J. Mol.

Biol.

(1991) 219, 103-108

Preparation of Human Manganese Superoxide Dismutase by Tri-phase Partitioning and Preliminary Crystallographic Data Harold F. Deutschl, Sakuo Ho&i’, Yukihiko Matsudal, Keiichiro Suzuki’ Kiyoshi Kawano 2, Yasuyuki Kitagawa3, Yukiteru Katsube3 and Naoyuki Taniguchi’t 1Department of Biochemistry, Osaka University Medical 2-2 Yamadaoka, Suita, Osaka 565, Japan 2The Osaka Rosai Hospital,

School

Sakai 591, Japan

31nstitute for Protein Research Osaka University, Suita 565, Japan (Received 16 October 1990; accepted 3 January

1991)

Human liver manganese superoxide dismutase has been purified by a short procedure that includes a tri-phase partitioning step to provide materials that can be crystallized from ammonium sulfate. X-ray diffraction studies at 3 A resolution show that the crystals belong to the hexagonal space group P6,22 or P6,22, with cell dimensions a= b = 81.1 A, c = 242.2 A. Manganese superoxide dismutase levels as determined by enzymatic assay as well as by enzyme-linked immunosorbent assay indicated that considerable variations occur in different livers but the total superoxide dismutase activity (Mn superoxide dismutase plus Cu,Zn superoxide dismutase) seems to be kept at const’ant values. Keywords:

Mn superoxide

dismutase; t&phase partitioning; X-ray data; human liver

1. Introduction The likely roles of superoxide dismutases (SODf, EC 1.15.1 .l) in ischemia and reperfusion injury (,Jolly it al., 1984; McCord, 1987), resistance to cytotoxity of tumor necrosis factor (Wong et al., 1989) and variations in their levels in tumors (Tshikawa et al., 1990) center in large part on the role of %-SOD. This widely distributed enzyme is localized mostly in the mitochondria (Weisiger & Fridovich, 1973a,b: Tyler, 1975; Panchenko et ccl., 1975; Auclair et al., 1977; Rest & Spitznagel, 1977: et al., 1989; Akai et Tizuka et al.. 1984: Kawaguchi al., 1990). The methods reported in detail for preparing this enzyme are rather involved (Salin et al., 1978; Matsuda et al., 1990). In the present study, the TPP fractionat,ion method (Niehaus & Dilts, 1982:

t Author to whom all correspondence should be addressed. $ Abbreviations used: SOD. superoxide dismutase: Mn-SOD, manganese superoxide dismutase: Cu.&r-SOT). (Ju,Zn-superoxide dismutase; TPP, tri-phase partitioning: p-APMSF. p-amidinophenylmethylsulfonyl fluoride.

crystallization;

preliminary

Odegaard et al., 1984; Lovrien et al., 1987; Jacobs et 1989; Pol et al., 1990) has been used in conjunction with several relatively simple chromatographic procedures to separate human liver Mn-SOD in a form that can be crystallized. Preliminary X-ray data for such material have been obtained.

al.,

2. Experimental (a) Activity

Procedures

measurements

Mn-SOlj (W-resistant) and Cu,Zn-SOD (CN-sensitive) activities were determined by the method of Weauchamp Kr Fridovieh (19Tl) using a 30 ml assay system. (b) Fractionation

proceduw

Livers that appeared grossly normal were obtained at autopsy. These or 2 to 3 cm cubes of them were frozen to effeet disruption of the mitochondria. From 300 to 500 g portions of the thawed tissue was homogenized at 4°C with 4 vol. (w/v) 915 M-NaCI, 1 rnM-benzamidine, 10 PM-p-APMSF. The suspension was centrifuged for 20 min at 0°C and the insoluble residue discarded. All subseyuent fractionation steps were carried out at 0°C. The supernatant was adjusted to pH 60( +@l) if necessary and t-butanol then added slowly with vigorous stirring to a concentration of 35% (v/v) and stirred for 30

