Lactobacillus casei

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 251, No. 11, Issue of June 10, pp. 3405-3410, Printed in U.S.A.

Transport

1976

and Utilization

Lactobacillus

of Methyltetrahydrofolates

casei* (Received

BARRY

SHANE

by

AND

E.

for publication,

October

17, 1975)

L. R. STOKSTAD

From the Department of Nutritional

Sciences, University of California, Berkeley, California 94720

Since the discovery of pteroylheptaglutamates in yeast (l), it has been recognized that pteroyl-y-polyglutamates are the major, and sometimes only, intracellular forms of the vitamin (2-g). The recent introduction of specific chromatographic procedures (10-13) coupled with methods for the synthesis of synthetic polyglutamates for use as reference standards (14-16) has made possible the exact determination of folate forms in bacteria and animal tissues (11-13, 15, 17-20). In vitro studies have shown that pteroylpolyglutamates are often better substrates than the corresponding monoglutamates for folate-requiring enzymes (21-33). These folate conjugates are also better retained by tissues (34, 35) while pteroylmonoand diglutamates are the preferred transport forms of the vitamin (34, 36-38). In this study, we have assessed the in vivo effectiveness of pteroylpolyglutamates in Lactobacillus casei by comparing bacterial growth rates with transport rates. EXPERIMENTAL

PROCEDURE

Materials[3H]PteGlu, I labeled in positions 9, 3’, and 5’ (specific activity, 0.5 or 37 Ci/mmol) and (d&5- [“Clmethyl-H,PteGlu (specific activity, 54 mCi/mmol) were obtained from Amersham/Searle, [“Clformaldehyde (specific activity, 10 mCi/mmol) and N-t-butylox*This research was supported by United States Public Health Service Grant AM-08171 from the National Institutes of Health. One of us (B.S.) is indebted to the Wellcome Trust for a travel grant. ‘The abbreviations used are: PteGlu, pteroylglutamic acid, folic acid; PteGlu,, pteroylmonoto pteroyl oligo-y+glutamic acid, n indicating the number of glutamic acid residues; H,PteGlu,, 5,6-7,8tetrahydropteroylmonoto oligo-y+glutamic acid. The symbols (1) and (d) are used to denote the natural and unnatural diastereoisomers of H,PteGlu., respectively, due to the asymmetrical center at position C-6.

ycarbonyl-L[“C]glutamic acid-a-benzyl ester (specific activity, 57.2 mCi/mmol) from New England Nuclear, and [“Clformate (specific activity, 47 mCi/mmol) from Schwa&Mann. Folic acid y-polyglutamates PteGlu,.,, [SH]PteGlu,., (specific activity, 4 mCi/mmol), and PteGlu,[“C]Glu-Glu (specific activity, 100 to 500 &i/mmol) were synthesized by the method of Baugh et al. (14). (l)-5-Methyl-H,[sH]PteGlu,., (specific activity, 37 Ci/mmol) was prepared from [SH]PteGlu by the biosynthetic method of Buehring et al. (15). (dl)-HQteGlu,,, was prepared by catalytic hydrogenation of PteGlu,,, (39). (dl)-5,10-[“ClMethylene-H,PteGlu,, prepared by mixing [‘C]formaldehyde with (dl)-H,PteGlu, (40), was reduced to (dl)-5-[“C]methyl-H,PteGlu, with sodium borohydride (41). (dl)-5. Methyl-H,[8H]PteGlu was prepared in a similar fashion except [8H]PteGlu was reduced to (dl)-H,[3H]PteGlu with sodium borohydride (42, 43) as catalytic reduction led to almost complete exchange of the tritium label. (l)-10.[“C]Formyl-H,P~~GILI,,~, prepared by incubating (dl)-H,PteGlu,,, with [“Clformate and purified Clostridium lo-formyltetrahydrofolate synthetase (43), were separated from the unnatural (d)-H,PteGlu,,, diastereoisomers by chromatography on QAE (quaternary aminoethyl).Sephadex A-25 (44). (l)-5-[“C]MethylHJ’teGlu,,, were prepared by sodium borohydride reduction of (13.5,10- [“Clmethenyl-H,pteGl~~,,~ formed by acidification of (&lo[“Clformyl-H,PteGlu,,, (45). 10.Formyl-PteGlu was prepared by formylation of PteGlu (46). All folate compounds were purified by chromatography on QAE-Sephadex A-25 (44) and were stored in phosphate buffer, pH 7, containing 0.2 M mercaptoethanol at 196”. Prior to their use, they were chromatographed on Sephadex G-25 (10). The identity of each compound was unambiguously confirmed by its chromatographic behavior on Sephadex G-25 and by differential microbiologic response before and after hog kidney folyl-y-glutamyl carboxypeptidase (conjugase) treatment (15, 47, 48). Organisms and Growth Conditions-Lactobacillus casei (ATCC 7469) and a chloramphenicol-resistant strain (NCIB 10463) were cultured by the procedure of Bird et al. (48) with slight modifications employed in this laboratory (47). PteGlu (2.3 nM) was added to the media except when growth response to folates was being measured. Measurement ofFolate Uptake-Bacteria were harvested by centrifugation from growth media in late log phase (20 to 24 hours at 37”) and

