (7). These results suggested that in vitro the metal-binding site for this type of SOD can accommodate and tightly bind metals other than manganese and iron. In the course of studying genetic diseases of copper metabolism, we have developed a blotting technique which identifies copper-binding proteins within ’ To whom correspondence should be addressed at National Institutes of Health, HGB, NICHD, Building 10, Room 98242, 9000 Rockville Pike, Bethesda, MD 20892. Fax: (301) 402-0234. 406 0885-4505/91 $3.00 Copyright 8 1991 by Academic Press, Inc. All rights of reproduction in any form reserved

DETECTION OF MnSOD IN COPPER BLOTS

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complex protein mixtures. This method, which is adapted from a zinc blotting technique (8), involves one- or two-dimensional SDS/PAGE, transfer to nitrocellulose, and subsequent exposure to radiolabeled copper. Development of an autoradiograph from the nitrocellulose filter identifies proteins to which the labeled copper has bound. When commercially obtained purified samples of known copper-binding proteins such as albumin, metallothionein, plasma amine oxidase, and copper/zinc SOD are tested in this technique, the proteins are readily detected. The mechanism of copper-binding presumably reflects renaturation of native metal binding sites after electrophoresis and transfer, analogous to that theorized for the zinc and antibody-binding domains of proteins studied in other blotting systems (V).

When we analyzed normal human fibroblast and liver proteins by this procedure, we observed an unknown 23 kDa copper-binding polypeptide (10). Based upon its molecular mass, estimated p1, and amino acid sequence of tryptic peptides, we now report the identification of this protein as MnSOD and characterize its copper-binding in copper blots. MATERIALS AND METHODS Muteriuls. For gel electrophoresis, standard commercial sources of chemicals were used. Purified samples of bovine erythrocyte copper/zinc superoxide dismutase and bovine plasma amine oxidase were obtained from Sigma (St. Louis, MO). 1251-labeled molecular weight standards were obtained from DuPont-New England Nuclear (Boston, MA). Prestained low molecular weight markers and amido black protein stain were obtained from Bio-Rad (Richmond, CA). Nitrocellulose 0.20-pm pore size was obtained from Schleicher & Schuell (Keene, NH). Manganese chloride and cupric chloride were obtained from Fisher (Fair Lawn, NJ). Sequencing grade bovine trypsin was obtained from Boehringer-Mannheim (Indianapolis, IN). HPLC grade trifluoroacetic acid was obtained from Applied Biosystems, Inc. (Foster City, CA), HPLC grade acetonitrile and water from Burdick & Jackson (Muskegon, MI), and Vydac HPLC columns from The Nest Group (Southboro, MA). Automated sequencer and analyzer reagents were provided by the manufacturer. Human fibroblast cell lines GM3529 and GM5757 were obtained from the Human Genetic Mutant Cell Repository (Camden, NJ). Sample preparation. Fibroblasts were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum (GIBCO BRL, Gaithersburg, MD), glutamine, and antibiotics as described (11). Cells were harvested with 0.25% trypsin, washed twice in 1 mM phenylmethylsulfonyl fluoride (PMSF) in phosphatebuffered saline (PBS), and lysed by suspension in 10 mM Tris/O.S% Nonidet P40/l mM PMSF on ice for 5 min followed by 10 strokes of a “B” pestle in a 2ml Dounce homogenizer tube. The mixture was centrifuged at 1OOOg for 5 min and the supernatant collected. Liver tissue was obtained from a postmortem specimen of an individual with nephropathic cystinosis. Solubilization in 1 mM PMSF/PBS was achieved by serial mincing, freeze-thawing, and sonication, followed by centrifugation at 15,OOOg for 4 min and collection of the supernatant. Protein concentration was determined by a bicinchoninic acid method with bovine serum albumin used as a standard (Pierce, Rockford, IL) (12). Sample aliquots

