Folic acid and vitamin B12: Transport and conversion to coenzyme forms

F O L I C A C I D A N D V I T A M I N B~2: T R A N S P O R T A N D C O N V E R S I O N TO COENZYME FORMS F. M. H ~ , P. M. DIGIROLAMO,K. Fum, G. B. HENDERSON,D. W. JACOBSn~,V. G. NF~F and J. I. RADn~* Department of Biochemistry, Scripps Clinic and Research Foundation, La Jolla, California 92037

I.

INTRODUCTION

TI~ nutritional and clinical importance of folic acid and vitamin B12 has been appreciated for several decades, but the molecular basis for this relationship is still not completely understood. A dietary insufficiency of these vitamins, or biochemical lesions which prevent their proper utilization, can produce a variety of disorders, particularly in the hematopoietic system (1). The extent of these physiological effects is remarkable considering that both vitamins are present at rather low levels (i.e., a few micrograms per gram) in mammalian tissues. However, by serving as components of enzyme systems which process large quantities of metabolites, coenzyme forms of folate and Bl2 function as biocatalysts and their effectiveness is amplified enormously. Because of these favorable logistics, folate- and B I 2-dependent enzymes would appear to be ideal targets for regulatory mechanisms in normal cells or for chemotherapeutic agents in malignant cells. Most, if not all, of the major folate-dependent enzymes have already been discovered. These enzymes utilize the coenzyme, tetrahydrofolate, to mediate the transfer of one-carbon (C t) units to and from various metabolites (e.g. thymidylate, inosinate, serine, methionine and histidine) whose importance to nucleic acid and protein synthesis is evident. The mechanism of these reactions is also well-understood: C~ groups occur at three different oxidation states (formate, formaldehyde and methanol) and they are linked to N 5 or N ~o (or both) of tetrahydrofolate (2). The individual reactions form a metabolic network (cf. Fig. 1 in ref. 3) which allows for the facile interconversion of the Cx units in the metabolltes. These interdigitated reactions are subject to various types of regulatory control (4). * Present address: Department of Biological Chemistry, University of Cincinnati, Cincinnuti, Ohio 45219. 131

132

F.M. HUENNEKENSet al.

Much less is known about B 12-dep endent enzymes, especially in mammalian cells. Only two enzymes, methylmalonyl CoA mutase and methionine synthetase, fit this category. It was thought that the mammalian ribonucleotide reductase might be a B 12-dependent enzyme like its counterpart in Laetobaeillus leichmannii, but present evidence indicates that it is probably more like the iron-dependent Escheriehia eoli enzyme (5). Recently, Barley et al. (6) have reported that glial cells grown in B t 2-deficient medium accumulate straight-chain C15 and C~7 fatty acids, and that the level of these fatty acids returns to normal upon addition of B 12. This important observation suggests that there may be an undiscovered B 12 enzyme concerned with the synthesis of complex lipids and that malfunction of this putative enzyme may be the basis for the symptoms of vitamin B x2 neuropathy (1). The paucity of information about the number of B ~2-dependent enzymes is matched by the uncertainty with regard to the nature and mechanism of action of the B ~2 coenzymes. Both adenosyl- and methyl-B t 2 (see Section II-A) fulfill the general criterion for coenzymes, i.e., they stimulate individual enzymatic reactions (7). However, the actual coenzyme that carries the mobile group may be a more reactive species such as B a 2r or B x2 s (reduced forms of B~ 2 in which the cobalt is assigned formal oxidation states of + 2 and + 1). In the methionine syntbetase-eatalyzed reaction, the mobile group is (CH3) +, but in the rearrangement of methylmalonyl CoA (or substrates for other isomerization reactions that are restricted to microbial systems) the mobile group is unknown. Since it is unlikely that the limited set of B~2-dependent enzymes will be integrated like the folate systems, the opportunities for regulation would appear to be more limited. It is known, however, that in E. coli mutants the level of the methionine synthetase is repressed when excess methionine is present (8). The B12-containing methionine synthetase, which provides the only known metabolic link between folate and B x2 (el. equation 1), may also contribute to the regulation of tetrahydrofolate-dependent systems in mammalian cells by generating the coenzyme as needed from the stable, storage form, 5-methyl tetrahydrofolate (9): 5-Methyl tetrahydrofolate + homocysteine B~2°.zymo_~ tetrahydrofolate +methionine

(1)

Enzymes that utilize folate and B12 coenzymes (step 3 in Fig. 1) are not, however, the only targets for regulatory processes or chemotherapeutic attack. Other possibilities include enzymes responsible for conversion of these vitamins to their coenzyme forms (step 2) and transport systems which allow entry of the vitamins into the cell (step 1). There is a good possibility that these consecutive processes may be linked and, therefore, be mutually regulatory. Since folate- and B I 2-dependent enzymes have been reviewed

FOLIC ACID AND VITAMIN B 12

133

extensively (e.g., refs. 2 and 7), the present discussion will be concerned only with steps 1 and 2 in Figure 1, viz., transport of these vitamins and conversion to their coenzyme forms. Most of the work described from this laboratory has been carried out with L1210 cells, although in some instances corollary information has been obtained from microbial systems.

FIG. 1. Targets for regulation and chemotherapeuticattack. (1)Coenzyme-mediated reactions; (2) Conversionof vitamin to coenzyrae;and (3) Transport of vitamin into cell. Abbreviations: V, vitamin; C, coenzyme;M and M', metabolites.

II.

A.

