Transport and Metabolism of Folates by Bacteria*

Transport of labeled folic acid (PteGlu), pteroylpolyglutamates (PteGlu3-5), 5-methyl-tetrahydrofolate (5-methyl-H4PteGlu), and methotrexate in late-log phase cells of Lactobacillus casei was active, and subject to inhibition by unlabeled pteroylmonoglutamates, pteroylpolyglutamates, and iodoacetate, but not glutamate or glutamate dipeptides. Pteroylpolyglutamates were transported without prior hydrolysis and shared a common uptake system with pteroylmonoglutamates. The affinity and maximum velocity of PteGlun uptake decreased with increasing glutamate chin length (Km:PteGlu1, 0.03 mum; PteGlu3, 0.32 mum; PteGlu4, 1.9 mum; PteGlu5, 3.7 mum) and comparisons with growth response curves suggested that polyglutamates were more effectively utilized by L. casei, once transported, than monoglutamate. No concentration of 5-methyl-H4PteGlu3-8 inside the cells was observed. The major folate metabolites found in L. casei preloaded with high levels of [3H]PteGlu (0.5 mum) were 10-formyl-H4PteGlu2 and 10-formyl-PteGlu. Both compounds were released, the monoglutamate more rapidly. Pteroyltriglutamate formation appeared to be a rate-limiting step in intracellular metabolism. No 10-formyl-Pte-Glu was found in iodoacetate-treated cells and efflux was inhibited. Cells preloaded with low levels of [3H]PteGlu (7 nm) metabolized the vitamin to polyglutamate forms, the major derivatives being H4PteGlun. First order exit rates of labeled folate from preloaded L. casei indicated an inhibition of PteGlu uptake with time. Exit rates dropped from 0.05 min-1 to greater than 0.002 min-1 as intracellular folate was metabolized from monoglutamate to polyglutamate derivatives (n larger than or equal to 3). In the latter case, materials lost by efflux were breakdown products and no folate of glutamate chain length greater than two was released. Pediococcus cerevisiae actively transported 5-methyl-H4PteGlu but did not take up to 5-methyl-H4PTeGlu3-8. No active accumulation of 5-methyl-H4PteGlu was observed in Streptococcus faecalis.

PteGlus, 3.7 PM) and comparisons with growth response curves suggested that polyglutamates were more effectively utilized by Z. casei, once transported, than monoglutamate. No concentration of 5-methyl-HPteGlus.s inside the cells was observed.
The major folate metabolites found in L. casei preloaded with high levels of [3H]PteGlu (0.5 pM) were IO-formyl-HsteGluz and IO-formyl-PteGlu. Both compounds were released, the monoglutamate more rapidly. Pteroyltriglutamate formation appeared to be a rate-limiting step in intracellular metabolism. No IO-formyl-PteGlu was found in iodoacetate-treated cells and efflux was inhibited. Cells preloaded with low levels of [3H]PteGlu (7 m) metabolized the vitamin to polyglutamate forms, the major derivatives being HPteGlu,.
First order exit rates of labeled folate from preloaded L. cosei indicated an inhibition of PteGlu uptake with time. Exit rates dropped from 0.05 mm1 to <0.002 min+ as intracellular folate was metabolized from monoglutamate to polyglutamate derivatives (n 2 3). In the latter case, materials lost by efflux were breakdown products and no folate of glutamate chain length greater than two was released.
Since the discovery of pteroylheptaglutamates in yeast (2) (3), algae (4), bacteria (5, 6), and animal tissues (7-9). The recent introduction of specific chromatographic methods for determining polyglutamate chain length (10, 11) coupled with methods for the preparation of synthetic polyglutamates for use as reference standards (12, 13) has made possible the exact determination of polyglutamate forms in bacteria and animal tissues (11,13,14). For instance, the main folate forms in rat tissues, with the exception of plasma, have been shown to be pentaglutamates (11,14), while those in Lactobaci/lus casei and Streptococcus juecalis were the octa-and tetraglutamates, respectively (13).