408

KALER,

MARAIA,

AND

GAHL

were frozen at - 20°C for up to 4 months. Purified protein samples were dissolved in distilled deionized water. Prior to electrophoresis, samples were diluted 1: 1 in sample buffer (100 mM Tris-HCl), pH6.8/10% glycerol/l% NaDodS04/2% pmercaptoethanol/ 0.05% bromophenol blue) and boiled for 4 min. Gel electrophoresis and electrophoretic transfer. Sample proteins were separated in 15% NaDodS04/polyacrylamide gels according to the protocol described by Laemmli (13) or by two-dimensional PAGE (14). Proteins were electroblotted in a Bio-Rad transblot system onto nitrocellulose as described (9) for 8 to 12 h at 70 mA. Proteins immobilized on nitrocellulose after transfer were visualized by staining with amido black after completion of copper blotting and autoradiography. Copper blotting and autoradiography. Nitrocellulose filters containing transferred proteins were equilibrated in a metal binding buffer (MBB) of 100 mM Tris-HCl, pH7.5/50 mM NaCl for at least 1 h. The filters were probed with 50100 &i of [67Cu]C12 (peak sp act 5.7 mCi/pg Cu, Brookhaven National Laboratory, Upton, NY) in 100 ml MBB for 30-45 min. The filters were then washed three times with 300 ml MBB over 20-30 min, placed in transparent plastic folders, and exposed to XAR film (Eastman-Kodak, Rochester, NY) with an intensifying screen (DuPont Cronex Lightning Plus) at -70°C for 0.5 to 24 h. In metal competition experiments, the MBB included 10m3 M concentration of the competing metal at each step (equilibration, probing, rinsing). The concentration of the [67Cu]C12 probe solution in the latter experiments was 2 x 10T9 M. Protein purification. Fifty grams of postmortem liver from an individual with nephropathic cystinosis was suspended in 150 ml of 10 mM Tris-HCl, pH 7.4/l mM PMSF/leupeptin lOpg/ml and homogenized in a Waring blender. The homogenate was spun at 5OOOg for 30 min and the supematant collected. Serial ammonium sulfate precipitations were performed at 0°C. The fractions were centrifuged at 6OOOg for 30 min and the precipitated proteins were dialyzed against 20 mM Tris-HCl, pH 7.4/50 mM NaCl for 6-12 h at 0°C. Aliquots of the dialyzed samples were frozen at -20°C. Proteins precipitated by 60-80% ammonium sulfate saturation were subjected to two-dimensional electrophoresis which indicated that the copper-binding protein was the major species of molecular mass 23 kDa. Approximately 50 pmol of this protein was subsequently electrotransferred to nitrocellulose from a one-dimensional gel. In situ proteolytic

digestion, separation

by HPLC,

and peptide sequence analysis.

These were performed by R. Robinson and W. S. Lane, Harvard Microchemistry Facility, Harvard University. In situ tryptic digestion of the protein electroblotted to nitrocellulose was performed essentially as described (15), omitting the NaOH wash to minimize loss of protein. After digestion the solution was immediately stored at -20°C. The resultant peptides were separated on a Hewlett-Packard 1090 HPLC equipped with a 1040 diode array detector, using a Vydac 2.1 x 150 mm Cl8 column. The gradient employed was a modification of that described (16). Briefly, where buffer A was 0.06% trifluoroacetic acid/H20 and buffer B was 0.055% trifluoroacetic acid/acetonitrile, a discontinuous gradient of 5% B at 0 min, 33% B at 63 min, 60% B at 95 min, and 80% B at 105 min with a flow rate of 150 p/min was used. Chromatographic data at 210 and 277 nm, and UV spectra from 209 to 321 nm of each peak were obtained. While monitoring ab-