FOLIC ACID

Transport

Folio acid and its derivatives are essential nutrients for a variety of cells, and folate antagonists such as amethopterin (Methotrexate) have been used extensively in cancer chemotherapy. For these reasons, considerable attention has been devoted to the mechanism by which folate compounds are transported into cells. L1210 cells, maintained in culture or carried in the ascitic form in mice, have been utifized for many of these investigations. Cell suspensions are exposed to labeled substrates, and the rate and extent of uptake is followed as a function of time. Representative data for folate, 5-methyl tetrahydrofolate and amethopterin are shown in Figure 2. The continued uptake of the first two compounds (panels A and 13) after the rapid, initial phase is due to their metabolism in the cell. Folate is reduced via dihydrofolate reductase and 5-methyl tetrahydrofolate is probably metabolized via methionine synthetase. Conversely, the inability of L1210 cells to metabolize amethopterin is responsible for the plateau in its uptake curve (panel C). Although the initial rate of folate transport is comparable to the rates of the reduced folate and amethopterin, the abnormally high K~, for folate (Table 1) suggests that L1210 cells (and perhaps other extrahepatic cells) are not designed to utilize this substrate in vivo. The uptake of folate compounds is energy-dependent and carrier-mediated (10). Several lines of evidence suggest that there are actually two separate systems for the transport of folate compounds into L1210 ceils

134

F. M. HUENNEKENSet aL

(A) Substrate Competition. Reduced folates (e.g., 5-formyl and 5-methyltetrahydrofolate) and amethopterin are mutually inhibitory, but they do not inhibit the uptake of folate; conversely, folate does not inhibit the uptake of the reduced folates or amethopterin (11). Km and Ks values, obtained in several laboratories, for the uptake of folate compounds by L1210 cells are compiled in Table 1.

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FIG. 2. Transport of folate (A), 5-methyl tetrahydrofolate (B), and amethopterin (C) into L1210 cells. Uptake of folate compounds at 37° was measured in samples containing 5>
(B) Transport Mutants. Although most L1210 mutants selected for resistance to amethopterin are characterized by elevated levels ofdihydrofolate reductase, resistant sublines defective in transport of the drug are occasionally encountered. One of the latter, designated L1210/R3T, takes up amethopterin at an initial rate which is only 50% of that seen with the parental, amethopterin-sensitive cells (L1210/S). The R3T cells are equally defective (ca. 50%) in their ability to transport 5-methyl tetrahydrofolate, but they transport folate at the normal rate (R. C. Jackson, D. Nicthammer and F. M. Huennekens, in preparation).

135

FOLIC ACID AND VITAMINB12 TAme 1. K I N R r I C C O N g r A N T S F O R ~ T OF FOLATeCOt~OUNVSINTOL1210 O~Js

/G

Compound

Ref. /~M

100 200

Folate 5-Methyl tetrahydrofolate*

5-Formyl tetrahydrofolate*

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2 5 0.4

* Value for/,L-diastereoisomer. t K~value with respect to inhibitionof amethopterin uptake. Ks value with respect to inhibition of 5-formyl tetrahydrofolate uptake. § J. I. Rader, D. Niethammer and F. M. Huennekens, unpublished restflts.

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FIo. 3. Effect of p-chloromercuriphenyisuifonate (pCMS) upon transport of 5-methyl tetrahydrofolate (O) and amethoptedn ( 0 ) into LI210 cells. Uptake system as in Fig. 2. Concentrations of dl, L-5-methyl tetrahydrofolate and amethopterin were 4.2 and 1.9/ad, respectively; p-cldoromercuriphenylsulfonate at the indicated concentrations was added at time zero and uptake was measured after 10 mill. Results are expressed as pervert of control, i.e. uptake in the absence ofpCMS.

136

F.M. mJENNEKENSet al.

(C) Sensitivity to Sulfhydryl Inhibisors. p-Chloromercuriphenylsulfonate and other sulfhydryl reagents inhibit the transport of 5-methyl tetrahydrofolate and amethopterin, but have no effect upon the uptake of folate (13). More important, the reduced relate and the drug are both inhibited to exactly the same extent by various concentrations of p-chloromercuriphenylsulfonate (Fig. 3), which demonstrates that they must share the p-chloromercuriphenylsulfonate-sensitive transport system. p-Chloromercuriphenylsulfonate inhibits the influx of amethopterin with a Ks value of approximately 10/~M. Iodoacetate also inhibits this process, although it is considerably less efficient (K, -~ 2000 IZM). Iodoacetate also exerts a curious, and as yet unexplained, enhancement effect when it is added after amethopterin uptake has reached the steady-state level (Fig. 4).

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FIG. 4. Effect of iodoacetate (IAA) and p-chloromercuriphenylsulfonate (pCMS) (inset) upon steady-state level of amethopterin in L1210 cells. Uptake system as in Fig. 2. Concentration of amethopterin was 2.0 pM. Control (0). IAA (1.0 rnM)orpCMS (50/~) was added (as indicated by arrows) at 16 rain (©). Both azide (10) and vincristine (14) have been reported previously to produce the same effect. Since the steady-state level of intraceUular amethopterin is the result of two components (free and dihydrofolate reductase-bound), it would appear as if each agent is interacting with a metabolic system inside the cell, and that the result is an enlargement of one (or both) of these components. In contrast, p-chloromercuriphenylsulfonate affects the amethopterin plateau in a more conventional manner (inset, Fig. 4), in that it appears to be restricted to inactivation of the carrier-protein at the outer suNace of the cell. This would allow effux, but not influx, to proceed until all of the carrier molecules has been inactivated or the intracellular free amethopterin had been depleted. That the latter has probably occurred is

FOLIC ACID AND VITAMIN B 12

137

shown by the fact that the new plateau corresponds to about I nmole of amethopterin/10 9 cells, which is essentially the amount of dihydrofolate reductase present. Transport of folate compounds into L. easel proceeds by way of a mechanism which affords an interesting contrast to that employed by the mammalian ceils. Maximum uptake is obtained when the cells are harvested in late-log phase (15-19 hr); the transport ability declines as the ceils enter the stationary phase. Uptake curves for folate, 5-methyl tetrahydrofolate and amethopterin

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Time, rain FIG. 5. Transport of folate (A), 5-methyl tetrahydrofolate (B) and amethopterin (C) into L. casei cells. Uptake of folate compounds at 37° was measured in samples(6 x 10s celis/ml)

suspended in 0.05M potassium phosphate, pH 6.8, containing 10 mM glucose. Reactions were stopped by the addition of cold 0.15MKCI. Cells were collected and washed on a millipoxefilter (0.22/:) and the radioactivitydetermined using a liquid scintillationcounter. Concentrations of [glu-3H]folate, [14C] (dl, L)-5-methyl tetrahydrofolate and [Y, 5"-3H]amethopterin were 1/a~. in the bacterial system (Fig. 5) are similar to those shown previously for L1210 ceils (cf. Fig. 2), except that each substrate appears to reach a plateau. On a per cell basis, the rate and extent of uptake of these folate substrates is lower for the bacterial system. Folate transport into L. easel is energy-dependent and carrier-mediated In the absence of glucose, uptake of folate is reduced significantly. There is also a stimulation of transport by for 2-mercaptoethanol, particularly in ceils that have been stored at 5° for several days. Omitting the thiol in the assay system causes a 2- to 3-fold reduction in uptake. Cysteine or dithiothreito] (but not ascorbate) can substitute for mercaptoethanol. The thiol

