Characterisation of the Carboxypeptidase G2 Catalytic Site and Design of New Inhibitors for Cancer Therapy.

The enzyme carboxypeptidase G2 (CPG2) is used in antibody-directed enzyme prodrug therapy (ADEPT) to catalyse the formation of an active drug from an inert prodrug. Free CPG2 in the bloodstream must be inhibited before administration of the prodrug in order to avoid a systemic reaction in the patient. Although a few small-molecule CPG2 inhibitors have been reported, none has been taken forward thus far. This lack of progress is due in part to a lack of structural understanding of the CPG2 active site as well as the absence of small molecules that can block the active site whilst targeting the complex for clearance. The work described here aimed to address both areas. We report the structural/functional impact of extensive point mutation across the putative CPG2 catalytic site and adjacent regions for the first time, revealing that residues outside the catalytic region (K208A, S210A and T357A) are crucial to enzyme activity. We also describe novel molecules that inhibit CPG2 whilst maintaining the accessibility of galactosylated moieties aimed at targeting the enzyme for clearance. This work acts as a platform for the future development of high-affinity CPG2 inhibitors that occupy new chemical space and will advance the safe application of ADEPT in cancer treatment.

Effect of Tea Catechins on Folate Analysis in Green Tea by Microbiological Assay.

Green tea is thought to be a primary source of folate in the Japanese diet, based on folate content analyzed by a microbiological assay. Green tea also contains high amount of catechins, in particular, epigallocatechin gallate (EGCg), which was demonstrated to be able to inhibit the digestive enzyme activities and microbial growth in the folate assay. In the present study, we examined whether tea catechins interfered with components of the folate assay for green tea. A marked inhibitory effect of EGCg on microbial growth was observed at an inhibitory concentration of higher than 10 µg/mL. Tea catechins without the galloyl moiety did not show an inhibitory effect. EGCg inhibited the activity of the three enzymes used for assay sample preparation at an inhibitory concentration of higher than 750 µg/mL for α-amylase, 1,000 µg/mL for protease, and 50 µg/mL for conjugase. However, with each step of the assay, the actual concentration of EGCg was decreased to below the inhibitory concentration of each analytical step. Lack of influence of EGCg on green tea folate assay was confirmed by an addition of folate standard in tea infusion. These results suggested that tea catechins have no practical impact on folate analysis in green tea, using the general microbiological assay.

γ-Glutamyl hydrolase modulation and folate influence chemosensitivity of cancer cells to 5-fluorouracil and methotrexate.

BACKGROUND: γ-Glutamyl hydrolase (GGH) regulates intracellular folate and antifolates for optimal nucleotide biosynthesis and antifolate-induced cytotoxicity, respectively. The modulation of GGH may therefore affect chemosensitivity of cancer cells, and exogenous folate levels may further modify this effect. METHODS: We generated a novel model of GGH modulation in human HCT116 and MDA-MB-435 cancer cells and investigated the effect of GGH modulation on chemosensitivity to 5-fluorouracil (5FU) and methotrexate (MTX) at different folate concentrations in vitro and in vivo. RESULTS: Overexpression of GGH significantly decreased chemosensitivity of MDA-MB-435 cells to 5FU and MTX at all folate concentrations as expected. In contrast, in HCT116 cells this predicted effect was observed only at very high folate concentration, and as the folate concentration decreased this effect became null or paradoxically increased. This in vitro observation was confirmed in vivo. Inhibition of GGH significantly increased chemosensitivity of both cancer cells to 5FU at all folate concentrations. Unexpectedly, GGH inhibition significantly decreased chemosensitivity of both cancer cells to MTX at all folate concentrations. In both GGH modulation systems and cell lines, the magnitude of chemosensitivity effect incrementally increased as folate concentration increased. CONCLUSION: Modulation of GGH affects chemosensitivity of cancer cells to 5FU and MTX, and exogenous folate levels can further modify the effects.

Mechanism-based inhibitors of folylpoly-gamma-glutamate synthetase and gamma-glutamyl hydrolase: control of folylpoly-gamma-glutamate homeostasis as a drug target.

