Proc. Natl. Acad. Sci. USA Vol. 83, pp. 7142-7146, October 1986 Biochemistry High-level expression of enzymatically active human Cu/Zn superoxide dismutase in (bacterial expression vector/A PL/ribosome binding site) JACOB R. HARTMAN, TANIA GELLER, ZIVA YAVIN, DANIEL BARTFELD, Dov KANNER, HAIM Aviv, AND MARIAN GORECKI Bio-Technology General () Ltd., Kiryat Weizmann, Rehovot 76326 Israel Communicated by Richard Axel, May 13, 1986

ABSTRACT Expression of human Cu/Zn superoxide dis- interest has evolved in the therapeutic potential of SOD. A mutase (SOD) with activity comparable to the human eryth- wide range of clinical applications has been suggested. These rocyte enzyme was achieved in Escherichwa coli by using a include prevention of oncogenesis and tumor promotion, vector containing a thermoinducible X PL promoter and a reduction of the cytotoxic and cardiotoxic effects of ,8-lactamase-derived ribosomosal binding site. The recombi- anticancer drugs (5), anti-inflammatory action (6), and pro- nant human SOD was found in the cytosol ofdisrupted bacteria tection against reperfusion damage of ischemic tissues (7). In and represented >10% of the total bacterial . The addition, there is much interest in studying the effects ofSOD enzyme was purified to homogeneity by salt precipitation, gel on the aging process (8). filtration chromatography, and ion exchange chromatography. The exploration ofthe therapeutic potential ofhuman SOD The active enzyme was obtained in high yield only when 1 mol has been hindered by its limited availability. The enzyme is of copper and 1 mol of zinc were incorporated into each mol of a dimeric metalloprotein composed of identical noncovalent- subunit during bacterial growth or by reconstitution of the ly linked subunits, each of 16 kDa and containing one atom apoenzyme. Human Cu/Zn SOD produced in bacteria has an ofcopper and one atom ofzinc (9). Each subunit is composed apparent subunit molecular massof19 kDaon NaDodSO4/poly- of 153 amino acids of known sequence (10, 11). Recently, a acrylamide gels. The native enzyme behaves as a dimer of 32 cDNA clone containing the entire coding region of human kDa as determined by gel filtration. Sequence analysis of the SOD was isolated and sequenced (12, 13). The gene coding NH2 terminus revealed that the first 14 amino acids corre- for human SOD was introduced by us into an efficient sponded to authentic human SOD except that the NH2-terminal bacterial expression vector. We report here the production of alanine was not acetylated. Thus, the bacterial processing gram quantities of enzymatically active human Cu/Zn SOD system readily removes the NH2-terminal methionine residue in Escherichia coli. from recombinant human SOD. MATERIALS AND METHODS The possible biological role of superoxide dismutase (SOD; Bacterial Growth and Induction Protocol. Construction of superoxide:superoxide oxidoreductase, EC 1.15.1.1) as an human SOD-expressing plasmid pSOD/31Tll was carried out oxygen free radical (O2j) scavenger was proposed in 1968 by according to published procedures (14) and will be described McCord and Fridovich (1) and has provoked considerable in detail elsewhere. Plasmid pSODJ31T11 was propagated in interest within the scientific community since that time. E. coli strain A1645 [c600 r- m+ gal' thr- leu- lac- bl (X Superoxide radicals and other highly reactive oxygen species cI857AHJ ABamHI N+)] (15), which produces X cI857 are produced in every respiring cell as by-products of repressor and the N gene product. Overnight cultures were oxidative metabolism, and they have been shown to cause grown at 30°C in L broth supplemented with tetracycline extensive damage to a wide variety of macromolecules and (12.5 ,ug/ml) and used to inoculate a 50-liter fermentor cellular components (for reviews, see refs. 2 and 3). A group containing casein hydrolysate (20 g/liter), yeast