138

F.M. m ~ r ~ , m ~ s et aL

must be present, however, during transport of the substrate; cells treated with mercaptoethanol show the same low activity after washing as untreated cells. K,, values for the transport of folate, 5-methyl tetrahydrofolate and amethopterin into L. casei cells are summarized in Table 2. These values (particularly for folate) are considerably lower than their counterparts in the L1210 system. Another major difference between these cells is that L. casei has only a single system for the transport of all folate compounds. This has been established by the following lines of evidence: (A) Substrate Competition. Folate, dihydrofolate, tetrahydrofolate, 5-formyl tetrahydrofolate, 5-methyl tetrahydrofolate, amethopterin and aminopterin competitively inhibit the influx of folate; K~ values are given in Table 2. TABLE2. Ka-NETIC CONSTANTS FOR TRANSPORT OF FOLATE COMPOUNDS INTO L. CASEI CELLS

Compound Folate Amethopter~ Aminopterin

Dihydrofolate Tetrahydrofolate 5-Methyl tetrahydrofolate 5-Formyl tetrahydrofolate Pteroie acid 2-Hydroxy-4-aminopteridine p-Aminobenzoylglutamate

Km

Ks

/2M

/ZM

0.35 0.21

0.90

0.38 0.30 2.0 0.093 0.28 0.68 0.36 > 200 > 200 > 200

Uptake system as described in Figure 5. Inhibition constants (Ks values) were determined in a system containing 1/a~ folate. Fragments of the relate molecule, e.g., pteroie acid, 2-hydroxy-4-aminopteridine and p-aminobenzoylglutamate, are poor inhibitors. The inhibition constants for 5-methyl tetrahydrofolate and amethopterin are essentially the same as the Km values obtained when these compounds serve as substrates. (B) Counter-transport of 5-Methyl Tetrahydrofolate. Folate, dihydrofolate, 5-formyl tetrahydrofolate and amethopterin promote the countertransport of 5-methyl tetrahydrofolate. (C) Stimulation by Mercaptoethanol. In a given lot of aged cells, mereaptoethanol produces the same degree of stimulation for the uptake of folate, 5-methyl tetrahydrofolate and amethopterin. (D) Inhibition by Iodoaeetate. The uptake of folate, 5-methyl tetrahydrofolate and amethopterin is inhibited to the same degree by varying con-

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ACID AND VrrA~nN B~2

139

centrafions of iodoacetate (Fig. 6); the K~ value for this process is about 1.5 mM. The general characteristics of the transport of folate compounds into mammalian and bacterialcellsare now reasonably well-established.However, an understanding of the detailed molecular mechanism by which these compounds actually traverse cell membranes will require: (A) A n identification of the energy-rich molecule(s) responsible for driving this process; (B) Knowledge of the manner in which energy is coupled to the translocation of the substratc; and (C) Isolation of the carrierprotein(s).Attempts to achieve the firsttwo objectives may be facilitatedby the abilityof L. casei cellsto be transformed into membrane vesiclesvia the general procedure of Kaback (I5).

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Fxo. 6. Effect of iodoacetate (IAA) upon transport of folate (O), 5-methyl tetrahydrofolate (O) and an~opterin (['-l) into L. casei cells. Uptake system as in Figure 5. Iodoacetate at the indicated concentrations was added to the cell suspension10 rain prior to introduction of the substrates and mercaptoethanol (20 raM). Uptake was measured after 10 rain; results are expressed as percent of control, i.e., uptake in the absence of IAA. Preliminary experiments h a w indicated that these vesicles are able to bind (but not transport) folate compounds; loss of the latter function is due, presumably, to some defect in the energy-coupling system. L. casei cells, because of their availability in large quantifies, are also the preferred source of starting material for isolation of carrier proteins. The most difficult aspect of this latter problem is to devise a reliable method for identifying a carrier protein once it has been detached from the membrane. The ability is a to bind labeled substrate (as measured, for example, by equilibrium dialysis) necessary but not sufficient criterion. A preferable method would be to fix a labeled folate molecule to the carrier prior to isolation. This might be accomplished if the folate compound also contained a reactive group (e.g., an azido stru~ure that could be photoactivated (16) or a nitrophenyl ester (17)) capable of formlng a covalent link with the protein.

140 B.

r.M. HUENNEKENSet al. Conversion to the Coenzyme (Tetrahydrofolate)

Folic acid is converted to its coenzyme form, 5,6,7,8-tetrahydrofolate, by the TPNH-linked dihydrofolate reductase (EC 1.5.1.3) (equation 2). The enzyme also reduces 7,8-dihydrofolate to tetrahydrofolate (equation 3), but at a much more rapid rate. In addition to this responsibility for the Folate + 2TPNH + 2H + Dihydrofolate + TPNH + H +

>tetrahydrofolate + 2TPN + >tetrahydrofolate + TPN +

(2) (3)

R = p-aminobenzoylglutamate N maintenance of the folate coenzyme level, dihydrofolate reductase has a special relationship with thymidylate synthetase. The reaction catalyzed by the latter enzyme (equation 4) involves both a C1 transfer and oxidation of 5,10-Methylene tetrahydrofolate + deoxyuridylate > dihydrofolate + thymidylate

(4)

the coenzyme to dihydrofolate. The cooperative action of the reductase is then required in order to regenerate tetrahydrofolate which, in turn, is the precursor of 5,10-methylene tetrahydrofolate. Dihydrofolate reductase appears to be the principal target of amethopterin, while the 5-methyl tetrahydrofolate transport system is probably a secondary site of action of the drug. K l for the interaction of most dihydrofolate reduetases with amethopterin is of the order of I0-9 M. The enzyme has been isolated from a variety of sources (2, 18). In addition to the conventional techniques of protein fractionation, affinity chromatography (using amethopterin linked eovalently to soluble or insoluble matrices) has been employed for this purpose (19). The enzyme from an amethopterin-resistant strain ofL. casei has been crystallized as a ternary complex containing equimolal amounts of TPNH and amethopterin (20). Dihydrofolate reductases have several interesting properties which are treated in detail elsewhere (2, 18): (A) The enzyme generally has a molecular weight of about 20,000, although both lower ( 15-16,000) and higher (28-30,000) molecular weights have been encountered (21). It appears to consist of a single polypeptide chain; no evidence has been obtained for the occurrence of subunit aggregates. (B) KMvalues of 10- ~ to 10- 7 M are found for interaction with the substrates (dihydrofolate and TPNH). (C) The catalytic activity of most reductases is increased several-fold by treatment with mercurials (or other agents

