Eur. J. Biochem. 60, 9-15 (1975)

Dihydrofolate Reductase from Bovine Liver Enzymatic and Structural Properties Heinz BAUMANN and Kenneth J WILSON Biochemisches Institut der Universitat Zurich (Received May 28/Septernbei 8, 1975)

Dihydrofolate reductase from bovine liver has been purified 5000-fold employing conventional techniques and methotrexate/aminohexyl/Sepharose affinity chromatography. Electrophoresis of the isolated enzyme on polyacrylamide gels resulted in the separation of two enzymatically active protein components which were not interconvertible by treatment with dihydrofolate and/or the coenzyme. The two forms, present in a ratio of 20: 1, were found by isoelectric focusing to have isoelectric points of 7.15 and 5.94. They had identical specific activities towards dihydrofolate (26.127.0 Ujmg) and folate (1.3 - 2.2 Ujmg), and h identical molecular weights (23 500) and amino acid compositions. Due to the small quantity of the acidic form and the similarity of the two forms, the amino-terminal sequence (19 residues) was determined on a mixture of carboxymethylated reductase. The single sulfhydryl group of the enzyme can be modified by several sulfhydryl reagents in the native enzyme without loss of activity. Modification of the same residue occurs in the denaturated state and partially inhibits renaturation to the fully active enzyme. One disulfide bridge was detected by reduction and alkylation. The cleavage of this bond did not effect the enzymatic activity.

Dihydrofolate reductase catalyzes the reversible NADPH-dependent reduction of dihydrofolate to tetrahydrofolate which is required at various stages in DNA, RNA, and amino acid biosynthesis [1]. Due to the central role of this enzyme in cellular metabolism during growth various substrate analogs were developed which have been successfully employed as cancer chemotherapeutic agents [2,3]. At the same time numerous investigations of the enzymatic and physical properties of dihydrofolate reductases isolated from both procaryotes and eucaryotes have been reported (see [4] for a review). However to date all detailed structural investigations have been restricted to the dihydrofolate reductases isolated from the inhibitor-resistant strains of Esclzevichia coli [5] and Streptococcusfuecium [6]. With the recently developed preparative methods based on affinity chromatography [7] sufficient amounts of vertebrate enzymes can now also be easily isolated for such structural studies. Utilizing this method we have purified the dihydrofolate reductase from bovine liver. In this communication we extend the structural characterization of the enzyme, first investigated by Rowe and Russel [8], by Enzyme. Dihydrofolate reductase or 5,6,7,8-tetrahydrofolate :NADP+ oxidoreductase (EC 1.5.1.3

reporting its amino acid composition, isoelectric properties and amino terminal sequence.The effects of sulfhydryl reagents on the enzymatic activity and stability of this enzyme are also reported. MATERIALS AND METHODS Bovine liver dihydrofolate reductase was isolated employing a combination of the methods previously outlined by Rowe and Russel [8] and Kaufman and Pierce [9]. Table 1 illustrates the isolation procedure. Steps 1- 4 are identical to those used by Rowe and Russel in their isolation of dihydrofolate reductase by convential chromatographic methods. The preparation of the methotrexate/aminohexyl/Sepharose and the procedures for adsorbing, washing, and stripping the column were performed according to Kaufman and Pierce. The assay methods for determining enzymatic activities were identical to those described by Rowe and Russel [8], i.e. for folate reduction the assay mixture (1 ml) contained 0.1 mM NADPH, 0.01 mM folate and 5 mM 2-mercaptoethanol in 50 mM sodium acetate, pH 4.25; for dihydrofolate reduction 0.1 mM NADPH, 0.014 mM dihydrofolate and 50 mM 2mercaptoethanol in 0.1 M potassium phosphate, pH 6.25. Assays, initiated by the addition of 5 - 10 1-11 of

