Proc. Nat. Acad. Sci. USA Vol. 71, No. 4, pp. 1334-1338, April 1974

Structural and Functional Similarities Between Mitochondrial Malate and L-3-Hydroxyacyl CoA Dehydrogenase (subunit structure/immunological cross-reactivity/amino-acid composition/ specificity of hydrogen transfer/evolution)

BARBARA E. NOYES*, BEAT E. GLATTHAAR, JOHN S. GARAVELLI, AND RALPH A. BRADSHAWf Division of Biology. and Biomedical Sciences, Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110 Communicated by P. Roy Vagelos, December 27, 1973

ABSTRACT Pig heart mitochondrial malate dehy- In view of these findings, it is of interest to examine the drogenase (EC 1.1.1.37), which has been obtained free of electrophoretic subforms, has been shown to have a structural relationships of mitochondrial , molecular weight of67,000 and to be composed of two poly- both among themselves and with the cytoplasmic forms. peptide chains. Comparison of these and other properties, Toward this end, the mitochondrial L-3-hydroxyacyl such as amino-acid composition, isoelectric point, and CoA dehydrogenase (EC 1.1.1.35) and malate dehydroge- keto-substrate inhibition, with those of L-3-hydroxyaeyl nase (EC 1.1.1.37) have been isolated in preparative amounts, CoA dehydrogenase (EC 1.1.1.35), another NAD+-depen- dent dehydrogenase of mitochondrial origin, suggests entirely free of undefined subforms, and their molecular prop- structural similarities of the type associated with pro- erties compared. From these results, it is concluded that these teins possessing common evolutionary origins. This con- enzymes probably possess the same order of structural simi- clusion is supported by immunological crossreactivity. larity that has been observed for the cytoplasmic malate and In view of these observations, the dissimilarity in the lactate dehydrogenases. stereospecificity of hydrogen transfer from to substrate catalyzed by the two enzymes is attributed to EXPERIMENTAL PROCEDURE 1800 rotation-in the binding orientation of the nicotinam- ide moiety of the NAD+, rather than to gross differences Homogeneous L-3-hydroxyacyl CoA dehydrogenase and mito- in the geometry of the of the two enzymes. chondrial from pig heart muscle were prepared as described previously (13, 14). In each case, the Comparisons of the structure-function relationships of numer- principal component was isolated free of the more acidic sub- ous proteins have provided a clearly emerging picture of the forms that have been present in the preparations of other evolution of biological function within a class of proteins workers. Amino acid analyses, N-terminal analyses, sedi- through selective genetic alterations leading to local changes in side chain character and environment without appreciably affecting the conformation of the polypeptide backbone. TABLE 1. Amino acid composition of pig heart The family of serine proteases, where similarities in primary mitochondrial matate dehydrogenase* and three-dimensional structure are manifested in a variety of proteolytic enzymes with differing specificities from diverse Gregory phylogenetic origins (1-5), is the best documented example of Thorne Anderton et al. This this phenomenon. Altho~h less well documented at the three- (19) (20) (21) study dimensional level, Iyaouyme and a-lactalbumin (6, 7) and ex- Lysine 54.3 48.8 48.6 51.6 nerve growth factor and insulin (8) represent additional 12.7 12.4 14.1 10.5 amples of structurally related proteins with unique functions. 14.7 20.0 14.6 16.5 More recent investigations have revealed that an entirely 46.9 48.8 47.5 50.1 parallel situation exists for the intracellular cytoplasmic de- Threonine 44.2 35.4 37.1 42.1 hydrogenases. Hill et al. (9), and Adams et al. (10) have dem- Serine 36.8 38.3 36.4 36.8 onstrated by means of x-ray crystallographic techniques that 46.3 45.0 46.6 49.8 cytoplasmic malate dehydrogenase and Proline 50.3 33.5 43.1 46.4 possess subunits of very similar polypeptide conformation. Glycine 59.0 54.5 54.4 57.8 In addition, it has been shown that the amino-terminal por- Alanine 71.0 54.5 60.9 65.6 tions of these which contain the NAD + cofactor Cysteine 14.7 14.4 14.7 13.8 molecules, Valine 47.6 43.0 56.0 53.6 , are closely related in conformation to the cor- Methionine 10.7 7.7 11.9 11.3 responding segments of glyceraldehyde-3-phosphate dehydro- Isoleucine 38.9 38.2 42.1 42.3 genase (11) and (12), suggesting an Leucine 55.6 52.6 53.4 56.5 even more distant evolutionary relationship involving all four Tyrosine 9.4 9.2 9.3 9.9 enzymes. Phenylalanine 21.5 23.0 20.2 22.3 - 1.6 *Present address: Department of Biochemistry, Stanford Uni- Total 634.6 580.9 610.9 636.9 versity School of Medicine, Palo Alto, Calif. 94305. t To whom correspondence should be addressed. * Residues/67,000 daltons of protein. 1334 Downloaded by guest on September 28, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Comparison of Mitochondrial Dehydrogenases i335

