Enzymatic reduction and methylation of folate following pH-dependent, carrier-mediated transport in rat jejunum.

Intestinal transport of [3H] folate was studied using everted sacs of rat jejunum. The proximal small intestine transports folate against a concentration gradient by a system which is saturable, pH-dependent, energy-dependent, sodium-dependent, sensitive to temperature, and appears to be a common transport system for folate compounds. Chromatographic analysis of folate compounds in the serosal compartment after a 60 min incubation with folate in the mucosal medium in sodium phosohate buffer indicated that metabolism of folate to 5-methyltetrahydrofolate was extensive at pH 6.0 and negligible at pH 7.5. The percent conversion of folate to 5-methyltetrahydrofolate at pH 6.0 was reduced by increasing the concentration of folate in the mucosal medium, thus indicating saturation of the reduction and methylation process. These findings indicate that folate transport in rat jejunum occurs by an energy-dependent, carried-mediated system and that both folate transport and intestinal conversion of folate to 5-methyltetrahydrofolate are pH-dependent.     

Cyclic adenosine-3',5'-monophosphate and folate transport in rat jejunum

The intestinal transport of 5-methyltetrahydrofolate and pteroylmonoglutamate was examined in everted sacs of rat jejunum exposed to compounds which increase intracellular cyclic adenosine-3', 5'-monophosphate. Adenyl cyclase stimulators (hydrocortisone and prostaglandin), phosphodiesterase inhibitors (3-isobutyl-l-methylxanthine, aminophylline and papaverine), and dibutyryl adenosine-3',5'-cyclicmonophosphate added to the mucosal medium inhibit the mucosal-to-serosal transport of physiological concentrations of 5-methyltetrahydrofalate and pteroylmonoglutamate. Transport inhibition is correlated with the ability of these agents to increase cellular cyclic adenosine-3', 5'-monophosphate. The active, carrier-mediated transport system of folate compounds is highly sensitive to the increase in cyclic adenosine-3', 5'-monophosphate level, while the diffusion system is insensitive. These data indicate that the active transport system of folates is modulated by cellular cyclic adenosine-3', 5'-monophosphate.     

Characteristics of transport of fluoresceinated methotrexate in rat small intestine

The small intestinal damage induced by the methotrexate (MTX) treatment results in malabsorption and diarrhea. The fluoresceinated methotrexate (F-MTX) may possibly be useful to study such effects of MTX on the small intestine. The purpose of this study is to characterize the transport of F-MTX in the small intestine in order to use it as a membrane transport and cellular marker of MTX. The transport of F-MTX in the rat small intestine (jejunum) was examined in the in vitro everted segments of the intestine. The uptake was pH-dependent and showed a maximal effect at pH 6.0, which was the same as the results of MTX previously reported. Further, it was temperature-dependent and was inhibited by metabolic inhibitors, dinitrophenol and sodium azide, and by MTX. The transport kinetics at pH 6.0 in the mucosal solution and at pH 7.4 in the serosal solution was saturable with Km of 0.48 +/- 0.23 microM and Vmax of 0.66 +/- 0.24 pmol/cm/min and in addition, the passive diffusion was observed there. These results suggested that the transport of F-MTX was energy-dependent and was mediated by the same transporter as that of MTX, although, in addition to it, other transport mechanism might contribute to the F-MTX transport. Therefore F-MTX will be of great use to investigate the MTX transport system in the normal and diseased states of small intestine, using various fluorescence techniques like visualization of membrane-associated transport proteins.     

Inhibition of intestinal absorption of 5-methyltetrahydrofolate by fluoxetine

Thein vitro uptake of 5-methyltetrahydrofolate (5-MeTHF) by rat and human intestine is dose-dependently inhibited by the antidepressant drug fluoxetine (FLX). In rat jejunum rings, 0.2 mM FLX inhibited the uptake of 5-MeTHF (0.25 μM) by 32% (15 min) and 49% (45 min). In brush border membrane vesicles (BBMV) from rat jejunum, 0.2 mM FLX inhibited the folate uptake at the overshoot (90 s) by 40 %. Similar inhibition was observed with human Caco-2 cells and duodenal biopsies. FLX action is exerted on the active transport component of the folate uptake, since the drug has no effect when the passive diffusion component becomes prominent by high substrate concentration, or by 0-4 ºC incubation or by addition of the folate transport inhibitor DIDS (1mM). The kinetic analysis with rat BBMV suggests a non-competitive inhibition of the 5-MeTHF transport by FLX, with apparent values for K_M = 0.89 μM, Vmax = 1.89 pmol/mg prot./10 s, and K_I = 0.21 mM. After 21 days of treatment with FLX (10 mg/kg/day), the folate uptake by jejunum rings or by BBMV from the treated rats was diminished, and the folate levels in erythrocytes and serum were also decreased.     

