THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 239, No. 8, August 1964

Printed in U.S.A.

The Role of Vitamin B,, in Methionine Biosynthesis

in Avian Liver

HERBERT DICKERMAN,* BETTY G. REDFIELD, J. G. BIERI, AND HERBERT WEISSBACH

From the Laboratory of Clinical Biochemistry, National Heart Institute, and the Laboratory of Nutrition and Endocrinology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health,

Bethesda, 14 Maryland

(Received for publication, January 24, 1964)

A consideration of numerous studies in viva indicates that the metabolism of methionine, folic acid, and cyano-B1$ is inter- related. The vitamin has a sparing effect on the methyl group requirement of young chickens, and, conversely, methionine has a sparing effect on the cyano-Blz requirement in chickens and rats (l-4). A more direct demonstration of an impairment in methyl group biosynthesis de novo was the inability of cyano-B12- deficient rats and pigs to convert 3-14C-serine and 2-14C-glycine to the trimethylamine moiety of choline (5, 6). This effect was not upon transmethylation (7). The formimino group of formininoglutamic acid, a catabolic product of histidine, is nor- mally transferred to folate-H4 so that little, if any, excretion of FGA occurs in response to a histidine load. As a consequence of cyano-Blz deficiency in rats and chickens, an increased FGA excretion occurs which is diminished by an increased dietary intake of methionine (8, 9). Elevated FGA excretions in re- sponse to histidine loads have also been reported in patients with pernicious anemia and megaloblastic anemia of pregnancy (10, 11).

The elucidation of methionine biosynthesis in Escherichia coli mutants has indicated a site at which the methionine, folic acid, and cyano-Blz interrelationship may occur. The methyl transfer from methylfolate-H4 to homocysteine (12), the terminal step of the biosynthetic pathway, is one of the known metabolic reactions which requires a cobamide prosthetic group (13-16). Other cofactors required for this reaction are reduced flavin (15) and Sadenosylmethionine (17, 18). Guest et al. (19) demonstrated that methyl-BIz could replace the cyano-BIp requirement in the enzyme system obtained from methionine-cyano-Blz auxotrophs of E. coli W, and, of more significance, the methyl group of methyl-B12 was transferred enzymatically to homocysteine to form methionine. The active form of the vitamin in the methyl- folate-H4-transferase2 reaction is as yet unknown, but recent

* This study was undertaken during the tenure of an Established Investigatorship of the American Heart Association, Inc.

1 The abbreviations used are: cyano-Blz, 5,6-dimethylbenzi- midazolylcobamide cyanide; Blz,, a reduced form of Blz having cobalt in a valence state of 2; Factor B, cobinamid hexa-amide; Factor VI,, cobyrinic acid a, b, c, d, e, g, hexamide (or cobyric acid) ; deoxyadenosyl-Blz, 5,6-dimethylbenzimidazolylcobamide 5’.deoxyadenosyl;methyl-Blz, 5,6-dimethylbenzimidazolylcobam- ide methyl; AMe, S-adenosylmethionine; FGA, formimino- glutamic acid; FMN, riboflavin phosphate, “flavin mononucleo- tide.”

2 In this study, the enzymatic activity, catalyzing the transfer of the methyl of methylfolate-Ha to homocysteine to form methi- onine, is designated methylfolate-H4 transferase. Similarly, the

studies suggest that it is a reduced cobamide derivative on the enzyme (16, 20).

Various investigators have demonstrated the formation of methionine from homocysteine by extracts of pigeon, chicken, sheep, and hog liver with serine or formaldehyde as donor of the 1 carbon unit (21-24). Sakami and Ukstins (25) have shown that, in hog liver, as in E. co& (12), the transfer of the methyl group from methylfolate-H4 to homocysteine is the terminal step in this biosynthesis. A cobamide requirement in animal tissues in vitro has not been reported, but AMe (17) and a reduc- ing system (26) are known requirements. Until recently, none of the reported enzyme systems of animal tissues has been exten- sively purified. During the progress of this study, Buchanan et al. (26) reported the purification of methylfolate-H4 homo- cysteine transferase from hog liver, which was identical to the bacterial system in substrate specificity and cofactor require- ments. The cyano-Blz content of the enzyme fractions was determined with Lactobacillus leichmannii as the test organism. The cobamide content was found to be proportional to the methylfolate-H4 transferase activity throughout the purification.

The present studies were undertaken to elucidate the role of cyano-B1, in methionine biosynthesis in animal tissues. They include the partial purification and characterization of methyl- folate-H4 transferase and methyl-B12 transferase activities from chicken liver and nutritional factors affecting these enzyme levels.