extract (10 of metalloproteins known as superoxide dismutases catalyze g/liter), glucose (10 g/liter), K2HPO4 (2.5 g/liter), the oxidation-reduction reaction MgSO4-7H2O (1 g/liter), NaCl (5 g/liter), and tetracycline (12.5 mg/liter). The pH was maintained at 7.0 ± 0.2 with 20j + 2H+ -- H202 + 02 NH3. Induction was started when cell concentration reached an OD6w of =10.0. At that stage, an additional 10g ofglucose and thus provide a defense against oxygen toxicity. There are per liter was added, the temperature was raised, and the three known forms of SOD that contain different metals- fermentation was allowed to continue at 42°C for 75 min. namely, iron, manganese, or both copper and zinc. All of Analysis of Bacterial Extracts. Bacterial cells were harvest- these catalyze the same reaction with high efficiency, and all ed by centrifugation and suspended in 50 mM potassium operate by a similar mechanism in which the metal is the phosphate buffer (pH 7.8). Aliquots were lysed in 3 x sample catalytic factor in the active site. These enzymes fall into buffer [30% (vol/vol) glycerol/9% NaDodSO4/2.1 M 2-mer- several evolutionary groups. The Fe-containing SODs are captoethanol/187.5 mM Tris HCl, pH 6.8/0.5% bromo- found primarily in prokaryotic cells, while Cu/Zn SODs have phenol blue), heated at 100°C for 5 min, and were been demonstrated in all higher eukaryotes. Mn SODs exist analyzed on 15% NaDodSO4/polyacrylamide gels (16, 17). throughout the phylogenetic range, from microorganisms to Proteins separated on polyacrylamide gels were electropho- humans (reviewed in ref. 4). retically transferred to nitrocellulose sheets (18) (0.45 ,um; Since every biological macromolecule can serve as a target Schleicher & Schuell) and underwent reaction with rabbit for the damaging action of the abundant oxygen radical, anti-human SOD IgG (affinity-purified from rabbit antisera raised against commercial human erythrocyte SOD; Sigma) The publication costs of this article were defrayed in part by page charge followed by incubation with 1251I-labeled protein A. The blots payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: SOD, superoxide dismutase; bp, base pair(s).

7142 Downloaded by guest on September 27, 2021 Biochemistry: Hartman et A Proc. Natl. Acad. Sci. USA 83 (1986) 7143 were thoroughly washed, air-dried, and exposed to Agfa- Gevaert curix RP2 x-ray film. Purification of the Recombinant Human SOD. Purification ofthe enzyme was accomplished by the following procedure: Induced bacteria were suspended in 50 mM potassium phosphate (pH 7.8) and disrupted by sonication. The lysate was centrifuged and the clear supernatant was passed through a cushion of DEAE-cellulose to remove nucleic acids. The eluate was treated with 2% NaDodSO4 at 370C for 1 hr. This procedure was shown to inactivate eukaryotic Mn SOD (19), and we found that it also inactivates the bacterial SOD activities (unpublished observations). Excess of NaDodSO4 was then removed by addition of potassium chloride to a final concentration of 0.3 M. The precipitate containing KDodSO4 was centrifuged out and the solution was treated with ammonium sulfate to 60% saturation. The precipitated proteins were removed by centrifugation. The supernatant was dialyzed against 20 mM potassium phos- phate (pH 7.8), concentrated by ultrafiltration, and separated by gel chromatography on a Fractogel 55 column. Fractions containing human SOD were identified by gel electrophore- sis, pooled, dialyzed, and brought to 2 mM potassium FIG. 1. Schematic description of clone pSOD,31T11. Construc- phosphate (pH 7.8) and loaded on a DE-52 column, which tion ofthe clone expressing human SOD (hSOD) is described in detail was developed with a linear gradient of 2-150 mM potassium elsewhere. Plasmid pSOD,/1T11 is 3700 bp long, contains the X PLOL phosphate (pH 7.8). The human SOD peak was collected and