With the exception of L. casei, which is able to grow on oxidized pteroylpolyglutamates of chain length at least up to seven (15), all folate-requiring bacteria so far investigated require mono-or diglutamates for growth (16). Intestinal absorption of folate is also limited to mono-and diglutamates (17) and human marrow cells will not transport triglutamates (18). From the limited studies carried out, the reason for intracellular metabolism of pteroyl compounds to polyglutamate forms would seem to be that these compounds have higher affinities for folate-dependent enzymes than the corresponding monoglutamates (19)(20)(21)(22)(23)(24)(25)). Another possibilit,y, suggested by lsuehring et al. (13), is that polyglutamates are better rctaincd by cells than nrc monoglutamate derivatives. They showed that folate in the growth medium of bacteria was apparently of shorter length than that found intracellularly.
To investigate some of these problems, uptake, efflux, and metabolism of various mow and polyglutamyl folate derivatives were studied in nonproliferating bacteria. Initially, uptake of 5-methyl-II~I'teGlu,i was studied in S. jueculis and Pediococcus ceretisiae as these organisms have been reported to actively trans- 5-Methyl-HaPteGlu was prepared by reduction of 5, IO-methenyl-HaPteGlu with sodium borohydride (28), HaPteGlu was preoared bv reduction of PteGlu in glacial acetic acid with sodium hithion;e (29), and 10.formyl-PteGlu was prepared by formylation of PteGlu (30).
The identity of each'compound was unambiguously confirmed by its chromatographic behavior on Sephadex G-25 and by differential microbiologic response after hog kidney conjugase treatment (13,15,31).
r-L-Glutamyl-L-glutamic acid was obtained from Sigma Chemical Co. and or-n-glutamyl-L-glutamic acid was obtained from Schwartz-Mann. In some experiments, S. juecalis medium contained thymidine (2 pgm per ml) instead of PteGlu.

Measurement of Folate
Uptake-Bacteria were harvested by centrifugation from growth media in late log phase (20 to 24 hours at 37"), washed twice with double-strength buffer, resuspended in buffer, and stored at O-4" during the course of an experiment until used. Unless indicated otherwise, the buffer used (single strength) was 50 mM KtIIPOd-100 mM sodium acetate-HaPOd, pH 6, containing glucose (lyO)). Cell concentration (dry weight) was estimated by absorbance at 640 nm.
Cells (0.02 to 1.0 mg per ml) were preincubated in a shaking water bath at 37" for 5 min before addition of labeled vitamin. Aliquots (0.5 or 1.0 ml) were removed at various intervals and filtered on HA filters (Millipore Corp., 25.mm diameter, 0.45~pm pore size). After washing with cold buffer (twice with 1 ml), cells plus filter were added directly to counting vials. Aquasol (New England Nuclear) or Triton X-lOO-toluene (1:2) scintillation mixture containing 2,5-diphenyloxazole (PPO) and 1,4-bis[2-(5phenyloxazolyl)]benzene (POPOP) (10 ml) was added to each vial. Counting efficiencies were determined by external standardization or by a channels ratio method and were about 80% for r4C and 25% for tritium. Eflux and Metabolism of Intracellular Folate-L.
casei (0.2 mg per ml) was preloaded with [H]PteGlu for various times, filtered, washed with buffer, resuspended in fresh buffer (2 volumes for exit rate studies; 0.5 volume for metabolic studies), and incubated with shaking at 37". In some experiments, resuspension buffer contained iodoacetate (20 mM) plus azide (50 mM) or unlabeled PteGlu (50 PM).
For exit rate studies, aliquots were removed at 2,4,6, and 8 min and filtered, and cell-free filt,rate (1 ml) was added to counting vials to determine released vitamin.
For metabolic studies, aliquots (5 ml) were removed at 5,20, and 60 min and filtered, and cells plus filter were resuspended in buffer (5ml) containingmercaptoethanol (0.2 M). Intracellular folate was extracted by boiling for 5 min and cell debris and filter were removed by centrifugation.