Effect of human milk folate binding protein on folate intestinal transport

The present study examined the effect of human milk folate binding protein (FBP) on the intestinal transport of 5-methyltetrahydrofolate (5-CH3H4PteGlu). This was performed by examining the transport of radiolabeled 5-CH3H4PteGlu bound to FBP using everted sacs of rat intestine. In the jejunum at pH 6, transport of 27 nM bound 5-CH3H4PteGlu was linear with time for 30 min of incubation. Transport of 13 nM bound 5-CH3H4PteGlu was higher in the jejunum than in the ileum at both pH 6 (2.1 +/- 0.3 and 0.36 +/- 0.03 pmol/g wet wt/25 min, respectively) and pH 8 (1.9 +/- 0.3 and 0.32 +/- 0.02 pmol/g wet wt/25 min, respectively). In the jejunum, transport of 13 nM bound 5-CH3H4PteGlu at pH 6 was less than transport of an equimolar concentration of free 5-CH3H4PteGlu (2.1 +/- 0.3 and 5.1 +/- 0.5 pmol/g wet wt/25 min, respectively) but was similar at pH 8 (1.9 +/- 0.3 and 2.47 +/- 0.3 pmol/g wet wt/25 min, respectively). In the ileum transport of bound and free 5-CH3H4PteGlu was similar at pH 6 (0.36 +/- 0.03) and 0.41 +/- 0.06 pmol/g wet wt/25 min, respectively) and pH 8 (0.32 +/- 0.02 and 0.43 +/- 0.1 pmol/g wet wt/25 min, respectively). The transport process of bound 5-CH3H4PteGlu in the jejunum was energy, temperature, and Na+ dependent, but not pH dependent, and was competitively inhibited by sulfasalazine. Ninety-two percent of the transport substrate that appeared in the serosal compartment following incubation with bound 5-CH3H4PteGlu was found to be free (unbound) 5-CH3H4PteGlu. These results show that human milk FBP decreases the rate of transport of 5-CH3H4PteGlu in the jejunum and suggest that FBP-bound 5-CH3H4PteGlu may utilize the same transport system as free 5-CH3H4PteGlu. The results also suggest a role for human milk FBP in regulating the nutritional bioavailability of folate.     

Source of Methyl Groups in Brain and Nerve Tissue in the Rat

Previous studies that demonstrated that mouse brain accumulated significantly more radioactivity from subcutaneously administered 5-methyltetrahydrofolate labelled in the methyl group compared to the label in the folate moiety are open to two interpretations. The methyl group could have been transferred to another compound (probably methionine) prior to its transport into the brain. Alternatively, if plasma 5-methyltetrahydrofolate per se is significantly involved in the provision of methyl groups to brain and nerve tissue it would be expected that the folate moiety would be returned to the plasma to complete the cycle and thus would appear not to have been taken up. In this article, using competition experiments that exploit the differences in the mechanism of transport of methionine and 5-methyltetrahydrofolate into brain and nerve, evidence is presented that in the rat the methyl group of 5-methyltetrahydrofolate is transported after its conversion to methionine.     

Binding properties of the 5-methyltetrahydrofolate/methotrexate transport system in L1210 cells

A binding component with a high affinity for 5-methyltetrahydrofolate (K_D = 0.11μm) is present on the external surface of L1210 cells. The amount of binder (1 pmol/mg protein) corresponds to 8 × 10⁴ sites per cell. The participation of this component in the high-affinity 5-methyltetrahydrofolate/methotrexate transport system is supported by similarities in the K_D values for 5-methyltetrahydrofolate and methotrexate binding and the K_t values of these compounds for transport. Relative affinities for other folate substrates (aminopterin, 5-formyltetrahydrofolate, and folate) and various competitive inhibitors (thiamine pyrophosphate, ADP, AMP, arsenate, and phosphate) are also similar for both the binding component and the transport system. The measured binding activity does not represent low-temperature transport of substrate into cells, since it is readily saturable with time and is eliminated by either washing the cells with buffer or by the addition of excess unlabeled substrate.     