103 0 1991 Academic l’ress Limited

104

H. F. Lkutsch

to 60 min following completion of this addition. The suspension was then centrifuged for 20 min at 10,000 g and the volume of the supernatant was measured. The amount of the liver extract, i.e. the volume of the aqueous portion of this supernatant, was calculated and sufficient solid ammonium sulfate was added to bring it to 75% saturation. After stirring of the mixture for 30 to 60 min. it was centrifuged for 20 min at 10,ooO g. The upper (t-butanol) and lower (ammonium sulfate) phases were carefully decanted and the intermediate phase precipitate was suspended in a small volume of ice-water containing about 5 ml of @5 M-K,HPO,. The suspension was dialyzed for 12 to 16 h against @02 M-pOhSSiUUI phosphate (pH 68)/ice mixture and additional dialysis was then carried out at 4°C to 10°C against repeated changes of the buffer until conductivity measurements indicated that equilibrium had been obtained. The dialyzed protein was centrifuged at 0°C for 20 min 10,000 g and the supernatant was applied to a column of phospho-cellulose (Whatman) equilibrated with &02 M-pOtat+f3iUfII phosphate (pH 68). Following this, the column was washed with 1 column volume of the buffer and a gradient elution to O-3 M-NaCl instituted. The amount of the elution buffer was 4 to 5 times that of thr column volume. The Mn-SOD-containing fractions could be detected by the purple color which, in initial experiments, was confirmed by enzymatic assays. These fractions were pooled, precipitated with saturated ammonium sulfate, suspended in a small volume of @l M-potassium phosphate (pH 7.0) and dialyzed for a short time aga.inst this buffer to effect solution. Following clarification by centrifugation for 10 min at 10,000 g at 0°C. the solution was applied to an S-300 Agarose (Pharmacia) column equilibrated with the above buffer.

Both spectrophotometric and immunological methods were employed. For solutions of purified protein, an absorption coefficient E&‘~;b’z” of 21-4 determined by lowangle laser light-scattering studies (Matsuda el al., 1990) was used. This may be compared with a value of 204 calculated from the amino acid levels reported by Rarra rf al. (1984). The latter figure is based on the calculated molecular weight of 22,200 and the assumption that the !I tyrosine and 6 tryptophan residues show no absorption perturbations in solutions of the protein. The amounts of Cu,Zn-SOD and Mn-SO11 in liver. homogenates were determined by enzyme-linked immunosorbent assays (Oka et al., 1989; Kawaguchi et al.. 1990). These results and those obtained for enzyme activity permitted calculation of specific enzyme activities in the starting material. (d) Crystallization

experiments

Two general methods were used. One utilized dialysis procedures, the other the vapor diffusion method. Protein from the S-306 column that had been first dialyzed against 55% saturated ammonium sulfate (pH 8) usually formed small amounts of amorphous precipitate. Their supernatants, when dialyzed against 60% saturated ammonium sulfate at room temperature, formed different types of crystals that appeared to be dependent on t.hr rapidity of crystallization and the protein concentration. When the vapor diffusion method was employed small droplets of protein (40 to 50 ~1) were suspended over 60 to 62% saturated ammonium sulfate solutions that had been adjusted to pH 8. These were maintained at 25°C

et al.

and small additions of saturated ammonium sulfate were made daily unt.il cryst.allization was initiated.

The preliminary unit cell parameters were calculated from pre(,ession photographs t,aken at room temperature with I%filtered (‘LIKE radiation with an Errraf-7%oniuh precession camera equipped with a Rigaku rot.ating anotk X-ray generator (Rigaku Co. T,td. Tokyo). ‘l’htt diffraction studies were made to 3 A resolution (1 is,=().I um).

