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PubMed1999

Characterization of the enzymatic activity of PSM: comparison with brain NAALADase.

BACKGROUND: The prostate cancer marker prostate-specific membrane antigen (PSM) is highly homologous to the brain enzyme N-acetylated alpha-linked acidic dipeptidase (NAALADase). NAALADase is known to cleave terminal carboxy glutamates from both the neuronal peptide N-acetylaspartylglutamate (NAAG) and folate polyglutamate. In this report, we compare the NAAG hydrolyzing activity of NAALADase and the prostate enzyme PSM. METHODS: Using a NAAG hydrolytic radioenzymatic assay, we compared the pharmacological and kinetic properties of the brain and prostate enzymes. RESULTS: Eight normal prostate tissues from different species exhibited NAAG hydrolyzing activity. Among 14 cancer cell lines examined, activity was observed in human LNCaP, PC-82, and rat Dunning G and AT-1 cells. Brain exhibited membrane-localized activity exclusively, while the prostate enzyme had activity in both membrane and cytosolic fractions. The only observed pharmacological difference was the sensitivity to their putative substrates, folate polyglutamate and NAAG. Kinetically, the soluble form of the prostate enzyme had two catalytic sites, while the membrane-bound form exhibited single site kinetics with a lower Vmax than the brain enzyme, which may suggest a less active hydrolase in the prostate. CONCLUSIONS: The brain enzyme NAALADase and the prostate enzyme PSM are remarkably similar. The importance of the differences in substrate specificities and kinetic parameters remains to be elucidated.

Animals↗
PubMed2006

Gamma-glutamyl hydrolase and drug resistance.

Gamma-glutamyl hydrolase (GGH) is a lysosomal enzyme involved in the metabolism of folates and anti-folates. It acts as an endo- and/or exo-peptidase to cleave gamma-polyglutamate chains that are attached to folates and anti-folates after they enter a mammalian cell. Whereas the addition of multiple glutamates is necessary to enable the cell to retain folates and anti-folates, hydrolysis of the polyglutamate tails by GGH has the opposite effect of making (anti)-folates exportable again. Thus, GGH plays an important role in the cellular homeostasis of folate. Furthermore, high levels of GGH have been associated with cellular resistance to anti-folates, in particular methotrexate. Consequently, GGH also has pharmacological importance. In addition to the intracellular GGH, carboxypeptidase II (also called intestinal folate conjugase, prostate specific membrane antigen or N-acetyl-alpha-linked acidic dipeptidase) is another enzyme with gamma-glutamyl hydrolase activity; it resides, however, in the cellular membrane. Although genetically and biochemically distinct, this enzyme too appears to play a major role in folate homeostasis, by cleaving polyglutamates from extracellular folate-polyglutamates, so that they can be imported into the cell. Finally, there have been reports suggesting that gamma-glutamyl hydrolase plays a role as a tumor marker in breast and lung cancer.

Animals↗
PubMed1987

Dissociation of the octameric bifunctional enzyme formiminotransferase-cyclodeaminase in urea. Isolation of two monofunctional dimers.

Partial denaturation of the circular octameric bifunctional enzyme formiminotransferase-cyclodeaminase in increasing urea concentrations leads to sequential dissociation via dimers to inactive monomers. In potassium phosphate buffer, dissociation to dimers in 3 M urea coincides with loss of both activities and a major decrease in intensity of intrinsic tryptophan fluorescence. In the presence of folic acid, these dimers retain the deaminase activity, but with folylpolyglutamates, both activities are protected and the native octameric structure is retained. The protection profiles with polyglutamates are cooperative with a Hill coefficient greater than 2, suggesting that binding of more than one folylpolyglutamate per octamer is required to stabilize the native structure. In triethanolamine hydrochloride buffer, transferase-active dimers that retain the intrinsic tryptophan fluorescence can be obtained in 1 M urea and stabilized at higher urea concentration by the addition of glutamate. Deaminase-active dimers are obtained by the protection of folate in 3 M urea. Proteolysis of the two kinds of dimers by chymotrypsin leads to very different fragmentation patterns, indicating that they are structurally different. We propose that the two dimers retain different subunit-subunit interfaces, one of which is required for each activity. This suggests that the native octameric structure is required for expression of both activities and therefore for "channeling" of intermediates.