High-performance liquid chromatographic measurement of 5,10-methylenetetrahydrofolate in liver.

The folate coenzyme 5,10-methylenetetrahydrofolate is an important folate metabolite which cannot be determined directly by HPLC near neutral pH because it dissociates to formaldehyde and tetrahydrofolate. A method for the determination of 5,10-methylenetetrahydrofolate in liver is described. This method involves (1) determination of liver 5-methyltetrahydrofolate; (2) chemical reduction of liver 5,10-methylenetetrahydrofolate (stabilized at pH 10) to 5-methyltetrahydrofolate; and (3) determination of total liver 5-methyltetrahydrofolate. Subtraction of (1) from (3) gives the concentration of 5,10-methylenetetrahydrofolate in liver.     

Binding of radiolabeled folate and 5-methyltetrahydrofolate to cow's milk folate binding protein at pH 7.4 and 5.0. Relationship to concentration and polymerization equilibrium of the purified protein

Binding of folate (pteroylglutamate) and 5-methyltetrahydrofolate, the major endogenous form of folate, to folate binding protein purified from cow's milk was studied at 7°C to avoid degradation of 5-methyltetrahydrofolate. Both folates dissociate rapidly from the protein at pH 3.5, but extremely slowly at pH 7.4, most likely due to drastic changes in protein conformation occurring after folate binding. Dissociation of 5-methyltetrahydrofolate showed no increase at 37°C suggesting that protein-bound-5-methyltetrahydrofolate is protected against degradation. Binding displayed two characteristics, positive cooperativity and a binding affinity that increased with decreasing concentrations of the protein. The binding affinity of folate was somewhat greater than that of 5-methyl tetrahydrofolate, in particular at pH 5.0. Ligand-bound protein exhibited concentration-dependent polymerization (8-mers formed at 13 M) at pH 7.4. At pH 5.0, only folate-bound forms showed noticeable polymerization. The fact that folate at pH 5.0 surpasses 5-methyltetrahydrofolate both with regard to binding affinity and ability to induce polymerization suggests that ligand binding is associated with conformational changes of the protein which favor polymerization.     

Rodent intestinal folate transporters (SLC46A1): secondary structure, functional properties, and response to dietary folate restriction

This laboratory recently identified a human gene that encodes a novel folate transporter [Homo sapiens proton-coupled folate transporter (HsPCFT); SLC46A1] required for intestinal folate absorption. This study focused on mouse (Mus musculus) PCFT (MmPCFT) and rat (Rattus norvegicus) PCFT (RnPCFT) and addresses their secondary structure, specificity, tissue expression, and regulation by dietary folates. Both rodent PCFT proteins traffic to the cell membrane with the NH(2)- and COOH-termini accessible to antibodies targeted to these domains only in permeabilized HeLa cells. This, together with computer-based topological analyses, is consistent with a model in which rodent PCFT proteins likely contain 12 transmembrane domains. Transport of [(3)H]folates was optimal at pH 5.5 and decreased with increasing pH due to an increase in K(m) and a decrease in V(max). At pH 7.0, folic acid and methotrexate influx was negligible, but there was residual (6S)5-methyltetrahydrofolate transport. Uptake of folates in PCFT-injected Xenopus oocytes was electrogenic and pH dependent. Folic acid influx K(m) values of MmPCFT and RnPCFT, assessed electrophysiologically, were 0.7 and 0.3 microM at pH 5.5 and 1.1 and 0.8 microM at pH 6.5, respectively. Rodent PCFTs were highly specific for monoglutamyl but not polyglutamyl methotrexate. MmPCFT mRNA was highly expressed in the duodenum, proximal jejunum, liver, and kidney with lesser expression in the brain and other tissues. MmPCFT protein was localized to the apical brush-border membrane of the duodenum and proximal jejunum. MmPCFT mRNA levels increased approximately 13-fold in the proximal small intestine in mice fed a folate-deficient vesus folate-replete diet, consistent with the critical role that PCFT plays in intestinal folate absorption.     