EXPERIMENTAL PROCEDURE

Nutritional Studies

Female Arbor Acres chickens were obtained from a com- mercial hatchery 1 day after hatching and kept eight in a cage. Food and water were supplied ad lib&m, and weights were determined at weekly intervals. The diet employed was de- scribed by Spivey Fox et al. (27). The cyano-B12-supplemented diet contained 0.1 mg of the vitamin per kg of diet.

When the animals were killed, the livers were excised and kept at approximately 0” for subsequent procedures. The tissues were homogenized in a Waring Blendor for 1 minute, and the resultant homogenates were centrifuged for 60 minutes at 105,000 x g. The supernatant fraction was assayed for methyl- folate-H4 and methyl-B12 transferase activities. The methyl-B,,

enzymatic activity, catalyzing the methyl transfer from methyl- B,z to homocysteine to form methionine, is designated methyl-B,2 transferase.

2545

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2546 Methionine Biosynthesis in Liver Vol. 239, No. 8

transferase reaction incubations were carried out for 2 hours while the methylfolate-H4 transferase reaction incubations were done for periods up to 1 hour.

PuriJication of Methyljolate-H4 and Methyl-Blz Transferases

Because both enzyme activities were found to fractionate in the same manner, a common purification procedure was de- veloped. Chicken livers from a commercial source (40 g) were homogenized in 2.5 volumes of 0.1 M Tris, pH 7.4, and the homoge- nate was centrifuged at 105,000 x g for 60 minutes. The superna- tant fraction was treated with a neutralized saturated solution of ammonium sulfate, and the protein that precipitated between 30 and 50% saturation was dissolved in 0.01 M Tris buffer, pH 7.4. This fraction was dialyzed against 4 liters of 0.005 M Tris buffer, pH 7.4, for 3 hours, and adjusted to a protein concentra- tion of 20 mg per ml. The dialyzed fraction (60 ml) was then adsorbed onto calcium phosphate gel, 1 mg of gel per mg of pro- tein. Following washing of the gel with 0.01 M potassium phos- phate buffer, pH 7.4, enzyme activity was eluted from the gel with 60 ml of 0.2 M potassium phosphate, pH 7.4. The gel eluate was fractionated with neutralized saturated ammonium sulfate, and the precipitate formed between 35 and 50% satura- tion was dissolved in 0.01 M Tris buffer, pH 7.4. This fraction (50 mg of protein per ml) was diluted lo-fold with 0.005 M Tris buffer, pH 7.4. The diluted enzyme solution was applied slowly onto a DEAE-cellulose column (1 X 9 cm) which had been equilibrated with 0.005 M Tris buffer, pH 7.4. Following pre- liminary washings of the column with 10 ml of 0.01 M potassium phosphate buffer, pH 7.4, and 20 ml of 0.01 M potassium phos- phate buffer, pH 7.4, containing 0.1 M NaCl, the enzyme was eluted with 30 ml of 0.01 M potassium phosphate buffer, pH 7.4, containing 0.3 M NaCI. The eluate (1.4 mg of protein per ml) was treated with neutralized saturated ammonium sulfate, and the protein that precipitated between 0 and 66% saturation was dissolved in 2 ml of 0.01 M Tris buffer, pH 7.4 (11 mg of protein per ml).

Methods and Materials

Methyl-Blz was synthesized from methyl iodide by the general procedure of Smith et al. (28). In the same manner 14C-methyl- Bi2 was prepared from 14C-methyl iodide. nn-Methylfolate-H4 was prepared according to the procedure of Keresztesy and Donaldson (29). Solutions of methylfolate-H4 were stored in 0.1 M mercaptoethanol.

The methylfolate-Hd and methyl-B12 transferase assay pro- cedures have been described in previous publications (16, 30). The former involves passage of the reaction mixture through a Dowex l-chloride resin bed which retains the substrate, 14C- methyl-labeled methylfolate-H4, and permits the product, i4C-methyl-labeled methionine, to pass through. The methyl- Blz-transferase assay is based on the retention of 14C-methionine on a Dowex 50-H+ resin and its subsequent elution after pho- tolytic decomposition of the substrate, 14C-methyl-Blz.

The standard incubation mixture for the methylfolate-H4- transferase reaction included, in a total volume of 0.2 ml, enzyme, 0.01 to 0.1 ml; i4C-methyl-labeled methylfolate-H4 (specific activity, 800 to 1800 c.p.m. per mpmole), 30 mpmoles; cyano-Blt, 10 mpmoles; FMN or FAD, 1 mpmole; AMe, 5 mpmoles; n-homocysteine (prepared from the thiolactone derivative), 50 mpmoles; ,&mercaptoethanol, 40 pmoles; and potassium phos-

phate buffer, pH 7.4, 10 pmoles. Anaerobic experiments were performed in Thunberg tubes in a nitrogen atmosphere.