region, including the nutL site, the P-lactamase gene (of pBR322) dialyzed, and the ionic strength was adjusted to 2 mM promoter and ribosome binding site, the entire human SOD coding potassium phosphate (pH 7.8) and then subjected to a second sequences, and confers resistance to tetracycline (see text). The DE-52 column. Homogeneous human SOD was eluted with DNA sequence at the junction between the 5' end of the SOD gene 15 mM potassium phosphate (pH 7.8). and the ribosomal binding site derived from 3-lactamase are pre- sented with the two possible start codons underlined. The Analysis of Induced Human SOD. SOD activity was mea- ribosome binding site is indicated with a dotted line under the sured by monitoring the inhibition of reduction of fer- nucleotide sequence. Five amino acids corresponding to the amino ricytochrome c, as described by McCord and Fridovich (1). terminus of authentic human SOD are also shown. Bacterial and human SOD activities were differentiated by using 1 mM KCN (20). Protein concentration was determined by the method of Lowry et al. (21) using bovine serum 1). Construction and cloning of the various expression albumin as a standard. Cu and Zn content in homogeneous vectors that resulted in the generation of plasmid SOD preparations were determined by atomic absorption. pSOD,31T11 will be described in detail elsewhere. SOD apoenzyme was prepared according to Weser and Expression of Human Cu/Zn SOD in E. coli. Plasmid Hartmann (22). Reconstitution was performed by simulta- pSODB1T11 was propagated in anE. coli strain that produces neous addition of Cu2+ and Zn2+ to highly purified constitutively the thermolabile repressor cI857 (25) and the apoenzyme (23). Human Cu/Zn SOD (Sigma) and bovine transcription antiterminator Ngene product (26). At 30°C, the Cu/Zn SOD (Grunenthal, Aachen, F.R.G.) were used as repressor binds to OL and blocks transcription from the standards. strong PL promoter. Nevertheless, a low level of expression Amino Acid Sequencing. NH2-terminal amino acid se- of human SOD is detected by immunoblot analysis (Fig. 2B, quence was determined by Edman degradation (24) using an Applied Biosystems (Foster City, CA) gas-phase protein A B sequencer (model 470A) followed by HPLC to identify the 2 34 56 23456 residues. phenylthiohydantoin of the cleaved amino acid 94- 0~ RESULTS 67- w-_ I Description of the Expression Vector pSODflT1l. We have 43- No1:# . . used a human Cu/Zn SOD cDNA clone, designated pS61-10, 30- which was constructed and sequenced by Groner and co- -" workers (12, 13), to generate a series ofrecombinant plasmids hSOD that can express the enzyme in E. coli. The most efficient 20.1-,m expression, as judged by polyacrylamide gel electrophoresis . .k, of bacterial lysates, was achieved with clone pSODPlT11, 14.4- _ which effects production of human Cu/Zn SOD correspond- ing to -13% of the total cellular protein (see below). Clone pSODBlT11 (Fig. 1) is 3700 base pairs (bp) long and is FIG. 2. Production ofhuman SOD (hSOD) in E. coli. (A) Extracts composed of the following segments: a 245-bp fragment of bacteria containing plasmid pSOD,31T11 were analyzed on a 15% containing the X phage PLOL regulatory region and the N NaDodSO4/polyacrylamide gel before and after induction (lanes 3 gene utilization site (nutL); a 200-bp fragment derived from and 4, respectively). A suspension ofinduced bacteria was sonicated for 2 min, cleared by centrifugation, and the pellet (lane 5) and pBR322 that contains the 3-lactamase gene promoter and the a that supernatant (lane 6) were analyzed. Each lane contained equiv- ribosome binding site sequences; and 520-bp fragment alent of one-fifth of a 1-ml culture at OD60 = 1. Lanes: 1, 1 ,ug of includes the entire coding region of human Cu/Zn SOD. The human Cu/Zn SOD (Sigma); 2, protein size markers (in kDa) plasmid contains the tetracycline-resistance (TetR) gene