Mercaptoethanol (0.2 M) was also added to the cell-free medium.

Identification of Labeled
Folates-Folates were identified bv their ch"romatographic behavior on Sephadex G-25 and DEAEcellulose before and after hog kidney conjugase treatment (13).
The chromatographic behavior of folates on Sephadex G-25 has been described in detail bv Shin et al. (10. 11). Columns (200 X 0.75 cm) were eluted with 6.1 in potassium phosphate buffer; pH 7, containing 0.2 M mercaptoethanol. In general, polyglutamates could be separated according to size. The aromatic and heterocyclic moieties of the folate compounds caused a slower passage through the gel than would be expected by their molecular sizes (for instance, PteGlua was eluted later than glutamic acid). Ilowever, formyl derivatives were retarded less than PteGlu, HaPte-Glu, and 5-methyl-HaPteGlu.
This was also true for polygluta-mates of folic acid and for lo-formyl-HdPteGlu, which eluted approximately at the position of I'teGlu,+r.
[3H]Folate samples were chromatographed on Sephadex G-25 columns that. had been previously standardized with authentic folate standards and individual peaks were rerun on Sephadex together with appropriate r4C-labeled or unlabeled folate standards.
Further identification of the various folate derivatives was achieved by chromatography of [3H]folate samples, before and after hog kidney conjugase treatment, on DEAE-cellulose as described by Buehring et al. (13). Columns (25 X 0.9 cm) were eluted by an exponential phosphate gradient formed with 0.01 M potassium phosphate, pH 6.0 (100 ml), in a closed mixing chamber attached to a reservoir containing 0.6 h* potassium phosphate, pH 6.0. All buffers contained 0.2 M mercaptoethanol.
A standard solution containing unlabeled PteGlu and IO-formyl-PteGlu and 5-[14C]methyl-H,PteGlu was added to each [3H]folate sample before application to the column. The 5-[14C]methyl-H4PteGlu standard had been stored for several months at 4' and, under these conditions, about 50% had been converted to a degradative product of unknown composition, indicated by X in Figs. 6, 7, and 9. The elution position of this compound, which had no growth-promoting activity for L. casei (13),was useful in the interpretation of data from DEAE-cellulose chromatoaraahv. as IO-formvl-HdPte-Glu was elutcd midway between X and iO~formyl-PteGl"u.
-Treatment of Kinetic Data-K, and V,,, values for folate transport were calculated by the statistical method of Wilkinson (32). Estimates of K,, and V,,, were obtained by a least squares weighted treatment of the Lineweaver-Burk double reciprocal plot (33) and "best-fitting" the provisional values to a hyperbola. A similar treatment was applied to determine KI values for folate analogs. All data were inspected visually to ensure that they followed Michaelis-Menten-type kinetics. V,,, values were checked in inhibition studies to ensure that they were unaffected by the presence of inhibitors and that competition was competitive. V,,, values are expressed as micromolar per min increase in intracellular vitamin concentration and assume an intracellular water volume of 4 ml per g (dry weight) of cells (34).
As loss of intracellular vitamin followed first order kinetics, rate constants were determined by a least squares log-linear regression method.

5-dlethyl-H4PteGlu
Uptake by Streptococcus faecalis-Although intracellular levels of 5-[r4C]methyl-H4PteGlu slowly equilibrated with extracellular vitamin at 37", the only concentration effects noted could bc explained by intracellular breakdown of labeled vitamin to the degradative product X. These effects were noted in a variety of glucose-containing buffers (20 mM potassium phosphate, pH 6; acetate-phosphate buffer, pH 6), and prcincubation of cells in buffer, addition of excess unlabeled PteGlu (50 PM) or azide (50 mM) plus iodoacetate (10 mM), and culturing cells in thymidine instead of PteGlu did not significantly change this uptake pattern. Uptake rates were approximately proportional to substrate concentration over the range tested (0.01 to 5.0 PM) and were reduced slightly at 0". A slight pH effect on uptake with a maximum at pH 6.0 was noted, but as low levels of relatively low specific activity vitamin was being measured, it was difficult to distinguish whether this was a transport phenomenon or a nonspecific binding effect.