DETECTION

OF MnSOD IN COPPER BLOTS

409

sorbance at 210 nm, fractions were manually collected by peak into 1.5ml microfuge tubes and immediately stored without drying at -20 degrees C. The peptide samples selected for amino acid sequence analysis were applied directly to a polybrene precycled glass fiber filter and placed in the reaction cartridge of an ABI Model 477A protein sequencer. The samples were subjected to automated Edman degradation using the program NORMAL-l, which was modified using the manufacturer’s recommendations for faster cycle time (37 min) by decreasing the dry-down times and increasing the reaction cartridge temperature to 53°C during coupling. The resultant phenylthiohydantoin amino acid fractions were manually identified using an on-line ABI Model 120A HPLC and Shimadzu CR4A integrator. The sequences identified were searched for in the NBRF protein database using the FASTA program (University of Wisconsin Genetic Computer Group). RESULTS AND DISCUSSION Purified samples of known copper-binding proteins albumin, plasma amine oxidase, and copper/zinc superoxide dismutase (Cu/Zn SOD) were readily detectable in copper blots (Fig. 1). Proteins used as negative controls (phosphorylase b, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, and lysozyme) were not detected (Fig. lb, lane 1). Copper-binding by Cu/Zn SOD (Fig. lb, lane 3) was less intense than by albumin and amine oxidase (Fig. lb, lanes 1 and 2). When the copper blotting technique was applied to normal human fibroblast lysates, a single copper-binding polypeptide of relative molecular mass 23 kDa was readily detectable using as little as 15 pg of total cytoplasmic protein (Fig. 2a, lane 1). This band was also present in copper blots of human liver lysates in addition to three other major copper-binding proteins (Fig. 2b, lane 1). When amido black was used to stain the nitrocellulose filters from which copper blots were made and the stained filter (Fig. 2c) was compared to its corresponding autoradiograph (Fig. 2b), it was apparent that the vast majority of fibroblast and liver proteins immobilized on nitrocellulose failed to bind copper under the conditions of this assay, implying that those which did bind copper did so with high specificity. One of the hepatic copper-binding polypeptides (M, = 67 kDa) comigrated with purified bovine serum albumin. Partial amino acid sequencing indicated that another of these bands (M, = 14 kDa) contained (Y and /3 hemoglobin (data not shown). The fourth hepatic copper-binding protein (M, = 30 kDa) may represent a dimer of hemoglobin chains, since both a 30- and a 1CkDa band are evident when purified hemoglobin is tested in copper blots (data not shown). The detection of these hemoglobin polypeptides may relate to their high histidine composition (>6%), since this amino acid has been associated with copper-binding in other proteins (17). The 23-kDa copper-binding polypeptide detected in both fibroblasts and liver was purified using copper blotting as a detection assay throughout several steps. The 23-kDa polypeptide was enriched in the 60-80% ammonium sulfate fraction. Two-dimensional electrophoresis (isoelectric focusing followed by SDS/PAGE) indicated that it was a basic protein (measured ~18.0, Fig. 3). After in situ tryptic digestion of the purified protein and separation of the resultant peptides by HPLC,

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123 1. Detection of known copper-binding proteins in a copper blot. (a) Nitrocellulose filter with immobilized proteins visualized by amido black. Lane 1, 10 pg each of phosphorylase b, albumin (arrow), ovalbumin, carbonic anhydrase, trypsin inhibitor, and lysozyme; lane 2, 10 pg plasma amine oxidase; lane 3, 10 pg copper/zinc superoxide dismutase. (b) Copper blot autoradiograph showing detection of albumin (lane l), plasma amine oxidase (lane 2) and with a less intense signal, Cu/Zn SOD (arrow, lane 3). The negative control proteins in lane 1 are not detected. FIG.

two peptides were selected for sequence analysis. When the sequences identified (Fig. 4) were searched for in a protein sequence database, they were found to match perfectly with human MnSOD. The first sequence includes a known polymorphic marker for MnSOD, a Thr to Ile substitution at residue No. 58 which corresponds to a C to T substitution in the cDNA (l&19). In order to further characterize its copper-binding capacity, MnSOD was compared to the other major copper-binding proteins in blots using [67Cu]C12 concentrations ranging between 10e9 and lOPro M (Fig. 5). MnSOD was detectable at all copper concentrations tested, and its binding response to concentration changes was similar to that of other polypeptides which bound copper in this assay. We demonstrated that the amount of radioactivity was not limiting in

