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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The model of a reversible reaction catalyzed by an oligomeric enzyme has been investigated. The regions of parameter values have been estimated at which the model describes "unidirectional" or alternative effects of allosteric modifiers on the rates of forward and backward reactions. The plausible explanation and mathematical description are offered for kinetic and regulatory peculiarities of reversible reactions catalyzed by NAD-dependent isocitrate dehydrogenase and catabolic ornithine carbamoylphosphate transferase. The phenomena of unidirectional catalysis of alternative directions of a reversible reaction by different enzymes are considered.
Mol Biol (Mosk)
PMID:[Kinetic and regulatory properties of reversible enzymic reactions]. 725 8

The ability of Fasciola hepatica to produce proline was assessed by measuring activity of the proline biosynthesis enzyme ornithine-delta-transaminase, and the arginine-dependent production of proline in vitro. This was related to the changes in bile proline of rats during the course of infection. It was found that the total production of proline and total enzyme activity per fluke increased as the fluke increased in size. However, while the specific enzyme activity remained constant throughout development, there was a preferential increase in proline production relative to protein content of the flukes just prior to their migration into the bile ducts. This early amplification of proline production could provide the stimulus to induce hyperplasia in the bile duct in preparation for fluke entry as proline production relative to protein content of fluke dropped markedly after migration into the bile ducts and never again reached the pre-migration level.
Mol Biochem Parasitol 1981 Jun
PMID:Proline biosynthesis by Fasciola hepatica at different developmental stages in vivo and vitro. 725 49

Several mechanisms have been demonstrated to be independently involved in methotrexate (MTX) resistance, including increased dihydrofolate reductase (DHFR) activity, decreased membrane transport, and decreased conversion to noneffluxing polyglutamates by folylpolyglutamate synthetase. We conducted the present study to test the hypothesis that nonpolyglutamatable antifolates with an N delta-hemiphthaloyl-L-ornithine side chain could be more potent than MTX against MTX-sensitive and -resistant human carcinoma cells via tighter DHFR binding, more efficient cellular uptake, the ability to bypass defective polyglutamation, or a combination. Two nonpolyglutamatable antifolates, N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine (PT523) and the new B-ring analogue N alpha-[4-[N-(2,4-diamino-5-chloroquinazolin-6-yl)methyl]amino] benzoyl-N delta-hemiphthaloyl-L-ornithine (PT619), were tested as inhibitors of purified recombinant human DHFR and were found to bind somewhat better to the enzyme than MTX as determined by competitive radioligand binding assay. PT523 and PT619 were 9- and 14-fold, respectively, more active than MTX as inhibitors of parental SCC25 human and neck squamous carcinoma cell growth in 72-hr cultures. Moreover, there was an even greater increase in relative potency against two previously described MTX-resistant cell lines with an increased DHFR content and a decreased ability to convert MTX to polyglutamates: SCC25/R1 (selected with MTX) and SCC25/CP (selected with cisplatin but collaterally resistant to MTX). Both PT523 and PT619 very efficiently inhibited [3H]MTX uptake by SCC25 cells in a 1-hr assay, with PT523 being 11-fold more potent and PT619 being 17-fold more potent than MTX. Greater inhibition of [3H]MTX uptake with PT523 and PT619 than with MTX was also observed in SCC25/R1 and SCC/CP cells. However, the increase in activity of PT523 and PT619 relative to MTX in uptake experiments was less than that in growth-inhibition assays, especially for SCC25/CP cells. This suggested that additional cytotoxicity determinants may exist in these resistant cells.
Mol Pharmacol 1995 Oct
PMID:Dihydrofolate reductase binding and cellular uptake of nonpolyglutamatable antifolates: correlates of cytotoxicity toward methotrexate-sensitive and -resistant human head and neck squamous carcinoma cells. 747 4