The standard incubation mixture for the methyl-B12-transferase reaction included, in a total volume of 0.2 ml, enzyme, 0.04 to 0.14 ml; 14C-methyl-labeled methyl-B12 (specific activity, 1800 c.p.m. per mpmole), 15 mpmoles; L-homocysteine, 50 mpmoles; and potassium phosphate buffer, 10 pmoles, pH 7.4.

A unit of methylfolate-H4 transferase or methyl-Blz transferase catalyzes the formation of 1 mpmole of methionine per hour at 37”. Protein concentrations were determined by the method of Warburg and Christian (31).

Cyano-Blz, FMN, FAD, L-homocysteine thiolactone, DPNH, and TPNH were purchased from the California Corporation for Biochemical Research. Deoxyadenosyl-B,, and Factor B were gifts from Dr. David Perlman, Squibb Institute of Medical Research. Highly purified intrinsic factor was the gift of Dr. Leon Ellenbogen of the Lederle Division of the American Cyan- amid Corporation. Factor V1, was the gift of Dr. J. L. Peel, Agricultural Microbiological Unit, Oxford University.

All radioactive determinations were done in a Packard Tri- Carb liquid scintillation counter with a naphthalene-dioxane scintillation fluid (32).

RESULTS

Nutritional Studies

Methylfolate-H4 and Methyl-B12 Transferase Levels Following Cyano-Biz Omission and Replenishment-Prior nutritional studies indicated that growth was impaired when newborn chickens were fed a diet suboptimal in methionine content, high in fat content, and devoid of cyano-B,s (4,27). In the present experi- ments also, a marked difference in weights was observed between animals on basal and cyano-Bit-deficient diets; the 4-week weights of the control animals averaged about 450 g, and those on the deficient diet averaged 100 to 200 g less. Previous studies in which this diet was used indicated that, under similar experi- mental conditions, FGA excretion was increased in response to a histidine load (9), but there was no decrease in hemoglobin content, red blood cell number, or hematocrit (33).

The effect of a cyano-Blz-deficient diet on the two transferase activities is shown in Table I. There was a distinct reduction in the level of both transferase specific activities in liver extracts from cyano-Blz-deficient animals. Similar results were obtained when the incubations were done under anaerobic conditions. In addition, there was no evidence of the presence of inhibitory factors in the liver extracts of cyano-Bis-deficient animals.

Restoration in vitro of either the methylfolate-H4 or methyl-Blr transferases from the livers of deficient chickens was not observed when cyano-Blz, deoxyadenosyl-B12, methyl-Biz, or Biz, were added to incubation mixtures.

The failure to restore enzyme activities in vitro led to an at- tempt at restoration of the activities of the enzyme or enzymes by administration of the vitamin in vivo. Table II records the results of one such experiment. The specific activity of the methylfolate-H4 transferase was increased 6-fold over that seen in cyano-Bit-deficient animals if cyano-Blz was administered at 3 and 24 hours before death. Animals which received only a single dose of cyano-B,n at 3 hours prior to sacrifice show no increase in enzyme level as compared to animals maintained on the deficient diet. Cyano-Bn injected at 3, 24, and 48 hours before death gave the same results as in animals that received

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August 1964 Dickerman, Redjield, Bieri, and Weissbach 2547

TABLE I Enzyme levels following nutritional omission of cyano-B12

The incubation mixtures and assay procedures are described in “Experimental Procedure.” The numbers in parentheses represent the range of specific activities. The standard deviations presented in this and following tables are derived from the equation

Diet No. of animals Methylfolate-HI transferase specific activity No. of animals Methyl-BIZ transferase specific activity

Basal. . . . . . . . . 39 5.35 f 1.52 (3.0-9.3) 18 0.51 f 0.10 (0.41-0.73) Cyano-Bt,-deficient . . . 30 0.55 f 0.28 (0.1-1.2) 16 0.10 f 0.04 (0.03-0.16)

TABLE II TABLE III

Eject of parenteral administration of cyano-B12 on methylfolate-Hd and methyl-Blz transferase activities

The incubation mixtures and assay procedures are described in “Experimental Procedure.” In the methylfolate-H, transferase determinations, each value represents the mean of eight samples derived from 16 animals. In the methyl-Blz homocysteine trans- ferase determinations, each value represents the mean of four samples derived from 8 animals.