DETECTION

411

OF MnSOD IN COPPER BLOTS

b

a

C

-116 -67

23 kD *

12

12

12

2. Detection of copper-binding proteins of fibroblast and liver. (a) Copper blot autoradiograph of fibroblast cytoplasmic proteins. Lane 1, 15 Fg of total protein (normal human tibroblast line GM3529); lane 2, ‘~I-labeled molecular weight standards. Labels denote M, x lo-‘. (b) Copper blot of human liver and fibroblast proteins. Lane 1, 35 pg total liver protein; lane 2, 30 gg fibroblast protein (GM3529) run in same gel. (c) Nitrocellulose filter corresponding to (b) stained with amido black. Arrows indicate the 23-kDa copper-binding polypeptide. FIG.

experiments of this type by determining that less than 6% bound to the filters, even at the lowest copper concentrations (data not shown). We also compared the effects of competition with manganese and copper on the detection of MnSOD in copper blots. MnCl* (1 mM, a lo*-fold excess relative to [67Cu]C1,) failed to compete, while 1 mM unlabeled CuCIZ competed efficiently against 67Cu binding (Fig. 6). Except for the enzyme reconstitution studies in which copper and manganese presumably competed for the same binding site (7), the capacity to bind copper has not been associated with MnSOD. Two possible interpretations of our results are (1) that under the conditions of this technique, the manganese-binding site of MnSOD has stronger affinity for copper than for manganese, and (2) that copper-binding by MnSOD in copper blots occurs at a site which is distinct from its manganese-binding site. Since MnSOD is not known to bind copper in vivo, if an independent copper-binding site is responsible for its detection in this technique, it may be due to alteration of the protein during denaturation and immobilization on nitrocellulose, and not present in the native enzyme. Alternatively, MnSOD could have an unrecognized role as an intracellular copper transport molecule related to the capacity for high affinity copper-binding illustrated in this system. Finally, our data do not formally exclude the possibility that the 23-kDa protein represents a MnSOD-like polypeptide whose metal-binding site is specific for copper. When we tested commercially obtained E. coli MnSOD in copper blots, the intensity of the band detected was less pronounced (data not shown). Comparison of the bacterial and human MnSOD sequences (41% homologous) shows that

412

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AND GAHL

4- pl +

8.2

4.7

FIG. 3. Two-dimensional (isolectric focusing, SDS/PAGE) separation of liver proteins. (a) Twodimensional copper blot of human liver proteins precipitated after 60-80% ammonium sulfate saturation. Arrow indicates faint autoradiographic signal corresponding to protein spot of estimated molecular mass 23 kDa and estimated p1 8.0. (b) Nitrocellulose filter corresponding to (a) stained with amido black for protein visualization. Position of the copper-binding protein is indicated.

only the human enzyme contains cysteine residues (two) and also has two more histidine residues (a total of 10) than the bacterial forms (1,ZO). Since ,cysteine as well as histidine has been implicated in copper-binding motifs in other proteins (21,22), these differences may be related to the higher affinity for copper of human MnSOD in copper blots. MnSOD is synthesized as a slightly larger molecular weight precursor which Peptide

I

52

58

GLY

Peptide

2

ASP

VAL

THR

ALA

GLN

ILE

64 ALA

LEU

GLN

180 ILE

PRO

ALA

LEU

191 _

ASN

VAL

ILE

ASN

_

GLU

ASN

VAL

MR

GLU

4. Amino acid sequences of two tryptic peptides from the 23-kDa copper-binding protein. Numbers denote the positions of these amino acids in human MnSOD (21). Residue No. 58 reflects a known DNA polymorphism. FIG.