Intracellular folate pools consist primarily of gamma-glutamyl isopeptide conjugates of reduced forms of the vitamin, folic acid. Biosynthesis of these oligomeric isopeptides is catalyzed by the enzyme folylpoly-gamma-glutamate synthetase (FPGS). The highly anionic character of the oligomers renders them unable to cross the cell membrane and, therefore, these forms of reduced folates (and certain antifolates) accumulate in cells to high concentration. gamma-Glutamyl hydrolase (GH) catalyzes the hydrolysis of the oligo-gamma-glutamates derivatives to monoglutamyl forms, which are substrates for the reduced folate carrier and able to exit the cell. This chapter describes research, primarily from our laboratories, on the design, synthesis, and biochemical evaluation of several novel analogues of glutamic acid, gamma-glutamyl peptides, and derivatives of folic acid as well as of antifolate drugs. These include a series of fluoroglutamic acids, fluoroglutamate-containing isopeptides, phosphorus-containing pseudopeptides, and epoxide-containing peptidomimetics. The fluoroglutamic acids and fluoroglutamate-containing folates and antifolates exhibit position-dependent effects on the reactions catalyzed by FPGS and GH, thus providing insight into the catalytic mechanism and control of these enzymes. The phosphinic acid-containing pseudopeptides are the most potent inhibitors of FPGS identified to date, and were designed based on mechanistic enzymology data from our laboratories and others, prior to the publication of any structural information about the targeted enzymes.

gamma-Glutamyl hydrolase, not glutamate carboxypeptidase II, hydrolyzes dietary folate in rat small intestine.

Dietary polyglutamyl folates are hydrolyzed to monoglutamyl folate derivatives prior to intestinal transport. In humans and pigs, the reaction occurs at pH 6.5 at the jejunal brush border membrane by folate hydrolase and is encoded by the glutamate carboxypeptidase II (GCPII) gene. Intracellular folate hydrolase with an optimal pH of 4.5 is encoded by the gamma-glutamyl hydrolase (gamma-GH) gene and predominates in rats. We determined the respective roles of GCPII and gamma-GH in dietary folate hydrolysis in rat small intestine. Duodenal, jejunal, and ileal mucosa, pancreas, and duodenal luminal fluid were collected from 10 Sprague-Dawley rats that had not been food deprived. Folate hydrolase was assayed at pH 4.5 and 6.5 with and without parahydroxymercuribenzoate (pHMB), an inhibitor of intracellular folate hydrolase. Folate hydrolase activity occurred at pH 4.5 in all tissues, was significantly inhibited by the addition of pHMB at both pH 4.5 and 6.5, and was virtually absent from brush border fractions at pH 6.5. The highest activity was in the postprandial duodenal luminal fluid at pH 4.5. Rat-specific primers for GCPII and gamma-GH were used to detect mRNA expression in pancreas, jejunal mucosa, and liver. GCPII expression was detected only in the liver, whereas gamma-GH was expressed in all 3 tissues. These results suggest that the hydrolysis of polyglutamyl folates in rats requires the intracellular folate hydrolase that is expressed by pancreatic gamma-GH, in contrast to GCPII that is expressed in the jejunal mucosal brush border in pigs and humans. gamma-GH folate hydrolase is abundant in rat postprandial pancreatic secretions and appears to hydrolyze dietary folates in the intestinal lumen prior to intestinal absorption.

Update on antifolate drugs targets.

Antifolate drugs are molecules directed to interfere with the folate metabolic pathway at some level. They can be recognized among the first rationally designed compounds applying the principle of structural analogy with the substrate developing the antimetabolite strategy. This strategy has taken advantage of the basic different features of the microbial and human folate metabolism and therefore allows targeting the pathway at different steps generating a specificity tools for Medicinal Chemists. Two main problems are giving renewed importance to such targets and therefore improving the efforts to discover new targets in the folate metabolism area. The first one is the increasing resistance to the present drugs due to different mechanisms such as the enzyme modification and the increased production of enzymes with not well recognized importance. The second one is the development of techniques directed to highlight the interference at genetic level of molecular probes as antifolate drug to develop new enzymes previously unknown. This approach is defined as genetic approach to drug discovery, from gene to drugs. The present article describes the importance in drug design and discovery of some antifolate targets among the best known at the present status of research such as thymidylate synthase (TS), dhydrofolate reductases, (DHFR) serine hydroxymethyltransferase (SHMT), folyilpolyglutamyl synthetase (FPGS), gamma-glutamyl hydrolase (gamma-GH), glycinamide-ribonucleotide transformylase (GARTfase), amino-imidazole-carboxamide-ribonucleotide transformylase (AICARTfase) and Folate transporters. Discovery, known functions, structure/function studies and inhibition will be described.