FOLIC A O D AI~D vrrA~N BI 2

141

that bind to, or oxidize sulfhydryl groups), chaotropic agents such as urea or guanidininm ion, and cations. The increased susceptibilityof the activated enzyme to proteolysis or heat denaturation suggests that activation involves an unfolding of the structure. Regulation of the conversion of folate to tetrahydrofolate is accomplished in two ways: (A) By the amount of dihydrofolate reductasc present; and (B) By factorswhich affectthe specificactivityof the enzyme. Radioimmunoassays to quantitate the amount of enzyme have yet to be developed, but this firstparameter can be calculated from the catalyticactivity(ifthe turnover number and molecular weight of the pure enzyme are known) or from a titration of the activity with amethoptedn (assuming a 1:1 stoichiometry) (22). The amount of enzyme present at any given time is a steady-statelevel resultingfrom synthesisand degradation of the protein. It has been observed repeatedly that,when cellsare treatedwith amethoptcrin, the levelof dihydrorelate reductase risesmarkedly in subsequent generations; this response may be an important factor in the development of resistanceto the drug. Bertino and Hillcoat (23) suggested that the increased enzyme level is due to the protection against intraceUular proteolysis afforded to the enzyme by the tightly-bound drug. Experimental support for this hypothesis was provided by Jackson and Huennekcns (24),who used 14C_leucine to label the enzyme in LI210 cells and showed that in the presence of amethopterin the halflifeof the enzyme was increased from 18 to 39 hr. A similar protective effect was observed when the L. casei enzyme was complexed with T P N H , i.e. the half-lifeof form II (see below) was 9 hr, as compared to 3 hr for form (I). Factors that affectthe catalyticactivityof dihydrofolate reductase provide a second way of regulating tetrahydrofolate synthesis.Although such factors have been sought extensively, the results have been essentially negative. N o physiological counterpart to the activation described above has yet been discovered. Mechanisms commonly encountered for regulation of enzyme activity (allostericeffects,subunit ~-- aggregate transitions,etc.)do not appear to be operative for this enzyme. Although the small molecular weight of this enzyme, its scnsitization by mercurials, and its sigmoidal kinetics with Hill coc~cients greater than unity all indicate that the 20,000 M W unit may be part of a larger structure, ultracentdfugation and gel filtrationof the enzyme, in the presence and absence of substrates (or inhibiters) and under various conditions of activation, have failed to reveal any aggregation. End-products of metabolic sequences involving the reductase, e.g., tetrahydrofolate, S-methyl tetrahydrofolate or thymidylate, do not appreciably repress the activityof the enzyme. Dihydrofolate reductases are susceptible,however, to a type of regulation which depends upon the absence or presence of T P N H . This phenomenon was discovered several years ago when it was observed that dihydrofolate reductase preparations from several sources showed multiple forms which

142

v.M. HUENNEKENSet al.

could be separated electrophoretically or chromatographically (25). The enzyme from L. casei, for example, is resolved into two distinct bands (Rs 0.54 and 0.84) when preparations are electrophoresed on polyacrylamide gel at pH 8.5 (26). The faster-moving band (form II)had an absorbance maximum at 340 nm and a visible fluorescence. Heat denaturation of II liberated TPNH in amounts approximately equimolal with the protein. Treatment of form I (the slower band) with an equimolal amount of TPNH prior to electrophoresis resulted in its conversion to II; conversely, II could be reconverted to I by treatment with dihydrofolate (27). The L. easel enzyme, however, did not form sufficiently stable binary complexes with TPN, dihydrofolate or amethopterin to allow their separation by electrophoresis. The multiple forms of the L. easel dihydrofolate reductase took on added importance when it was found that II had a much higher affinity for amethopterin than I (20). This was demonstrated by two different experiments: (A) Treatment of I with amethopterin did not produce an electrophoreticaUystable complex, while under the same conditions II was transformed into a faster-moving band (Rs = 0.93); the latter was shown to be a ternary complex containing equimolal amounts of enzyme, TPNH and drug. (13)Titration of I and II with dihydroaminopterin (which undergoes changes in its fluorescence upon binding to the protein) revealed that only the latter reacted appreciably with the drug. These results help to explain why amethopterin has a K~ value of 10-9 M for interaction with the enzyme under the usual assay conditions (TPNH present) and a K, value of 10- 6 M when it is used to titrate the fluorescence of tryptophan residues in the enzyme (21). Recently, a similar investigation has been undertaken with the dihydrofolate reductase from an amethopterin-resistant subline (R6) of cultured L1210 cells. This enzyme, isolated in homogeneous form, is able to form stable complexes with a number of substrates and inhibitors. As shown in Figure 7, bands corresponding to these binary complexes have been obtained when the enzyme (Rs = 0.21) is allowed to interact, prior to electrophoresis with dihydrofolate (Rs = 0.26), amethopterin (Rs = 0.26) and TPNH (Rs = 0.44). Although not shown in thisfigure, stable binarycomplexes can also be obtained with TPN (Rs = 0.40) or acid-modified TPNH (Rs = 0.51); the latter is the 290 nm-absorbing compound obtained by exposure of TPNH to pH 3 (28). Also, two catalytically-inactive, ternary complexes can be demonstrated by this technique, one with TPNH and amethopterin (Rs = 0.51) (Fig. 7) and the other with acid-modified TPNH and dihydrofolate (Rs = 0.60). The electrophoretic mobility of these complexes provides some interesting information about the structure of the protein. The small increase in mobility that accompanies the addition of dihydrofolate or amethopterin to the L1210 enzyme (el. gels 1-3 in Fig. 7), or addition of the drug to the enzymeTPNH complex (cf. gels 4 and 5), would be expected from the presence of two additional negative charges (i.e. from the carboxyl groups of the folate

Origin

Front FIG. 7. Gel electrophoresis of enzymesubstrate and -inhibitor complexes. Complexes were prepared by admixing L1210 dihydrofolate reductase with excess substrate or inhibitor at the indicated pH, followed by exhaustive dialysis. Experimental conditions for electrophoresis (at pH 8.5) have been described previously (26). Gels from left to right: (1) free enzyme Q; (2) E-dihydrofolate (pH 6.0); (3) Eamethopterin (MTX) (pH 6.0); (4) E-TPNH (PH 8.5); and (5) E-TPNHamethopterin (PH 8.5).