mentation equilibrium analyses, and isoelectric focusing were Immunological Cross-Reactivity of 'Mitochondrial Malate performed as described previously (13, 15). Dehydrogenase and i-3-Hydroxyacyl CoA Dehydrogenase. Rabbit antibodies against the enzymes were produced by The results of quantitative precipitin analyses of L-3-hy- injecting 0.25 ml of 2 mg/ml of samples of L-3-hydroxyacyl droxyacyl CoA dehydrogenase and mitochondrial malate CoA dehydrogenase or mitochondrial malate dehydrogenase, dehydrogenase, presented in Fig. 2, illustrate cross-reactivity in complete Freund's adjuvant (DIFCO), into all four foot- between the two pig heart dehydrogenases. In the case of anti- pads of the animal. Two rabbits were injected with each pro- serum to L-3-hydroxyacyl CoA dehydrogenase, mitochondrial tein. Five weeks after the initial injection, an additional mg of malate dehydrogenase can precipitate about 2% of the anti- protein in incomplete Freund's adjuvant was injected into body at the equivalence point. L-3-Hydroxyacyl CoA de- each rabbit either subcutaneously or in the footpads. One hydrogenase, on the other hand, precipitated 11-12% of the week after the booster injection, the rabbits were bled from antibody in the antimitochondrial malate dehydrogenase the marginal ear vein on three successive days. Bleeding was serum at the equivalence. Cross-reactivity was observed with repeated on three successive days of each week until the anti- sera from all four rabbits. In control experiments utilizing body titer of the serum dropped. The serum was collected nonspecific rabbit sera, mitochondrial malate dehydrogenase after centrifugation of the clotted blood and stored at -20°. and L-3-hydroxyacyl CoA dehydrogenase precipitated less Quantitative precipitin tests were performed as described by than 10% and 20% the amount of protein precipitated from Kabat and Mayer (16). Total protein in the precipitate was the heterologous antiserum. In addition, cytoplasmic malate determined using the biuret reaction (17). dehydrogenase did not precipitate significant amounts of pro- The stereospecificity of hydrogen transfer was determined tein from either antiserum. for both enzymes using the procedures of Davies et al. (18). Stereospecificity of Transfer Catalyzed by Mito- [4-3H]NAD+ was a product of New England Nuclear Corp. Hydrogen chondrial Malate Dehydrogenase and L-3-Hydroxyacyl CoA and yeast alcohol dehydrogenase was purchased from Sigma Chemical Co. Dehydrogenase. -catalyzed transfer of hydrogen be- tween substrate and cofactor has been reported to be stereo- RESULTS specific for the A-side (pro-R hydrogen) of the nicotinamide Characterization of the Principal Component of Pig Heart ring for mitochondrial malate dehydrogenase and specific for Mitochondrial Malate Dehydrogenase: Amino-Acid Composi- the B-side (pro-S hydrogen) of the ring for L-3-hydroxyacyl tion. The amino-acid composition of the principal mitochon- CoA dehydrogenase (18, 28). However, in each case, the mea- drial malate dehydrogenase of pig heart is summarized in surements were performed with impure enzyme. Consequently, Table 1, along with other compositions previously reported. the experiments were repeated with the homogeneous prepara- In each case, the