Glutamyl hydrolase: properties and pharmacologic impact.

Glutamyl hydrolase cleaves the poly-gamma-glutamate chain folate and antifolate poly-gamma-glutamates. Its cellular location is lysosomal with large amounts of the enzyme constitutively secreted. The highest levels of glutamyl hydrolase mRNA in humans is found in the liver and kidney. Baculovirus-expressed human enzyme has been used to evaluate the method of hydrolysis of methotrexate-gamma-glu4 and MTA-gamma-glu4. In both cases, the substrates are hydrolyzed by removal of the outer two gamma-glutamate linkages, yielding glu and gamma-glu2 as the glutamate products. Cell lines resistant to 5,10-dideazatetrahydrofolate (lometrexol) have sevenfold higher activities of glutamyl hydrolase. These cultures have a 60% to 90% reduced amount of antifolate polygamma-glutamates and 30% reduced folyl poly-gamma-glutamates. These results suggest the possibility of using glutamyl hydrolase to favorably modulate the activity of antifolate therapy.

Site-directed mutagenesis establishes cysteine-110 as essential for enzyme activity in human gamma-glutamyl hydrolase.

Gamma-glutamyl hydrolase (GH), which hydrolyses the gamma-glutamyl conjugates of folic acid, is a key enzyme in the maintenance of cellular folylpolyglutamate concentrations. The catalytic mechanism of GH is not known. Consistent with earlier reports that GH is sulphydryl-sensitive, we found that recombinant human GH is inhibited by iodoacetic acid, suggesting that at least one cysteine is important for activity [Rhee, Lindau-Shepard, Chave, Galivan and Ryan (1998) Mol. Pharmacol. 53, 1040-1046]. Using site-directed mutagenesis, the cDNA for human GH was altered to encode four different proteins each with one of four cysteine residues changed to alanine. Three of the mutant proteins had activities similar to wild-type GH and were inhibited by iodoacetic acid, whereas the C110A mutant had no activity. Cys-110 is conserved among the human, rat and mouse GH amino acid sequences. The wild-type protein and all four mutants had similar intrinsic fluorescence spectra, indicating no major structural changes had been introduced. These results indicate that Cys-110 is essential for enzyme activity and suggest that GH is a cysteine peptidase. These studies represent the first identification of the essential Cys residue in this enzyme and provide the beginning of a framework to determine the catalytic mechanism, important in defining GH as a therapeutic target.

Novel inhibitors of carboxypeptidase G2 (CPG2): potential use in antibody-directed enzyme prodrug therapy.

The design and synthesis of potent thiocarbamate inhibitors for carboxypeptidase G2 are described. The best thiocarbamate inhibitor N-(p-methoxybenzenethiocarbonyl)amino-L-glutamic acid 6d, chosen for preliminary investigations of in vitro antibody-directed enzyme prodrug therapy (ADEPT), abrogated the cytotoxicity of a combination of A5B7-carboxypeptidase G2 conjugate and prodrug PGP (N-p-{N,N-bis (2-chloroethyl)amino}phenoxycarbonyl-L-glutamate) toward LS174T cells. This is the first report of a small-molecule enzyme inhibitor proposed for use in conjunction with the ADEPT approach.

Mechanism-based inhibition of human folylpolyglutamate synthetase: design, synthesis, and biochemical characterization of a phosphapeptide mimic of the tetrahedral intermediate.