Comparison of various cobamide derivatives in restoration of transferase specijk activities in vivo

This table presents a summary of results obtained in three experiments. For each compound a total of 24 animals were evaluated for the methylfolate-H4 transferase percentage of restoration and 20 animals for the methyl-B12 transferase restora- tion. The animals were kept on a cyano-Bit-deficient diet for 4 weeks. At 24 and 48 hours prior to death, 7.75 mfimoles of the indicated compounds were injected. The activity observed in extracts of control chicks (maintained on a diet with cyano-Brt present) was used as the 100% value. Diet

Basal......................... Cyano-Blp-deficient . . . Cyano-B12-deficient + injec-

tion of cyano-Brz*

- Methylfolate-H4

transferase specific Methyl-Bn

transferase specific activity activity

4.40 f 1.1 0.53 f 0.4

3.36 f 0.6

0.48 f 0.05 0.10 f 0.03

0.23 f 0.06

* At 3 and 24 hours prior to death, 7.75 mpmoles of cyano-Brz were injected.

cyano-B,, at 3 and 24 hours. There was no significant weight difference between the repleted animals (those receiving the vitamin for 24 hours) and those maintained on a deficient diet. A 2- to a-fold increase was observed in the methyl-B,, transferase activity following administration of cyano-B,, in viva.

Table III shows the effect of several cobamides on the restora- tion of activities of the enzyme or enzymes in viva in deficient animals. It is apparent that those cobamides which have a nucleotide moiety, i.e. cyano-Bn, deoxyadenosyl-Brz, and methyl- Bn, were more effective than Factor B, which is devoid of a nucleotide moiety. This observed specificity agrees with studies in viva in a variety of organisms (34) which have shown that the nucleotide-free Blz analogues are generally ineffective.

InJluence of Dietary Methionine on Methyljolate-Ha and Methyl- BIz Transferase-The sparing effect of methionine on the growth rates of cyano-Bn-deficient newborn chickens has been demon- strated in previous studies (1, 4). In the present studies, methionine supplements comparable to those reported (4) were fed to young chickens in order to study the effect of the amino acid on the specific activities of the two transferases. Of particu- lar interest is the fact that methionine is the product of both transferase reactions. Four groups were studied: those on com- plete diets with and without a methionine supplement, and those on a cyano-B,,-deficient diet with and without a methionine supplement. The results are summarized in Table IV.

The addition of methionine to the cyano-Bn-deficient diet did not lead to a restoration of either methylfolate-H’a or methyl-B12 transferase activities, although the animals on a diet supple-

Restoration of specific activity

Derivative Methylfolate-HI Methyl-Bn

transfera% transferase

% %

Cyano-BIP. . . . . . . . . . . . 40 31 Methyl-Brn . . . . . 65 46 Deoxyadenosyl-Blz . . . 49 36 Factor B..................... 9 5

TABLE IV

Effect of dietary methionine on methylfolate-H4 and methyl-B12 transferase activities

A 3.week interval from birth was used as the experimental period in this study. Control intake was the basal diet (27), which included 3 g nL-methionine per kg of diet; in the nn-methi- onine-supplemented group, an additional 6 g of methionine per kg of diet were fed. The body weight represents the mean weight of each group of eight animals. The incubation mixtures and assay procedures are described in “Experimental Procedure.”

I I Specific activity

Diet Bod .x wag t

Basal..................... Basal + methionine sup-

plement................. Cyano-Blz-deficient . . Cyano-Brz deficient + me-

thionine supplement.

g

279*

355 251

324

4.22 f 0.36 0.32 f 0.05

1.29 f 0.24 0.31 f 0.06 0.38 f 0.12 0.07 f 0.04

0.22 f 0.14 0.06 f 0.02

* In this experiment the animals on the basal diet had a slightly lower average weight than was usually observed at the 3-week period.

Methylfolate-H4 transferase

Methyl-B11 transferase

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2548 Methionine Biosynthesis in Liver Vol. 239, No. 8

TABLE V

Effect of varied amounts of dietary D.+methionine on methylfolate-Hb and methyl-B,2 transferase of

chicken liver

The experimental feeding period was 4 weeks. Basal diet in- cluded 3 g of nL-methionine per kg of diet while the appropriate DL-methionine supplements are indicated in the left-hand col- umn. Body weight represents the mean of eight animals in each experimental group. The incubation procedures and assay pro- cedures are described in “Experimental Procedure.”

Diet

Basal........................ Basal + 6 g of methionine per

kg of diet.. Basal + 12 g of methionine

per kg of diet. . Basal + 18 g of methionine

per kg of diet. .

I Smcific activity

1.

g 486 6.2 f 0.50

507 2.4 f 0.09

528 1.8 f 0.35

418 1.4 f 0.36

Methyl-B,z transferase

0.39 i 0.05

0.40 f 0.05

0.32 f 0.08

0.31 f 0.05

TABLE VI

Purijication of methylfolate-Hb-homocysteine and methyl-B12-homocysteine transferase

Incubation and assay procedures were used as described in “Experimental Procedure.”