Characteristics of transport of L-arginine were studied in Leishmania donovani promastigotes grown in vitro in a defined medium. The promastigotes exhibited a time-dependent, temperature-sensitive, pH-dependent and saturable uptake of arginine. Metabolic inhibitors caused 81-92% inhibition, indicating that arginine influx in promastigotes is an energy requiring process. The presence of Na+ ions was necessary for full activity. Considerable inhibition was also noticed with valinomycin, gramicidin and amiloride. The transporter seems to involve an -SH group at the active site. The most distinctive feature of the leishmanial transporter was that lysine and ornithine did not show significant competition with arginine transport. Other neutral and acidic amino acids, as well as polyamines were also ineffective. The arginine analogues, viz., nitro-L-arginine methyl ester, N-nitro-L-arginine, aminoguanidine, agmatine and D-arginine were not recognised by the transporter, while N-methyl-L-arginine acetate and phospho-L-arginine showed competition, indicating stereo-specificity of the transporter and recognition of both the guanidino group, as well as the arginine side chain by the transporter. No exchange of intracellular [14C]arginine taken up by the promastigotes was noticed during incubation with 2 or 5 mM arginine in the extracellular medium. Eighty percent of the arginine taken up remained in the trichloroacetic acid-soluble fraction. Pentamidine caused competitive inhibition of arginine transport, exhibiting an IC50 value of 40 microM. Results indicate the presence of a novel distinct arginine transporter in Leishmania promastigotes.
Mol Biochem Parasitol 1995 May
PMID:Kinetics and molecular characteristics of arginine transport by Leishmania donovani promastigotes. 747 1

Studies on the mode of action of PT523 [N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine], a potent nonpolyglutamatable antifolate, were carried out in sensitive and resistant H35 rat hepatoma cell lines in culture, to compare it with other antifolates, including three dihydrofolate reductase (DHFR) inhibitors, i.e., methotrexate (MTX), gamma-fluoro-MTX, and trimetrexate (TMQ), two thymidylate synthase inhibitors, i.e., N10-propargyl-5,8- dideazafolate (PDDF) and 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolate (dmPDDF), and the glycinamide ribonucleotide formyltransferase inhibitor 5,10-dideaza-5,6,7,8-tetrahydrofolate. PT523 was the most active compound in this group against the parental H35 cells, with an IC50 ranging from 2.5 nM for 72 hr of treatment to 0.21 microM for 2 hr of treatment. Sublines resistant to MTX by virtue of a transport defect or a combination of defective transport and increased DHFR activity were resistant to PT523 and MTX but not to PDDF, whereas sublines resistant to fluoropyrimidines by virtue of increased thymidylate synthase activity were resistant to PDDF but not to PT523, TMQ, or MTX. Inhibition of H35 cell growth by PT523 was associated with a concentration- and time-related decrease in de novo dTMP and purine biosynthesis. Growth inhibition by PT523, MTX, and TMQ was prevented by leucovorin or a combination of thymidine (dThd) and hypoxanthine but not by dThd or hypoxanthine alone; in contrast, growth inhibition by dmPDDF was prevented by dThd alone. Intracellular reduced folate polyglutamate pools were markedly altered by PT523 treatment, with the most pronounced effect being an increase in 7,8-dihydrofolate mono- and polyglutamates and a decrease in 5,10-methylene-5,6,7,8-tetrahydrofolate mono- and polyglutamates, 5,6,7,8-tetrahydrofolate mono- and polyglutamates, and 10-formyl-5,6,7,8-tetrahydrofolate mono- and polyglutamates. This pattern was qualitatively similar to that observed with MTX and TMQ but different from that observed with dmPDDF or 5,10-dideaza-5,6,7,8-tetrahydrofolate, which resulted in little or no change in the folate species. Uptake of [3H]MTX and [3H]folinic acid, but not [3H]folic acid, by H35 cells was inhibited in a dose-related manner by PT523, suggesting that penetration of the cell probably involves, at least in part, active transport by the MTX/reduced folate carrier. To determine whether the potent cellular effects of PT523 might be due to chemical or enzymic clevage to N'-(4-amino-4-deoxypteroyl)-L-ornithine, a potent inhibitor of folylpolyglutamate synthetase, the formation of [3H]MTX polyglutamates in CCRF-CEM lymphoblasts pulsed with [3H]MTX after preincubation with PT523 was examined.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1994 Apr
PMID:Biochemical studies on PT523, a potent nonpolyglutamatable antifolate, in cultured cells. 751 64