3405

Downloaded from www.jbc.org by guest, on July 10, 2011

Transport of labeled 5-methyltetrahydrofolate (5.methyl-H,PteGlu) and 5-methyl-H,PteGlu, by Lactobacillus casei was via a carrier-mediated system. Both the natural (I) and unnatural (d) diastereoisomers were transported. Active transport was only demonstrated with the monoglutamate. Comparisons of growth rates with transport rates demonstrated that pteroylpolyglutamates were more effectively utilized by L. casei than the monoglutamate derivatives. The rate-limiting step in the utilization of higher polyglutamates was their transport into the cell, and in the case of the oxidized polyglutamates, their reduction. Utilization of monoglutamates was not limited by their transport but by metabolism, which also explains the different types of growth response curves observed between monoglutamates and higher polyglutamates. This phenomenon, sometimes called positive drift, prevents normalization of these growth curves. The rate-limiting step with the monoglutamates appears to be either the metabolism of 5-methyl-H,PteGlu or their conversion to polyglutamate forms.

Transport

3406

and Utilization

RESULTS

Uptake of Methyltetrahydrofolates by Lactobacillus casei-Kinetic data for methyltetrahydrofolate uptake are shown in Table I. The (d&5[“Clmethyl-H,PteGlu,, 5 diastereoisomers used in this study were 1:l mixtures of the (d) and (1) isomers. Excess purified &methyltetrahydrofolatehomocysteine methyltransferase from rat liver (23) transferred 50% of their “C label to methionine compared to a complete transfer with the (1) forms. As reported previously (34, 52), 5-methyl-H,PteGlu was actively transported by L. casei. Kinetic constants for the monoglutamate were assessed after 1 and 2 min of uptake. Chromatography of intracellular extracts on Sephadex G-25 (not shown) indicated that less than 5% of the transported vitamin was metabolized in this time period. Similar kinetic constants were found for the (I) isomer and the (dl) diastereoisomers indicating that both isomers were transported and at approximately the same rate. Transport of the unnatural (d) isomer was also demonstrated by the ability of the cells TABLE