Absorption of 5-methyltetrahydrofolate in rat jejunum with intact blood and lymphatic vessels

Transport results from in vitro studies may not be applicable to in vivo situations. In this study, we extended our previous in vitro observations regarding the intestinal transport of 5-methyltetrahydrofolate to in vivo studies in the unanesthetized rat and examined the effect of the unstirred water layer on the absorption process. We used a well defined intestinal perfusion technique. Absorption of 0.5 and 5 microM 5-methyltetrahydrofolate proceeded in a linear manner for 40 min of perfusion at 0.31 and 1.74 nmol/100 cm per min, respectively. Absorption of 0.5 microM 5-methyltetrahydrofolate increased with increasing perfusate flow-rate from 0.5 to 2 to 4 ml/min, indicating an unstirred water layer influence on the absorption rate. Absorption of the substrate was saturable with an apparent Kt of 5.7 microM and Vmax of 3.45 nmol/100 cm per min. Absorption was pH-dependent, and was inhibited by structural analogues. In contrast to the in vitro data, addition of glucose (20 mM) to the perfusate was unnecessary for in vivo absorption to proceed. Unconjugated cholic (5 mM) and deoxycholic (1 mM) acids and the organic anion rose bengal (0.1 mM) inhibited the absorption of 0.5 microM 5-methyltetrahydrofolate when added to the perfusate. Conclusions: the results of previous in vitro studies of 5-methyltetrahydrofolate intestinal transport are applicable to in vivo situations, except that luminal glucose was found to be unnecessary in the latter. The unstirred water layer modulated the absorption of 5-methyltetrahydrofolate, while unconjugated bile acids and rose bengal inhibited it.     

Transport of folate compounds into Lactobacillus Casei

Transport of folate, 5-methyl tetrahydrofolate, and amethopterin into Lactobacillus casei occurs against a concentration gradient, is pH and temperature dependent, requires glucose, exhibits saturation kinetics, is maximal when cells are harvested in late-log phase, and is repressed by excess folate in the growth medium. K_m values are 0.35, 0.90, and 0.21 μm for the influx of folate, 5-methyl tetrahydrofolate, and amethopterin, respectively. Dihydrofolate, tetrahydrofolate, 5-formyl tetrahydrofolate, 5-methyl tetrahydrofolate, aminopterin, and amethopterin are inhibitors of folate influx. Countertransport of 5-methyl tetrahydrofolate is enhanced by various other folate compounds. Uptake of folate, 5-methyl tetrahydrofolate, and amethopterin is inhibited to the same degree by increasing concentrations of iodoacetate. These results indicate that a single system is responsible for transport of a variety of folate compounds into L. casei.     

Carrier affinity as a mechanism for the pH-dependence of folate transport in the small intestine

A mildly acidic pH in the lumen of the small intestine markedly enhances the transport of folate. This study investigated the relationship between pH and the affinity between folic acid and the apical membrane transporter using brush border membrane vesicles from rat jejunum and differentiated monolayer cultures of the colon carcinoma cell line, CaCo-2. Uptake studies with BBMV were conducted at folic acid concentrations of 0.1 to 50 mumol/l, conditions which were suitable for analyzing uptake data based on the Michaelis-Menten equation modified to include a nonsaturable component. These analyses yielded apparent Km values of 0.6 and 12.3 microM at pH 5.5 and pH 7.4, respectively (P less than 0.05). Values for Vmax were lower at pH 5.5 than at pH 7.4 (0.8 vs. 1.6 pmol/mg protein per 10 s, P less than 0.05). The studies with CaCo-2 cells employed folic acid concentrations of 0.1 to 5 mumol/l. Under these conditions the apparent Km for folic uptake was lowest at pH 6.0, where the Km was 0.7 mumol/l. The apparent Km increased sharply as a neutral pH was approached; reaching a value of 13.9 mumol/l at pH 7.1. These data suggest that the prominent pH effect on intestinal folate transport is, in part, explained by an increased affinity of the folate substrate for its membrane transporter.     

Methotrexate transport in variant human CCRF-CEM leukemia cells with elevated levels of the reduced folate carrier. Selective effect on carrier-mediated transport of physiological concentrations of reduced folates