McElwee and Scott (26) suggested that S. juecalis concentrated 5-methyl-H~l'teGlu as Lactobacillus casei was unable to grow on used medium containing low levels of PteGlu and 5methyl-H4PteGlu in which S. fuecalis had previously been grown. As 5-methyl-H&eGlu will support growth of L. casei but not S. jaecalis, it appeared that S. juecalis had removed all of the 5-methyl-H41'teGlu from the medium. We were able to confirm these growth observations but, using 5-[14C]methyl-H41'teGlu, found practically all the radioactive label in the used medium. It appeared that the lack of growth by L. casei was due to the removal of a non-folate growth factor by S. juecalis or excretion of a growth inhibitor, as has been suggested for Pediococcus ceretisiae (35), rather than lack of folate. Uptake of Folates by P. cerewisiae-As S. jaecalis did not appear to actively transport 5-methyl-H&eGlu, uptake studies were carried out with P. ceretisiae, an organism that requires reduced folates, with the exception of 5-methyl-HJ'teGlu, for growth. Table I shows typical effects of time on the uptake of various mono-and polyglutamyl folates by P. cerevisiae. As shown by Mandelbaum-Shavit and Grossowicz (27), the organism will actively transport 5-methyl-H4PteGlu but will not concentrate PteGlu.
It was hoped that this organism could be used for transport studies on 5-methyl-H$teGlu polyglutamates, but only low levels of the Glus to Glus forms were taken up (Table I) and it is possible that these results reflect binding to the cells and filter rather than transport as such.
Uptake of Folate ~1Ionoglutamates by L. case%- Fig. 1 shows typical effects of time on the uptake of [3H]I'teGlu and 5-[i4C]methyl-H&eGlu by L. casei. Intracellular concentrations of greater than 2000.fold over that in the medium were found when low folate concentrations were used, and in the first 5 min at least, most of this transported vitamin had not been metabolized to different forms. The 5.[i"C]met.hyl-HJ'teGlu was a mixture of two diastereoisomers, of which only the (+)-form is biologically active. When heavier cell suspensions (0.5 to 1.0 mg per ml) were used, practically half of the radioactivity in the medium was taken up (Table II). Under comparable conditions, L. casei was unable to deplete the medium of [3H]I'teGlu, and an overshoot effect was often noted in intracellular tritium levels. Uptake of 5-methyl-HJ'teGlu and PteGlu was proportional to cell concentration up to 0.05 mg per ml (dry weight). Above this concentration, a falling off from linearity was observed.
[3H]Methotrexate was also concentrated by L. casei ( Fig. 2A). A rapid initial uptake or binding occurred within 60 s and was followed by a gradual accumulation of tritium label.

5-[i4C]Methyl-H4PteGlu
or [3H]PteGlu uptake by L. casei was similar in buffers containing sodium or potassium, phosphate, or acetate, and was reduced in citrate buffer. Uptake was stimulated by increased buffer concentrations, maximal rates were found with dipotassium phosphate ( 2 100 mM) or sodium acetate ( 2200 mM). This stimulation was most apparent when low substrate concentrations were used. A broad pH optimum between pH 5 and 6.5 with a peak at pH 5.5 was observed when uptake was measured in the presence of excess 5-[14C]methyl-H41'teGlu or [3H]PteGlu (0.5 PM). Uptake dropped off more rapidly outside this pH range. With lower folate levels (0.03 PM), similar effects were noted, with a more pronounced drop below pH 5 and above pH 6.5, suggesting a reduced affinity of the uptake system(s) for folates in these pH ranges. During growth of L. casei, the medium pH drops from 6 to about 5.2. It appears that this change in pH has little effect on the ability of the organism to transport folates.