Uptake

Methyltetrahydrofolates to remove over one-half the labeled vitamin from the medium. At higher cell concentrations (1 mg/ml), over 80% of the (dZ)-5-[“Clmethyl-H,PteGlu (initial concentration, 105 nM) was transported within 2 min. 5- [“C]Methyl-HQteGlu, uptake by L. casei also exhibited saturation kinetics but the uptake rate was considerably slower than with the monoglutamate (Table I). The (d) isomer was transported at a faster rate than the (I) isomer but demonstrated a lower affinity for the uptake system (Table I). At an extracellular concentration of 50 nM, (I)-5-[“ClmethylH&eGlu, was concentrated about 12-fold in 60 min by L. casei (initial uptake rate 0.19 min-‘) and the (dl) diastereoisomers about 17-fold (initial uptake rate 0.35 min-I). At high extracellular concentrations (50 PM), intracellular vitamin concentrations reached 18 and 45 /*M with the (1) and (dl) forms, respectively, after 60 min. The apparent intracellular concentration of low levels of the pentaglutamate conflicted with data we previously reported on the transport of (1)-5methyl-H,[SH]PteGlu, (34). The 3H-labeled compound (0.3 nM) was concentrated about 1.5.fold in 60 min, which could have been a result of intracellular binding, as its uptake was not inhibited by iodoacetate (10 mM). To investigate this apparent anomaly further, L. casei was incubated with 5 MM (1).5-[“Clmethyl-H,PteGlu, for 60 min, by which time the apparent intracellular 14C-labeled vitamin concentration had reached 22 pM, and the intracellular extract and medium were chromatographed on Sephadex G-25. The results, shown in Fig. 1, indicate that by 60 min, over one-half the “C label had I -7 8 I

L

- 250

To x

CELLS

5’ -200

a k!

I

parameters

for methyltetrahydrofolate transport in Lactobacillus casei Cells (0.1 mg/ml for monoglutamates, 0.2 mg/ml for pentaglutamates) were preincubated at 37” for 5 min in 50 mM K,HPO,/lOO mM sodium acetate/HJ’O, buffer, pH 6, containing glucose (1%) and mercaptoethanol (5 mM) before addition of 5. [“Clmethyltetrahydrofolates. Uptake was measured at 1, 2, 5, 10, 30, and 60 min. Folate

K,

+ S.E.

pMmmin-’

w

(I)-5-CH,-HQteGlu (do-5-CH,-HSteGlu (d)-5-CH,-H,PteGlu” (O-5.CH,-H,PteGlu, (do-5-CH,-H,F’teGlu, (d)-5-CH,-H,PteGlu,”

0.035 0.028 0.023

* +

0.005(5) 0.002(5)

1.69 + 0.31 (8) 2.75 7.4

V _~~ i S.E.

zt 0.62(S)

20.3 17.5 15.7 0.283

i

1.0(5)

*

0.3(5)

+

0.014(S)

1.09 *

0.08 (8)

4.6

“Kinetic constants for the (d) isomers are derived from those obtained with the (1) and (dl) isomers and assume that (d) and (1) isomers are transported by the same uptake system.

201\ A FRACTION

B

13op C D NUMBER

150

40

E (1.7ml

)

FIG. 1. Metabolism of (1).55[“Clmethyl-H,F’teGlu, by Lactobacillus casei. Cells (1 mg/ml) were preincubated at 37” for 5 min in 50 mM K,HPOJlOO mM sodium acetate/HzO, buffer, pH 6, containing 1% glucose and 5 mM mercaptoethanol before the addition of labeled vitamin (5 PM). After 60 min (intracellular “C-labeled vitamin concentration 22 PM), t,he cells were filtered, washed, and extracted with boiling 0.1 M phosphate buffer, pH 7, containing 0.2 M mercaptoethanol and a portion of the intracellular extract (equivalent to 2 mg of cells) and cell-free medium (2 ml) applied to Sephadex G-25 columns (200 x 0.7 cm). The columns were eluted with 0.1 M phosphate buffer, pH 7, containing 0.2 M mercaptoethanol and fractions were collected after the void volume (V,) as indicated by the elution position of blue dextran 2000. Standards used to calibrate the column were (I)-5-methyl-HQteGlu. (A), methionine (B), SH,O (C); S-adenosylmethionine (D), and 5-methyl-HQteGlu (E).