Ammonia-Lyases↗
PubMed1996

Antirheumatic agents. I. Novel methotrexate derivatives bearing an indoline moiety.

Various novel methotrexate (MTX) derivatives bearing an indoline moiety were synthesized and tested for biological activities using human peripheral blood mononuclear cell (hPBMC) and human synovial cells (hSC) derived from patients with rheumatoid arthritis (RA). Compounds having potent activity in vitro were further evaluated using an adjuvant arthritis model in vivo. N-[1-(2,4-Diamino-6-pteridinylmethyl)indoline-5-carbonyl]-L-glutam ic acid 2f showed more potent activities than MTX in vitro and in vivo, and N-[1-(2,4-diamino-6-pteridinylmethyl)-indoline-5-carbonyl]-L-2-ami noadipic acid 2d exhibited fairly good activities in vitro and considerable activity in vivo. Compound 2d was, as expected, not sensitive to folyl-polyglutamate synthetase (FPGS) and did not undergo polyglutamation, a process which may be responsible for a side-effect during MTX therapy.

Animals↗
PubMed1985

DL-threo-4-fluoroglutamic acid. A chain-terminating inhibitor of folylpolyglutamate synthesis.

The effects of 4-fluoroglutamate on the reaction catalyzed by partially purified rat liver folylpolyglutamate synthetase have been investigated. DL-threo-4-Fluoroglutamate was an effective, concentration-dependent inhibitor of polyglutamylation of both tetrahydrofolate and methotrexate, while the erythro isomer was weakly inhibitory. 4-Fluoroglutamate acted as an alternate substrate; the DL-threo isomer was incorporated only slightly less effectively than L-glutamate, while the erythro isomer was poorly incorporated. The resulting product, a pteroylglutamyl-gamma-(4-fluoro)glutamate, was a very poor substrate for further glutamylation. Thus, when tetrahydrofolate and 4-fluoroglutamate were substrates, the sole Zn/HCl cleavage product co-chromatographed on high performance liquid chromatography with chemically synthesized p-aminobenzoylglutamyl-gamma-(4-fluoro)glutamate. When [3H]methotrexate (4-NH2-10-CH3PteGlu) and 4-fluoroglutamate were the substrates, one product was obtained which co-chromatographed on high performance liquid chromatography with chemically synthesized 4-NH2-CH3PteGlu-gamma-(4-fluoro)glutamate. Further evidence that the product from [3H]methotrexate was a dipeptide came from gamma-glutamyl hydrolase digestion experiments and quantitative amino acid analysis. The appearance of trace amounts of a product having properties consistent with the addition of a second 4-fluoroglutamate occurred only under forcing conditions. The chemically and enzymatically synthesized fluoroglutamate-containing products were at least 15 times poorer than the analogous diglutamyl compound as substrates for rat liver folylpolyglutamate synthetase. These results are consistent with inhibition of polyglutamate synthesis by 4-fluoroglutamate through a "leaky" chain termination mechanism.

Animals↗
PubMed1999

Roles of folylpoly-gamma-glutamate synthetase in therapeutics with tetrahydrofolate antimetabolites: an overview.

Folylpoly-gamma-glutamate synthetase (FPGS) catalyzes the addition of several equivalents of glutamic acid to the gamma-carboxyl group in the side chain of folate cofactors and analogs. Folylpoly-gamma-glutamate synthetase has three functions in folate homeostasis in mammals: polyglutamation prevents efflux of folate cofactors from the cell, it increases the binding of folate cofactors to some of the enzymes of folate interconversion and biosynthesis, and it appears to allow the accumulation of folates in the mitochondria that are required for glycine synthesis. The efficient substrate activity of the newer generations of tetrahydrofolate analogs results in levels of intracellular accumulation of cytotoxic drug in any cell expressing FPGS in which the enzyme activity is not suppressed by feedback, and the binding of folate inhibitors of thymidylate synthase and glycinamide ribonucleotide formyltransferase is substantially increased by polyglutamation. Resistance to these drugs appears to be most frequently due to mutations that change the level of polyglutamation of parent compound, a clear indication of the centrality of the process to the cytotoxicity of these drugs. Folylpoly-gamma-glutamate synthetase is widely expressed in human tumors and is tightly linked either to proliferation or to a lack of differentiation. The cytotoxicity of both thymidylate synthase and purine inhibitors requires continued inhibition of target for greater than one generation time, so that the integrative function of FPGS adds considerably to the efficiency of folate antimetabolites.

