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At least 289 records · Page 16Linked to original sources
PubMed1988

p-Aminobenzoylpolyglutamates with hydrophobic end groups. A new class of inhibitors of hemoglobin S polymerization.

We have synthesized and tested a new series of compounds that inhibit the polymerization of deoxyhemoglobin S by noncovalent interaction. They consist of three structural elements: A p-aminobenzoyl residue to anchor the compound in the central cavity between the beta chains, a number of glutamates in gamma linkage to provide tight binding, and one or two hydrophobic amino acid residues which block the intermolecular hydrophobic interaction of valine beta 6. The most active compound was p-aminobenzoyl-(gamma-Glu)5-Phe-Phe. It increases the solubility of deoxy-HbS by a factor of 1.3 at a concentration of only 5-6 mM and is effective even in the presence of physiological concentrations of 2,3-diphosphoglycerate.

4-Aminobenzoic Acid↗
PubMed1985

Formation of 7-hydroxymethotrexate polyglutamyl derivatives and their cytotoxicity in human chronic myelogenous leukemia cells, in vitro.

The rapid synthesis of poly-gamma-glutamyl derivatives of 7-hydroxymethotrexate (7-OH-MTX) and their selective intracellular retention are reported in human chronic myelogenous leukemia cells, K-562. After a 30-min exposure to 5 microM [3H]7-OH-MTX, three different polyglutamyl derivatives were detected by high-performance liquid chromatography. When extracellular 7-OH-MTX was removed, the 7-OH-MTX diglutamate level declined slowly in comparison to the monoglutamate, but the higher polyglutamyl derivative levels increased. Within 10 min after exposure of cells to 7-OH-MTX, the level of these polyglutamyl derivatives far exceeds the dihydrofolate reductase binding capacity. Gel filtration or charcoal binding analysis followed by high-performance liquid chromatography analysis of the bound component showed intracellular binding of virtually all 7-OH-MTX tetraglutamate at a level 4-fold higher than that of the dihydrofolate reductase binding capacity. No bound 7-OH-MTX diglutamate or triglutamate could be detected. Treatment of the 7-OH-MTX tetraglutamate: protein complex with 100 microM unlabeled methotrexate (MTX) for 15 min resulted in only a partial dissociation of this complex to an extent compatible with the dihydrofolate reductase level. The residual 7-OH-MTX tetraglutamate remained bound to a site with a molecular weight of approximately 25,000 to 35,000 as assessed by Bio-Gel P-60 analysis and could not be displaced by folic acid, 5-formyltetrahydrofolate, 7-OH-MTX, or the tetraglutamate of MTX. 7-OH-MTX and MTX cytotoxicities were compared by clonogenic assay in agar and by their effects on cell growth. After a 2-hr exposure, the 50% inhibitory concentrations for 7-OH-MTX and MTX in cells growing in agar were 10(-5) and 10(-6) M, respectively. A 10-fold difference in cytotoxicity was also observed in cells growing in suspension. Continuous exposure to glycine: adenosine: thymidine completely protects cells from a sustained exposure to 7-OH-MTX over the entire period of clonal growth. However, even a brief exposure to 7-OH-MTX also requires continuous exposure to glycine: adenosine: thymidine for protection. This suggests that, as observed for MTX, the 7-OH-MTX polyglutamyl derivatives that are retained within the cells have a sustained cytotoxic effect after the monoglutamate is removed.

Cell Division↗
PubMed2003

Loss of folylpoly-gamma-glutamate synthetase activity is a dominant mechanism of resistance to polyglutamylation-dependent novel antifolates in multiple human leukemia sublines.