Characterization of Bovine Dihydrofolate Reductase

10

enzyme solution, were done at 30 "C and the activities (pmol substrate utilized/min) were calculated using absorption coefficients of {340 = 18.4 x lo3 M-lcm-' [lo] for folate reduction and 5340 = 12.3 x lo3 M-' cm-' [Ill for dihydrofolate reduction. When active site titrations were performed the enzyme was preincubated for 5 min in the presence of known concentrations of methotrexate (5307 = 22.1 x lo3 M-' cm-l ) [12] before addition of the substrate. Protein concentrations prior to the affinity chromatographic step in the isolation procedure (see Table 1) were determined by the method of Lowry et al. [I31 using bovine serum albumin as the standard; on homogenous preparations enzyme concentrations were determined by amino acid analyses. Polyacrylamide gel electrophoresis in the absence of dodecylsulphate was performed according to Ornstein [I41 in 7.5% gels; in the presence of dodecylsulphate, 10 % or 15% gels were employed according to Laemmli [15]. Staining for enzyme activity using 3 - (43 - dimethylthiazolyl- 2)- 2,5 - diphenyltetrazolium bromide was carried out as described by Nakamura and Littlefield [16]. For isoelectric focusing a 110-ml column from LKB was used at 4 "C with 1.2% Ampholine (LKB) of pH 5-9. Following 5- 10 days of electrophoresis at 1200 V and 2.5 mA the column was eluted by pumping and the individual fractions were tested for the presence of dihydrofolate reductase activity. Prior to amino acid analysis the pooled fractions were passed over a 0 . 9 ~ 6 0 - c mcolumn of Sephadex G-25 equilibrated with 0.05 M potassium phosphate, pH 6.8. Amino acid analyses were performed on a Durrum D-500 analyzer following sample hydrolysis in 6 N HC1 under vacuum at 110 "C for 22 h. Minimum molecular weight and number of residues were calculated according to Delaage [17]. Automatic sequencing was performed in a Beckman sequencer model 890 B (updated) using the dimethylbenzylamine buffer system [18] and the Beckman peptide program (111374). Direct identification of the phenylthiohydantoin amino acid derivatives was carried out by gas chromatography on a Beckman gas chromatograph, model GC-65, equipped with 10% SP-400 columns. Indirect identification of the derivatives was made by amino acid analyses of the free amino acids following HI hydrolysis [19]. Sepharose 4B, Sephadex G-75 and G-25 fine were purchased from Pharmacia; dithiothreitol and 5 3 ' dithiobis-(2-nitrobenzoate) from Pierce. Methotrexate was from Nutritional Biochemistry, folate and dihydrofolate from Sigma, and NADPH from Boehringer. p-Chloromercuribenzoate, N-ethylmaleimide and iodoacetic acid were from Fluka; N-[2,3-14C]ethylmaleimide (2.1 Ci/mol) from Amersham. All chemicals used in the sequencer were purchased from Beckman.

Table 1. Purification of dihydrofolate reductase from bovine liver The table presents the average values from 12 separate purifications. For step 1, 800 g from a single bovine liver was homogenised in an equal volume of 0.1 M potassium phosphate buffer, pH 6.8. For step 4, column size, 12 x 120 cm; buffer, 0.05 M potassium phosphate, pH 6.8; for step 7, column size, 0.9 x 20 cm; column prepared and eluted according to Kaufman and Pierce [9]; for step 7, column size, 2.6 x 30 cm; buffer, 0.05 M potassium phosphate, pH 6.8 Purification step

Protein Activity Specific Total Yield activity activity

U

%

0.006 0.009

320.0 308.2

96.3

0.837

0.011

264.8

82.8

0.150

0.104

232.1

72.5

1.010

0.103

202.0

63.1

4.153

24.700

149.5

46.7

2.600

29.690

145.6

45.5

mg/ml

U/ml

1. Glass-wool filtrate 53.4 2. Protamine sulfate 37.2 3. 35-85% ammonium sulfate fraction 75.0 4. Sephadex G-75 1.4 chromatography 5. 90 % ammonium sulfate fraction 9.8 6 . Methotrexate,' aminohexyl/ Sepharose affinity chromatography 0.168 7. Sephadex G-25 chromatography 0.088