from (Pharmacia). The gel was stained with Coomassie brilliant blue. (B) pBR322. At the junction between the 4-lactamase regulatory A parallel gel was blotted onto a nitrocellulose filter and underwent sequences and the SOD coding region, there are two alter- reaction with rabbit anti-human SOD IgG and 125I-labeled protein A native translation start codons (ATG) located in-phase (Fig. as described. Downloaded by guest on September 27, 2021 7144 Biochemistry: Hartman et al. Proc. Natl. Acad Sci. USA 83 (1986) lane 3), probably due to the presence of active A-lactamase Table 1. Enzymatic activity and metal content of promoter. This band was undetected in control bacteria SOD preparations (without the expression plasmid) or in uninduced E. coli bearing an expression plasmid that includes the ribosome Activity, mol/subunit binding site region derived from clI followed by the human SOD preparation units/mg Cu Zn SOD gene (unpublished data). Upon temperature shift and Human SOD* 167 0.07 1.62 incubation at 420C, a strong induction of human SOD is Apoenzyme 0 <0.01 <0.02 observed (Fig. 2A, lane 4). This induction is due to repressor Reconstituted human SOD 2931 0.81 0.88 inactivation, which allows transcription to proceed from PL. Human SODt 2730 0.88 0.90 The amount of human SOD produced within the 75-min Bovine SOD 2805 0.97 1.01 induction period corresponds to 13.3% of total bacterial Human SOD (authentic)* 1606 ND ND protein as determined by densitometric scanning of lane 4 in Values were determined with highly purified SOD preparations Fig. 2A. The concentration of human SOD in extracts of (see Fig. 3) as described in Materials and Methods. ND, not induced bacteria was 10.14 ug per ml of culture (OD600 = 1) determined. as determined by radioimmunoassay using 125I-labeled stan- *Human SOD prepared from E. coli grown on standard medium. dard human SOD (data not shown). This value represents a tHuman SOD prepared from E. coli grown on medium supplemented >30-fold increase in human SOD compared to the amount with 2 ppm of Zn2+ and 200 ppm of Cu2+. present in uninduced cultures. tObtained from Sigma; contains 90% pure enzyme by gel Human SOD expressed in E. coli readily reacts with electrophoresis. rabbit-anti-human SOD (Fig. 2B), has an apparent molecular mass of 19 kDa, and comigrates with an authentic Table 1. Purified human SOD produced in bacteria under human SOD on NaDodSO4/polyacrylamide gels (Figs. 2 and standard growth conditions possessed only fractional enzy- 3). Since the subunit molecular size of human SOD as matic activity (-5%) as compared to bovine Cu/Zn SOD. calculated from its amino acid sequence is =16 kDa (10, 11, Analysis ofmetal content revealed that the enzyme produced 13), the above observation indicates an anomalous migration. in this manner contained only 8% of the expected amount of Purification of Human SOD from Induced E. coli Cells. Cu. Apparently, most of the Cu sites were occupied by Zn2+ Sonication of bacteria producing human SOD and clearing of ions since the protein contained almost twice the expected the cellular debris revealed that essentially all of the enzyme level of Zn2+. However, the completely metal-free apoen- remained in the cytosol (Fig. 2, lanes 5 and 6). This behavior zyme (Table 1) regained essentially full specific activity upon indicates that human SOD is highly soluble in bacteria, rather reconstitution in solution containing both Cu2' and Zn2+, than being deposited and compartmentalized. Pure human each at a concentration of 1.2 mol ofion per mol ofactive site SOD was obtained by using a combination of salt precipita- (Table 1). tion, gel filtration, and ion exchange chromatography. Details Despite the successful reconstitution, we wanted to pro- are provided in Materials and Methods. Fig. 3 (lanes 4, 7, and duce enzymatically active human SOD directly in bacteria. 