We have studied the molecular basis of drug resistance in human CCRF-CEM leukemia cells exposed to high dose intermittent pulses of novel polyglutamatable antifolates that target various folate-dependent enzymes. These include the dihydrofolate reductase (DHFR) inhibitors edatrexate, methotrexate and aminopterin, the thymidylate synthase (TS) inhibitors ZD1694 and GW1843, the glycinamide ribonucleotide formyltransferase (GARTF) inhibitor DDATHF as well as the multitargeted antifolate LY231514 inhibiting both TS, DHFR and GARTF. Fourteen antifolate-resistant sublines were isolated, 11 of which displayed a drug resistance phenotype that was based on impaired folylpoly-gamma-glutamate synthetase (FPGS) activity as these cell lines: 1) typically lost 90-99% of parental FPGS activity; 2) expressed 1.4-3.3-fold less FPGS mRNA (only 4 cell lines); 3) displayed up to 10(5)-fold resistance to polyglutamylation-dependent antifolates including ZD1694 and MTA; 4) retained sensitivity to polyglutamylation-independent antifolates including ZD9331 and PT523; 5) were up to 19-fold hypersensitive to the lipid-soluble antifolates trimetrexate and AG377; 6) had a normal or a small decrease in [(3)H]MTX transport; and 7) had a 2.1-8.3-fold decreased cellular folate pools and a consequently increased folate growth requirement. The remaining 3 antifolate-resistant sublines lost 94-97% of parental [(3)H]MTX transport and thus displayed a high level resistance to all hydrophilic antifolates. To screen for mutations in the hFPGS gene, we devised an RT-PCR single strand conformational polymorphism (SSCP) assay. RT-PCR-SSCP analysis and DNA sequencing showed that only a single FPGS-deficient subline harbored an FPGS mutation (Cys346Phe). Three-dimensional modeling of the human FPGS based on the crystal structure of Lactobacillus casei FPGS suggested that this mutation maps to the active site and interferes with the catalytic activity of the enzyme due to a putative bulky clash between the mutant Phe346 and a native Phe350 within alpha-helix A10 in a highly conserved C-terminal hydrophobic core. This was consistent with a 23-fold decreased affinity of the mutant Cys346Phe FPGS for L-glutamate. We conclude that decreased FPGS activity is a dominant mechanism of resistance to polyglutamylation-dependent novel antifolates upon a high-dose intermittent exposure schedule. The finding that cells may exhibit 5 orders of magnitude of resistance to polyglutamylation-dependent antifolates but in the same time retain parental sensitivity or hypersensitivity to polyglutamylation-independent antifolates or lipophilic antifolates offers a potentially promising treatment strategy in the overcoming of FPGS-based anticancer drug resistance.

Base Sequence↗
PubMed1986

Folylpolyglutamate synthetase inhibition and cytotoxic effects of methotrexate analogs containing 2,omega-diaminoalkanoic acids.

The properties of a series of methotrexate analogs containing 2,omega-diaminoalkanoic acids have been investigated. The compounds were potent inhibitors of dihydrofolate reductase but, unlike methotrexate, they were also inhibitors of mammalian folylpolyglutamate synthetases. The potency of synthetase and reductase inhibition increased with increasing length of the 2,omega-diaminoalkanoate moiety. The most cytotoxic compound and the most potent inhibitor of both dihydrofolate reductase (I50 = 2.5 to 4 nM) and folylpolyglutamate synthetase (Ki ca. 4 microM) contained 2,5-diaminopentanoic acid (ornithine). These compounds were 70- to 100-fold less cytotoxic than methotrexate to human leukemia cell lines; however, they retained their potency against sublines resistant to methotrexate via defective transport. Their dual loci of enzyme inhibition and their efficacy against methotrexate transport-defective cell lines indicate that these compounds may be an important new class of antifol.

Aldehyde Oxidase↗
PubMed2003

Differences in effects on DNA gyrase activity between two glutamate racemases of Bacillus subtilis, the poly-gamma-glutamate synthesis-linking Glr enzyme and the YrpC (MurI) isozyme.

Bacillus subtilis possesses two isogenes encoding glutamate racemases, the poly-gamma-glutamate synthesis-linking Glr enzyme and the YrpC isozyme, and produces abundant amounts of the Glr enzyme. The YrpC isozyme, but not the Glr enzyme, was found to influence the activity of DNA gyrase, as did the MurI-type glutamate racemase of Escherichia coli, which is involved in peptidoglycan synthesis during cell division.

Amino Acid Isomerases↗
PubMed1978

Evidence for an essential arginine recognition site on adenosine 3':5'-cyclic monophosphate-dependent protein kinase of rabbit skeletal muscle.