0.340 0.346

RESULTS AND DISCUSSION

Isolation Results obtained at various stage of purification are illustrated in Table 1. The final recovery (46%) and specific activity (29.7 Ujmg) of the enzyme isolated by the present procedure are similar to the values recently published by Kaufman for the isolation of the same enzyme from calf liver homogenates [7]. The last step in the purification of dihydrofolate reductases from numerous sources [7,9,20- 221 has involved adsorption chromatography which results in a purification as well as the removal of bound substrate. When a sample of purified bovine liver enzyme was subjected to hydroxyapatite chromatography under conditions identical to those of Kaufman and Pierce [9] no differences were noted in either the specific activity, the amino acid composition, or the patterns on polyacrylamide gels. However, the was) ob) absorption coefficient of the enzyme (i& served to decrease from 52.5 x lo3 M-' cm-' to 29.9 x lo3 M-lcm-'. This difference reflects the removal of bound dihydrofolate (5282 = 28.0 x lo3 M-' cm-' [23]) which was employed for eluting the enzyme from the affinity matrix. The purified enzyme was found to be unstable and inactivated to varying degrees by dialysis, concentration by membrane filtration under nitrogen, ammonium sulfate precipitation, and lyophilization. Instability was also noted when the enzyme was al-

11

H. Baumann and K. J. Wilson

Fig. 1. Polyacrylamide gel electrophoresis of purified bovine liver dihydrofolute reductme. (A) Electrophoresis in 7.5 % gels in the absence dodecylsulphate : (I) enzyme after methotrexate/aminohexyl/Sepharosechromatography ; (11, 111), enzyme from peaks I and 11 from isoelectric focusing (Fig. 2), respectively. Coomassie blue (left) and activity staining (right). (B) Electrophoresis in 15 % acrylamide gels in the presence of 0.1 % sodium dodecylsulphate and stained with Coomassie blue: (I, 11, 111), same as under A. For eachpair of gels the one on the left was heated in the absence of 2-mercaptoethanol and the one on the right in the presence of 2-mercaptoethanol

lowed to remain at temperatures higher than 4 "C for extended lengths of time. Dihydrofolate in all cases protected against inactivation to a greater extent than did NADP' and NADPH. Separation of the Two Forms of the Enzyme

As found for the same enzyme from hamster cells [24], LM 4 mouse lymphoma cells [25], Lactobacillus casei [26], and chicken liver [27] polyacrylamide gel electrophoresis of the purified reductase indicated two protein components both possessing enzymatic activity (Fig. 1A). Incubation in an excess (10 mM) of NADPH or dihydrofolate, a method which interconverts the two forms of the dihydrofolate reductase of L. casei [26], did not alter the gel patterns. These results contrast with the increased electrophoretic mobilities observed when both dihydrofolate reductases from methotrexate-resistant hamster cells bind the coenzyme [24]. Artificial production of multiple bands with dihydrofolate reductase activity, found to occur when the enzyme isolated from LM 4 mouse lymphoma cells was incubated with bromphenol blue [25], can not, in this case, account for the presence of two active species since electrophoresis was performed in the absence of the dye. A transformation of one form into the second during isolation also can

be excluded since liver extracts were shown to contain both enzymes with mobilities and relative concentrations identical to those of the purified mixture. The two forms, either in the presence or absence of dihydrofolate, were found by column isoelectric focusing to have isoelectric points of 5.94 0.06 and 7.15 & 0.05 (mean and S.E. of 6 determinations) (Fig. 2). The isolated enzymes exhibited either in the presence or absence of NADPH and/or dihydrofolate the same mobilities on polyacrylamide gels as those in the original mixture (see Fig. 1A). Both enzymes possessed identical absorption coefficients = 3 1.O x lo3 M-lcm-') and similar specific activities for dihydrofolate (26.1 -27.0 Ujmg) and folate (1.3 2.2 Ujmg) reduction. On the basis of similar enzymatic properties, identical amino acid compositions and molecular weights (see below), and in view of the observation that they are not interconvertible by substrate or coenzyme we conclude that both forms represent the same enzyme which differ only in overall charge. Structural Properties

Electrophoresis in dodecylsulphate (Fig. 1B) performed either on a mixture or on the isolated forms of the enzyme revealed protein components with