This study reports the isolation and characterization of a variant of the human CCRF-CEM leukemia cell line that overproduces the carrier protein responsible for the uptake of reduced folates and the folate analogue methotrexate. The variant was obtained by adapting CCRF-CEM cells for prolonged times to stepwise decreasing concentrations of 5-formyltetrahydrofolate as the sole folate source in the cell culture medium. From cells that were grown on less than 1 nM 5-formyl-tetrahydrofolate, a variant (CEM-7A) was isolated exhibiting a 95-fold increased Vmax for [3H]methotrexate influx compared to parental CCRF-CEM cells. The values for influx Km, efflux t0.5, and Ki for inhibition by other folate (analogue) compounds were unchanged. Affinity labeling of the carrier with an N-hydroxysuccinimide ester of [3H]methotrexate demonstrate an approximately 30-fold increased incorporation of [3H] methotrexate in CEM-7A cells. This suggests that the up-regulation of [3H]methotrexate influx is not only due to an increased amount of carrier protein, but also to an increased rate of carrier translocation or an improved cooperativity between carrier protein molecules. Incubation for 1 h at 37 degrees C of CEM-7A cells with a concentration of 5-formyltetrahydrofolate or 5-methyltetrahydrofolate in the physiological range (25 nM) resulted in a 7-fold decline in [3H]methotrexate influx. This down-regulation during incubations with 5-formyltetrahydrofolate or 5-methyltetrahydrofolate could be prevented by either the addition of 10-25 nM of the lipophilic antifolate trimetrexate or by preincubating CEM-7A cells with 25 nM methotrexate. The down-regulatory effect was specifically induced by reduced folates since incubation of CEM-7A cells with 25 nM of either methotrexate, 10-ethyl-10-deazaaminopterin, aminopterin, or folic acid, or a mixture of purines and thymidine, had no effect on [3H]methotrexate influx. Similarly, these down-regulatory effects on [3H]methotrexate transport by 5-formyltetrahydrofolate, and its reversal by trimetrexate or methotrexate, were also observed, though to a lower extent, for parental CCRF-CEM cells grown in folate-depleted medium rather than in standard medium containing high folate concentrations. These results indicate that mediation of reduced folate/methotrexate transport can occur at reduced folate concentrations in the physiological range, and suggest that the intracellular folate content may be a critical determinant in the regulation of methotrexate transport.     

Uptake of oxidized folates by rat liver mitochondria

Folate, dihydrofolate, and methotrexate are rapidly taken up by rat liver mitochondria. The apparent maximal matrix folate concentration is about 2.5-fold that of the suspending medium, whereas dihydrofolate and methotrexate equilibrate across the inner membrane. Fully reduced folates, including tetrahydrofolate, 5-methyltetrahydrofolate, and 5,10-methylenetetrahydrofolate penetrate only the intermembrane space. Addition of dihydrofolate or methotrexate effects a rapid release of pre-loaded folate, and external methotrexate promotes the release of pre-loaded dihydrofolate. The extent of dihydrofolate uptake is enhanced by addition of folate. These results suggest that oxidized folates are transported to the matrix by a carrier-mediated mechanism.     

Transport of 5-formyltetrahydrofolate into primary cultured rat astrocytes

Transport of 5-formyltetrahydrofolate (5-FTHF) into primary cultured rat astrocytes was studied. Uptake of 5-FTHF into astrocytes was linear in the first 60 min and is saturable with K(m)=3.3 microM and V(max)=27.5 pmol/mg protein/45 min in pH 7.4 medium. Uptake of 5-FTHF displayed the characteristics of countertransport. Uptake of 5-FTHF was inhibited by the structural analogs 5-methyltetrahydrofolate, methtrexate, and folic acid (K(i)=3.8, 2.7, and 18.4 microM, respectively). Uptake was significantly decreased by sodium azide but was increased by high concentration of sodium cyanide and low concentration of sodium arsenate. Uptake was also inhibited by p-chloromercuriphenylsulfonate and by the anions probenecid and 4,4(')-diisothiocyanostilbene-2,2(')-disulfonic acid. Acute exposure of the cells to ethanol (100mM) inhibited the uptake for 90 min of the experimental duration. It is concluded that astrocytes have a system for the uptake of 5-FTHF and folate analogs which is carrier mediated, this system is sensitive to energy inhibitors and alcohol exposure.     

Mediated uptake of folate by a high-affinity binding protein in sublines of L1210 cells adapted to nanomolar concentrations of folate

Summary An L1210 cell line (JT-1), which can grow in medium supplemented with 1nm folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37°C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23±0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [³H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37°C remained as unmetabolized folic acid. Binding was also rapid at 0°C but uptake at the plateau was only one-half the value obtained at 37°C. Half-maximal saturation of the binding component (K _D) occurred at a folate concentration of 0.065nm at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 (K _D=2.0nm). 5-Methyltetrahydrofolate was also bound by this component (K _i=13nm at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate (K _i=45nm) and methotrexate (K _i=325nm). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500nm caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein. An additional low-affinity, high-capacity transport system for folate that had been proposed previously was not observed under a variety of experimental conditions in either the adapted or parental cells.     