3. Results and Discussion

Solutions of’ protein prepared 11~ thr TPP methoci when applied to a phospho-cellulose column yavcl elution patterns of the type shown in Figurr l(s) and (b). Various amounts of a,n early elating. rnzymatically active fraction denot’ed I’- I \vas seen prior to t~he major P-2 fraction. The P-I mutcriai was usualI\: a minor fraction in experiments sncsh as that shown in Figure l(a), but with somr livers rrprtxsent,ed almost WC),, of the acativity isolated ill ttrih chromatographic step. An cxarnplr of thr I:\ticLr typr of expt~rimc~nt is shown in Figurr I(b). ‘T’t~t~sc~ Mn-SOL) fracbt ions c:onc*ent,rated as tlesc~riht~cl ut~l~~~. Experimrntal Procrtlurrs to 1 to SO,, protein. \vhtln applied to an S-300 Agarose column gave an elution profile as shown in Figure 1((+). Tt is i~l>parr~~t that most of the protein CJlutes as a single c~ornpont~ttt. The t~nz~rn(~ t~lutrtl at about 0+X of’ t hr (.olun11) v0lnrn~~. at14 thr P-l a11t1 t’-? f’r.ac+iotls show\:t~l no disc*erniblr diffrrenc*c>. Fnrtherrnorc~. I)OI h frwcst ioIls N)Llltl bC c~r~stallizt~tl follow~irr~ thrir \)iLSSiiKl‘ ovt’r itll s-300

cY~lllInIl.

105

Mn Superoxide Dismutase

0

100

200

300

400

0

500

5

Volume (ml)

IO

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IO 8

P6 < 4

P2g” I-

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I 200

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I 400

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Volume (ml)

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(b)

(ei

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: .c

40

: .?.

60

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(c 1

(f)

Figure 1. Results for the chromatography of the TPP extract of (a) 290 g of liver on a 1.8 cm x 57 cm column of phospho-cellulose (P-l and P-2 show fractions possessing Mn-SOD activity); (b) 500 g of liver on a 2.5 cm x 57 cm column of phospho-cellulose (P-l and P-2 show fractions possessing Mn-SOD activity). (c) Gel permeat,ion result for fraction P-2 of(b) on a 1.8 cm x 57 cm column of S-300 Agarose. Results for the chromatography of purified Mn-SOD on phenyl-boronat,e Agarose (d) of 34 mg in 2.9 ml on a 1-2 ml column, (e) of 27 mg in 1.2 ml on a 40 ml column. (fj Chromatographic result. for 2 mg of Mn-SOD on a 1-Oml column of Butyl-Toyopearl (the ammonium sulfate concentration is shown by the dotted line). was performed. Protein isolated from the S-300 column was employed. In one of these experiments, 34 mg of Mn-SOD in 2.9 ml of 25 mM-glycylglycine (pH SO) buffer was applied to a 1.2 ml column of phenyl-boronate Agarose (Amino PAB-60, Amicon). A small volume column was employed, since it appeared that if glycated protein was indeed present it would likely be in only small amounts.

The result for this overloaded column is shown in Figure l(d). Two major components are seen to have been eluted. Both had specific activities of 5000 units per milligram. The first component eluted slightly after the void volume and represented about 40% of the protein applied to the column. Essentially no protein eluted upon the application of 1 o/o (w/v) sorbitol. The major com-

106

Ii. F. Deutsch et al.

--

Table 1 The enzymatic activities and the immunoreactive contents of CM-sensitive A’OI, (Cu,Zn-SOD) and CN-resistant SOD (Mn-SOD) in different human livers SOD actkit\ (units/mg p&in) Liver sample

Total SOD

(‘N-resistant SOD

SOD content (pg/mg protein) (‘N-sensitivrt SOD

tThese values are based on; total SOD activity determined.