On the basis of the chemical and structural features of the amino acid sequences in the vicinities of phosphorylatable hydroxyamino acid residues in several of the well-known protein substrates for skeletal-muscle cyclic AMP-dependent protein kinase, it is hypothesized that the phosphorylatable residue at position i and arginine residue at position i-3 of these protein substrates are located on a peptide turn on the hydrophilic protein surface. It is further hypothesized that there is an arginine-recognition site near the active centre on the protein kinase. This site is essential for the function of cyclic AMP-dependent protein kinase, for, not only does it recognize specifically the exposed arginine residue of the protein substrate, but, more importantly, via the interaction with arginine-(i--3), it may help to steer the topologically adjacent serine-i into proper orientation on the nearby active centre for phosphorylation. Model-building and kinetic data that provide support for the proposed hypotheses are presented.

Adenosine Triphosphate↗
PubMed2000

Polyglutamylation of folate coenzymes is necessary for methionine biosynthesis and maintenance of intact mitochondrial genome in Saccharomyces cerevisiae.

One-carbon metabolism is essential to provide activated one-carbon units in the biosynthesis of methionine, purines, and thymidylate. The major forms of folates in vivo are polyglutamylated derivatives. In organisms that synthesize folate coenzymes de novo, the addition of the glutamyl side chains is achieved by the action of two enzymes, dihydrofolate synthetase and folylpolyglutamate synthetase. We report here the characterization and molecular analysis of the two glutamate-adding enzymes of Saccharomyces cerevisiae. We show that dihydrofolate synthetase catalyzing the binding of the first glutamyl side chain to dihydropteroate yielding dihydrofolate is encoded by the YMR113w gene that we propose to rename FOL3. Mutant cells bearing a fol3 mutation require folinic acid for growth and have no dihydrofolate synthetase activity. We show also that folylpolyglutamate synthetase, which catalyzes the extension of the glutamate chains of the folate coenzymes, is encoded by the MET7 gene. Folylpolyglutamate synthetase activity is required for methionine synthesis and for maintenance of mitochondrial DNA. We have tested whether two folylpolyglutamate synthetases could be encoded by the MET7 gene, by the use of alternative initiation codons. Our results show that the loss of mitochondrial functions in met7 mutant cells is not because of the absence of a mitochondrial folylpolyglutamate synthetase.

Amino Acid Sequence↗
PubMed1993

A depletion-repletion folate bioassay based on growth and tissue folate concentrations of rats.

To improve standardization of a folate bioassay, folate-depleted rats were repleted with a folate-free amino acid-based diet supplemented with 29 levels of folic acid. Growth was the main response variable and body tissue folate concentrations were also assessed. Because a positive correlation was observed between low levels of dietary folic acid and growth and little or no correlation was observed between high levels and growth, six regression models with a steep slope for low levels and a shallow or zero slope for high levels of dietary folic acid were evaluated. The model referred to as the "two-phase regression" or "change-point" model best described the relationship. Depleted rats needed 674 +/- 71 nmol folic acid/kg diet to reach their full growth potential. This value is biologically sensible, and this regression model is well established in the statistical literature. The change-point model is highly recommended to characterize the growth response, because growth is a functional response and, in the range of 226 to 680 nmol folic acid/kg, this response is linear, which is an additional advantage. Linear responses are easier to interpret because of complicated issues of interpretation and confidence intervals with nonlinearities. Linear regressions described serum and liver folate responses, whereas exponentials described whole blood and carcass folate responses. Depleted rats needed 5920 and 5780 nmol folic acid/kg diet to maximize their whole blood and carcass folates, respectively.

Animals↗
PubMed1994

Construction of a new teichuronopeptide-defective derivative from alkaliphilic Bacillus sp. C-125 by cell fusion.

Cell walls of facultative alkaliphilic Bacillus sp. C-125 are composed of peptidoglycan, teichuronic acid, and teichuronopeptide. A mutant lacking teichuronic acid, or defective in both teichuronic acid and teichuronopeptide has been isolated from the organism. We now constructed another type of a cell-wall defective mutant that was defective in teichuronopeptide but had teichuronic acid, by cell fusion using protoplasts prepared from a wild-type strain and a mutant defective in teichuronopeptide and teichuronic acid. This mutant grew more poorly than wild type or a teichuronic acid-defective strain of C-125. The growth, however, was faster than that of the parental strain defective in both teichuronic acid and teichuronopeptide.