12

Characterization of Bovine Dihydrofolate Reductase

molecular weights of 23000 and 46000 (Fig.3A), the former comprising > 95 % of the stainable protein. The addition of 2-mercaptoethanol (2 %) prior to heating of the sample in dodecylsulphate resulted in the disappearance of the higher molecular weight component (Fig. 1B), thus suggesting that dimerization might occur through the single titratable sulfhydryl groups of the enzyme (see below). These molecular weight estimations were confirmed by gel chromatography on Sephadex G-75

6

5 4 -E

. 3

3 c ,

-> 29 c

1

0

Effluent (ml) Fig. 2. Isoelectric focusing of bovine er dihydvofolate reductase Enzyme (3.5 mg in 8 ml of 0.05 M potassium phosphate, pH 6.8, and 10% sucrose) was applied to the column and focusing carried out as described in Methods. Determinations of pH (O), absorbance (e), and enzymatic activity (0) were performed on individual fractions following column elution

sium phosphate, pH 6.8. During the chromatography we frequently observed the presence of an inactive protein component (< 10 % of the total protein) having a molecular weight of 46000 and the same amino acid composition as the active enzyme. The component was shown by dodecylsulphate polyresis to have molecular bsence and 23000 in the 01. of the enzyme assuming a showed a single binding site per molecule for methotrexate (Fig. 3B). This finding confirms the homogeneity of our final preparation. Rowe and Russel [S], using an enzyme solution with approximately 30-fold lower specific activity, also found by methotrexate titration a molecular weight in the range of 20000. A possible explanation for the result could be a contamination by catalytically inactive dihydrofolate reductase which was however capable of binding methotrexate. Our titrations of homogenous enzyme preparations with different specific activities support this assumption. In spite of decreased enzymatic activity methotrexate binding appeared to be unaffected (Fig. 3 B). Table 2 presents the amino acid composition of bovine dihydrofolate reductase. The minimal molecular weights calculated from all amino acid analyses were consistently 23500 & 1000 thus supporting the molecular weight values determined by the other methods (see above). The amino acid compositions of the two forms differing in their isoelectric points (Fig. 2) were identical to that listed in Table 2. The amino-terminal sequence of bovine dihydrofolate reductase is shown in Fig.4 and compared to the amino-terminal regions of the same enzymes form E. coli and S. fnecium. The data represent the results of four degradations performed on the bovine enzyme from four different enzyme preparations

A 7 -

E

6 -

f

5 -

2u

4

p

3 -

-m

tate aminotransferase

Dihydrofo ate -reductase dim ctate dehydrogenase

‘;f

0 Dihydrofolate reductase

I 01 0

L 0.1

0.2

0.3

0.4

0.5

0.6

Relative rnigration

Fig. 3. Molecular weight estimations of bovine liver dihydrofolate reductase. (A) Electrophor 10 % polyacrylamide gels In the presence of 0.3 % sodium dodecylsulphate; (B) methotrexate titration carried out on enzyme with 13.7 U/mg (A) and 29.0 U/mg (O), respectively. The assay mixtures contained 74.0 pmol (A) and 21.7 pmol (0)of enzyme/nil with inhibitor concentratlons ranging from 1- 150 pmol/ml

13

H. Baumann and K. J. Wilson 1

Bovine

5 10 15 ___ G1y Arg- Pro -Leu-Asn- Ser - Ile -Val - Ala -Val- Ser -Gln- Asn -Met-Gly- Ile Va 1

-

-

-

Gly -Lys - Asn -

Met -Trp - Ala -Gln- Asp -Lys- Asn -Gly -Leu- Ile - Gly -Lys - Asp -

E ~oli

Met -Phe-Ile

S. faecium

N e t -1le -Ser - Leu - Ile -Ala - Ala -Leu- Ala -Val - Asp -Arg-Val - Ile - Gly -Met - Glu -

Ser

Fig 4 Comparison of the amino terminal sequence of bovine liver, E. coli [3] and S. faecium [4] dihydrofolate reductases. Carboxymethylated enzyme was subjected to automatic sequence degradation and the phenyltlnohydantoin deiivatives identified as described in Methods Identical amino acid residues are indicated by bold-face type