Folylpolyglutamate synthetase (FPGS) catalyzes anATP-dependent ligation reaction that results in the synthesis of poly(gamma-glutamate) metabolites of folates and some antifolates. We have synthesized and characterized the prototype of a new class of mechanism-based FPGS inhibitor in which a phosphonate moiety mimics the tetrahedral intermediate formed during the ligation reaction. This phosphonate, 4-amino-4-deoxy-10-methyl-pteroyl-L-glutamyl-gamma-[Psi¿P(O)(OH)-O¿] glutarate (4-NH2-10-CH3-Pte-L-Glu-gamma-[Psi¿P(O)(OH)-O¿]glutarate), is not a substrate for human FPGS, but is a linear, competitive inhibitor (Kis = 46 nM) with respect to methotrexate as the variable substrate. Inhibition is not time-dependent and preincubation of FPGS with this phosphonate does not increase the degree of inhibition, suggesting that it is not a slow, tight-binding inhibitor involving a time-dependent isomerization, EI --> EI*. Substructures containing the phosphonate moiety but lacking the pterin are much less inhibitory to FPGS, indicating that a significant portion of the inhibitor binding energy is derived from the pterin moiety, a feature also observed in substrate binding. 4-NH2-10-CH3-Pte-L-Glu-gamma-[Psi¿P(O)(OH)-O¿]glutarate is also an analog of a proposed tetrahedral intermediate in the reaction catalyzed by gamma-glutamyl hydrolase (gamma-GH), another enzyme of importance in controlling folate homeostasis in cells. This intermediate would arise from direct attack of H2O on the dipeptide, 4-NH2-10-CH3-Pte-L-Glu-gamma-L-Glu. The fact that 4-NH2-10-CH3-Pte-L-Glu-gamma-[Psi¿P(O)(OH)-O¿]glutarate is not an inhibitor of gamma-GH strongly suggests that hydrolysis of poly-gamma-glutamates catalyzed by gamma-GH does not involve the direct attack of water at the scissile amide bond. Methotrexate, its gamma-glutamyl dipeptide metabolite, and 4-NH2-10-CH3-Pte-L-Glu-gamma-[Psi¿P(O)(OH)-O¿]glutarate are equipotent as inhibitors of human dihydrofolate reductase (the primary target of methotrexate), but the phosphonate does not significantly inhibit another important folate-dependent enzyme, thymidylate synthase. Thus, the phosphonate moiety in this analog represents an important new lead in the development of FPGS inhibitors.

Evaluation of phosphorus-containing inhibitors of gamma-glutamyl hydrolase.

Several putative, phosphorus-containing inhibitors of gamma-glutamyl hydrolase were synthesized and evaluated for inhibitory activity. The phosphonamidoic acids were shown to be weak competitive inhibitors while both a phosphoramidate diester and a phosphonamidate ester were shown to be potent time-dependent inactivators, presumably through irreversible phosphorylation of an active site nucleophile.

gamma-Glutamyl hydrolase from human sarcoma HT-1080 cells: characterization and inhibition by glutamine antagonists.

Elevated gamma-glutamyl hydrolase (GGH) activity as a contributing factor in mechanisms of acquired and intrinsic antifolate resistance has been reported for several cultured cell lines. Despite this, little is known about this enzyme, especially the human species. Using the human HT-1080 sarcoma line, we observed the secretion of GGH activity into media during culture (a phenomenon that could be markedly stimulated by exposure to NH4Cl) and an acidic pH optimum for in vitro catalytic activity of the enzyme. These properties are consistent with a lysosomal location for the enzyme. Unlike rodent GGH, preparations of HT-1080 enzyme (purified < or = 2000-fold) displayed exopeptidase activity in cleaving successive end-terminal gamma-glutamyl groups from poly-L-gamma-glutamyl derivatives of folate, methotrexate (MTX), and para-aminobenzoic acid substrates and a marked preference for long-chain polyglutamates (Km values for glu4 versus glu1 derivatives were 17- and 15-fold lower for folate and MTX versions, respectively). Using an in vitro assay screen, several glutamine antagonists [i.e., 6-diazo-5-oxo-norleucine (DON), acivicin, and azaserine] were identified as human GGH inhibitors, with DON being the most potent and displaying time-dependent inhibition. In cell culture experiments, simultaneous exposure of DON (10 microM) and [3H]MTX for 24 hr resulted in modest elevations of the long-chain gamma-glutamyl derivatives of the antifolate for HT-1080 and another human sarcoma line. These compounds may serve as useful lead compounds in the development of specific GGH inhibitors for use in examining the relationship between GGH activity and antifolate action and may potentially be used in clinical combination with antifolates that require polyglutamylation for effective cellular retention.

Galactosylated antibodies and antibody-enzyme conjugates in antibody-directed enzyme prodrug therapy.