Animals↗
PubMed1990

Tetrahydrohomofolate polyglutamates as inhibitors of thymidylate synthase and glycinamide ribonucleotide formyltransferase in Lactobacillus casei.

In order to determine the mechanism for the effects of homofolates on growth of Lactobacillus casei, polyglutamated derivatives of homofolate (HPteGlu), dihydrohomofolate and tetrahydrohomofolate (H4HPteGlu) were synthesized and tested as inhibitors of folate-requiring enzymes. The following L. casei enzymes were examined: thymidylate synthase (TS), glycinamide ribonucleotide formyltransferase (GARFT), aminoimidazolecarboxamide ribonucleotide formyltransferase, serine hydroxymethyltransferase and dihydrofolate reductase. Polyglutamates of (6R,S)-H4HPteGlu are potent inhibitors of TS and GARFT. For example, the IC50 values of (6R,S)-H4HPteGlu6 are 0.7 microM for TS and 0.3 microM for GARFT. By contrast, the value for HPteGlu6 is greater than 10 microM for both TS and GARFT. Inhibition of TS and GARFT by (6R,S)-H4HPteGlu derivatives increases with polyglutamate chain length. For TS, the Glu5 and Glu6 derivatives of (6R,S)-H4HPteGlu are 20 and 30 times more potent than the monoglutamate, respectively. For GARFT, the Glu2-6 derivatives are 2-3 times more potent than Glu1. Inhibition of TS and GARFT by (6R,S)-H4HPteGlu polyglutamates is almost entirely due to the unnatural (6R) diastereomer at C-6. Homofolate derivatives are only weak inhibitors of aminoimidazolecarboxamide ribonucleotide formyltransferase, serine hydroxymethyltransferase, and dihydrofolate reductase. We conclude that both TS and GARFT are potential targets of (6R)-H4HPteGlu polyglutamates.

Acyltransferases↗
PubMed2002

Rapid method for the determination of total 5-methyltetrahydrofolate in blood by liquid chromatography with fluorescence detection.

A liquid chromatographic method is described for the determination of total 5-methyltetrahydrofolate (5-MTHF) in whole-blood samples. The method was applied to a survey of whole-blood total 5-MTHF levels of women at child-bearing age. To determine whole-blood total 5-MTHF content, a whole-blood sample was frozen and thawed to break red blood cells and the 5-MTHF polyglutamates were released and hydrolyzed into 5-MTHF monoglutamate by endogenous polyglutamates hydrolase in the plasma. In brief, an aliquot of 0.1 ml whole-blood sample was mixed with 0.3 ml 57 mmol/l ascorbic acid and incubated at 37 degrees C for 60 min, then diluted with 0.6 ml buffer solution (0.2 mol/l potassium phosphate dibasic and 30 mmol/l mercaptoethanol, pH 8.5). After the sample was heated at 100 degrees C for 10 min and centrifuged, the supernatant was analyzed by reversed-phase liquid chromatography with fluorescence detection. The recoveries from spiked samples were from 95 to 105% with within-day and day-to-day relative standard deviations less than 6.5%. The detection limit was estimated to be 30 nmol/l based on three times the noise level (peak to peak). Application of the method to a survey of whole-blood total 5-MTHF levels of women at child-bearing age showed that the method was reliable and suitable for the determination of blood total 5-MTHF.

Chromatography, Liquid↗
PubMed1989

Folate cycle kinetics in human breast cancer cells.