Methylfolate-HI Methyl-Bn transferase transferase

Fraction

‘p+? Recovery sp’?cific Recovery actw1ty actw1ty

% % Supernatant................... 0.41 100 0.12 100 30 to 50% (NHJzSOd. 1.28 52 0.61 86 Calcium phosphate eluate.. 3.5 58 0.73 42 35 to 55% (NH&Sod. . 4.1 32 1.6 42 DEAE eluate . 13.6 24 5.2 31 0 to 66% (NH,)zSOh 15.9 19 6.2 26

TABLE VII

Requirements for methylfolate-Hd-homocysteine transferase

The incubation mixtures and assay were performed as described in “Experimental Procedure.” As an enzyme source, 190 pg of the third ammonium sulfate fraction were used. The anaerobic determinations were done in Thunberg tubes in a nitrogen at- mosphere.

Reaction mixture Methionine formed

Aerobic Anaerobic

I m,moles/60 min

Complete ......................... - o-Mercaptoethanol. .......... - Cyano-Bit ................... - S-Adenosylmethionine ....... - Homocysteine ............... - FMN. .......................

2.11 3.17 0.50 2.16 0.33 1.65 0.37 0.38 0.30 0.18 1.90 2.78

mented with methionine had a higher average body weight than those which did not receive methionine. This indicated that the enzyme alterations observed with cyano-Blz depletion were not the result of the over-all impairment in cellular biosyntheses, but rather a specific effect on the enzyme activities which were studied.

A difference was observed in the specific activities of methyl- folate-H4 transferase obtained from chickens on t.he basal diet (with cyano-Blz present) and those which received added methio- nine. As seen in Table IV, the average specific activity of the methionine-supplemented group was 30% of the average of those on the basal diet, a suggestion that methionine supplementation in viva may inhibit synthesis or activation of the methylfolate-Hl transferase. Methionine did not inhibit the transferase reaction in vitro. The difference in the specific activities of the enzyme occurred despite a 20% greater average body weight in the methionine-supplemented group. In contrast, there was essen- tially no significant difference in the specific activities of methyl- Blz transferase of the two groups. Both activities in t.he cyano- Blz-deficient groups, with or without methionine additions, were too low for an evaluation of the methionine effect.

The response of the methylfolate-H4 and methyl-B12 transferase activities in the liver of chickens on diets containing varied amounts of methionine is indicated in Table V. At all levels tested, there was an extensive reduction in the methylfolate-H’r transferase specific activity without a comparable decrease in the methyl-Blz t.ransferase specific activity. Recombinations in vitro of homogenates obtained from chickens on a basal methio- nine intake and from those on an intake of 21 g of methionine per kg of diet (Groups I and IV) did not indicate the presence of .nhibitory or stimulatory factors. 1

Purijication and Properties of Methyljolate-HI and MethyGBlz Transjerases of Chicken Liver

The purification of both enzyme activities was developed con- currently with the nutritional studies which have been described. Table VI is a summary of a representative purification. The low initial specific activity of the methylfolate-H., transferase is characteristic of commercially obtained chicken livers. It should be stressed that the two activities were distributed in the same fractions at all steps of the purification procedure. How- ever, in two purifications, the methyl-B12-transferase activity was purified 50- to 60-fold over the original extract whereas the methylfolate-H4 transferase activity was purified 30- to 40-fold.

The requirements for the transfer of the methyl group of methylfolate-HI to homocysteine to form methionine are indi- cated in Table VII. Besides the methyl group acceptor, homo- cysteine, AMe, cyano-Blz, and a reducing system are required for the complete reaction. These requirements, as well as the stimulation of the reaction by anaerobiosis, are the same as previously described for the transferase activity studied in cruder fractions (16,35).

During the course of the present study, ,&mercaptoethanol was found to stimulate markedly the methyl transfer from methyl- folate-Hd aerobically with the more purified enzyme preparations (calcium phosphate eluate or beyond). Even under anaerobic conditions, there was a 30% stimulation in the presence of this sulfhydryl reagent. In the presence of /?-mercaptoethanol, the stimulations of FMN, FAD, DPNH, or TPNH under aerobic or anaerobic conditions are relatively inconsistent and of a small

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magnitude.3 The reducing system may be needed to keep the enzyme-bound cobamide in a reduced state, but this is as yet unproven in animal systems.

The enzymatic reaction was linear with time up to 60 minutes and was also proportional to enzyme concentration (Fig. 1). The pH optimum of the methylfolate-H4 transferase activity was between 6.8 and 7.8. K, determinations of the required com- pounds under aerobic conditions indicated the following values: nn-methylfolate-Ha, 2.25 X low5 ~\r; AMe, 7.5 X 10e6 M; and n-homocysteine, 9 x 10h6. The requirements for the enzymatic transfer of the methyl group of methyl-Bit to homocysteine were only the methyl donor, methyl-Blz, and the methyl acceptor, homocysteine. The optimal pH range was between pH 6.8 and 8.0. The K, for homocysteine was 2 x 10e4 M, while that for methyl-Blz was 5.2 X 10B4 M.