ponent was obtained by washing the column with 65 M-Tris . HCl (pH 8.0). To possibly gain some insight into the nature of the Mn-SOD active fractions eluting before the 011 chromatography component upon main phospho-cellulose, i.e. fractions denoted P-1 1 a 1.2 ml pool of these isolates containing 27 mg of protein was applied to a 4-ml phenyl-boronatr column. The protein level was calculated using a 1 y0 extinction value at 280 nm of 21.4 and is thus an approximation. The elution pattern is shown in Figure l(e). A brown “drop-through” fraction was seen but no protein eluted upon the application of addition of the sorbitol. Following 1% 65 ivr-Tris . HCI (pH &O) buffer, two components eluted. The major one comprised about 80% of this protein. This fraction had a specific activity of 6300 units per milligram as determined by the xanthine/ tetrazolium assay oxidase-nitroblue xanthine (Barra et al., 1984), but the minor Tris fraction was 10,000 units per milligram. The drop-through fraction contained 14,000 units per milliliter. If this latter material had an extinction of 21.4, its specific activity would have been about 12,000 units per milligram. SDS/polyacrylamide gel electrophoresis of it showed that Mn-SOD comprised less than half of the total protein. This indicates that the enzyme in this fraction had an extremely high specific activity. These variations are of interest. They suggest that human Mn-SOD has molecules of variant activity and that inhibitors or activators of this enzyme are present. However, this is unlikely for, as will be described, the levels of immunoreactive and enzymatically active SODS in liver homogenates were roughly correlated. The results obtained from chromatography on the phenyl-boronate columns cannot be explained by ion-exchange interactions or the binding of glycoidcontaining material but rather appear to be due to hydrophobic interactions. To pursue this possibility, 2 mg of Mn-SOD in 50 mM-phosphate buffer (pH 7.5) containing 40% saturated ammonium sulfate was applied to a Butyl-Toyopearl (TOSOH,

hln-SOD

- CN-resistant

t’u.%n-SOL)

SOD specific activit! (units/mg SOD) Nr&iOf)

(:u.%,i~SOf)

SOD = C‘N-sensitive SOD. Y.D., not

Japan) column and eluted by a gradient made by adding 50 mM-phosphate (pH 7.5). The protein applied to the column had a specific activity of 5700 units per milligram. The results for these experiments are shown in Figure l(f). A single major component with a small amount of late elution protein is seen. Since the major component possessed 9000 enzyme units per milligram, hydrophobic chromatography resulted in a marked increase in activity. Attempts to crystallize the protein eluted from the Butyl-Toyopearl column were unsuccessful. The increases in activity of Mn-SO11 that, often accompanies hydrophobic chromat~ography are of interest and need further exploration. To pursue the reasons for the activity noted. it appeared desirable to determine why different human livers show such variations in yields and activities of Mn-SOD. Such variations have been noted elsewhere (Tizuka et nl., 1984; Marklund et nl.. 1982). In our attempts. eight different livers wertl employed and the CN-sensitive SOD (Cu,Zn-SOD) and CN-resistant SOD (Mn-SOD) activities as well as the enzyme-linked immunosorbent assay levels (Oka et al., 1989; Kawaguchi et al., 1990) of their homogenates were measured. The dat,a in Table 1 show that in the six liver homogenates assayed for Mn-SOD, the immunochemical levels varied over sixfold. This suggests that, prior to fractionation a liver should be assayed to determine if it. possesses an adequate level of this enzyme. The immunoreactive SOD levels, which were determined by specific monoclonal antibodies, were roughly but not exactly correlated to the enzymatic activities. The specific activities in the liver homogenates shown in Table 1 are fairly constant. except for liver 2. They ranged from IO:000 to 18,600 and from 72,000 to 152,000 units per milligram. for Mn-SOD and Cu,Zn-SOD, respect’ively. The values for Mn-SOD appear to be high compared to those reported previously (MeCord et al.. 1977). However. they are in the range of some of t)he Mn-SOD isolates on a boronate affinity column. Even though the activities of the CN-sensitive SOD (Cu,Zn-SOD)