Studies of the role of a particulate folate-binding protein in the uptake of 5-methyltetrahydrofolate by cultured human KB cells

The characteristics of the uptake by human epidermoid carcinoma (KB) cells of 5-methyltetrahydrofolate at extracellular concentrations in the physiologic range and the possible role of a membrane-associated folate binder in folate uptake by KB cells have been investigated. Uptake of 5-methyltetrahydrofolate was specific, saturable, and time-, temperature-, and concentration-dependent. Trypsin treatment released 50% of the 5-methyltetrahydrofolate accumulated by KB cells at 4 degrees C, but only 12% at 37 degrees C, indicating that most of the accumulated ligand was intracellular at 37 degrees C, thus demonstrating transport. Accumulated 5-methyltetrahydrofolate was bound to a membrane-associated protein which required detergent for its solubilization, and a significant amount of which was oriented to the cell exterior as demonstrated by its release by trypsin treatment of intact KB cells. The membrane-associated folate binder was immunoprecipitated by antiserum to purified human placental folate receptor, and this antiserum inhibited 5-methyltetrahydrofolate uptake by intact KB cells in a concentration-dependent manner. These data support the hypothesis that the membrane-associated folate-binding protein of human cells participates in the transport of folates under physiologic conditions.     

Transport of folinate and related compounds in Pediococcus cerevisiae

The properties of folinate and 5-methyltetrahydrofolate (5-CH(3)-H(4)PteGlu) transport mechanism of Pediococcus cerevisiae were studied. The uptake was dependent on temperature, pH (optimum for both compounds at pH 6.0), and glucose. Iodoacetate, potassium fluoride, and sodium azide inhibited the uptake. 5-CH(3)-H(4)-PteGlu was apparently not metabolized but folinate was metabolized. Metabolism of folinate was reduced by preincubation of cells with fluorodeoxyuridine. The transport system for folinate and 5-CH(3)-H(4)PteGlu were specific for the l-isomers. Pteroylglutamate, aminopterin, and amethopterin did not interfere with the uptake. Tetrahydrofolate competed with the uptake of folinate. The transport of folinate and 5-CH(3)-H(4)PteGlu at 37 C conformed to Michaelis-Menten kinetics; apparent K(m) for both compounds was 4.0 x 10(-7)m, and the V(max) for folinate was 1.0 x 10(-10) moles per min per mg (dry weight) and for 5-CH(3)-H(4)PteGlu it was 1.6 x 10(-10) moles per min per mg (dry weight). Both compounds accumulated in the intracellular pool at a concentration about 80- to 140-fold higher than that in the external medium. Folinate inhibited competitively the uptake of 5-CH(3)-H(4)PteGlu with a K(i) of 0.4 x 10(-7)m. Unlike 5-CH(3)-H(4)PteGlu, which accumulated only at 37 C, folinate was also taken up at 0 C by a glucose- and temperature-independent process, which was not affected by the metabolic inhibitors mentioned above. Since at 0 C the intracellular concentration of folinate was also considerably higher than the external, binding of the substrate to some cellular component is assumed. The finding of an efficient transport system for l-5-CH(3)-H(4)PteGlu is of special interest, since this compound has no growth-promoting activity for P. cerevisiae.     

The preparation and properties of folate-binding protein from cow''s milk.

An improved affinity-chromatographic method for the preparation of folate-binding protein from cow's milk is described. Under dissociating conditions the protein appeared homogeneous in the ultracentrifuge, with a molecular weight of 35 000 +/- 1500, but it was heterogeneous on electrophoresis and ion-exchange chromatography and evidently consisted of several glycoproteins with similar molecular weights that all bound folic acid. Overall, the protein contained a high proportion of half-cystine (18 residues/molecule) and 10.3% of carbohydrate. At saturation it bound approx. 1 mol of folate/mol of protein at pH 7.2. Equilibrium-dialysis measurements of the binding of folic acid and 5-methyltetrahydrofolate to the purified protein gave non-linear Scatchard plots, the shapes of which depended on pH. The results were interpreted in terms of ligand binding to a polymerizing system in which the affinity of ligand for monomer was greater than its affinity for polymer. When the protein concentration was similar to that in cow's milk, dissociation constants (Kd) for folate and 5-methyltetrahydrofolate were 3 nM and 5 nM respectively at pH 7.2 and 37 degrees C, whereas Kd for the binding of folate to monomer was about 50 pM. The properties of the binding protein are discussed in relation to its possible role in folate absorption in the gut.