FOLIC ACID AND VITAMINBl2

143

molecule). A similar increase in mobility is observed when dihydrofolate reductases containing a single accessible sulfhydryl group are complexed withp-chloromercuribenzoate. When TPNH is added to the protein, however, the mobility of the complex is increased (of. gels 1 and 4) more than can be accounted for by the negative charges of the pyridine nucleotide. (This effect is even more striking with the L. casei enzyme--see Figure 5 in ref. 27.) It appears that binding of TPNH produces major conformational changes in the protein which expose otherwise buried negative groups. In this connection, it is of interest that Male and Kozloff (29) have postulated that interaction of the T-4 phage dihydrofolate reductase witl~TPNHproduces conformational changes in the taft plate enzyme which may be necessary for attachment of the phage to the host cell. These enzyme=substrate or enzyme=inhibitor complexes can also be characterized by means of their distinctive absorbance spectra. The stability of these complexes is evident from the fact that well-dialyzed preparations contain equimolal amounts of enzyme and substrate (or inhibitor). Figure 8A shows the spectra of the LI210 enzyme (1__ at 278 nm, shoulder at 290 nm), its dihydrofolate complex (increased absorbance below 300 nm and new shoulders in the 300--350 nm region) and its amethopterin complex (increased absorbance below 300 nm and new maxima at 350 and 370 nm). Similarly, the enzyme-TPNH complex (Fig. 8B) shows the characteristic maximum of the reduced pyridine nucleotide at 340 nm, and the enzyme= TPNH-amethopterin complex contains recognizable spectral features of all three components. The ability to create and manipulate multiple forms of dihydrofolate reductase in vitro raises questions about the occurrence and relative amounts of these forms in vivo, and their possible physiological significance. Amethopterin=resistant L. casei contains approximately equal amounts of forms I and II. L1210 cells also contain both forms, but the relative amounts vary with the degree of resistance to amethopterin (R. C. Jackson, D. Niethammer and F. M. Huennekens, in preparation). When L1210/R6 cells are grown in the presence of radioactive amethopterin, the faster-moving band seen on gel electrophoresis at RI = 0.S (inset, Fig. 9) is actually not form II, but is the complex of form II with amethopterin (Fig. 9). In chicken liver, form I of the enzyme predominates (25), but this may be due (o the lability of II during its isolation from the tissue and electrophoretic analysis. Multiple forms of the reductase from other tissues can often be seen in the elution profiles published in connection with purification procedures. However, the tightness of binding of the pyridine nucleotide to the particular enzyme and the procedures used for its purification may influence the apparent ratio of forms I and II. At present, there is no reliable way of determining this ratio in intact cells. One possible approach to this problem is to use amethopterin to "freeze" the TPNH onto form II.

F.M. HUENN~K~S et aL

144

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FIG. 8. Absorbanc¢ spectra ofcnzym¢-substrate and enzyme=inhibitorcomplexes. Complexes wcrc prcparod as described in Figurv 7. (A) Spectra of enzyme (~) and binary complcxe~ of enzyme with dihydrofolate (- - -) and amethopterin (. • .). (B) Spectra of enzyme (--), enzymc-TPNH complex ( - - - ) and the ternary complex of enzyme with TPNH and amethopterin (" • -).

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FIG. 9. In vivo formation o f enzyme-TPNH-amethopterin complex. L1210/R6 cells were grown in culture in the presence o f [3H]amethopterin. Inset, gel electrophoresis o f an a m m o n i u m sulfate preparation stained for protein (left panel) and enzyme activity (right panel) (26). Radioactivity measurements ( 0 - - - O ) were performed on each o f the gel slices.

Fox~c ACIDAND W T ~ N at2

145

The purpose of II in the cell is still obscure. As both TPNI-I and dihydrofolate (as well as the enzyme) exist in relatively low concentrations in the cell, there is little possibility of forming a ternary complex by simultaneous collision of the three components. However, ff the enzyme were to pre-exist as theTPNH complex, reaction of this complex with a dihydrofolate molecule would be kinetically more favorable. The amount of dihydrofolate reductase in cells is considerably less than the amount of TPNH, but of course the latter is used for many other purposes. At any given time, the TPNH:TPN ratio in the cell may determine the amount of form II present. Some evidence has been obtained which suggests that the ratio of forms I and H of the enzyme can be correlated with the transition of cells from amethopterin-sensitivity to amethopterin-resistance. Using the methods then available, Harding et al. (30) found that, as L1210 ceils/n vivo were challenged with amethopterin, the emerging population of resistant cells contained progressively larger amounts of form I. Similar results were obtained subsequently using L1210 cells maintained in culture (R. C. Jackson, D. Niethammer and F. M. Hueunekens, in preparation). Thus, it appears that the resistant cell is attempting to make use of the decreased affinity for amethopterin of the TPNH-free enzyme (form I) as a means of minimizing the toxic effects of the drug. III.

A.