values have been normalized to 67,000 tions known to be free of all subforms. [4-3H]NADH syn- molecular weight to facilitate comparison. With the exception thesized from [4-'H]NAD+ in the presence of and of the composition reported by Anderton (20), the values ob- yeast alcohol dehydrogenase is labeled on the B-side of the tained with the homogeneous protein (column 4) are in close nicotinamide ring, since alcohol dehydrogenase stereospecif- agreement with the preparations that also contain the more ically transfers the hydrogen from unlabeled ethanol to the acidic subforms (columns 1 and 3). This finding is consistent A-side of the ring (29). In subsequent conversion of the tri- with previous observations (14, 22). tiated NADH to NAD+ by L-3-hydroxyacyl CoA dehydro- genase or mitochondrial malate dehydrogenase, the tritium Molecular Weight. The molecular weight of the principal should remain in the NAD+ if the enzyme is A-specific, but component of mitochondrial malate dehydrogenase was deter- should be transferred to the substrate if the enzyme is B- mined by sedimentation equilibrium, in the presence and specific. In the case of mitochondrial malate dehydrogenase, absence of denaturing solvents, gel filtration, and sodium 99% of the initial radioactivity was accounted for in the dodecyl sulfate electrophoresis. The values obtained for the native protein are in excellent agreement with previously re- TABLE 2. Molecular weight data for pig heart ported numbers (Table 2). The subunit molecular weights, mitochondrial dehydrogenases which have not been previously reported, are consistent with a dimeric structure composed of polypeptide chains of equal MW native enzyme' size. Method 3-HADH mMDH Amino-Terminal Analyses. Amino-terminal analysis of mitochondrial malate dehydrogenase by the cyanate method Sedimentation equilibrium 65, OOOb 66, 400; 70,000c of Stark and Smyth (25) gave 1.8 residues of alanine per mol of Gel filtration 75, OOOb 74, 000; 67, OOOd protein. Following correction for blank analyses, carried out Amino acid composition 67, OOOb 67, 000; 70,000e on protein not exposed to urea or cyanate, no other significant MW enzyme subunit end groups were found. These results are in qualitative agree- Sedimentation equilibriumf 31,000b 35,700 ment with previous analyses (19) and in quantitative agree- SDS gel electrophoresis 32, 000b 34,500 ment with a dimeric subunit structure (26). Isoelectric Focusing. The principal component of pig heart a Abbreviations: 3-HADH, i,3-hydroxyacyl CoA dehydroge- nase; mMDH, mitochondrial malate dehydrogenase; MW, mitochondrial malate dehydrogenase gives a single uniform molecular weight; SDS, sodium dodecyl sulfate. boundary on isoelectric focusing as shown in Fig. The 1A. b Taken from refs. 13 and 15. isoelectric point, determined from the measured pH of the 0 Calculated from sedimentation and diffusion analyses (23). eluted fractions, is 9.3. This value is entirely consistent with d Taken from ref. 24. the observed behavior of the enzyme on CM-cellulose and o Taken from refs. 20 and 21. starch gel electrophoresis (14, 27). f Analysis in 6 M guanidine . HC1. Downloaded by guest on September 28, 2021 1336 Biochemistry: Noyes et al. Proc. Nat. Acad. Sci. USA 71 (1974)