Bacillus↗
PubMed1996

Polyglutamylation of the dihydrofolate reductase inhibitor gamma-methylene-10-deazaaminopterin is not essential for antitumor activity.

As part of a continuing program aimed at developing nonpolyglutamylatable inhibitors of dihydrofolate reductase that are less toxic and more specific in their action, we herein report the therapeutic efficacy and toxicity of gamma-methylene-10-deazaaminopterin (MDAM) in athymic nude mice bearing advanced human HCT-8 ileocecal xenografts and its antitumor activity in C57BL/6 x DBA/2 F1 (hereafter called B6D2F1) mice bearing P388 murine leukemia. For the xenograft study, MDAM was administered at the maximum tolerated dose by the following dose schedules: (a) 5-day continuous i.v. infusion at 1.0 mg/kg/day (schedule I); and (b) i.v. push, daily for 5 days at 50 mg/kg/day (schedule II). The maximum tolerated dose values for methotrexate (MTX) under these conditions were 0.2 and 1.0 mg/kg/day for schedule I and schedule II, respectively. MTX did not exhibit any significant antitumor activity in this model system by both schedules; however, MDAM induced complete responses of 13 and 25% and partial responses of 25 and 50% by schedules I and II, respectively. MDAM also exhibited antitumor activity significantly superior to that of MTX in the P388 tumor model. One of the enantiomers of MDAM, which possesses the natural configuration at the gamma-methyleneglutamate moiety (l-MDAM), has been shown to be a better inhibitor of human recombinant dihydrofolate reductase and H35 hepatoma cell growth than D,L-MDAM. L-MDAM inhibited the uptake of radiolabeled folinic acid to H35 hepatoma cells eight times more efficiently than MTX. The results indicate that the superior activity of MDAM relative to MTX may be partially due to a combination of enhanced transport to tumor cells and slower deactivation by aldehyde oxidase.

Aminopterin↗
PubMed2001

Isolation of Bacillus subtilis (chungkookjang), a poly-gamma-glutamate producer with high genetic competence.

A bacterium with high poly-gamma-glutamate (PGA) productivity was isolated from the traditional Korean seasoning, Chung-Kook-Jang. This bacterium could be classified as a Bacillus subtilis, but sporulation in culture was infrequent in the absence of Mn2+. It was judged to be a variety of B. subtilis and designated B. subtilis (chungkookjang). L-Glutamate significantly induced PGA production, and highly elongated PGAs were synthesized. The volumetric yield reached 13.5 mg ml(-1) in the presence of 2% L-glutamate. The D-glutamate content was over 50% in every PGA produced under the conditions used. During PGA production, glutamate racemase activity was found in the cells, suggesting that the enzyme is involved in the D-glutamate supply. Molecular sizes of PGAs were changed by the salt concentration in the medium; PGAs with comparatively low molecular masses were produced in culture media containing high concentrations of NaCl. B. subtilis (chungkookjang) harbors no plasmid and is the first B. subtilis strain reported with both naturally high PGA productivity and high genetic competence.

Alanine Transaminase↗
PubMedDate not supplied

Protease-catalyzed regioselective polymerization and copolymerization of glutamic acid diethyl ester.

Protease-catalyzed polymerization and copolymerization of L-glutamic acid diethyl ester hydrochloride (1) have been performed in a buffer of high concentration. Papain and bromelain showed high catalytic activity toward the polymerization. H-H COSY NMR analysis of the product showed the exclusive formation of poly(alpha-peptide), which was further confirmed by comparison with NMR spectra of poly(alpha-methyl gamma-L-glutamate). The papain-catalyzed polymerization of gamma-methyl L-glutamate did not occur under the similar reaction conditions, supporting the regioselective production of the polymer having an alpha-peptide linkage from 1. The effects of the reaction parameters have been systematically investigated. The copolymerization of 1 with various amino acid esters took place by the papain catalyst to give peptide copolymers.

Amino Acids↗