VITAMIN B12

Transport

Transport of vitamln B12 and its derivatives (see section IH-B) has been studied by the same methodology that has been used for folate compounds. The amount of this vitamin taken up is extremely small, being measured in picomoles per 109 ceils. For this reason, 57Co.labeled substrates of high specific activity must be employed in order to obtain measurable values. Extensive studies which have been performed on B12 transport into E. coli (31) have revealed that the process is energy-dependent and carrier-mediated. Uptake consists of an initial rapid phase in which B12 is bound to a membrane receptor (ca. 200/cen), followed by a slower entry into the cell. The / ~ for B12 (cyanocobalamin) was found to be about 4nM. B12a (Ks = 0.3 riM), methyl cobalamin (Ks = 1.2 riM) and adenosyl cobalamin (Kl.~ 4.2 riM) were competitive inhibitors of B12 uptake and are themselves probably transported into the cells. Uptake of B12 into mammalian ceils requires, in addition to whatever transport components are present in the Ceil membrane, the presence of an external protein, transcobalamin-II (TC-II) (32). In this connection, it should be recalled that transport of this vitamin across the intestinal wall likewise requires an accessory protein, intrinsic factor. It is not yet clear P

146

F. M. H U E N N E K E N S

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whether these external carrier proteins are the means for identifying the material to be transported at the cell membrane, or whether they cover some structural feature on the B t 2 molecule which would otherwise interfere with transport. TC-II, which is present only at very low levels in serum and ascitic fluid, has been purified to homogeneity ( 2 x 106-fold) from h u m a n serum by Alien and Majerus (33). These investigators made use of an affinity chromato-

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FIG. 10. Transport of B12 into L1210 cells. Uptake at 37° was measured in samples containing 5 x l0 s cells/ml, suspended in phosphate-buffered saline, pH 7.4, supplemented with 1% glucose. A concentration of 30 pM vitamin Bx, was obtained by adding an appropriate quantity of human serum saturated with [s 7Co]]3t 2. Reactions were stopped at the indicated times by' addition of cold 0.9 % NaCI, cells were isolated by centrifugation and radioactivity was determined using a fiquid scintillation counter. The control sample contained n o SCrUlTL

graphy technique in which B t2 was linked covalently to Sepharose. The molecular weight was found to be 53,900 by ultracentrifugation and 60,000 by gel filtration. The Bt 2-aaYmity column technique has not been entirely satisfactory, however, owing to the difficulty in recovering transportactive TC-II, even when the adsorbent is treated with 7.5 M guanidinium hydrochloride. F o r this reason, our studies on Bx2 transport into L1210 cells have made use o f partially fractionated serum as the source of TC-II. Figure 10 illustrates the results of a representative experiment showing the

FOLIC ACID AND VITAMINB12

147

rate and extent of uptake of B i2 in the presence and absence of serum. Release of B12 can also be demonstrated by incubating pre-loaded cells in fresh medium devoid of TC-II (Fig. II). The labeled B12 recovered from the supernatant in rids experiment still appears to be bound to TC-II, as shown by its behavior during chromatography on ion-exchangers and gel filtration. Uptake of B~2 into L1210 cells occurs at about the same rate (ca. 0.3 picomole/min/109 cells) as it does in E. coli (31). The K, for Bz2 uptake in the mammalian cells is about 0.5 n~. In long-term experiments (48 hr uptake), Bi2 transported into these cells is converted to adenosyl-Bi, (36%) and methyl-B~2 (6%). In an attempt to devise inhibitors for the uptake of B~ 2, a series of analogs of the BI 2 coenzyme, adenosyl cobalamin, have been synthesized in which various nucleosides and alkyl groups provide

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the top axial ligand. Nucleosides used included: adenine arabinoside, cytosine arabinoside, homoadenosine, inosine, 6-mercaptopurine riboside, formycin, 8-adenosine and 2-fluoroadenosine. Alkyl groups included: --CH3, ---CCI2F and --CC1F2. Methyl and adenosyl cobinamides and diaquocobinamide (analogs of methyl and adenosyl cobalamin and Bt2a, respectively, in which the lower axial figand is water instead of dimethylbenzimidazole) were also prepared. When tested as inhibitors of TC-IImediated uptake of B12 by L1210 cells, all of the nucleoside and alkyl cobalamins were about equally effective (three representative results are shown in Fig. 12). In contrast, methyl cobinamide and adenosyl cobinamide (not shown in the figure) were relatively poor inhibitors, while diaquocobinamide was intermediate in its effect. It is likely that these analogs compete with Bl2 for the binding site on TC-H. Whether the analogs are actually transported into cells remains to be determined with labeled compounds.

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B.

Conversion to the Coenzymes (Adenosyl- and Methyl Cobalamin)

Cyanocobalamin, the commonly-used form of the vitamin, reacts slowly in both chemical and enzymatic systems. For laboratory use, it is converted to the more reactive B t 2, in which the cyano group has been replaced by a hydroxyl. The conversion of B12a to the two coenzyme forms, adenosyl cobalamin and methyl cobalamin, occurs via the enzymatic pathway outlined below:

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FOLIC ACIDAND VITAMINBx~_

149

B12 a is reduced first to BI2 r by a DPNH-dependent flavoprotein (fP)x, and B~ 2r is reduced further to B 12. by another DPNH-dependent flavoprotein

(fP)2 (34). An "adenosylating enzyme" then catalyzes a reaction between B12 , and ATP to yield adenosyl cobalamin 05). A comparable reaction in which adenosyl methionine (AM) 'supplies the methyl group is probably responsible for the synthesis of the other B12 coenzyme, methyl cobalamin. Bl2~ and B12r reductases, (fP)l and (fP)2, have been partially purified from bacterial sources. It is likely that the mechanism in each case involves the DPNH-dependent reduction of the flavin, and that the latter reacts nonenzymatically with the B12 moiety. Reduction of B12r to B120 is so highly endergonic (36) that it must be coupled to a second reaction. In the terminal

nPFH4] "" BI2,,d '"RS-m I I. . . . . . . . . . .