DETECTION 1000

1

OF MnSOD IN COPPER BLOTS 650

pM

2

1

pM

100

2

1

413

pM

2

5. Effect of different [67Cu]CIz concentrations on copper blots of liver and fibroblast proteins. Lanes 1, 35 pg total liver protein; lanes 2, 30 pg total fibroblast protein (GM3529). FIG.

includes an N-terminal leader peptide (18). It appears that the form we detect in copper blots of fibroblast and liver proteins is the fully processed enzyme, whose molecular mass has previously been estimated by SDS/PAGE to be 24 kDa (23). The p1 of the processed enzyme from rabbit liver has been reported as 7.8 (23) which agrees approximately with our result of 8.0 for human MnSOD. Because we studied cytoplasmic extracts of human fibroblasts and liver, our data do not indicate the precise subcellular localization of MnSOD in these tissues. We have, however, detected MnSOD in copper blots of purely cytosolic fractions of fibroblasts, i.e., the lOO,OOOg supernatant following cell rupture by nitrogen cavitation (data not shown). For investigations of the amount of MnSOD present in complex mixtures of human tissues (as opposed to expression or activity), the copper blotting method may be useful as a simple and sensitive assay. For example, in studies of normal and transformed human lung fibroblasts, MnSOD was detectable in Western blots using 250 pg of cell protein per lane (24). In copper blots, approximately 10 times less cellular protein is needed to detect MnSOD in the identical normal cell line, WI38 (data not shown), or other human fibroblast cell lines (Fig. 2a). The amount of immunoreactive MnSOD measured by Western blotting was significantly lower in the transformed fibroblasts, a finding of interest to us because during preliminary characterization of the 23-kDa protein we had observed that HeLa cells, another transformed cell line, contained decreased amounts of this band in copper blots

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AND GAHL

mM MnCl2

1 mY Cut32

FIG. 6. Competition of MnSOD copper-binding. MnSOD from normal human fibroblast line GM5757 competed in 67Cu blots with unlabeled MnQ and CuQ.

(data not shown). The reason for decreased amounts of MnSOD in transformed cells is not known (24). In addition to use for MnSOD detection, the copper blotting technique should find application in studies of copper-binding by other biological molecules including authentic copper-dependent enzymes, transcription factors known to bind copper (22), various copper proteins found in nature (25), and in the investigation of human disorders of copper metabolism. Copper blotting can be considered as an adjunct to gel filtration and ion exchange chromatography, two methods previously used to study copper-binding molecules (25,26). REFERENCES 1. Bannister JV, Bannister WH, Rotilio G. Aspects of the structure, function, and applications of superoxide dismutase. Crit Rev Biochem 22:111-180, 1987. 2. Smith M, Turner BM, Tanigaki N, Hirschhorn K. Regional localization of HLA, MEs, and SOD,,, on chromosome 6. Cytogenet Cell Genet 22428-433, 1978. 3. Levanon D, Lieman-Hurwitz J, Dafni N, Wigderson M, Sherman L, Bernstein Y, Laver-Rudich Z, Danciger E, Stein 0, Groner Y. Architecture and anatomy of the chromosomal locus in human chromosome 21 encoding the Cu/Zn superoxide dismutase. EMBO J 477-84, 1985. 4. Slot JW, Gueze HJ, Freeman BA, Crap0 JD. Intracellular localization of the copper-zinc and manganese superoxide dismutases in rat liver parynchymal cells. Lab Invest 5363-371, 1986. 5. Stallings WC, Pattridge KA, Strong RK, Ludwig ML. The structure of manganese superoxide dismutase from Thermus thermophilus HB8 at 2.4 A resolution. J Biol Chem 26096424-16432, 1985. 6. Carlioz A, Ludwig ML, Stallings WC, Fee JA, Steinman HM, Touati D. Iron superoxide dis-