Table 2 Amino acid composition of bovine dihydrofolate reductase Mean and S.E. values of analyses from 22 different enzyme preparations, values after 22 h hydrolysis The assumed integral values are given in parentheses Amino acid

Occurrence in the enzyme residues/mol

Lysine Histidine Arginine Aspartic acid Threonine“ Serine” Glutamic acid Proline Glycine Alaiiine Cysteineb Valine Methionine Isoleucine Leucine Tyrosine” Phenylalanine Tryptophan Total

16.0 f 0.3 3.3 f 0.1 9.5 f 0.1 21.6 f 0.2 7.3 0.2 10.6 f 0.2 27.3 0.3 12.7 i 0.3 15.7 & 0.3 10.4 f 0.3 3.1 f 0.1 16.1 0.4 4.6 f 0.2 10.6 f 0.1 17.6 F 0.1 6.4 k 0.1 9.8 _+ 0.1

* *

206

a Integral number corrected for destruction occurring during hydrolysis. Determined following carboxymethylation [28] and oxidation ~91. Hydrolysis in p-toluene sulphonic acid [30] indicated the presence of 3.1 residues ; spectrophotometric determination [31J 4.0 residues.

originating from individual livers. Possible population heterogeneity was indicated by the observation that in three experiments only glycine and in the fourth only valine was identified as the amino terminal residue. No other heterogeneities were found at the other 18 positions analyzed.

Suljhydryl Modijhtions Chemical modification experiments performed on the dihydrofolate reductases from L. casei and S. faecium have implicated the involvement of both tryptophanyl [32] and methionyl [33] residues in

either coenzyme binding or catalysis. The enzyme from E. coli contains two cysteine residues one of which reacts in the native molecule with 5,5‘-dithiobis(Znitrobenzoate) without reduction of activity while the other can be modified by mercurials resulting in the loss of the activity [34]. Similarly, inhibition upon exposure to sulfhydryl reagents was noted with the enzyme from calf thymus [22]. Table 3 shows the effect of different sulfhydryl reagents on the enzymatic activity of bovine dihydrofolate reductase. Treatment of the native enzyme either in the presence or in the absence of dihydrofolate, resulted in the modification of one sulfhydryl group per enzyme molecule. When modification was performed in the presence of dihydrofolate a lower reactivity of the sulfhydryl was obgroup toward 5,5‘-dithiobis-(2-nitrobenzoate) served (0.4- 0.5 mol sulfhydryl group per mol enzyme under conditions identical to those described in Table 3 ) which may reflect partial substrate protection. One sulfhydryl group was also found to be modified with iodoacetic acid or 5,5’-dithiobis-(2-nitrobenzoate) when the reaction was carried out in a denaturant. Enzymatic activity was absent following modification with 5,5’-dithiobis-(2-nitrobenzoate)in dodecylsulphate ; it was present but substantially reduced following reaction in guanidine hydrochloride. Removal of the 5-thio-(2-nitrobenzoate) group from this partially active enzyme however restored full activity. Since denaturation in 6 M guanidine hydrochloride is totally reversible (i.e. enzymatic was totally absent when assays were performed in the presence of 6 M guanidine hydrochloride, but dilution into the standard assay mixture results in full renaturation of the enzyme), these results suggest that the modification of the sulfhydryl group hinders the refolding to the fully active enzyme. The presence of a single disulfide bond is suggested by the observation that following reduction 2.5 sulfhydryl groups can be carboxymethylated. Since full reduction with dithiothreitol in guanidine does not affect enzymatic activity (Table 3) this bridge appears to be non-essential for maintenance of the active site. Thus dihydrofolate reductase from bovine liver is similar to those from chicken [37] and sheep [38] liver in that sulfhydryl groups are not involved in catalysis.