Antibody directed enzyme prodrug therapy (ADEPT) has been studied as a two- and three-phase system in which an antibody to a tumor-associated antigen has been used to deliver an enzyme to tumor sites where it can convert a relatively nontoxic prodrug to a cytotoxic agent. In such a system, it is necessary to allow the enzyme activity to clear from the blood before prodrug injection to avoid toxicity caused by prodrug activation in plasma. To accelerate plasma clearance of enzyme activity, two approaches have been studied. The studies have been performed with a monoclonal anticarcinoembryonic-antigen antibody fragment A5B7-F(ab')2 conjugated to a bacterial enzyme, carboxypeptidase G2 (CPG2), in LS174T xenografted mice. In the first approach, a monoclonal antibody (SB43), directed at CPG2, was used, which inactivates CPG2 in vitro and in vivo. SB43 was galactosylated so that it had sufficient time to form a complex with plasma CPG2, resulting in the inactivation and clearance of the complex from plasma via the carbohydrate-specific receptors in the liver. Injection of SB43gal 19 hours after administration of the radiolabeled conjugate reduced the percentage of injected dose per gram in blood without affecting levels in the tumor. The second approach involved galactosylation of the conjugate so that it cleared rapidly from blood via the asialoglycoprotein receptors in the liver. Localization of the radiolabeled conjugate was achieved by blocking this receptor for about 8 hours with a single injection (8 mg/mouse) of an inhibitor that binds competitively to the receptor. This allowed tumor localization of the conjugate followed by a rapid clearance of the galactosylated conjugate from blood as the inhibitor was consumed. A tumor-to-blood ratio of 45:1 was obtained at 24 hours, which increased to 100:1 at 72 hours after the conjugate injection. These accelerated clearance mechanisms have been applied in antitumor studies in ADEPT.

Regulation of folate and one-carbon metabolism in mammalian cells. IV. Role of folylpoly-gamma-glutamate synthetase in methotrexate metabolism and cytotoxicity.

Chinese hamster ovary (CHO) cells expressing human and Escherichia coli folylpoly-gamma-glutamate synthetase (FPGS) activities were used as models to study factors regulating the cytotoxicity and metabolism of methotrexate (MTX). CHO cells expressing human FPGS metabolized MTX to polyglutamates characteristic of human cells. Cellular MTX accumulation and metabolism to polyglutamates were dependent on the level of FPGS activity and were unaffected by putative gamma-glutamyl hydrolase inhibitors. The sensitivity of cells continuously exposed to MTX was not influenced by FPGS activity. After short term exposure to MTX, cells expressing higher levels of FPGS were more sensitive to the drug. MTX was not transported into the mitochondria and MTX treatment had no effect on preexisting mitochondrial folates while cytosolic folates were converted to oxidized forms. Mitochondrial folate accumulation was significantly impaired by MTX treatment, suggesting that the mitochondrial folate transport system is specific for reduced folates.

The properties of the secreted gamma-glutamyl hydrolases from H35 hepatoma cells.

gamma-Glutamyl hydrolase has been partially purified and characterized from conditioned culture medium of H35 hepatoma cells. Evidence for heterogeneity of the enzyme is derived from its elution as three distinct peaks of enzymatic activity when the enzyme is purified by TSK-butyl-Sepharose column chromatography. These three enzyme fractions appear to have identical catalytic properties but, as yet, the basis for their resolution is not understood. A rapid, sensitive and simple assay based on reverse-phase HPLC fluorescent detection with pre-column derivatization using o-phthalaldehyde (OPA) was developed to separate OPA-derivatives of poly-gamma-glutamates and glutamic acid. Using this assay and the standard HPLC assay for pteroylpolyglutamates, the enzyme appears to be an endopeptidase with respect to pteroylpenta-gamma-glutamate (PteGlu5), methotrexate penta-gamma-glutamate (4-NH2-10-CH3PteGlu5) and p-aminobenzoyl-penta-gamma-glutamate (pABAGlu5). The initial products are PteGlu1 (or 4-NH2-10-CH3PteGlu1 or pABAGlu1) and intact tetra-gamma-glutamate, which is subsequently degraded to glutamic acid. When penta-gamma-glutamate is the substrate, the cleavage of the gamma-bonds by the enzyme is less ordered, with the early appearance of mono-, di-, tri- and tetraglutamate. Poly-alpha-glutamate is not a substrate nor are pABA-gamma-Glu5 or penta-gamma-glutamate covalently linked to albumin. 4-NH2-10-CH3PteGlu2 or Glu5 bound to dihydrofolate reductase is not a substrate for the enzyme, offering further evidence that protein-associated poly-gamma-glutamates are poor substrates for gamma-glutamyl hydrolase from H35 hepatoma cells.