were washed and resuspended in 50 mM K,HPO,/lOO mM sodium acetate/HQO, buffer, pH 6, containing glucose (1%) and mercaptoethanol (5 mM). Transport of labeled folates was measured as described previously (34). Cell concentration (dry weight) was estimated by absorbance at 640 nm. Metabolism of Intracellular Folate--L. casei suspensions in acetate/ phosphate buffer, pH 6 (containing 1% glucose and 5 mM mercaptoethanol) were incubated with labeled methyltetrahydrofolates for various times, filtered (HA filters, Millipore Corp., 0.45 am pore size), washed with buffer, and cells plus filter were resuspended in 0.1 M potassium phosphate buffer, pH 7, containing 0.2 M mercaptoethanol. Intracellular folate was extracted by boiling for 5 min, and cell debris was removed by centrifugation. Mercaptoethanol (0.2 M) was also added to the cell-free medium. Identification of Labeled Compounds-Intracellular and extracellular extracts were chromatographed on Sephadex G-25 and DEAE-cellulose (DE52, Whatman) before and after conjugase treatment as previously described (15, 34). Various folate and non-folate standards were applied with the samples. The chromatographic behavior of folates on Sephadex G-25 and DEAE-cellulose has been described in detail by Shin et al. (10, 11, 15). Treatment of Kinetic Data--K, and V,,,.. values for folate transport were calculated by an unweighted nonlinear regression method (49) with six cycles of reiteration (50). Lineweaver-Burk plots were also drawn to verify Michaelis-Menten-type kinetics. V,,,., values are expressed as micromolar increase in intracellular vitamin concentration per min and assume an intracellular water volume of 4 ml/g (dry weight) of cells (51).

of

Transport

and Utilization

L. casei

Response

to Methyltetrahydrofolates-Fig.

2 shows

typical growth curves of L. casei with various folate standards. On a molar basis, PteGlu (Curve 2, Fig. 2) was slightly less active and (dl)-&methyl-H,PteGlu (not shown) was about 50% as active as (I)-5-methyl-HQteGlu (Curve 1, Fig. 2) in promoting growth. The growth curve with (l)-5-methylHJ’teGlu,, plotted on a semilogarithmic scale (Curue 3, Fig. 2B), showed a steeper slope than was found with the monoglutamates. This phenomenon, known as positive drift, has been reported for PteGlu polyglutamates (47) as well as naturally occurring folylpolyglutamates (53), and complicates the microbiological assay of total folates unless all folates are first converted to the monoglutamate form. A comparison of the effectiveness of various folates in promoting half-maximal growth of L. casei is shown in Table II. Also shown, for each folate, is a dose-response slope which is the relative increase in absorbance per relative increase in folate concentration at half-maximal growth, and which is a measure of the positive drift. The transport data reported here and previous studies with oxidized pteroylpolyglutamates (34) have shown that the polyglutamates do not break down to the monoglutamate derivatives with prolonged incubation. The growth experiments described above were repeated with a chloramphenicolresistant L. casei strain, which eliminated the need for autoclaving of the folate derivatives. This strain showed a poorer response to all folates than the parent strain. However, the relative growth-promoting abilities of the monoglutamates compared to the polyglutamates were identical with the parent strain, demonstrating that the results obtained with the parent strain were not due to breakdown of polyglutamates to monoglutamates during autoclaving. Theories advanced to explain the positive drift phenomenon include an increased permeability of the cell to polyglutamates with prolonged incubation (47) and the induction of a conjugase to break down the polyglutamates (53). Neither of these, *Manuscript

in preparation.