A mathematical description of polyglutamated folate kinetics for human breast carcinoma cells (MCF-7) has been formulated based upon experimental folate, methotrexate (MTX), purine, and pyrimidine pool sizes as well as reaction rate parameters obtained from intact MCF-7 cells and their enzyme isolates. The schema accounts for the interconversion of highly polyglutamated tetrahydrofolate, 5-methyl-FH4, 5-10-CH2FH4, dihydrofolate (FH2), 10-formyl-FH4 (FFH4), and 10-formyl-FH2 (FFH2), as well as formation and transport of the MTX polyglutamates. Inhibition mechanisms have been chosen to reproduce all observed non-, un-, and pure competition inhibition patterns. Steady state folate concentrations and thymidylate and purine synthesis rates in drug-free intact cells were used to determine normal folate Vmax values. The resulting average-cell folate model, examined for its ability to predict folate pool behavior following exposure to 1 microM MTX over 21 h, agreed well with the experiment, including a relative preservation of the FFH4 and CH2FH4 pools. The results depend strongly on thymidylate synthase (TS) reaction mechanism, especially the assumption that MTX di- and triglutamates inhibit TS synthesis as greatly in the intact cell as they do with purified enzyme. The effects of cell cycle dependence of TS and dihydrofolate reductase activities were also examined by introducing G- to S-phase activity ratios of these enzymes into the model. For activity ratios down to at least 5%, cell population averaged folate pools were only slightly affected, while CH2FH4 pools in S-phase cells were reduced to as little as 10% of control values. Significantly, these folate pool dynamics were indicated to arise from both direct inhibition by MTX polyglutamates as well as inhibition by elevated levels of polyglutamated FH2 and FFH2.

Breast Neoplasms↗
PubMed1998

Cellular but not plasma pharmacokinetics of lometrexol correlate with the occurrence of cumulative hematological toxicity.

Lometrexol inhibits the first folate-dependent enzyme in de novo purine biosynthesis and is avidly polyglutamated and retained in tissues expressing folylpolyglutamate synthetase. Although clinical studies have been limited by cumulative toxicity, preclinical studies show that pretreatment with folic acid can protect normal tissue while maintaining tumor cytotoxicity. Therefore, a Phase I study of lometrexol every 21 days preceded by i.v. folic acid was initiated. Lometrexol was studied in six patients at 15 mg/m2, in six patients at 20 mg/m2, in three patients at 25 mg/m2, and in nine patients at 30 mg/m2. Patients received either 5 mg of folic acid 1 h before or 25 mg/m2 3 h before lometrexol. Blood samples were obtained around the first course and weekly thereafter for determination of plasma and erythrocyte (RBC) lometrexol concentrations. Bioactive folates in plasma and RBCs were determined in a subset of patients. Lometrexol pharmacokinetics were best described by a three-compartment model. Mean clearance and volume of distribution were 1.6 +/- 0.6 liters/h/m2 and 8.9 +/- 4.1 liters/m2. Mean half-lives were 0.23 +/- 0.1, 2.9 +/- 1.4, and 25.0 +/- 48.7 h. Pharmacokinetics were independent of either lometrexol or folic acid dose. In the weekly blood samples, RBC lometrexol levels rose, long after plasma lometrexol was undetectable. RBC lometrexol levels were independent of folic acid or lometrexol dose. Bioactive folates measured in plasma and RBCs during this same time period did not accumulate. Rising RBC levels were correlated with a fall in hematocrit, hemoglobin, and platelet count. This study indicates that the cumulative toxicity of lometrexol is related to tissue concentration and not plasma pharmacokinetics. RBC lometrexol may be an indicator of cumulative drug exposure and effect.

Antimetabolites, Antineoplastic↗
PubMed2004

Effects of novel peptides derived from the acidic tail of synuclein (ATS) on the aggregation and stability of fusion proteins.