.5 E

0 CYa

.4 U pH z 4 cr .3 0 I I 4 I Ii ?I

FRACTION NUMBER FIG. 1. Isoelectric focusing of the principal form of mitochondrial malate dehydrogenase as a homogenous preparation (A) and as a mixture with i,3-hydroxyacyl CoA dehydrogenase (B). Each experiment was carried out using 1% ampholyte, pH 8-10, and 10 mg of each protein. Malate dehydrogenase (- - -) and i.3-hydroxyacyl CoA dehydrogenase activity (- -) were assayed as described pre- viously (13, 14). The pH (. ** ) and absorbance at 276 nm (-) were monitored for each fraction.

NAD+ pool. For i-3-hydroxyacyl CoA dehydrogenase, how- 24). The molecular weights obtained in this study are consis- ever, no radioactivity was present in the NAD + pool following tent with this value. Several lines of evidence have also indi- DEAE-cellulose chromatography and 92% of the initial radio- cated that the quaternary structure was composed of two activity was recovered in L-3-hydroxybutyryl CoA. These re- identical polypeptide chains (26, 36, 37), although molecular sults confirm the previous reports that the two enzymes cata- weight studies under denaturing conditions and quantitative lyze the transfer of hydrogen from their respective substrates amino end group data were lacking. Such studies, described to opposite sides of the nicotinamide ring of the cofactor (18, here, confirm a dimeric subunit structure for this enzyme. 28). This size and distribution of component polypeptide chains of mitochondrial malate dehydrogenase is similar to that of DISCUSSION several other dehydrogenase enzymes. Considering the de- Preparations of mitochondrial malate dehydrogenase are veloping ideas that proteins possessing related biological usually characterized by the presence of multiple electro- functions possess related structures, at both the primary and phoretic forms. The origin of this heterogeneity has been three-dimensional levels, we have compared the properties of attributed to a variety of causes including conformational this enzyme with those of enzymes of related function. The differences (22), combination of very similar, but not identi- most striking relationship observed was with another mito- cal, subunits (30) and proteolytic degradation (31). Recently, chondrial enzyme, r-3-hydroxyacyl CoA dehydrogenase, the we described a preparation of this enzyme from pig heart that isolation and characterization of which has been recently is free of any subforms and showed that this principal com- reported (13, 15). The similarity in substrate specificity is ponent, which is the most basic form of the enzyme from this depicted schematically in Fig. 3, which indicates that the cat- tissue on starch gel electrophoresis, can give rise to the other alytic action of each enzyme is the NAD +-dependent oxidation subforms during preparative manipulations (14). This ap- of a hydroxyl group beta to a carbonyl function. Since both parently irreversible phenomenon is best explained by the enzymes possess molecular weights of about 67,000 (Table 2) successive desamidation of glutamine and asparagine residues. and have dimeric structures composed of identical or very In view of the uniqueness of this preparation, we have re- similar polypeptide chains, we have compared several other examined several of the physical and chemical properties of properties to ascertain the possible extent of similarity be- the prinkcipal component of pig heart mitochondrial malate tween the two enzymes. The subunit amino acid composition dehydrogenase to resolve earlier incomplete or controversial of the mitochondrial malate dehydrogenase and L-3-hydroxy- reports. acyl CoA dehydrogenase are given in Table 3. A decidedly Although several molecular weights for mitochondrial similar profile is evident, with only serine, proline, isoleucine, malate dehydrogenase of pig heart, or closely related species, and cysteine showing substantial differences. The subunit have been reported (32-35), the most consistent values have composition of the cytoplasmic malate dehydrogenase (38) indicated a molecular weight of 68,000 to 70,000 (20, 21, 23, and lactate dehydrogenase (39) from pig heart are also listed. Downloaded by guest on September 28, 2021 Proc. Nat. Acad. Sci. USA 71 (.1974) Comparison of Mitochondrial Dehydrogenases 1337 TABLE 3. Amino acid composition of mitochondrial and cytoplasmic dehydrogenases' Human Pig heart placenta