3407

Methyltetrahydrofolates

:

0.6

: u) 0,5 t =

0.4

v, g

0,3

0 4’

0,2

t 0

0.1 I NG

2 3 EQUIVALENTS

0.1 0.2 OF PteGlu

0.5 PER

I 2 TUBE

FIG. 2. Microbiological response of Lactobacillus casei to folates. Cell cultures were grown for 20 hours at 37” as described under “Experimental Procedure” in the presence of the indicated amounts of cl)-5-methvl-H,PteClu (Curue I), PteGlu (Curue 2). or t&5-methvlH,PteClu, (&rue 3). Cell growth was measured by absorbance at 840 nm and response curves were plotted in a linear-linear (A) or linear-log (B) fashion. TABLE

Relatioe

transport,

Folate

PteGlu, PteGlu, PteGlu, PteGlu, PteGlu. PteGlu, PteGlu, (I)-5CH,-HJ’teGlu (d&CH,-H,PteGlu (I)-5-CH,-H,PteGlu,

II

growth, and intracellular metabolism various folates by Lactobacillus casei Transport”

Growth0

100

100 100 100 66 20 3.5 2.4 129 64 29

31 9.6 1.4 0.32 0.067 0.048 114 123 0.033

DOS& response SlCp2 0.41 0.41 0.41 0.66 0.78 0.80 0.85 0.48 0.46 0.95

rates of

Metabolismd

3.2 10.4 47 63 52 50 1.13 0.52 879

“Relative

transport rates (PteGlu = 100) were calculated as as the folate concentrations required for growth were considerably lower than their K, values for transport. Transport data for PteGlu,., are from Shane and Stokstad (34). Values in italics were assessed from K, values (34) as K, values, in these cases, were not determined. *Relative growth is the reciprocal of the folate concentration required for half-maximal growth relative to PteGlu (= 100). Data for PteGlu,., are from Tamura et al. (47). ‘The dose-response slope is the relative increase in absorbance divided by the relative increase in folate concentration at half-maximal growth, i.e. if A = f(S), the dose-response slope is f’(S,,,)..S,,j Aaoso. Data for PteGlu,., are from Tamura et al. (47). d The relative metabolism rate is the relative growth rate divided by the relative transport rate.

V,,,.,IK,

however, are supported by the transport data for 5-methylH,PteGlu, shown here or for PteGlu, (34). A simpler explanation, that differences in growth response curves are due to differences in metabolism and transport of the various folates, has been overlooked. The growth curves for monoglutamates (Fig. 2) are approximately hyperbolic in nature and the dose-response slope for hyperbolic growth at half-maximum height is 0.5 (cf. Table II). If a linear relationship exists between growth and extracellular vitamin concentration, the dose-response slope would be 1.0 (cf. 5-methyl-H,PteGlu,, Fig. 2 and Table II). It can be seen that as the polyglutamate chain length increases, bacterial


been transferred to a variety of compounds. The major metabolite eluted from Sephadex at the same position as methionine. Some “C label eluted at the void volume which suggested incorporation into high molecular weight material. 5-Methyl-H,PteGlu, in the medium eluted as a symmetrical peak (Fig. 1) in a similar fashion to that observed with standard compound (not shown) while the major peak found with intracellular label was slightly askew suggesting the presence of another metabolite. A major labeled metabolite derived from 5- [“Clmethyl-HQteGlu elutes slightly later than the elution position of 5methyl-H,PteGlu,’ and its presence is indicated here. No labeled monoglutamate was detected (Fig. 1) and it was apparent that the pentaglutamate was transported without hydrolysis. The intracellular concentration of 5[“Clmethyl-H,PteGlu, was calculated to be 1.5- to 2-fold higher than in the medium, i.e. about the same as was found with 3H-labeled compound. In a similar manner, the apparent intracellular concentration of low levels of 5methyl-H,PteGlu, was shown to be due to the rapid metabolism of this compound with the consequent incorporation of the “C label into nonfolate compounds2 The elution positions of the “C-labeled metabolites were unaffected by conjugase treatment, and their elution positions after chromatography on DEAE-cellulose did not correspond to any folate monoglutamate standards. Thus, although 5methyllH,PteGlu, transport appears to be carriermediated, there was no evidence for an active accumulation.