Mn Superoxide

and of the (X-resistant SOD (Mn-SOD) vary, the total SOD activity in each liver sample was found to be in the range of 130 to 200 units per milligram of protein. These results suggested that in humans the total SOD activities of the liver are rather constant, even though these isozymes are located in different compartments. When one of the SOD isozymes decreases, the other SOD or SOD-like activity increases. Such compensation would serve to protect against the superoxide anion generated as the result of various oxidative stresses. Kelner & Bagnell (1990) reported that transfection of a human pSV2 Cu,Zn-SOD expression vector into murine fibroblasts resulted in an increased expression of Cu,Zn-SOD and decreased expression of the Mn-SOD. The consequent reduction in the intracellular steady-state superoxide concentrations appear to result in a decreased synthesis of Mn-SOD. These results are in keeping with our data indicating that the levels of Cu,Zn-SOD and %-SOD are coordinately expressed in human liver under physiological conditions. However, the mechanism by which such compensation occurs remains to be elucidated.

(b) Crystallographic

results

Dialysis procedures gave small needles, hexagonal rods or thin hexagonal plate-type crystals that were not large enough for crystallographic studies. A series of vapor diffusion, hanging drop experiments were employed to prepare adequate crystals for this purpose. The various experiments employed different concentrations of polyethylene glycol4000, %methyl-2,4-pentanediol and ammonium sulfate. The best conditions utilized 60 y0 to 62 y0 saturated ammonium sulfate at pH 8.0 to 8.2 and protein levels from 4 to 10 mg/ml. Symmetrical bihexagonal crystals as large as 1 mm x 0.4 mm could be obtained. The best crystals formed at controlled temperatures (25 “C). X-ray precession photographs are consistent with the hexagonal space group P6,22 or P6522. The unit cell parameters are a= b = 81.1 a and c =242.2 8. The corresponding unit cell volume is I.38 x lo6 83. If it is assumed that two 22,000 dalton molecules are present in an asymmetric unit, a V,,, value of 2.6 A3/dalton is obtained. An X-ray precession photograph of the h01 zone of a hexagonal P6,22 or P6522 single crystal of human &-SOD is shown in Figure 2. Wagner et al. (1989) crystallized human recombinant Mn-SOD using 2-methyl-2,4-pentanediol and reported that the crystal has an orthorhombic space group P2,2,2 with unit cell parameters a=7551 A, h = 79.00 A and c = 67.95 a. However, native human Mn-SOD seems to have a tetrameric structure

(Matsuda

et

al., 1989, 1991), whereas the recombi-

nant protein

is dimeric

(Wagner

et al., 1989).

While

present data indicate differences in the spatial grouping of native Mn-SOD and t,he recombinant the

form.

it cannot

be concluded

at present

that

major

Dismutase

107

Figure 2. An ‘X-ray precession photograph of the h01 zone of a hexagonal P6,22 or P6,22 single crystal of human Mn-SOD. The photograph was taken with N-filtered CuKcl radiation from a rotating anode X-ray generator operating at 40 kv, 100 mA for 40 h exposure. For this film, p = 15” (c&,,~,,= 3 A).

differences exist in the arrangement forms in their crystals.

of the two

This work was supported in part, by Grants-in Aid for Cancer Research from the Ministry of Science and Culture, Japan and from the Princess Takamatsu (lancer Research Fund. One of us (H.F.D.) thanks the Ministry of Science and Culture (Monbusho). Japan for support of a Visiting Professorship.