In- 3- s- 17-- MDHb HADHb MDHO LDHd EDHb,e Lysine 26 33 31 26 10 Histidine 5 7 4 12 7 E Arginine 8 8 10 11 22 C Aspartic 03 acid 25 27 39 32 21 Threonine 21 24 16 12 16 'U Serine 18 26 22 21 19 V Glutamic acid 25 27 27 28 28 Proline 23 13 12 13 19 Glycine 29 20 23 26 31 Alanine 33 28 32 20 36 Cysteine 7 1 5 -f 6 Valine 27 24 26 36 32 Methionine 6 7 8 8 4 Isoleucine 21 12 19 24 4 0 100 300 SOO 30 32 36 41 Leucine 28 ANTIGEN ADDED, PG/3 ML SERUM Tyrosine 5 6 8 7 6 Phenyl- FIG. 2. Precipitin curves of io3-hydroxyacyl CoA dehydroge- alanine 11 15 11 7 13 nase (-) and mitochondrial malate dehydrogenase (0) with anti- Tryptophan 0 1 5 -f 1 i-3-hydroxyacyl CoA dehydrogenase serum and of i-3-hydroxy- Total 318 309 330 (319) 316 acyl CoA dehydrogenase (0) and mitochondrial malate de- hydrogenase (U) with anti-mitochondrial malate dehydrogenase a Abbreviations: s- and m-MDH, cytoplastic (soluble) and serum. Each point represents the average of three determinations. mitochondrial malate dehydrogenases; 3-HADH, io3-hydroxyacyl CoA dehydrogenase, LDH, lactate dehydrogenase; 17-p-EDH, Burns et al. (40). This enzyme, which has a molecular weight of 17-,B-estradiol dehydrogenase. 67,000, was found to be composed of two identical or very b Residues/33,500 daltons of protein. o similar subunits, each possessing amino-terminal alanine. Residues/35,000 daltons of protein (38). With the exception of the pairs, lysine and arginine, and leu- d Residues/36,000 daltons of protein (41). cine and isoleucine, a close resemblance to mitochondrial e Ref. 40 f Values not reported. malate dehydrogenase can be seen. In each case, if the sum of these pairs is used in the comparison, the relationship be- comes even closer. Coupled with the low tryptophan content, It has already been established that the main chain conforma- which seems to be characteristic of the mitochondrial dehy- tions of these two cytoplasmic enzymes, as judged by x-ray drogenases, these observations suggest that 17-fl-estradiol analysis of pig cytoplasmic malate dehydrogenase and dogfish dehydrogenawe is also structurally related to this family of lactate dehydrogenase, are very similar (9, 10). Interestingly, enzymes. comparison with the two mitochondrial dehydrogenases shows Several other properties accentuate the similarity of mito- a rather similar profile, suggesting that all four enzymes may chondrial malate dehydrogenase and L-3-hydroxyacyl CoA possess homologous primary and three-dimensional struc- dehydrogenase. Both are basic proteins with similar pI values. tures. The last column of this Table lists the composition of However, they can be resolved on isoelectric focusing, as human placental 17-,B-estradiol dehydrogenase as reported by shown in Fig. 1B. Each possesses a low extinction coefficient