of

Transport and Utilization of Methyltetrahydrofolates

3408

DISCUSSION

5-Methyl-H,PteGlu and 5-methyl-H,PteGlu, were transported via a carrier-mediated system in L. casei. Although the monoglutamate was actively transported, intracellular concentrations of the pentaglutamate only slightly exceeded that in the medium. Intracellular 5-methyl-H,PteGlu,, however, was rapidly metabolized with the incorporation of its l-carbon unit into a variety of non-folate compounds. Consequently, an intracellular concentration of radioactive label was observed with 5- [“Clmethyl-H,PteGlu, but not with 5-methylH,[3H]PteGlu, (34). Unexpectedly, (d)-5-methyl-HQteGlu, the unnatural diastereoisomer, was transported at about the same rate as its natural (1) isomer. However, no inhibition of bacterial growth was observed when (dl)-5-methyl-H,PteGlu was used as the folate source, presumably because the levels of folate required for maximal growth are considerably lower than their K, values for uptake. Previous studies have shown that pteroylmonoglutamates (34, 52) and oxidized polyglutamates (34) are transported without prior hydrolysis by a common uptake system in L. casei and do not break down to monoglutamates as L. casei does not possess a conjugase enzyme (15, 34). The transport system for folates in this organism appears to have a wide specificity for pteroylglutamates. All monoglutamates are transported at about the same rate while pteroylpolyglutamates demonstrate a progressive decrease in affinity with increasing glutamate chain length. Pterin, pteroic acid, and p-aminobenzoyl glutamate (52) as well as glutamic acid

and its dipeptides (34) have no affinity for the uptake system. Although pteroyl mono- and diglutamates are the preferred transport forms of the vitamin (34, 36-38), in practically every case investigated pteroylpolyglutamates have been shown to be the predominant, if not only, intracellular forms of the vitamin (2--g). In L. casei, the major forms found are octaglutamates if the organism is grown in the presence of low levels of folate (15, 54); if high levels of folate are supplied in the medium, lower chain length polyglutamates are found (12, 15, 34), predominantly the tetraglutamate derivative. High levels of folate also repress the transport-binding protein in L. cusei (55). Pteroylpolyglutamates were originally thought to be intracellular storage forms of the vitamin. However, in vitro studies have shown them to be equally as, or more, effective than monoglutamates as enzyme substrates. Examples include lo-formylH&eGlu synthetase (21), the B,,-dependent 5-methylH,PteGlu-homocysteine transmethylase (22-24), 5,10methylene-H,PteGlu reductase (23), thymidylate synthetase (25), H,PteGlu reductase (25, 26), and 5,10-methyleneHJ’teGlu-a-ketoisovalerate hydroxymethyltransferase (27). In addition, many microorganisms possess a 5-methylH&eGlu,-homocysteine transmethylase (28-33). This enzyme, which has not been detected in mammalian tissues (22, 23) and is B,,-independent, will not utilize 5-methyl-H,PteGlu or will utilize it extremely poorly. As pteroylpolyglutamates are the major intracellular forms of folate, they must be considered the natural substrates for these enzymes. In addition, pteroylpolyglutamates are effective inhibitors of thymidylate synthetase (25, 56) and 5,10-methylene-H,PteGlu reductase (23) which suggests a regulatory role for them in folate-dependent reactions. In order to assess the in oiuo effectiveness of the various folates, growth rates with the different compounds were compared with their transport rates. It has been reported that L. casei will not grow on folates with glutamate chain length greater than three. However, 5-methyl-H,PteGlu, was an effective growth promoter and growth with oxidized folates of chain length at least up to seven has been reported by Tamura et al. (47). The effectiveness of each folate for L. cusei growth was assessed by the amount of folate required for half-maximal growth, as these data were available in the literature for the oxidized pteroylpolyglutamates (47). This is arbitrary, as maximal growth is not a function of folate concentration. A more accurate assessment would have been to use the growth rate at low folate concentrations, i.e. where growth was proportional to the extracellular folate concentration. This would not affect the values for the higher polyglutamates but would increase the values slightly for the lower polyglutamates as they exhibit a hyperbolic type growth curve. At low folate levels, (l)-5-methyl-H,PteGlu, is about 20% as effective as PteGlu as a growth promoter while its relative effectiveness ii 29% at half-maximal growth. The values shown in Table II for the intracellular effectiveness of the different folates are only meant as a rough guide so comparisons of growth-promoting abilities at half-maximal growth will suffice. Growth with PteGlu, (n 2 4) and 5-methyl-H,PteGlu, was proportional to their transport rates and the oxidized polyglutamates were all about equally effective as active intracellular folates. Intracellular 5-methyl-H,PteGlu, was considerably more effective than the oxidized polyglutamates, suggesting that the reduction of these compounds as well as their transport was rate-limiting in their utilization. L. cusei H,PteGlu reductase is equally active with pteroylmonoand polyglutamates (25).