8) shows three different human SOD preparations with >95% The data presented above suggested that the intracellular purity. Final yields were =2 g of homogeneous human SOD concentration of Cu2+ was insufficient to saturate the human from each 50 liters of E. coli harvested at OD6w = 20, SOD produced. In a series of experiments, we found that corresponding to -10% of the induced human SOD (data not increasing the Cu2+ concentration in the growth medium shown). The native enzyme has a size of 32 kDa as deter- increased the specific activity of human SOD (Fig. 4). mined by gel filtration on Sephadex G-100 (results not Moreover, the induced SOD was sensitive to 1 mM KCN, shown). Enzymatic Activity and Chemical Characterization. The enzymatic activity and metal content of various highly purified SOD preparations (see Fig. 3) are summarized in 2 3 4 5 6 7 8 20 - 2 0 E X 94- CO 67- 0co 43- _ E I lo 30- I H~~~~~~~~~ 1 00~~~~~~~co) 20.1 - _ - two

144-

Cu2+, ppm FIG. 3. Polyacrylamide gel electrophoresis of SODs. Highly purified SOD preparations were analyzed on a 15% NaDodSO4/ FIG. 4. Effect of Cu2+ ions on the activity ofhuman SOD (hSOD) polyacrylamide gel and stained with Coomassie brilliant blue. Lanes: produced in E. coli. Cells containing pSOD,31T11 were grown in L 1, bovine Cu/Zn SOD (Grunenthal) (3 Mg); 2, human Cu/Zn SOD broth supplemented with 2 ppm of Zn2+ and the indicated Cu2+ (Sigma) (2 ug); 3, protein size markers (in kDa) (Pharmacia); 4, concentrations (both ions were supplied as sulfate salts). The amount purified human SOD isolated from E. coli grown on standard medium ofhuman SOD induced was determined by scanning polyacrylamide (7 ,g); 5, bacterial human SOD apoenzyme (7 Mug); 6, reconstituted gels with purified bovine SOD as standard (open boxes). SOD bacterial human SOD (7 jig); 7 and 8, human SOD isolated from two activity was measured as described in Materials and Methods separate preparations ofE. coli grown on medium supplemented with (striped boxes) and in the presence of 1 mM KCN (black boxes). Data 2 ppm of Zn2+ and 200 ppm of Cu2+ (7 Mg each). presented are for 1-liter cultures at OD6w = 1. Downloaded by guest on September 27, 2021 Biochemistry: Hartman et al. Proc. Natl. Acad. Sci. USA 83 (1986) 7145 indicating that the activity corresponded to human SOD and ofthe strong inducible leftward promoterPL ofbacteriophage not the bacterial enzymes (4, 19). Data shown in Fig. 4 X and the 3-lactamase promoter-ribosomal binding site indicate that, in L broth, addition of 75 ppm of Cu2" resulted region. A small level of human SOD production in the in an increase of human SOD activity from 100 to 2000 absence of any induction was observed either as the result of units/mg as measured in crude bacterial lysates. Notwith- the constitutive 8-lactamase promoter or residual unre- standing, the amount of the induced enzyme did not depend pressed activity ofthe X PL promoter. Upon temperature shift on Cu2+ concentration. Similar results were obtained in to 420C and inactivation of the thermosensitive cI857 repres- casein hydrolysate medium. Furthermore, the presence of up sor, efficient transcription from the X PL promoter was to 200 ppm of Cu2+ in the growth medium did not affect the initiated followed by translation from the 3-lactamase ribo- growth rate of the bacteria. E. coli grown in casein hydroly- some binding site. In our earlier experiments, E. coli grown sate medium supplemented with 200 ppm of Cu2+ produced, on LB or casein hydrolysate medium expressed enzymati- after induction, fully active human SOD, which had the cally inactive human SOD. The protein so produced was expected metal composition (Table 1). successfully activated by reconstitution (Table 1). Bacteria We assume that certain components ofrich media (such as produced enzymatically active human SOD only when they L broth and casein hydrolysate) chelate copper