. | 3-NYDROXYACYL 'H ° I C*A DEHYDROGENASE RT+CH-CH2-C*jSCA -. R C-CH2-C± SCoA !- I I I I I

NAD + + NADH

OOC+CH-CH2-C4-O- a * -OOC4C-CH2-C-& I I MALATE I I L J .--- DENYDROGENASE L . FIG. 3. Schematic representation of the reactions catalyzed by ',3-hydroxyacyl CoA dehydrogenase and malate dehydrogease. Downloaded by guest on September 28, 2021 1338 Biochemistry: Noyes et al. Proc. Nat. Acad. Sci. USA 71 (1974) [mitochondrial malate dehydrogenase: A '%I cm, 278 nm = 2.88; 5. Stroud, R. M., Kay, L. M. & Dickerson, R. E. (1971) Cold I13-hydroxyacyl CoA dehydrogenase: A % cm, 278 nm = 4.39 Spring Harbor Symp. Quant. Biol. 36, 125-140. 6. Hill, R. L., Brew, K., Vanaman, T. C., Trayer, I. P. & (13)] reflecting their low tryptophan content. Kinetic analyses Mattock, P. (1968) Brookhaven Symp. Biol. 21, 139-152. indicate that both enzymes show pronounced substrate inhi- 7. Browne, W. M., North, A. C. T., Phillips, D. C., Brew, K., bition by the keto-form of their respective substrates (13, 41). Vanaman, T. C. & Hill, R. L. (1969) J. Mol. Biol. 42, Finally, each enzyme displays limited but significant immuno- 65-86. logical cross-reactivity with antiserum prepared against the 8. Frazier, W. A., Angeletti, R. H. & Bradshaw, R. A. (1972) Science 176, 482-488. other enzyme, indicating the presence of at least some related 9. Hill, E., Tsernoglou, D., Webb, L. & Banaszak, L. J. (1972) or identical immunological determinants. J. Mol. Biol. 72, 577-591. The two mitochondrial enzymes possess one feature of ap- 10. Adams, M. J., Ford, G. C., Koekoek, R., Lentz, P. S., parent dissimilarity, namely the stereospecificity of hydrogen McPherson, A., Rossmann, M. G., Smiley, I. E., Schevitz, transfer from R. W. & Wonacott, A. J. (1970) Nature 227, 1098-1103. the cofactor NADH to substrate. Reexamina- 11. Buehner, M., Ford, G. C., Olsen, K. W. & Rossmann, M. G. tion of this feature of the enzyme mechanism with the homQ- (1973) Abstr. 9th Int. Congr. Biochemistry, p. 66. geneous enzymes has confirmed earlier reports that both malate 12. Branden, C. -J., Eklund, H., Zeppezauer, E., Nordstr6m, dehydrogenases catalyze the transfer from the A-side of the B., Boime, T., Soderlund, G. & OhIsson, I. (1973) Abstr. nicotinamide ring, while i,3-hydroxyacyl CoA dehydrogenase 9th Int. Congr. Biochemistry, p. 37. 13. Noyes, B. E. & Bradshaw, R. A. (1973) J. Biol. Chem. 248, is B-side specific. This difference might indicate either that 3052-3059. there is a totally different orientation of cofactor in each 14. Glatthaar, B. E., Barbarash, G. R., Noyes, B. E., Banaszak, enzyme, which would undoubtedly also mean two unique pro- L. J. & Bradshaw, R. A. (1974) Anal. Biochem., 57, 432-451. tein structures, or that the nicotinamide ring is simply rotated 15. Noyes, B. E. & Bradshaw, R. A. (1973) J. Biol. Chem. 248, in 3060-3066. 1800 the manner that it is bound in one enzyme relative to 16. Kabat, E. A. & Mayer, M. M. (1961) Experimental Im- the other. This change could occur as the result of appropriate munochemistry (Charles C Thomas, Springfield, Ill.). mutations of amino acid side chains that are located in the 17. Itzhaki, R. F. & Gill, D. M. (1964) Anal. Biochem. 9, 401- vicinity of the 3 and 5 positions of the bound nicotinamide. 410. Although the evolutionary pressures that would produce such 18. Davies, D. D., Teixeira, A. & Kenworthy, P. (1972) Biochem. J. 127, 335-343. changes can only be speculated upon, it seems reasonable to 19. Thorne, C. J. R. (1962) Biochim. Biophys. Acta 59, 624-633. assume that changes required to accommodate the binding of 20. Anderton, B. H. (1970) Eur. J. Biochem. 