growth becomes more proportional to extracellular vitamin concentration. Under these circumstances, as the effective intracellular vitamin concentration is proportional to the transport rate, a measure of the effectiveness of the different folate compounds in intracellular metabolism can be assessed by the ratio of growth rate to transport rate (Table II). PteGlu,., have identical growth response curves (47) but are transported at different rates (Table II). With these compounds, their intracellular metabolism to “active” forms, rather than their transport, must be the limiting factor defining their growth-promoting abilities. This is also consistent with a “hyperbolic-type” growth curve. As (l)-5-methylH,PteGlu behaved in a similar fashion to PteGlu, the ratelimiting step in metabolism of these compounds does not appear to be the reduction of oxidized folates. Rather, the comparison of their effectiveness in intracellular metabolism would indicate that their metabolism to polyglutamates is rate-limiting and that monoglutamates are not nearly as effective as polyglutamates as enzyme cofactors or substrates. Bacterial growth with PteGlu, and higher polyglutamates becomes more linearly related to external vitamin concentration and their “effectiveness” in intracellular metabolism are approximately the same, implying that glutamate chain elongation is not necessary with these compounds for them to become active folates. (l)-5-Methyl-H,PteGlu, was slightly more effective than PteGlu, as a growth promoter but was transported at a considerably slower rate. Thus, its intracellular effectiveness was considerably greater than that of PteGlu,. It appears that with longer chain length polyglutamates, growth is limited by the reduction of PteGlu, as well as by its transport rate. This interpretation of the growth and transport data predicts that as the incubation time is increased, and monoglutamates are metabolized to polyglutamyl forms, the positive drift between polyglutamate and monoglutamate would decrease. This has been reported (47).

Transport

and Utilization

Acknowledgments-We would like to thank Joe Watson for his excellent technical assistance and Dr. J. Rabinowitz of the Department of Biochemistry for the gift of Clostridium lo-formyl-H,PteGlu synthetase. REFERENCES 1. Pfiffner, J. J., Calkins, D. G., Bloom, E. S., and O’Dell, B. L. (1946) J. Am. Chem. Sot. 68, 1392 2. Bohringer, R., Kim, W. K., and Samborski, D. J. (1969) Can. J. Biochem. 47, 1161-1169 3. Ericson, L. E., Widoff, E., and Banhidi, Z. G. (1953) Acta Chem. Stand. 7, 974-979 4. Hutchins, B. L., Stokstad, E. L. R., Bohonos, N., Sloane, N. H., and SubbaRow, Y. (1948) J. Am. Chem. Sot. 70, l-3 5. Sirotnak, F. M., Donati, G. J., and Hutchinson, D. J. (1963) J. Bacterial.

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8. Whitehead, V. M. (1971) Blood 38, 809 9. Rabinowitz, J. C., and Himes, R. H. (1960) Fed. Proc. 19,963%970 10. Shin, Y. S., Buehring, K. U., and Stokstad, E. L. R. (1972) J. Biol. Chem.