strongly. At were grown on Cu2+-supplemented medium. We therefore high levels of human SOD expression, not enough free monitored expression levels and purification steps by gel copper is available within the bacteria to fill the copper site electrophoresis and immunoblot analysis. A functional cor- in the enzyme. The addition of 75-200 ppm of Cu2+ ions to relation was made on the purified preparations; these data are the media apparently raises the intracellular Cu2+ concen- shown in Table 1. Cells containing pSOD,31T11 routinely tration to levels that are sufficient to saturate the active sites produced quantities of human SOD accounting for 13% of for the overproduced human SOD. In contrast, sufficient Zn total bacterial protein. is incorporated into human SOD when 2 ppm of Zn2+ is After submission of this manuscript, Hallewell et al. (28) present in the medium. reported the expression of human SOD in E. coli using the The sequence of 14 amino acids at the amino terminus of tacI promoter system. In contrast to our work, these authors purified human SOD was determined by Edman degradation reportedly obtained human SOD with normal specific activity (data not shown). The sequence was found to be identical to from E. coli cultivated in L broth. It is difficult to explain this the NH2 terminus ofCu/Zn human SOD, which confirms the discrepancy, since no details regarding medium supplemen- authenticity of the bacterial product (10, 11, 13). tation are provided by the authors; it is unlikely that this Absorption spectra ofvarious SOD preparations are shown difference in results is due to the different promoter systems in Fig. 5. It is worthwhile to note that while the maximum used. absorbance of the human SOD prepared from induced According to the DNA sequence of the cloned human one bacteria grown on standard medium is at 279 nm, both the SOD, initiation of protein synthesis could start at either at 5' the enzyme and the enzyme isolated from E. coli of two ATG codons located the end of gene (Fig. 1). reconstituted Protein studies prove that human SOD isolated grown on have an sequencing copper-supplemented casein hydrolysate from E. coli lacks an NH2-terminal methionine and begins absorbance peak at 265 nm. This maximum is characteristic with an alanine residue. This result suggests that protein of authentic human SOD (27). biosynthesis is initiated at the second ATG and that the DISCUSSION methionyl residue is removed by a bacterial peptidase. It is also possible that protein synthesis starts at the first ATG, The results presented above show that E. coli is capable of which would require removal of three amino acids. In any overproducing an active human SOD, which is under control case, it seems that the soluble recombinant human SOD is accessible to E. coli-processing enzymes that generate the mature protein. The only obvious structural difference be- tween authentic Cu/Zn human SOD and the bacterial pro- duced enzyme is that the former contains an acetyl group 04 blocking the NH2-terminal alanine residue (9-11). Authentic human SOD may be also glycosylated. Our results indicate that neither acetylation nor glycosylation is essential for enzymatic activity. It should be noted that the amino termi- 0.3 nus of all known mammalian Cu/Zn SODs is N-acetylated (4). However the Cu/Zn SODs of yeast (29), swordfish (11),