Ejects of Cobamides on Methyljolate-H4 Transjerase

Previous observations with extracts of chicken, rat, and hog liver indicated that addition of cyano-Bn and other cobamide compounds in vitro markedly stimulated the methylfolate-H4 transferase in an air but not in a nitrogen atmosphere (16, 26, 35). This was similar to the observations of Peel (37) on the cobamide stimulation of the COz-pyruvate exchange reaction. With the more purified enzyme preparation, the addition of cyano-B1z in vitro resulted in 50 to 100% stimulation in the reaction rate under anaerobic conditions (Table VII). This effect was only observed if /?-mercaptoethanol and either FMN or FAD were present in the incubation.4 Deoxyadenosyl-Bn, BJtr, methyl-Blz, Factor B, and Factor V1, all satisfied the co- bamide requirement under anaerobic conditions. Factor B had a maximal effect at lo-* M while cyano-Blz stimulated maximally at 10e5 M. Deoxyadenosyl-Blz, B12=, and methyl-Blz were essen- tially equivalent in effectiveness to cyano-Blz while Factor VI, behaved as Factor B. The effectiveness of a variety of cobamide derivatives, including those which lack a nucleotide, indicates that the stimulation in vitro is a nonspecific effect, such as re- ported by Peel (37), and is not related to a definitive coenzyme function. Highly purified intrinsic factor5 did not inhibit the methylfolate-Hd transferase reaction either when it was added directly or when it was preincubated with the partially purified enzyme.

Purification of 60Co-Cyanocobalamin-labeled Methyljolate-H4 Transjerase

Because a definite coenzymatic function of cyano-Blz or a BU derivative had not appeared with purification of the methyl- folate-H4 transferase, an alternative approach was undertaken to demonstrate the participation of a cobamide in methionine biosynthesis in chickens. Since the methylfolate-H4 transferase activity of chicken liver decreased during cyano-Blz deficiency and was partially restored with cyano-Blz replenishment in viva,

3 Recently, Buchanan et al. (26) have been able to satisfy par- tially the requirement for reduced FAD with fl-mercaptoethanol in the transferase from hog liver enzyme. Similar results have been obtained in E. coli 113.3W and type B (36, 30).

4 A paradoxical observation was that although flavin compounds stimulated the reaction in the presence of cobamide compounds (see Table VII), the reaction was inhibited by flavin compounds in the absence of cobamide compounds.

5 This fraction of intrinsic factor bound 4 pg of cyanocobalamin per mg of protein.

0 .2 .4 .6 .a 60 120

mg PROTEIN MINUTES

FIG. 1. A, effect of enzyme concentration on methylfolate-Ha transferase reaction. The incubations were performed for 60 minutes. B, effect of time on methylfolate-Ha transferase reac- tion. The source of enzyme was 180 fig of the third ammonium sulfate fraction, 0 to 66y0.

it was anticipated that 6aCo-cyano-Blz injected into deficient chicks would label the enzyme obtained from such animals, if a cobamide compound was attached to the enzyme. Sixteen chicks were maintained on a cyano-Blz-deficient diet, and, after a 3- week period, a total of 2.25 mpmoles of G°Co-labeled cyano-B,z (specific activity, 1.4 PC per mpmole) were injected per animal over a 4%hour period. Comparable animals which did not receive a cobamide supplement had a specific activity in the original supernatant fraction of 0.26 for the methylfolate-H4 transferase. The @Co-cyano-B12-treated animals had a specific activity for methylfolate-H4 transferase of 1.0, again showing the ability of cyano-Blz administration to restore the enzymatic activity in part (see Table II).

The liver supernatant fraction of G°Co-cyano-B1a-treated animals was purified according to the method described in “Experimental Procedure.” Table VIII records the distribution of radioactivity emitted by 6oCo-cyano-Blz, units of methyl- folate-H4 transferase, and the ratio of specific activities of these two components. Except for the 0 to 30% fraction of the first ammonium sulfate step, there was a definite correlation between units of enzyme activity and total radioactivity over the entire purification. The ratio of counts per minute of 60Co to number of enzyme units decreased abruptly with the first ammonium sulfate fractionation but then approach a constant value as would be expected if the G°Co-cobamide was attached to the enzyme. Further evidence in this respect is seen by the examination of the DEAE-cellulose (Table VIII, Step 6) fractionation (Fig. 2). A minor component of eluted 6oCo-cyano-B12 radioactivity was apparent in the pass-through and 0.01 M potassium phosphate wash. These fractions contained the bulk of the applied protein and a small fraction of the methylfolate-Hr transferase units. The bulk of 6OCo radioactivity emitted, and methylfolate-H4 and methyl-B12 transferase activities, were retained on the column and eluted identically with 0.01 M potassium phosphate, pH 7.4, containing 0.3 M sodium chloride. The peak tube represents a 9% recovery of original methylfolate-H4 transferase activity (30-fold purified) as well as 5% of the original supernatant GOCo-cyano-Blz. The achievement of a constant ratio of 60Co per enzyme unit as well as the retention of 6oCo-cyano-Blz through

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TABLE VIII Distribution of 6oCo radioactivity and methylfolate-Ha transferuse during purification

The nutritional experimental procedure and details of the GOCo-cyano-Bls administration are discussed in the text. The over-all purification was 30-fold.