The acidic tail of alpha-synuclein (ATSalpha) has been shown to protect the glutathione S-transferase (GST)-ATSalpha fusion protein from environmental stresses, such as heat, pH and metal ions. In this study, we further demonstrated that the introduction of ATSalpha into other proteins, such as dehydrofolate reductase and adiponectin, renders the fusion proteins resistant to heat-induced aggregation and that the acidic tail of beta- or gamma-synuclein can also protect the fusion proteins from heat-induced aggregation. Interestingly, the heat resistance of GST-ATSalpha deletion mutants, which contain shorter peptides derived from the highly charged regions of ATSalpha, was approximately proportional to the number of added Glu/Asp residues. However, the negative charges in the ATSalpha-derived peptides appear insufficient to explain the extreme heat resistance of the fusion proteins, since polyglutamates appeared to be much less effective than the ATSalpha-derived peptides in conferring heat resistance on the fusion proteins. These results suggest that not only the negatively charged residues but also the specific amino acid sequence of ATSalpha play an important role in conferring extreme heat resistance on the fusion proteins. Furthermore, the heat-induced secondary structural changes and thermal inactivation curves of GST-ATSalpha deletion mutants indicated that the introduction of ATSalpha-derived peptides does not significantly affect the intrinsic stability of the fusion proteins.

Adiponectin↗
PubMed1980

The bifunctional enzyme formiminotransferase-cyclodeaminase is a tetramer of dimers.

Formiminotransferase-cyclodeaminase, an octameric protein of identical, bifunctional polypeptides of Mr = 62,000, yields a transferase-active fragment of Mr = 80,000 upon proteolysis with chymotrypsin in the presence of the inhibitor folic acid. The purified fragment contains one size of polypeptide, Mr = 39,000, on dodecyl sulfate gels. Cross-linking with the bifunctional reagent dithiobis(succinimidyl propionate) confirmed the dimeric structure of the purified fragment. Reaction of the native octamer with the very short bifunctional reagent difluorodinitrobenzene yields dimer and tetramer in excess of trimer, thereby indicating two types of subunit interaction in the protein. The isolation of a dimeric fragment after proteolysis and the results of cross-linking support a tetramer of dimers structure for the native enzyme. The purified transferase fragment has approximately 68% of the activity of the native enzyme, but has lost specificity for the naturally occurring polyglutamate derivatives of tetrahydrofolate. This is illustrated by an increase in Km for tetrahydropteroylpentaglutamate from 3.4 microM with the native transferase to 89 microM with the fragment transferase. It is suggested that the bifunctional enzyme may have only one polyglutamate binding site/pair of transferase-deaminase sites.

Ammonia-Lyases↗
PubMed1997

Structure and regulation of expression of the Bacillus subtilis valyl-tRNA synthetase gene.

We have sequenced the valyl-tRNA synthetase gene (valS) of Bacillus subtilis and found an open reading frame coding for a protein of 880 amino acids with a molar mass of 101,749. The predicted amino acid sequence shares strong similarity with the valyl-tRNA synthetases from Bacillus stearothermophilus, Lactobacillus casei, and Escherichia coli. Extracts of B. subtilis strains overexpressing the valS gene on a plasmid have increased valyl-tRNA aminoacylation activity. Northern analysis shows that valS is cotranscribed with the folC gene (encoding folyl-polyglutamate synthetase) lying downstream. The 300-bp 5' noncoding region of the gene contains the characteristic regulatory elements, T box, "specifier codon" (GUC), and rho-independant transcription terminator of a gene family in gram-positive bacteria that encodes many aminoacyl-tRNA synthetases and some amino acid biosynthetic enzymes and that is regulated by tRNA-mediated antitermination. We have shown that valS expression is induced by valine limitation and that the specificity of induction can be switched to threonine by changing the GUC (Val) specifier triplet to ACC (Thr). Overexpression of valS from a recombinant plasmid leads to autorepression of a valS-lacZ transcriptional fusion. Like induction by valine starvation, autoregulation of valS depends on the presence of the GUC specifier codon. Disruption of the valS gene was not lethal, suggesting the existence of a second gene, as is the case for both the thrS and the tyrS genes.

Acylation↗
PubMed1980

Enzymatic synthesis of folylpolyglutamates. Characterization of the reaction and its products.