Uptake of 5-dlethyl-[3H]H.,PteGlu Polyglutamates by L. casei-The uptake of 5-methyl-[3H]H&eG1u3-a by L. casei is shown in Table II. Although a slow accumulation of tritium label was observed, intracellular levels never reached greater than 50% more than extracellular levels. At these low levels of uptake, metabolism or binding could explain this small concentration effect. Iodoacetate (20 mM) had no apparent effect on uptake rates so there was no evidence for an active accumulation of these compounds. However, unlike the results obtained with P. cerevisiae (Table I), it was apparent that 5-methyl-H&eGlu polyglutamates were being taken up by the bacteria. Essentially identical results were obtained when these uptake experiments were repeated utilizing 5-methyl-H&eGlu polyglutamates synthesized on different occasions.
Uptake of [14C]PteGlu Polyglutamates by L. case&The uptake of [%]PteGlu3-5 by L. casei is shown in Fig. 2B. Relatively large amounts of the polyglutamates had to be used as well as high cell concentrations (1 mg per ml) in order to accurately measure the uptake of the extremely low specific activity material. Even under these conditions, significant accumulation and intracellular concentration of vitamin occurred. In contrast to PteGlu upt,ake, within 60 min most of the PteGlus-5 had been taken up by the cells. Uptake of these compounds was also subject to inhibition by iodoacetate and was reduced at 0".
Extracts Uptake of 5-CHs-H4PteGlu,, and PteGlu by Pediococcus cerevisiae Cells (1 mg per ml) were preincubated at 37" for 5 min in 50 mM K,HPOb-100 mM sodium acetate-H8P01 buffer, pH 6, containing 1% glucose before addition of labeled folate compounds. Uptake was measured at the indicated times.   Table III. In each case, uptake followed typical Michaelis-Menten-type kinetics. Initially, Pte-Glu, uptake was measured at pH 6, the pH of the growth medium. The values obtained at the optimum pH, 5.5, were practically identical. It can be seen that increasing the glutamate chain length leads to a decrease in aflinity for the transport system and a lowering of maximal uptake rates. A comparison of these transport parameters with growth data for PteGlu, obtained previously by Tamura et al. (15), and making the assumption that growth of the bacteria was purely a function of intracellular vitamin at suboptimal folate concentrations, suggests that, once transported, the longer chain polyglutamates are better utilized by L. casei than is PteGlu.
(zt)-5-Methyl-HJPteGlu demonstrated a similar affinity for the uptake system as PteGlu (Table III) stereoisomers and the true affinity of the active (+)-stereoisomer would be about 14 nM, assuming no competition from the biologically inactive (-)-form. Of course, if the (-)-stereoisomer is a competitive inhibitor of ( +)-5-methyl-H4PteGlu. uptake, and has a similar affinity for the uptake system but is not transported, then the true K, for (+)-5-methyl-H4PteGlu uptake would be 27 nM and its V,,,,, value would be about 47 PM +min-1.
It can be seen that ionic (or buffer) strength, as noted earlier with 5-methyl-HJ'teGlu and PteGlu transport, affects the affinities for the transport system(s) but not maximal uptake rates (Table III).
An apparent K, value of approximately 1 PM was obtained statistically for [3H]methotrexate uptake ( Fig. 2A) (Table III), which suggests that all the compounds tested are transported by the same system. The slight difference between K, and Kr values for PteGlu, are easily explained by the high cell concentrations needed to measure uptake of these com-L-Glutamic acid, cr-r-glutamyl-L-glutamic acid, and y-n-glutamyl-L-glutamic acid (100 pM) had no effect on 5-[i4C]methyl-H41'teGlu or [3H]1%eGlu transport.