FIo, 13. Mechanism of methionine synthetase. Abbreviations: AM, S-adenosylmethionine; FH+, tetrahydrofolate; mSFH+, 5-methyl tetrahydrofolate; RSH, homocysteine; RS-m, methionine, R, FAD-containing flavoprotein; F, FMN-containing flavoprotein. Subscripts "o" and "r" refer to oxidized and reduced.

steps of B 12 coenzyme synthesis, the cleavage of ATP and the demethylation of adenosyl methionine provide the driving force. An interesting variant of this coupled reaction occurs during the operation of methionine synthetase which catalyzes reaction (1). As isolated from E. coli K-12, the synthetase itself contains one mole of B 12, per mole of protein (MW 186,000). Activation of the enzyme is carried out by TPNH acting via two flavoproteins (R and F) to reduce the B 12r t o B ~2 +, and the latter is methylated by adenosyl methionine (Fig. 13). R contains one mole of FAD per mole of protein (MW 27,000), while F contains one mole of FMN per mole of protein (MW 19,400). Once the B~2 has been fixed in the reduced state, however, it can function cyclically as a coenzyme transferring a methyl group from 5methyl tetrahydrofolate to homocysteine without the need of a reducing system or adenosyl methionine. Occasionally, unwanted oxidation of the

150

F. M. H U E N N E K E N S e t

aL

reduced coenzyme (dashed line in the figure) will again require these accessory systems (TPNH, R, F and AM) to reactivate the Bx 2 enzyme. It is probable that a similar set of reactions, i.e., reduction and adenosylation, occurs in mammalian cells for the conversion of vitamin B,2 to its coenzyme forms (37). However, the level of this conversion is so low that it has only been measured with the aid of labeled substrates. It is interesting that certain B12 deficiencies in man appear to result from a defect in the reductive steps or the adenosylation reaction (38, 39). This would suggest that this sequence might be under normal metabolic control, and that its inhibition might be useful in cancer chemotherapy. SUMMARY

Folate and B,2 coenzymes are involved in the biosynthesis of purines, pyrimidines and amino acids and thus play an important role in cell replication. Regulation of folate- and B 12-dePendent metabolism, and the possible exploitation of such regulation in cancer chemotherapy, can be achieved at several different levels: (a) transport of the vitamins into cells; (b) conversion of these vitamins to their coenzyme forms, and (c) reactions catalyzed by coenzyme-dependentsystems. Transport of relate compounds into mammalian cells is an energydependent, carrier-mediated process. Three separate lines of evidence (substrate competition, mutant sublines and sulfhydryl inhibitors) indicate that LI210 cells utilize two separate carrier systems for folate compounds. One is specific for folate, while the other is specific for 5-methyl tetrahydrofolate; the latter system also transports tetrahydrofolate, 5-formyl tetrahydrofolate (folinate) and amethopterin (Methotrexate). In contrast, ].~ctobacillus casei utilizes a single carrier system for all of the folate compounds. The intracellular conversion of folate to the eoenzyme, tetrahydrofolate, is mediated by the TPNH-dependent enzyme, dihydrofolate reductase. This enzyme is the target for folate antagonists such as amethopterin. Interaction with the drug, however, is markedly dependent upon prior binding of TPNH to the enzyme. Treatment of cells with amethopterin generally causes the level of dihydrofolate reductase to rise in subsequent generations. This has been attributed to a decreased rate of degradation of the enzyme (stabilized by complex formation with amethopterin) rather than to an increased rate of synthesis. Polyacrylamide gel electrophoresis and absorbance spectra have been used to study the interaction of dihydrofolate reductases from LI210 cells and L. co~ei with various substrates and inhibiters. Transport of vitamin B12 is an energy-dependent, carrier-mediated process. In mammalian cells transport requires, in addition to the membrane carrier system, an external protein, transcobalamin-II. Analogs of adenosyl

FOLIC ACID AND VITAMIN B12

151

cobalamin inhibit this process. Conversion of B12 to its coenzyme forms (adenosyl- and methyl cobalamin) is accomplished by the two-step reduction of B12a (Com) to B12r (Con) to B12s (CoI), followed by reaction of the latter with ATP or adenosyl methionine. A variant of this system has been encountered in the activation of the Bx2-contalning methionine synthetase by TPNH and two flavoproteins. ACKNOWLEDGEMENTS

This work was supported by grants from the American Cancer Society (BC-62) and the National Cancer Institute, National Institutes of Health (CA 6522). The authors are indebted to B. Bowen, P. Cramer, C. Lintner and D. Sampson for expert technical assistance, and to K. Vitols for aid in preparation of the manuscript. REFERENCES I. I. CHAN/acn~, The Megaloblastic Anaemias, Blackwell Scientific Publications, Oxford (1969). 2. F. M. HLm~m~Ns, Folate and B12 coenzymes, pp. 439-513 in Biological Oxidations (T. P. SmoEa, ed.), Interscience, New York (1968). 3. J. I. RAD~ and F. M. Hum~s]~sm, Folate coenzyme-mediated transfer of one-carbon groups, in The Enzymes, Vol. IX (P. D. BOYE~, ed.), Academic Press, New York, (1973) pp. 197-223. 4. R. Smart and A. MAtcsoup.x, Regulation of folate-dependent enzymes, Ann. N.Y. Acad. Sci. 186, 55-69 (1971). 5. R. L. B ~ and E. Vrrots, The control of nucleofide biosynthesis, Ann. Rev. Biochem. 37, 201-224 (1968). 6. F. W. BARLEY,G. H. S A ~ and R. H. Aeex~s, An effect of vitamin Bz2 deficiency in tissue culture, J. Biol. Chem. 247, 4270-4276 (1972). 7. H. A. B~dt~R, Corrinoid-dependent enzymic reactions, Ann. Rev. Biochem. 41, 55-90 (1972). 8. R. C. G m ~ s , R. D. Wn~tJ~s, H.-F. Ku~3, C. Sx~A~sand H. Wemse^cH, Effects of methionine and vitamin Bxa on the activities of methionine biosynthetic enzymes in met J mutants of Facherichio coli K-12, Arch. Biochem. Biophys. 158, 249-256 (1973). 9. V. HEReeRTand R. ZALUSKY,Interrelations of vitamin B~2 and relic acid metabolism: relic acid clearance studies,./. Clin. Investig. 41, 1263-1276 (1962). 10. I. D. GOLDMAN,The characteristics of the membrane transport of ametbopterin and the naturally occurring folates, Ann. N. Y. Acad. Sci. 186, 400-422 (1971). 11. A. N~mAs, P. F. N]xoN and J. R. BERTn~O,Uptake and metabolism of NS-formyltctrahydrofolate by L1210 leukemia cells, Cancer Res. 32,1416-1421 (1972). 12. F. M. S m O T ~ , S. KUmTA and D. J. H ~ N , On the nature of a transport alteration determining resistance to amethopterin in the L1210 leukemia, Cancer Res. 28, 75-80 (1968). 13. F. M. HUeNt~KmCS,J. I. RAD~, V. N ~ , F . OTTn~G,R. JACKSONand D. NI~'HAMMEi, Folate antagonists: transport and target site in leukemic cells, pp. 496-503 in Erythrocytes, Thrombocytes, Leukocytes (E. GERLACH,K. MOS~, E. ~ and W. WILM A ~ , eds.), Georg Thieme, Stuttgart (1973). 14. M.J. Prr~ and I. D. GOLDMAN,Characteristics of the vlncristine-induced augmentation of Methotrexate uptake in Ehrllch ascites tumor cells, J. Biol. Chem. 248, 5067-5073

(1973). 15. H. R, KAaACX,Bacterial membranes, Methods in Enzymology XXII, 99-120 (1971).