References Akai, F., Maeda, M., Suzuki, K., Tnagaki. S., Takagi. H. & Taniguchi, P;. (1990). Immunocytochemical localization of manganese superoxide dismutase (M n-SO I)) in the hippocampus of the rat. Neurosci. Letters, 115. 19-23. Arai, K., Maguchi, S., Fujii, S., Ishibashi, H., Oikawa. K. & Taniguchi, i\i. (1987). Glycation and inactivation of human Cu-Zn-superoxide dismutase: Tdentification of the in vitro glycated sites. J. Riol. (‘hum. 262. 16969-16972. Auclair, C., Hakim, J. & Boivin, P. (1977). Subcellular superoxide dismutase activity in phagocytozing human blood polymorphonuclear leucorytes. F’BHS Letters, 79, 390-392. Barra. D.; Schinina, M. E., Simmaco, M.. Rannis;ter, J. V.. Bannister, W. H., Rotilio. G. & Bossa. F. (1984). The primary structure of human liver manganese superoxide dismutase. J. Biol. Chem. 259, 12595-12601. Beauchamp, C. & Fridovich, I. (197 I). Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44. 27G287. Iizuka, S., Taniguchi, K. EL Makita, A. (1984). Enzyme-linked immunosorbent assay for human manganese-containing superoxide dismutase and its

H. F. Deutsch et al

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content in lung cancer. .J. Yat. Cancrr Inst. 72. 1043-1049. Ishikawa, M.: Yaginuma, Y.. Hayashi, H., Shimizu. T.. Endo, Y. & Taniguchi, N. (1990). Reactivity of a monoclonal antibody to manganese superoxide dismutase with human ovarian carcinoma. (‘nnw, Res. SO, 2538-2542.

Jacobs, G. R.. Pike, R. N. & Dennison. (‘. (19X!)). Isolation of cathepsin D using three-phase partitioning in t-butanol/water/ammonium sulfate. ATMJ!. Biochem. 180, 169-171. Jolly, S. R., Kane, W. J., Bailie, M. B., Abrams, G. I). B Lucchesi, B. R. (1984). Canine myouardial reperfusion injury. Its reduction by the combined administration of superoxide dismutase and catalasr. (‘ire. Res. 54, 277-285.

Kawaguchi, T.. Xoji, S.. ITda, T.. Nakashima. Y.. Takeyasu, A., Kawai, Y., Takagi. H.. Tohyama. $1. & Taniguchi, X‘. (1989). A monoclonal antibotl? against COOH-terminal peptide of human liver manganese superoxide dismutase .J. Biol. (‘h,rnr. 264. 5762-5767. Kawaguchi, T., Suzuki, K.. Matsuda. Y.. Kishiura. T.. Uda, T., Ono, M., Sekiya. (:.. Ishikawa. M.. Iino. S.. Endo. Y. & Taniguchi, N. (1990). Serum manganrsrsuperoxide dismutase: Normal values and increased levels in patients with acute myocardial infarction and several malignant diseases determined by an enzyme-linked immunosorbent assay using a monoclonal antibody. J. Immunol. Methods, 127, 24Ck254. Kelner, M. J. & Bagnell, R. (1990). Alteration of endogenous glutathione peroxidase, manganese superoxide dismutase, and glutathione transferase activity in cells transfected with a copper-zinc superoxide dismutase expression vector. Explanation for variations in paraquat resistance. J. Biol. Chem. 265. 10872-10875. Lovrien, R., Goldensoph, C., Anderson, P. C. & Odegaard, Micro to Macro, B. (1987). In Protein Pur@cution: pp. 131-148, A. R. Liss, Xew York. Marklund, S. L., Westman, X. G., Lundgren, E. & Roos, G. (1982). Copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase, and glutathione peroxidase in normal and neoplastic human cell lines and normal human tissues. Cancer Res. 42, 1955-1961. Matsuda, Y., Ishikawa, T., Higashiyama, S., Sugiyama. T., Kawata, S., Tarui, S. & Taniguchi, P;. (1989). An acidic variant form of Mn-superoxide dismutase in human hepatocellular carcinoma. In Medical Biochemical and Chemical Aspects of Free Radicals (Hayaishi, O., Xiki, E., Kondo, M. & Yoshikawa, T.. Elsevier Sci. Publishers, eds), pp. 751-754, Amsterdam. Matsuda, Y., Higashiyama, I’., Kijima, Y., Suzuki, K.. Kawano, K., Akiyama, Y., Kawata, S., Tarui, S.. Deutsch, H. F. & Taniguchi, N. (1990). Human liver manganese superoxide dismutase: purification and crystallization, subunit association and sulfhydryl reactivity. Eur. J. Biochem. 194, 713-720. Edited

&Cord. J. M. (1987). Oxygen-derived radicals: a link Fed. between reperfusion injury and inflammation. Proc. Fed. Amer.