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C. M., and Scott, J. M. (1972) Biochem. Biophys. Res. 48, 1675-1681 Baugh, C. M., Stevens, J. C., and Krumdieck, C. L. (1970) Biochim. Biophys. Acta 212, 116-125 Buehring, K. U., Tamura, T., and Stokstad, E. L. R. (1974) J. Biol. Chem. 249, 1081-1089 Godwin, H. A., Rosenberg, I. H., Ferenz, C. R., Jacobs, P. M., and Meienhofer, J. (1972) J. Biol. Chem. 247, 2266-2271 Shin, Y. S., Buehring, K. U., and Stokstad, E. L. R. (1974) Arch. Biochem. Biophys. 163, 211-224 Leslie, G. I., and Baugh, C. M. (1974) Biochemistry 13,4957-4961 Brown, J. P., Davidson, G. E., and Scott, J. M. (1974) Biochim. Commun.

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21. Curthoys, N. P., and Rabinowitz, J. C. (1972) J. Biol. Chem. 247, 1965-1971 22. Coward, J. K., Chello, P. L., Cashmore, A. R., Parameswaran, K. N., DeAngelis, L. M., and Bertino, J. R. (1975) Biochemistry 14, 1548-1552 E. L. R. (1975) Can. J. 23. Cheng, F. W., Shane, B., and Stokstad, Biochem.

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Growth with PteGlu,., and 5-methyl-H,PteGlu was not related to their transport rates. L. casei will utilize all pteroylmonoglutamates about equally well for growth and metabolizes a large proportion of all these compounds to the 5methyl form (15). Intracellular metabolism of these compounds seems to be the rate-limiting step in their utilization which would explain their hyperbolic growth curves and, by extension, the positive drift phenomenon observed between mono- and polyglutamyl folates. The rate-limiting step in their utilization does not seem to be their reduction as PteGlu was as effective as 5-methylH ,PteGlu. The increased intracellular effectiveness of polyglutamates compared to monoglutamates suggests that the limiting step in the utilization of monoglutamates is their metabolism to polyglutamyl forms. Little is known about the specificity of the enzymes responsible for the formation of pteroylpolyglutamates. Sakami et al. (57) reported the presence of two enzymes in Neurospora crassa, one specific for the formation of pteroyldiglutamate and the other for synthesis of longer chain polyglutamates, and both specific for HzteGlu, derivatives. Escherichia coli contains a 10.formyl-H,PteGlu, synthetase (58) which will also utilize a variety of other pteroylmonoglutamates but not 5-methyl-H,PteGlu. In L. casei, H,PteGlu, (34) appears to be a substrate for the polyglutamate synthetase but not 5methyl-H,PteGlu.’ These data suggest that either the metabolism of 5-methyl-HQteGlu, with the consequent formation of other folate coenzymes which are potential substrates for the polyglutamate synthetase, or the polyglutamate synthetase reaction is the rate-limiting step in the metabolism of monoglutamates. It may be a general feature of folate metabolism that the metabolism of 5-methylHzteGlu is rate-limiting. Nixon et al. (59), in studies with murine lymphoma cells, showed that 5- [“ClmethylH,[SH]PteGlu was rapidly metabolized with the incorporation of the [“Clmethyl group into methionine. However, a large proportion of the 3H label in the cell was identified as 5-methyl-H,PteGlu, demonstrating that its resynthesis rate was faster than its metabolism rate.

3409

of Methyltetrahydrofolates

3410

Transport and Utilization of Methyltetrahydrofolates

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Proc. 32, 471 58. Masurekar, M., and Brown, G. M. (1975) Biochemistry 14, 2424-2430 59. Nixon, P. F., Slutsky, G., Nahas, A., and Bertino, J. R. (1973) J. Biol. Chem. 248, 5932-5936