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16. H. KIE~ER, J. L~rDSTROM,E. S. LENNOXand S. J. Sn'~GER,Photo-affmity labeling of specific acetylcholine-binding sites on membranes, Prec. Natl. Acad. ScL 67, 1688-1694 (1970). 17. J. M. BECKER, M. WILCHEK and E. KATCHALSKI,Irreversible inhibition of biotin transport in yeast by biotinyl-p-nitrophenylester, Prec. Natl. Acad. Sci. 68, 2604-2607 (1971). 18. R. L. BLAKLEY, The Biochemistry of Folic Acid and Related Pteridines, Chapter 5, pp. 139-187, North-Holland, Amsterdam (1969). 19. J. M. WHrrEL~Y,Some aspects of the chemistry of the folate molecule, Ann. N.Y. Acad. Sci. 186, 29--41 (1971). 20. F. OffiNG and F. M. HtrE~,mrmNs, TPNH-dependent binding of amethopterin by dihydrofolate reductase from Lactobacillus casei, Arch. Biochem. Biophys. 152, 429-431 (1972). 21. F. M. HU'~N~KENS, R. B. DUNLAP, J. H. F I ~ I M , L. E. GU~ERSV~, N. G. L. HAnDn~O, S. A. LEWSONand G. P. MELL, Dihydrofolate reductases: structural and mechanistic aspects, Ann. N. Y. Acad. Sci. 186, 85--99 (1971). 22. W. C. W~RKH~S~R, Specific binding of 4-amino folic acid analogues by folic acid reductase, J. Biol. Chem. 236, 888-893 (1961). 23. J. R. BERTINOand B. L. HILLCOAT,Regulation of dihydrofolate reductase and other folate~requiring enzymes, Advances in Enzyme Regulation 6, 335-349 (1968). 24. R. C. JACKSONand F. M. Ht~NN~r~NS, Turnover of dihydrofolate reductase in rapidlydividing cells, Arch. Biochem. Biophys. 154, 192-198 (1973). 25. G. P. MELL, M. MARTELLI,J. KIRCHNERand F. M. HUENNEKENS,Multiple forms of dihydrofolate reductase, Biochem. Biophys. Res. Commun. 33, 74-79 (1968). 26. R. B. DUNLAP, L. E. GtrSVERSEN and F. M. HL~,~K~NS, Interconversion of the multiple forms of dihydrofolate reductase from amethopterin-resistant Lactobacillus casei, Biochem. Biophys. Res. Commun. 42, 772-777 (1971). 27. L. E. GUNVERSEN,R. B. DtmLAP, N. G. L. HJ~vr~G, J. H. l~g~.Sl-~nv~,F. OTrlNG and F. M. HUE~,rE~rS, Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei, Biochemistry 11, 1018-1023 (1972). 28. N. J. OPI"E~a-mlMER,The primary acid product of DPNH, Biochem. Biophys. Res. Commun. 50, 683-690 (1973). 29. C. J. MALEand L. M. KOZLO~T,Function of T4D structural dihydrofolate reductase in bacteriophage infection, J. Virol. 11, 840-847 (1973). 30. N. G. L. HARDING,M. F. MARTELLIand F. M. HUENNEKENS,Amethopterin-induced changes in the multiple forms of dihydrofolate reductase from L1210 cells, Arch. Biochem. Biophys. 137, 295-296 (1970). 31. J. C. WHITE,P. M. DIGIROLAMO,M. L. FU, Y. A. PROTONand C. BRADn~a%Transport of vitamin B12 in Escherichia coll. Location and properties of the initial B12-binding site. J. Biol. Chem. 248, 3978-3986 (1973). 32. A. E. FrNKLER and C. A. HALL, Nature of the relationship between vitamin B12 binding and cell uptake, Arch. Biochem. Biophys. 120, 79-85 (1967). 33. R. H. ALLEN and P. W. MAJERUS, Isolation of vitamin B~2-binding proteins using affinity chromatography. III. Purification and properties of human plasma transcobahmin-II, J. Biol. Chem. 247, 7709-7717 (1972). 34. G. A. WALKER,S. MURPHYand F. M. Htm~,mKENS,Enzymatic conversion of vitamin Bt2, to adeuosyl-Bl 2: evidence for the existence of two separate reducing systems, Arch. Biochem. Biophys. I34, 95-102 (1969). 35. E. VrrOLS, G. A. WALKERand F. M. HU~NN~NS, Enzymatic conversion of vitamin Bx2, to a cobamide coenzyme, ct-(5, 6-dimethylbenzirnidazolyl) deoxyadenosyl cobamide (adenosyl-Bx 2), J. Biol. Chem. 241, 1455-1461 (1966). 36. G. N. SCHRAUZER,J. W. SmERT and R.J. WINZ~ASS~N,The nucleophilicity of vitamin Bt2s, J. Am. Chem. Soc. 90, 2441-2442 (1968). 37. S. S. K~I~WAR,C. SP~_~,p.s,B. McAuSLAN and H. W~SSBACH,Studies on vitamin Bx2 metabolism in HeLa cells, Arch. Biochem. Biophys. 142, 231-237 (1971).

FOLIC ACID AND VITAMIN B12

153

38. S.H. MUDD, H. L. LEvYand R. H. A a ~ A derangement in B~2 metabolism leading to homocystinemia, cystathioninemia and methylmalonic aciduria, Biochem. Biophys. ICes. Commun. 35, 121-126 (1969). 39. L. E. Rosm,m~tG, A.-C. LH~_~QV~r, Y. E. I-IsL~and F. M. R ~ L O O M , Vitamin B12-dependent methylmalonicaciduria: defective B12 metabolism in cultured fibroblasts, Biochem. Biophys. Res. Commun. 37, 607-614 (1969).