Inactivation and clearance of an anti-CEA carboxypeptidase G2 conjugate in blood after localisation in a xenograft model.

Studies with a conjugate of carboxypeptidase G2 (CPG2) and the F(ab')2 fragment of monoclonal anti-CEA antibody, A5B7, have shown specific localisation in a human colon tumour xenograft, LS174T, growing in nude mice. The conjugate reaches a peak concentration in the tumour within 24 h but enzyme activity in blood remains above a critical value for therapeutic purposes for several days. Here we describe a new monoclonal antibody, SB43, raised against CPG2 which is capable of reducing enzyme activity in blood. In vitro studies demonstrated specific binding of SB43 to CPG2 causing inactivation. Moreover, in the nude mouse model SB43 was also capable of inactivating the enzyme in the circulation within minutes of administration. Radiolabelled native SB43 persisted in blood for several days and appreciable non-specific uptake into the xenograft was also observed. Uptake of SB43 by the tumour, with possible inactivation of CPG2 at this site, could be limited by first coupling the antibody to galactose. This ensured recognition and excretion of SB43 and SB43-enzyme complexes via the liver and their rapid removal from the circulation. Galactosylation had no effect on the ability of SB43 to inactivate the enzyme.

Biochemical and growth inhibition studies of methotrexate and aminopterin analogues containing a tetrazole ring in place of the gamma-carboxyl group.

The biological activities of novel analogues of methotrexate (MTX) and aminopterin (AMT) in which the gamma-carboxyl was replaced by a 1H-tetrazol-5-yl ring, an isosteric group with acidic properties similar to a carboxyl group, were investigated. The tetrazolyl analogues of MTX and AMT were more potent inhibitors of the growth of CCRF-CEM and K562 human leukemia cell lines during continuous (120 h) and 24-h pulse exposure than were the respective parent drugs; only when the exposure time was reduced to 6 h were the parent drugs more potent. These inhibitory effects on growth correlated with the onset of and recovery from inhibition of de novo thymidylate biosynthesis. Growth inhibition by the analogues was protectable by leucovorin. MTX-resistant CCRF-CEM sublines with decreased transport or increased dihydrofolate reductase (DHFR) levels were cross-resistant to the analogues. The analogues were as potent as their parent drugs in inhibiting DHFR activity in vitro and at displacing [3H]MTX from intracellular DHFR. Each analogue was more effective than its parent drug at inhibiting uptake of [3H]MTX into CCRF-CEM cells. The tetrazole analogue of AMT was a linear competitive inhibitor (Kis = 50 microM) of CCRF-CEM folylpolyglutamate synthetase, while the tetrazole analogue of MTX, unlike all other inhibitors, was linear noncompetitive (Kis = 51 microM, Kii = 321 microM). The data suggest that, compared with MTX or AMT, the tetrazole substituent, in place of the gamma-carboxyl group, allows more efficient transport into cells via the reduced folate/MTX carrier and the resulting greater uptake of the analogues leads to inhibition of DNA synthesis and cell death at lower extracellular concentrations during long exposures. The mechanism of cell death could involve inhibition at folypolyglutamate synthetase, but DHFR is the primary target. The low potency of the analogues during short exposure is presumably related to the inability to form the poly-gamma-glutamyl metabolites required for intracellular retention.

Inhibition by selected food components of human and porcine intestinal pteroylpolyglutamate hydrolase activity.

Studies were conducted to determine the in vitro effect of selected food components on activity of the brush border membrane pteroylpolyglutamate hydrolase (folate conjugase) of porcine and human intestine. Foods differed widely in their effects although the pattern of the effects on both porcine and human enzymes was similar. Extracts of legumes, tomatoes, and orange juice consistently inhibited the conjugase activity. Citrate was also inhibitory to some extent. In contrast, extracts of cereal grain flours, whole egg, milk, cabbage, cauliflower, and lettuce caused little inhibition. Purified phytohemagglutinins, soybean trypsin inhibitors, and bovine milk folate-binding protein had no effect on the conjugase activity at the concentrations tested. The food substances that inhibited the conjugase activity did not bind the polyglutamyl folate substrate or inhibit intestinal brush border membrane sucrase and alkaline phosphatase. These findings suggest that food composition may influence folate bioavailability by interfering with the intestinal deconjugation of dietary polyglutamyl folates.