E&z of Intracellular
Vitamin-The efflux of labeled vitamin was studied with L. casei that had been preloaded for various times wi .h [3H]l%eG1u. Exit followed first order kinetics, and rate constants are shown in Table V. With cells that had been preloaded for 5 min with relatively large amounts of [3H]I%eGlu (0.5 PM), considerable reuptake of vitamin lost by efflux took place, as evidenced by the increased exit rate when uptake was  Cells (0.02 mg per ml for monoglutamates, 1.0 mg per ml for polyglutamates) were preincubated at 37" for 5 min in 50 mM K~HPOI-100 mM sodium acetate-HaPOd buffer containing 1% glucose at the indicated pH before addition of labeled folate. Uptake was measured at 1, 2, 5, and 10 min. V,,, was calculated from 1 min uptake data.  7) 14.0 f 0.9 (7) 23.1 f 0.9 (7) 23.7 zt 0.7 (7)  inhibited by excess unlabeled PteGlu. Exit rates were very low in the presence of metabolic inhibitors. Reloading cells for 60 min with [3H]PteGlu (0.5 C(M) did not significantly affect the exit rate of labeled vitamin when reuptake was inhibited by excess unlabeled PteGlu, but the net exit rate in buffer alone was increased. It appeared that influx of vitamin was decreased compared to cells that had been preloaded for 5 min and suggests some metabolic control over the transport of PteGlu. This would also explain the overshoot effect noted earlier with PteGlu uptake.
Cells preloaded with low levels of [3H]PteGlu (7 nM) for 5 min exhibited a similar exit rate to that found with 0.5 PM PteGluloaded cells; so it was apparent that the exit system was not saturated under the conditions of these experiments. With longer preloading times with 7 pM [3H]1%eGlu, and consequent intracellular metabolism to polyglutamate forms, the exit rate dropped to very low levels. This was not a result of increased reuptake, as excess unlabeled PteGlu had little effect on these rates. Metabolic inhibitors increased the exit rates under these conditions but the rates were still very low. It should be noted that the intracellular vitamin concentration was similar irrespective of preloading time, so the change in exit rate must have been solely a function of folate form.
ilfetabolism rnM sodium acetate-HaP04 buffer, pH 6, containing 1% glucose were preloaded at 37" with [3H]PteGlu (0.5 FM; specific activity: 0.5 Ci per mmol). After 5 min, the cells were filtered, a portion extracted with boiling buffer, and intracellular extract (equivalent to 0.4 mg cells) and cell-free medium (1 ml) was applied to Sephadex G-25 columns min after resuspension in fresh media. Exit rates were checked to ensure that they corresponded to the rates shown in Table V.
The Sephadex G-25 elution patterns of cell extract and medium after a 5-min preloading with [3H]PteGlu (0.5 pM) are shown in cells incubated in buffer alone had metabolized I'teGlu to several compounds, including some polyglutamates eluting before standard PteGluo. One mctabolite (Peak A in Fig. 4B) eluted just after standard PteGlua at the approximate position of a loformyl-PteGlu2 derivative, while another (Peak B in Fig. 4B) eluted at the approximate position of a IO-formyl-PteGlu derivative. A similar elution pattern was seen in cells suspended in buffer containing excess unlabeled PteGlu although less labeled vitamin was retained by these cells as reuptake was inhibited (Fig. 4B). Conversely, cells resuspended in buffer plus iodoacetate contained higher levels of labeled vitamin as efflux was inhibited (Fig. 4B). In this case, a large part of the intracellular vitamin was metabolized to the compound resembling a loformyl-PteGluz derivative (Peak A in Fig. 4B) and a smaller polyglutamate fraction was observed. The Sephadex G-25 elution patterns of tritium in the media after resuspending the cells for 60 min are shown in on DEAE-cellulose as described in the legend to Fig. 7. A, the elution pattern obtained with folate derivative from the medium of cells suspended in buffer alone (Peak A, Fig. 5C); B, the pattern with intracellular derivative from iodoacetatetreated cells (Peak A, Fig. 5B).