Purification fraction

Supernatant*...........................................,..., First (NH,)&lOd fractionation

0 to 30%. :. 30 to 500/,*. 50 to 70%.

Calcium phosphate adsorption and elution Supernatant.............................................., 0.01 M phosphate buffer, pH 7.4, eluate . . 0.2 M phosphate buffer, pH 7.4, eluate*. 0.3 M phosphate buffer, pH 7.4, eluate

Second (NHd)*SOd fractionation 0 to 35%. 35 to 55%0*. . .

DEAE-cellulose, tube 15 (See Fig. 2).

* These fractions have been used for further purification.

I 6X104

1 I I I I , , ,

A

-

PASS THROUGH i i K-PO)

O.OlM K-PO,-NoCl K-PO,-N&l OOIM 0.M O.OlM 03M

2 4 6 8 10 12 14 16 IS

TUBE NUMBER

FIG. 2. DEAE-cellulose column chromatography of Wo-cyano- Blz, methylfolate-H4, and methyl-B12 transferase activities. Six milliliters of the second ammonium sulfate fraction, 35 to SO%, were applied to a DEAE-cellulose column (27 X 1 cm) and eluted as previously described in “Experimental Procedure.” Ten- milliliter fractions were collected. A, concentration of protein and counts per minute emitted by Wo per tube. B, units of methylfolate-Hc and methyl-B12 transferase activity per tube.

the purification suggests that a cobamide is attached to the apoenzyme in animal tissues. These results are similar to those obtained with the holoeneyme isolated from E. coli auxotrophs grown in the presence of cyano-Blz (38).

6TO

G.jLrn.

1.5 x 106

0.72 X 1Oj 6.5 X lo5 2.0 x 105

0.2 x 105 0.2 x 105 3.9 x 105 0.8 X lo5

0.2 x 105 2.5 X lo5 4.5 x 104

- Methylfolate-Hd

transferase

- I-

lotd unijs c.p.m.lenzyme wzil

4230 354

2090 34 3340 195 810 246

140 143 2580 150 1050 76

195 102 2520 102 400 101

Ratio

The above experiment was done with chickens on a cyano-Bn- deficient diet. It was felt that injection of GOCo-cyano-Blz into control chickens on a diet adequate in cyano-Blz would yield little, if any, significant activity in the protein since the enzyme sites would be expected to be saturated with the cobamide prosthetic group. Injection of G°Co-cyano-Blz into animals on a control diet did yield radioactivity in a protein fraction that was associated with the enzyme through a 40-fold purification. How- ever, with the chicks on an adequate cyano-Blz intake, the ratio of Wo radioactivity to number of activity units of the purified enzyme was only 16. This value was one-sixth of that obtained from the vitamin-deficient animals. The data suggest that even at dietary levels of 0.1 mg of cyano-Blz per kg of diet, the enzyme sites are not completely saturated with the cobamide prosthetic group.

DISCUSSION

Of the predicted functions of cyano-Blz in animal tissues, only its participation in the methylmalonyl-CoA isomerase reaction has been extensively elucidated by the demonstration of a de- crease in enzyme activity following vitamin deficiency, restora- tion of activity with vitamin replenishment in viva, purification of a cobamide-containing enzyme, removal of the cobamide prosthetic group in vitro, and restoration of activity in vitro by the addition of a cobamide, i.e. deoxyadenosyl-Blz (39-41).

The present study was undertaken to clarify in a similar manner the role of cyano-B12 in methionine biosynthesis in animal tissues. However, only three of the requisites which would indicate a cobamide participation have been fulfilled. These are the depletion of the methylfolate-H&ransferase concentra- tions in avian liver during cyano-Bn depletion, repletion of the specific activity with administration of cobamide derivatives 24 hours prior to death, and the intimate association of 6oCo-cyano- Blz radioactivity during purification of the methylfolate-Hr transferme. Further credence can be assigned to the participa- tion of a cobamide derivative in this reaction because of the structural specificity of the cobamides which were effective in replenishment. Factor B, which lacks the nucleotide moiety,

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August 1964 Dickerman, Redfield, Bieri, and Weissbach 2551

was essentially ineffective, while those which possessed the nucleotide, i.e. cyano-Blz, deoxyadenosyl-B,t, and methyl-Blp, were effective in restoration of liver enzyme specific activity. It should be stressed that the prosthetic group has not as yet been isolated from the holoenzyme in these animal studies and that the above data only suggest that a cobamide participates in the reactions. However, other points in favor of this view should be mentioned. The requirements of the reaction are essentially identical with those discussed for the cobamide-dependent reac- tion in E. coli, in contrast to the non-cobamide-dependent pathway that has been described (13). Also the ability to obtain methyl-B,, transfer is consistent with the observation seen in E. coli (19, 16).