DETECTION

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

OF MnSOD IN COPPER BLOTS

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mutase. Nucleotide sequence of the gene from Escherichiu coli K12 and correlations with crystal structures. J Biol Chem X%1555-1562, 1988. Ose DE, Fridovich I. Manganese-containing superoxide dismutase from Escherichiu coli: Reversible resolution and metal replacements. Arch Biochem Biophys 194~360-364, 1979. Schiff LA, Nibert ML, Fields BN. Characterization of a zinc blotting technique: Evidence that a retroviral gag protein binds zinc. Proc Nat1 Acad Sci USA 85:4195-4199, 1988. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Nat1 Acad Sci USA 76~4350-4354, 1979. Kaler SG, Maraia RJ, Gahl WA. A novel 23 kD copper-binding protein of human fibroblasts and liver identified by a copper blotting technique. Pediatr Res 29:194A/1150, 1991. Renlund M, Tietze F, Gahl WA. Defective sialic acid egress from isolated fibroblast lysosomes of patients with Salla disease. Science 232~759-762, 1986. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem lSO:76-85, 1985. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 222680-685, 1970. O’Farrell PM. High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:40@74021, 1975. Aebersold RH, Leavitt J, Saavedra RA, Hood LE, Kent SBH. Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc Nat1 Acad Sci USA 84~6970-6974, 1987. Stone KL, LoPresti MB, Williams ND, Crawford JM, DeAngelis R, Williams KR. Enzymatic digestion of proteins and HPLC peptide isolation in the sub-nanomole range. In Techniques in Protein Chemistry (Hugh TE, Ed.). San Diego: Academic Press, 1989, pp 377-391. Lau SJ, Sarkar B. Ternary coordination complex between human serum albumin, copper (II), and L-histidine. J Biol Chem 246~5938-5943, 1971. Ho YS, Crapo JD. Isolation and characterization of complementary DNAs encoding human manganese-containing superoxide dismutase. FEBS Lett 22!X256-260, 1988. Church SL. Manganese superoxide dismutase: Nucleotide and deduced amino acid sequence of a cDNA encoding a new human transcript. Biochim Biophys Actu 1087~250-252, 1990. Barra D, Schinina ME, Simmaco M, Bannister JV, Bannister WH, Rotilio G, Bossa F. The primary structure of human liver manganese superoxide dismutase. J Biol Chem 259:12595-12601, 1984. Nielson KB, Winge DR. Preferential binding of copper to the beta domain of metallothionein. J Biol Chem 2594941-4946, 1984. Furst P, Hu S, Hackett R, Hamer, D. Copper activates metallothionein gene transcription by altering the conformation of a specific DNA binding protein. Ceil 55~705-717, 1988. Wispe JR, Clark JC, Burhans MS, Kropp KE, Korfhagen TR, Whitsett JA. Synthesis and processing of the precursor for human mangano-superoxide dismutase. Biochim Biophys Acta 994:3036, 1989. Oberley LW, McCormick ML, Sierra-Rivera E, Kasemset-St Clair D. Manganese superoxide dismutase in normal and transformed human embryonic lung tibroblasts. Free Radic Biol Med 6:379-384, 1989. Thurman DA, Salt DE, Tomsett AB. Copper phytochelatins of Mimulus guttatus. In Metal Ion Homeostasis: Molecular Biology and Chemistry (Hamer DH, Winge DR, Eds.). New York: Alan R. Liss, 1989, pp 367-374. Palida FA, Ettinger MJ. Identification of proteins involved in intracellular copper metabolism. Low levels of a =48-kDa copper-binding protein in the brindled mouse model of Menkes disease. J Biol Chem 266~4586-4592. 1991.

Human manganese superoxide dismutase is readily detectable by a copper blotting technique.

Manganese-superoxide dismutase (MnSOD) in human fibroblast and liver lysates was found to bind copper avidly under the conditions of a copper blotting...
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