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McCord, J. M., Boyle, J. A., Day, E. D., Jr, Rizzolo, L. .I, & Salin, M. L. (1977). A manganese-containing superoxide dismutase (erythrocuprein) from human liver. In Superoxide and Superoxide Dismutases (Michelson, A. M.. McCord, J. M. k Fridovich, I.. eds) pp. 129138, Academic Press, New York. Niehaus, W. G. & Dilts, R. P.. Jr (1982). Purification and characterization of mannitol dehydrogenase from Aspergillus parasiticus. J. Bacterial. 151. 243-250. Odegaard, B. H., Anderson, P. C. & Lovrien, R. E. (1984). Resolution of the multienzyme cellulase complex of Trichoderma reesei QM9414. J. Appl. Biochem. 6. 15g-183. Oka, S.. Ogino. K., Matsuura, S., Yoshimura. S., Yamamoto, K., Okazaki, Y., Takemoto. T., Kato, K. & Uda, T. (1989). Human serum immuno-reactive copper, zinc-superoxide dismutase assayed with an enzyme monoclonal immunosorbent in patients with digestive cancer. Clin. Chim. Acta, 182, 209-220. Panchenko, L. F., Brusov, 0. S., Gerasimov. A. M. & Loktaeva, T. D. (1975). Tntramitochondrial localization and release of rat liver superoxide dismutace. FEBS Letters, 55, 84-87. Pike, R. pu’. & Dennison, C. (1989). Protein fractionation by three phase partitioning (TPP) in aqueous,’ t-butanol mixtures. Biotech. Bioeng. 33, 221-228. Pol. M. C., Deutsch, H. F. & Visser, I,. (1990). Purification of soluble enzymes from erythrocyte Int. .I. hemolysates by three phase partitioning, Biochem. 22, 179-185. Rest. R. F. & Spitznagel, J. K. (1977). Subcellular distribution of superoxide dismutases in human neutrophils. influence of myeloperoxidase on the measurement of superoxide dismutase activity. Biochem. J. 166, 145-153. Salin, M. I,.. Day, E. e., cJr & Crapo. .J. 11. (1978). Isolation and characterization of a manganrsecontaining superoxide dismutase from rat liver. ,4 rch. Bioche,m. Biophys. 187, 223-228. Tyler, D. D. (1975). Polarographie assay and intracellular distribution of superoxide dismutase in rat liver. Biochem. J. 147. 493-504. Wagner, U. (i.. Werber. $1. M.. Beck, Y.. Hartman. J. K.. Frolow. F. & Sussman, ,J. I,. (1989). Characterization of crystals of genetically engineered human mangallese superoxide dismutase. J. Mol. Biol. 206, 787-788. Weisiger, R. ‘4. & Fridovich, I. (1973a). Superoxidr dismutase: Organelle specificity. J. Biol. (‘hem. 248. 3582-3592.

Weisiger, R. A. & Fridovich. I. (19736). Mitochondrial superoxide dismutase: Site of synthesis and intra.J. Biol. (‘hem. 248. mitochondrial localization. 47934796.

Wong, G. H.. Elwell, J. H., Oberley, L. W. & Goeddel. D. V. (1989). Manganous superoxide dismuta,se is essential for cellular resistance to cytotoxicity of tumor necrosis factor. Cell, 58, 923-931.

by R. Huber

Preparation of human manganese superoxide dismutase by tri-phase partitioning and preliminary crystallographic data.

Human liver manganese superoxide dismutase has been purified by a short procedure that includes a tri-phase partitioning step to provide materials tha...
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