unlabeled PteGlu was, as expected, mainly in the monoglutamate form (Peak D) plus a compound with the chromatographic properties of a 10.formyl-PteGlu derivative (Peak B in Fig. 4C). Comparison of this elution pattern with the pattern obtained after a 5-min resuspension of cells in buffer containing excess unlabeled PteGlu (not shown) indicated that practically all of Peak B was released in the first 5 min. i\Iost of the intracellular folate released from the cells after a 60.min resuspension in buffer containing metabolic inhibitors eluted at the position of the loformyl-PteGluz derivative (Peak A in Fig. 4C). Fig. 5, A to C, shows comparable results for cells that had been preloaded for 60 min with [W]l'teGlu (0.5 PM) before resuspension in fresh buffer for 60 min. After preloading, the intracellular vitamin was mainly in the monoglutamate form (Fig. 5A) as was found after a 5-min preloading (Fig. 4A). However, resuspension of the cells led to further metabolism of intracellular vitamin (Fig. 5B). The lo-formyl-PteGlut and lo-formyl-1'teGlu derivatives built up (Peaks A and B, Fig. 5B) as well as a compound with the elution position of a PteGluz derivative (Peak C). A similar pattern was found with cells resuspended in buffer containing excess unlabeled PteGlu while most of the intracellular vitamin in iodoacetate-treated cells eluted at the position of a IO-formyl-PteGluz derivative (Peak A, Fig. 5B). The Sephadex G-25 elution patterns of label in the media of cells incubated in buffer alone or buffer plus excess unlabeled 1'teGlu ( Fig. 5C) showed that the main released forms after 60 min were lo-formyl-PteGlu and PteGluz derivatives (Peaks B and C) Fig. 5C) was applied to a DEAE-cellulose column (25 X 0.9 cm). The column was eluted by an exponential phosphate gradient formed with 0.01 M potassium phosphate buffer, pH 6, (100 ml) in a closed mixing chamber attached to a reservoir of 0.6 M potassium phosphate buffer, pH 6. The elution positions of reference compounds, applied with the [tH]folate sample, are indicated beneath the abscissae.
(Peak B) appeared to be complete by 5 min. No lo-formyl-PteGlu derivative was found in the medium of iodoacetatetreated cells (Fig. 5C), only a buildup of the IO-formyl-PteGluz derivative (Peak A) plus some monoglutamate.
The identity of the 10.formyl-PteGluz derivative (Peak A, Fig. 4, B and C; Fig. 5, B and C) was confirmed by DEAE-cellulose chromatography.
The folate eluted as a single peak after conjugase treatment at the position of lo-formyl-HdPteGlu ( Fig.  6) indicating it to be 10.formyl-HIPteGlut. Similarly, the loformyl-PteGlu derivative (Peak B, Figs. 4C and 5, B and C) was identified to be 10.formyl-PteGlu (Fig. 7). It was apparent that only a small amount of polyglutamate of chain length greater than two was formed under the described conditions and it appeared that triglutamate synthesis was a rate-limiting step in metabolism of PteGlu. It also appeared that iodoacetate inhibited the synthesis of polyglutamates.
In order to study the further metabolism of PteGlu and to study efflux of higher polyglutamates, cells were preloaded for 2 or 4 hours with low levels of [3H]PteGlu (7 nM). Although some variation in polyglutamate composition was found under these conditions, the results shown in Fig. 8, A to C, are typical of the effects noted.
The Sephadex G-25 elution pattern of intracellular labeled vitamin after preloading with [3H]PteGlu (7 nM) for 120 min (Fig. 8A) showed that most of the vitamin consisted of polyglutamates with chain length 23. Folate in the medium was of shorter glutamate chain length (Fig. 8A) with considerable amounts of mono-and diglutamate forms. Resuspension of these cells for 60 min in buffer alone led to further chain elongation of intracellular folate (Fig. 8B) and practically no net release of labeled vitamin (Fig. 8C). With cells incubated in buffer containing iodoacetate, a small release of mono-and diglutamates was observed as well as a folate which eluted just before standard PteGlua from Sephadex G-25 (Fig. 8C). There was less apparent intracellular chain elongation with iodoacetate-treated cells (Fig.  8B) and in some experiments no chain elongation was observed. Intracellular labeled folate from cells suspended in buffer alone for 60 min was treated with conjugase and chromatographed on DEAE-cellulose (Fig. 9A) Fig. 9A, 72%) with some loformyl-HdPteGlu (Peak B, 13%), 5-methyl-HdPteGlu (Peak C, lo%), and a trace of PteGlu (5%). Material eluted straight through the column (Peak A, Fig. 9) represented breakdown products of unknown composition. These breakdown products were always observed when high specific activity (37 Ci per mmol) PteGlu was used (13). Iodoacetate-treated cells, after conjugase treatment, also contained 10.formyl-HIPteGlu (Peak B, Fig. 9B, 19%), 5-methyl-HJ'teGlu (Peak C, Fig. 9B, 6%), HJ'teGlu (Peak D, Fig. 9B, 19%), as well as breakdown products (Peak A, Fig. 9B). However, most of the radioactive label eluted from DEAE-cellulose at the position of X (54oj,), the 5-methyl-H&eGlu breakdown product (Fig. 9B). The material eluted at Peak X was rechromatographed on Sephadex G-25 (Fig. 10) and, although it had been treated with conjugase, still eluted just prior to standard PteGlus. It was apparent from this that the chain lengths of polyglutamates found in iodoacetate-treated cells (Fig. 8B) were lower than their elution positions from Sephadex G-25 suggested.