0 and fruitfly (30) contain a free amino-terminal valine residue. The recovery of large quantities of pure human SOD with .0.2 full enzymatic activity permits the clinical evaluation of this enzyme. We have already commenced studies on the thera- peutic effectiveness of human SOD in the reperfusion of ischemic heart tissue, in kidney transplantation, and in the prevention of bronchopulmonary dysplasia in premature neonates. Thus, experimental data on some clinical implica- tions ofsuperoxide dismutase administration will be available I, in the near future. 250 270 290 310 We thank Y. Groner for providing the SOD cDNA plasmid; Y. nm Burstein, M. Zeevi, and H. Ben-Artzi for help in amino acid FIG. 5. Absorption spectra of native enzyme, apoenzyme, and sequencing; S. Blumberg for valuable discussions; and F. Naider for reconstituted human SOD produced in E. coli. Concentrations of all critical reading of the manuscript. The expert assistance of C. human SOD preparations were 1 mg/ml in H20. -, Recombinant Luxemburg, E. Fishel, S. Rorlik, and M. Landsberg is gratefully human SOD isolated from E. ccli grown on standard medium; acknowledged. apoenzyme; , reconstituted enzyme; ----, recombinant human SOD isolated from E. coli grown on medium supplemented with 2 1. McCord, J. M. & Fridovich, I. (1969) J. Biol. Chem. 244, ppm of Zn2+ and 200 ppm of Cu2+. 6049-6055. Downloaded by guest on September 27, 2021 7146 Biochemistry: Hartman et al. Proc. Natl. Acad. Sci. USA 83 (1986)

2. Fridovich, I. (1979) in Advances in Inorganic Biochemistry, A. B. (1985) Gene 36, 131-141. eds. Eichhorn, G. L. & Marzilli, L. G. (Elsevier/North-Hol- 16. Laemmli, U. K. (1970) Nature (London) 227, 680-685. land, New York), pp. 67-90. 17. Maizel, J. V., Jr. (1971) in Methods in Virology, eds. Mara- 3. Freeman, B. A. & Crapo, J. D. (1982) Lab. Invest. 47, morosch, K. & Koprowski, H. (Academic, New York), Vol. 5, 412-426. pp. 179-246. 4, Steinman, H. M. (1982) in Superoxide Dismutase, ed. Oberley, 18. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl. L. W. (CRC, Boca Raton, FL), Vol. 1, pp. 11-68. Acad. Sci. USA 76, 4350-4354. 5. Oberley, L. W. & Buettner, G. R. (1979) Cancer Res. 39, 19. Geller, B. L. & Winge, D. R. (1983) Anal. Biochem. 128, 1141-1149. 86-92. 6. Huber, W. & Menander-Huber, K. B. (1980) Clin. Rheum. 20. Crapo, J. D., McCord, J. M. & Fridovich, I. (1978) Methods Dis. 6, 465-498. Enzymol. 53, 382-393. 7. Fridovich, I. (1983) Annu. Rev. Pharmacol. Toxicol. 23, 21. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, 239-257. R. J. (1951) J. Biol. Chem. 193, 265-275. 8. Talmasoff, J. M., Ono, T. & Cutler, R. G. (1980) Proc. Natl. 22. Weser, U. & Hartmann, H. J. (1971) FEBS Lett. 17, 78-80. Acad. Sci. USA 77, 2777-2781. 23. Jewett, S. L., Latrenta, G. S. & Beck, C. M. (1982) Arch. 9. Hartz, J. W. & Deutsch, H. F. (1972) J. Biol. Chem. 247, Biochem. Biophys. 215, 116-128. 7043-7050. 24. Edman, P. & Begg, G. (1967) Eur. J. Biochem. 1, 80-91. 10. Jabusch, J. R., Farb, D. L., Kerschensteiner, D. A. & 25. Sussman, R. & Jacob, F. (1962) C. R. Hebd. Seances Acad. Deutsch, H. F. (1980) Biochemistry 19, 2310-2316. Sci. Ser. A 254, 1517-1519. 11. Barra, D., Martini, F., Bannister, J. V., Schinina, M. E., 26. Roberts, J. W. (1969) Nature (London) 224, 1168-1174. Rotilio, W. H., Bannister, W. H. & Bossa, F. (1980) FEBS 27. Briggs, R. G. & Fee, J. A. (1978) Biochim. Biophys. Acta 537, Lett. 120, 53-56. 86-99. 12. Lieman-Hurwitz, J., Dafni, N., Lavie, V. & Groner, Y. (1982) 28. Hallewell, R. A., Masiarz, F. R., Najarian, R. C., Puma, Proc. Natl. Acad. Sci. USA 79, 2808-2811. J. P., Quiroga, M. R., Randolph, A., Sanchez-Pescador, R., 13. Sherman, L., Dafni, N., Leiman-Hurwitz, J. & Groner, Y. Scandella, C. J., Smith, B., Steiner, K. S. & Mullenbach, (1983) Proc. Natl. Acad. Sci. USA 80, 5465-5469. G. T. (1985) Nucleic Acids Res. 13, 2017-2034. 14. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular 29. Johansen, J. T., Overballe-Peterson, C., Martin, B., Hase- Cloning: A Laboratory Manual (Cold Spring Harbor Labora- mann, V. & Svendsen, I. (1979) Carlsberg Res. Commun. 44, tory, Cold Spring Harbor, NY). 201-217. 15. Honigman, A., Mahajna, J., Altuvia, S., Koby, S., Teff, D., 30. Lee, Y.-M., Friedman, D. J. & Ayala, F. J. (1985) Proc. Natl. Locker-Giladi, H., Hyman, H., Kronman, C. & Oppenheim, Acad. Sci. USA 82, 824-828. Downloaded by guest on September 27, 2021