15, 562-567. otherwise rather different substrates would be the most likely 21. Gregory, E. M., Yost, F. J., Rohrback, M. S. & Harrison, causative agent. It may be significant that the L-3-hydroxy- J. H. (1971) J. Biol. Chem. 246, 5491-5497. 22. Kitto, G. B., Wassarman, P. M. & Kaplan, N. 0. (1966) acyl CoA ester substrates are considerably more bulky than Proc. Nat. Acad. Sci. USA 56, 578-585. the four-carbon dicarboxylic-acid substrates of malate de- 23. Thorne, C. J. R. & Kaplan, N. 0. (1963) J. Biol. Chem. 238, hydrogenase, and their binding with the proper alignment 1861-1868. of the remainder of the catalytic apparatus may have re- 24. Murphey, W. H., Kitto, G. B., Everse, J. & Kaplan, N. 0. quired a displacement of the amide group of the nicotinamide (1967) Biochemistry 6, 603-610. 25. Stark, G. R. & Smyth, D. G. (1963) J. Biol. Chem. 238, 214- ring. In view of the numerous similarities, this latter explana- 226. tion for the differences in stereospecificity of hydrogen trans- 26. D6venyi, T., Rogers, S. J. & Wolfe, R. G. (1966) Nature 210, fer by mitochondrial malate dehydrogenase and io3-hydroxy- 489-491. acyl CoA dehydrogenase is favored. 27. Thorne, C. J. R., Grossman, L. I. & Kaplan, N. 0. (1963) of of Biochim. Biophys. Acta 73, 193-203. The final resolution the extent structural relationships 28. Marcus, A., Vennesland, B. & Stern, J. R. (1958) J. Biol. between the mitochondrial dehydrogenases themselves and Chem. 233, 722-726. with the related cytoplasmic enzymes will only be attained by 29. San Pietro, A., Kaplan, N. 0. & Colowick, S. P. (1955) J. the determination of the complete primary and three-dimen- Biol. Chem. 212, 941-952. sional structure of each enzyme. It is when this 30. Kulick, R. J. & Barnes, F. W. (1968) Biochim. Biophys. Acta hoped that, 167, 1-8. information is available, it will be possible to describe the 31. Cassman, M. & King, R. (1972) Biochim. Biophys. Acta evolution of the pyridine-nucleotide-dependent dehydroge- 257, 143-149. nases. 32. Wolfe, R. G. & Neilands, J. B. (1955) J. Biol. Chem. 221, 61-69. The authors would like to thank Mr. Gary R. Barbarash for 33. Heyde, E. & Ainsworth, S. (1968) Biochem. J. 109, 663-668. assistance with the enzyme preparation, Ms. Lita Lowry for 34. Siegel, L. & Englard, S. (1961) Biochim. Biophys. Acta 54, assistance with the ultracentrifuge measurements, and Drs. 67-76. George R. Drysdale and Leonard J. Banaszak for helpful discus- 35. Grimm, F. C. & Doherty, D. G. (1961) J. Biol. Chem. 236, sions. This work was supported by U.S. Public Health Service 1980-1985. Grant AM 13362. B.E.N. was, and J.S.G. is, a USPHS Pre- 36. Chilson, 0. P., Kitto, G. B. & Kaplan, N. 0. (1965) Proc. doctoral Trainee, Grant GM 1311. B.E.G. is a Fellow of the Swiss Nat. Acad. Sci. USA 53, 1006-1014. National Fund for Scientific Research. R.A.B. is a USPHS Re- 37. Kitto, G. B. & Lewis, R. G. (1967) Biochim. Biophys. Acta search Career Development Awardee, AM 23968. 139, 1-15. 38. Wade, M. J. (1971) Ph.D. Dissertation, Washington Uni- 1. Neurath, H., Walsh, K. A. & Winter, W. P. (1967) Science versity, St. Louis, Mo. 158, 1638-1644. 39. Wachsmuth, E. D., Pfleiderer, G. & Wieland, T. (1969) 2. Hartley, B. S. (1970) Phil. Trans. Roy. Soc. London Ser. B Biochem. Z. 340, 80-94. 257, 77-87. 40. Burns, D. J. W., Engel, L. L. & Bethune, J. L. (1972) 3. Shotton, D. M. & Watson, H. C. (1970) Nature 225, 811-816. Biochemistry 11, 2699-2703. 4. Sigler, P. B., Blow, D. M., Matthews, B. W. & Henderson, 41. Pfleiderer, G. & Hohnolz, E. (1959) Biochem. Z. 331, 245- R. (1968) J. Mol. Biol. 35, 143-164. 253. Downloaded by guest on September 28, 2021