Rat liver folylpolyglutamate synthetase was partially purified and its reaction and products were characterized. The preparation contained no conjugase activity. Gel filtration analysis revealed a molecular weight of 69,000. The synthetase was optimally active at pH 8.4 (37 degrees C), required mercaptoethanol and a monovalent cation, and was highly specific for L-glutamate. Only purine nucleoside triphosphates served as the energy source for the reaction; ATP and dATP gave the best activity. All naturally occurring folates (including 5-methyl-tetrahydrofolic acid) as well as a number of folate analogs (including methotrexate) served as substrates. The unnatural diastereoisomer of at least one folate, 5,6,7,8-tetrahydrofolic acid, was also a substrate. Modifications of the terminal, acceptor glutamate led to loss of substrate activity, as well as loss of binding. High pressure liquid chromatography analysis, conjugase digestion, double radiolabel studies, and amino acid analysis of acid-hydrolyzed product confirmed that folylpoly-gamma-glutamates were synthesized. High concentrations of (dl)-5,6,7,8-tetrahydrofolic acid favored accumulation of short chain (predominantly diglutamate) products while low concentrations favored accumulation of longer chains (predominantly tetraglutamate). This inverse relationship between concentration and chain length may be of regulatory significance. Synthesis of pentaglutamate forms, the predominant chain length of rat liver folates in vivo, was detected at low (dl)-5,6,7,8-tetrahydrofolic acid, but hexaglutamate was not detected. Synthetic (l)-5,6,7,8-tetrahydropteroylpentaglutamate was a poor substrate for the synthetase but it inhibited formation of polyglutamates from monoglutamates. These observations indicate that the predominant chain length of folates in rat liver may be determined solely by the substrate specificity of the rat liver synthetase. Inhibition by the specificity of the rat liver synthetase. Inhibition by the pentaglutamate derivative offers a means by which folylpolyglutamates could regulate their own synthesis.

Adenosine Triphosphate↗
PubMed1982

Folate oligoglutamate:amino acid transpeptidase.

A new and previously unrecognized enzyme activity folate oligoglutamate:amino acid transpeptidase (folate transpeptidase), was detected in rat liver extracts. This activity involves the exchange of the terminal gamma-glutamyl residue (where PteGlu represents pteroylglutamic acid) of H4PteGlu-gamma-Glu, PteGlu-gamma-Glu, or p-aminobenzoyl-Glu-gamma-Glu for an amino acid such as [14C]glutamic acid, [14C]methionine, [14C]glutamine, or [14C]glycine. In the case of substrates PteGlu2 and [14C]Glu, the product is PteGlu-gamma-[14C]Glu. The enzyme responsible for catalyzing this activity was purified 39,000-fold by precipitation of inactive protein, fractionation on anionic and molecular sieve columns, and chromatography on diaminohexane-Sepharose. The enzyme functions maximally at pH 7.9, shows an absolute requirement for sulfhydryl reductants, and appears to have a molecular weight of 65,000. The function of folate transpeptidase is not known. A second, though much weaker, enzyme activity, tetrahydrofolic acid:amino acid ligase (folate ligase), co-purified with folate transpeptidase through all steps. Folate transpeptidase and the associated folate ligase activity were separated from folate polyglutamate synthetase during the initial steps of the purification procedure.

Acyltransferases↗
PubMed1989

Random tyrosine and glutamic acid-containing polymers are very powerful inhibitors of casein kinase-2.

The random heteropolymers Glu/Tyr(4:1) and Glu/Tyr(1:1) that are widely used as substrates for tyrosine protein kinases, are very powerful competitive inhibitors of casein kinase-2, but not of casein kinase-1, with respect to the protein substrate, their Ki values being one to two orders of magnitude lower than those of polyglutamates of similar size. The inhibitory power is reduced if tyrosine is partially replaced by alanine, as in the polymer Glu/Ala/Tyr(6:3:1) and it disappears upon inclusion of lysine, the polymer Glu/Ala/Lys/Tyr(2:6:5:1) actually behaving as a stimulator. These data indicate that non-phosphorylatable hydroxylic residues in addition to acidic ones are required in order to optimize the binding of pseudo-substrates to the catalytic site of casein kinase-2.

Casein Kinases↗