14

Characterization of Bovine Dihydrofolate Reductase

Table 3. Effect of suljhydryl reagents on bovine liver dihydrofolate reductase Experiments with iodoacetic acid and 5,5'-dithiobis-(2-nitrobenzoate)(Nbs,) were carried out on enzyme preparations in the absence of bound dihydrofolate; experiments with N-ethylmaleimide and p-chloromercuribenzoate (CIHgBzOH) were in the presence of substrate; enzyme concentrations were 5-50 nmol/ml. Buffers employed: 0.1 M Tris-HCI, pH 8.0, with iodoacetic acid; 0.1 M sodium phosphate, pH 8.0, with N-ethylmaleimide and Nbs,; 0.01 M sodium phosphate, pH 7.0, with ClHgBzOH. Molar excess of the reagents over total sulfhydryl content shown in parentheses. Incubations were carried out in the presence ofthe reactants and/or denaturant for the indicated periods of time. Iodoacetic acid was added only after 3 h ofpreincubation of the enzyme in the reagents indicated. Residual activity (ui)was determined by removing an aliquot and diluting directly into the assay mixture in the absence of 2-mercaptoethanol (see Methods). Similarly, control activity (0,)was determined from a sample incubated under the same conditions except in the absence of sulfkydryl reagent and denaturant ~