Despite the failure to resolve the purified enzyme into a cobamide coenzyme and apoenzyme, the addition of a variety of cobamide compounds in vitro did stimulate the methyl transfer from methylfolate-H4 to homocysteine under aerobic and, to a lesser extent, anaerobic conditions. The characteristics of this effect, (a) the lack of cobamide specificity, (6) the decrease in the magnitude of the effect during anaerobiosis, (c) the requirement of /3-mercaptoethanol for the cobamide stimulation, and (d) the relative efficacy of Factors B and VI, at low concentrations, suggest that the stimulation is similar to the nonenaymatic catalysis of monothiol oxidation by cyano-Blz derivatives (42).

The relationship of the methyl-B,2 transferase activity to the methylfolate-H4 transferase is still in doubt. One major differ- ence between the two transferase activities was the suppression of methylfolate-H4 transferase levels by increased dietary methio- nine without a comparable effect on the levels of methyl-B,, transferase. On the other hand, the enzyme activities were not separated during purification although the methyl-B12 transferase activity was purified to a slightly greater extent. This appeared to be due to a greater lability of the methylfolate-H4 transferase. In addition, there was a comparable response of the two transfer- ase activities to cyano-Blz depletion and repletion.

The suppression of avian liver methylfolate-H4 transferase by increased dietary methionine would be expected to result in an accumulation of methylfolate-H4 in z&o. On the contrary, dietary methionine has been reported to lower the levels of methylfolate-Ha in rat liver with a corresponding increase in the concentration of formylated folate-H., compounds (43). It is difficult to reconcile these two findings. One possibility is that methionine might lead to a reduction in the synthesis of methyl- folate-H4, as well as a reduction in the methylfolate-H4 transfer- ase.

SUMMARY

1. Nutritional deficiency of 5,6-dimethylbenzimidazolyl- cobamide cyanide (cyano-Blz) in young chicks leads to a marked reduction of N5-methyltetrahydrofolate and 5,6-dimethyl- benzimidazolylcobamide methyl (methyl-B,,) transferase ac- tivities; replenishment with cyano-Brz, 5,6-dimethylbenzimida- zolylcobamide 5’-deoxyadenosyl (deoxyadenosyl-Blz) , and methyl-Blz resulted in partial restoration of both activities, while cobinamid hexa-amide (Factor B) was not effective.

2. Feeding of methionine supplements at levels of 6 g of methionine per kg of diet resulted in reduction of methyltetra- hydrofolate transferase but not of methyl-Blz transferase ac- tivity.

3. The methyltetrahydrofolate and methyl-B12 transferase activities of chicken liver have been partially purified and char-

acterized. The activities were not separated at this stage of purification. The purified methyltetrahydrofolate transferase required homocysteine and S-adenosylmethionine, and P-mercap- toethanol as a reducing compound. Methyl-Blz transferase required only the methyl group acceptor, homocysteine.

4. Cyano-Biz, deoxyadenosylLBl2, methyl-Blz, Factor B, cobyrinic acid a, b, c, d, e, g, hexamide (Factor V,,), and a re- duced form of cyano-Bia (B,,) all stimulated the methyltetra- hydrofolate transferase reaction aerobically and, to a lesser extent, anaerobically. The anaerobic cobamide stimulation was effective only in the presence of &mercaptoethanol and riboflavin phosphate “flavin mononucleotide” or flavin adenine dinucleotide. This is suggestive of the nonenzymatic catalysis by cobamides of the oxidation of thiols. A cobamide compound has not been isolated from the purified transferases.

5. ‘joCo-Cyanocobalamin, when injected into vitamin-Blz- deficient chicks, was incorporated into a protein fraction which was found during purification in the same fractions as methyl- tetrahydrofolate transferase during a 30-fold purification. In the nondepleted animal, there was evidence of ‘j@Co-cyano- cobalamin incorporation, but to a much lesser extent than in the replenished animals.

AcknowledgmentsThe authors are indebted to Mrs. Esther M. Hurley and Mr. Woodrow Duvall for assistance in the nutri- tional studies. In addition, Mr. Todd Wheeler provided able technical assistance during the course of this study. The authors wish to thank Dr. Anthony Mead for his generosity in helping to prepare the tetrahydrofolate.

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Herbert Dickerman, Betty G. Redfield, J. G. Bieri and Herbert Weissbach in Methionine Biosynthesis in Avian Liver12The Role of Vitamin B

1964, 239:2545-2552.J. Biol. Chem. 

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