The small amounts of labeled vitamin released from cells SUS-A : .
known folate forms, and appeared to be breakdown products.
2 5 c D 5-Methyl-HaPteGlu and PteGlu uptake by nonproliferating A cells of Lactobacillus casei possessed characteristics of a carriermediated active process (34,36,37). Vitamin was taken up and retained against a concentration gradient and the transport process was dependent on energy, pH, temperature, ionic effects, 6 t I and displayed structural specificity and saturation kinetics.
peaks were found, equivalent to the two peaks of Fraction A in Fig. 9. The reason for this conjugase effect was unclear. However, none of the radioactive label released from cells containing polyglutamates of chain length 2 3 corresponded to any of the polyglutamates were transported without prior hydrolysis. Although Cooper (38) suggested that PteGlu and 5-methyl-HdPteGlu were transported by separate uptake systems in L. casei, the similarities we found between K, values for uptake and K, values measured against both [3H]l'teGlu and 5-[i4C]methyl-HJ'teGlu uptake suggests that all folates tested were transported by the same system. The K, value of 0.03 PM for PteGlu uptake was similar to that reported by Cooper (38) (0.045 PM). The only qualitative difference between uptake of PteGlu compared to other folates was an inhibition of uptake with time. This apparent metabolic control was probably a consequence of culturing the cells with PteGlu as folate source.
A decrease in transport system affinities and maximal transport rates was observed with increasing pteroylpolyglutamate chain length and glutamic acid dipeptides did not affect uptake. It appears that the specificity of the uptake system was for the pteroyl moiety of folate compounds.  8C) and the material eluting just before standard PteGlur (Fig. 8C) were chromatographed on DEAE-cellulose before and after conjugase treatment. Each sample, before conjugase treatment, eluted straight through the column at the position of the The study of the exit mechanism was complicated by metabolism and reuptake of vitamin. Exit was inhibited by iodoacetate, suggesting a carrier-mediated system. When cells were loaded with relatively large amounts of [3H]PteGlu, very little folate containing more than 2 glutamate residues was formed and it appeared that triglutamate formation was rate-limiting. Sakami et al. (40) This latter compound was probably the "early eluting folate" described by Cooper (38) which had similar properties to, but was distinct from, 10.formyl-PteGlu, and whose formation was inhibited by methotrexate.
These results are also in agreement with the observation of Ohara and Silber (41) that the specific activity of L. casei 10.formyl-H4PteGlu synthetase was highest in late-log phase cells. Although some first part of Peak A, Fig. 9. After conjugase treatment, two differences in exit rates with iodoacetate-treated cells might be Comparisons of transport parameters with growth data obtained previously by Tamura et al. (15) suggests that, although pteroylpolyglutamates are transported at a slower rate, once transported they are more effectively utilized by L. casei for growth than is PteGlu.
[3H]Methotrexate uptake did not follow Michaelis-Mententype kinetics, probably because the compound is rapidly broken down and released by L. casei (39).