Buffer

Sulfhydryl reagents

Temperature

~~~

~~~~~~~~

Reaction time

~

~

~~

~

~~~

Percent residual activity

~~

~

~~~

~~

~

~

~~~-

SH groups reacted

0,

-' 100 0,

+ 6 M Guanidine hydrochloride + 6 M Guanidine hydrochloride and dithiothreitol(l0) + Dithiothreitol(l0) + 6 M Guanidine hydrochloride + 6 M Guanidine hydrochloride and

Iodoacetic acid (10) Nbs, (200) ClHgBzOH (200) N-Ethylmaleimide (20) -

Iodoacetic acid (10)

dithiothreitol

+ 6 M Guanidine hydrochloride + 0.2 % Sodium dodecylsulphate

Nbs, (200)

"C

h

0 30 30 30

3 0.5 1 22

94 94 99 100

mol/molenzyme

0/30

3

95

0/30 0 0

3 3 3

100 98 32

0.7" 0.9"

0 30 30

3 -

30 41 0

2.5" 0.9 0.9

0.8" 0.8b 1.3' OXd

-

a Determined as carboxymethylcysteine by amino acid analysis following removal of excess reagents by dialysis, versus H,O and hydrolysis as described in Methods. Determined spectrophotometrically according to Ellman [35] ' Determined spectrophotometrically [36]. Determined as incorporated N-['4C]ethylmaleimide following removal of excess reagent by chromatography on Sephadex G-25 in the same buffer as used for the incubation.

We thank Ursula Schneider for performance of polyacrylamide gel electrophoresis and Yvonne Werner for her excellent technical assistance. This work was supported in part by SNSF-Grant 3.409.74.

REFERENCES 1. Huennekens, F. M. (1963) Biochemistry, 2, 151- 159. 2. Huennekens, F. M. (1969) in The Biological Basis of Medicine (Bittar, E. E. &Bittar, N.,eds)vol. 3,pp. 129- 161, Academic Press, New York. 3. Friedkin, M. (1973) Fed. Proc. 32, 2148-2153. 4. Huennekens, F. M., Dunlap, R. B., Freisheim, J. H., Gundersen, L. E., Harding, N. G. L., Levison, S. A. & Mell, G. P. (1971) Ann. N . Y. Acad. Sci. 186, 85-99. 5. Bennett, C. D. (1974) Nature (Lond.) 248, 67-68. 6. Gleisner, J. M., Peterson, D. L. & Blakley, R. L. (1974) Proc. Natl Acad. Sci. U.S.A. 71, 3001 - 3005. 7. Kaufman, B. T. (1974) Methods Enzymol. 34, 272-281. 8. Rowe, P. B. & Russel, P. J. (1973) J. Biol. Chem. 248,984-991. 9. Kaufman, B. T. & Pierce, J. V. (1971) Biochem. Biophys. Res. Commun. 4,608-613. 10. Hillcoat, B. L. & Blakley, R. L. (1966) J. Biol. Chem. 241, 2995-3001. 11. Hillcoat, B. L., Nixon, P. F. & Blakley, R. L. (1967) Anal. Biochem. 21, 178-189.

12. Seeger, D. R.,Cosulich, D. B., Smith, J. M. & Hultquist, M. E. (1949) J. Am. Chem. SOC.71, 1753-1758. 13. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J . Biol. Chem. 193,265-275. 14. Ornstein, L. (1964) Ann. N . Y. Acad. Sci. 121, 321 - 349. 15. Laemmli, U. K. (1970) Nature (Lond.) 227, 680-685. 16. Nakamura, H. & Littlefield, J. W. (1972) J . Biol. Chem. 247, 179- 187. 17. Delaage, M. (1968) Biochem. Biophys. Acta, 168, 573 - 575. 18. Hermodson, M. A., Ericsson, L. H., Titani, K., Neurath, H. & Walsh, K. A. (1972) Biochemistry, 11, 4493-4502. 19. Smithies, O., Gibson, D., Fanning, E. M., Goodfliesh, R. M., Gilman, J. G. & Ballantyne, D. L. (1971) Biochemistry, 10, 4912-4921. 20 Perkins, J. P., Hillcoat, B. L. & Bertino, J. R. (1967) J. Biol. Chem. 242,4771 - 4776. 21 Nixon, P. F. & Blakley, R. L. (1968) J . Biol.Chem. 243,47224731. 22 Greenberg, D. M., Tam, B. D., Jenny, E. & Payes, B. (1966) Biochim. Biophys. Acta, 122, 423 - 435. 23 Greenfield, N. J., Williams, M. N., Poe, M. & Hoogsteen, K. (1972) Biochemistry, 11, 4706- 4711. 24 Hanggi, U. J. & Littlefield, J. W. (1974) J. Biol. Chem. 249, 1390- 1397. 25 Hiebert, M., Gauldie, J. & Hillcoat, B. L. (1972) Anal. Biochem. 46,433-437. 26 Dunlap, R. B., Gundersen, L. E. & Huennekens, F. M. (1971) Biochem. Biophys. Res. Commun. 42,772- 777.

15

H. Baumann and K. J. Wilson 27. Mell, G. P., Martelli, M., Kirchner, J. & Huennekens, F. M. (1968) Biochem. Biophys. Res. Commun. 33, 74-79. 28. Hirs, C. H. W. (1967) Methods Enzymol. 11, 199-203. 29. Hirs, C. H. W. (1967) Methods Enzymol. 11, 197-199. 30. Liu, T. Y. & Chang, Y. H. (1971) J . Bid. Chem. 246, 28422848. 31. Edelhoch, H. (1967) Biochemistry, 6, 1948- 1954. 32. Liu, J. K. & Dunlap, R. B. (1974)Biochemistry,13,1807- 1814.

33. Gleisner, J. M. & Blakley, R. L. (1975) J . Biol. Chem. 250, 1580- 1587. 34. Williams, M. N. & Hoogsteen, K. (1974) Fed. Proc. 33, 1310. 35. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77. 36. Boyer, P. D. (1954) J . Am. Chem. Soc. 76, 4331-4337. 37. Osborn, M. J. & Huennekeus, F. M. (1958) J. Biol. Chem. 233, 969 - 974. 38. Morales, D. R. & Greenberg, D. M. (1964) Biochim. Biophys. Acta, 85, 360- 376.

H. Baumann and K. J. Wilson, Biochemisches Institut der Universitat Zurich, Ziirichbergstrasse 4, CH-8032 Zurich, Switzerland

Note Added in Proof (November 14, 1975). After submission of this paper the details concerning a similar investigation on bovine dihydrofolate reductase [Peterson, D. L., Gleisner, J. M. & McNamer, A. (1975) Fed. Proc. 34,6831 were kindly provided by Prof. R. L. Blakley. Their sequence results differ at positions 6 (Ala), 11 @:), 12 (Glu), 13 (Asp), and 19 (Asp). In our investigation, amide assignments are based on relative increases in ammonia following acid hydrolysis of the phenylthiohydantoin amino acid derivatives. The identifications of the Ser residues at positions 6 and 11 are derived from both gas chromatography and amino acid analysis results. On the basis of an amino terminal sequence analysis of [14C]-carboxymethylatedreductase we excluded the presence of a Cys residue at either of these positions.