Airway wall remodeling, including hyperplasia of airway smooth muscle, is regarded as an important contributor to airway hyperresponsiveness in asthmatic patients. The effects of the proinflammatory cytokine, tumor necrosis factor alpha (TNF alpha) on the mitogenic responses of human cultured airway smooth muscle have been investigated. Lower concentrations of TNF alpha (0.3 to 30 pM) had a small, delayed (48-h incubation), stimulatory effect on DNA synthesis that was blocked by dexamethasone (1 microM), aspirin (100 microM), or primaquine (30 microM) pretreatment, indicating that this effect was secondary to the release of cyclooxygenase products. TNF alpha (300 pM; 24- to 48-h incubation) alone had no effect on cell number or DNA or protein synthesis, but markedly reduced the stimulatory effects of thrombin (0.3 U/ml). TNF alpha (300 pM) also inhibited mitogenic responses to fetal calf serum (10%), epidermal growth factor (300 pM), and the thromboxane A2 mimetic U46619 (100 nM), indicating a nonselective effect. The inhibitory effects of TNF alpha (300 pM) were not blocked by pretreating the cells with the cyclooxygenase inhibitor aspirin (100 microM), the 5-lipoxygenase inhibitor CGS 8515 (3 microM), or the nitric oxide synthase inhibitor nitro-iminoethyl-L-ornithine (100 microM), suggesting that neither arachidonic acid metabolites nor nitric oxide were mediators of the inhibitory effect. The phospholipase A2 inhibitor primaquine (30 microM) had no effect on the inhibitory responses to TNF alpha, whereas the anti-inflammatory steroid dexamethasone (1 microM) prevented TNF alpha inhibition of mitogenic responses. Thus, concentrations of TNF alpha, within the range detected in bronchoalveolar lavage fluid from asthmatics, suppress mitogenic responses by a mechanism that is sensitive to inhibition by anti-inflammatory steroids, but does not appear to involve established targets for modulation by steroids, including arachidonic acid metabolism or induction of nitric oxide synthase.
Am J Respir Cell Mol Biol 1995 Jan
PMID:Tumor necrosis factor alpha modulates mitogenic responses of human cultured airway smooth muscle. 752 28

Three genes called rocD, rocE and rocF were found near the rocR gene in B. subtilis. The product of rocD is similar to eukaryotic ornithine aminotransferases. The product of rocE shares similarity with the product of B. subtilis rocC and with the product of E. coli lysP. The rocE gene may encode an arginine permease. The rocF gene encodes a polypeptide similar to several arginases. Heterologous expression in E. coli indicated that rocD encodes an ornithine aminotransferase and that rocF encodes an arginase. Arginine utilization was abolished in both rocD and rocF mutants of B. subtilis confirming the role of these genes in arginine catabolism. The rocDEF genes form an operon transcribed from a -12, -24 promoter almost identical to the -12, -24 promoter of the rocABC operon. The expression of the rocDEF operon was induced by the presence of arginine, ornithine or proline in the growth medium and depended on the presence of the sigma factor SigL. Transcription of this operon was also abolished in a B. subtilis strain containing a null mutation in the regulatory gene rocR. Two tandemly repeated upstream activating sequences very similar to those previously identified in the rocABC system were found centered at positions -120 and -70, respectively, upstream from the transcription start site of rocDEF. Deletion analysis showed that at least one upstream activating sequence is involved in the expression of the rocDEF operon. These sequences are probably the target of RocR. Analysis of a rocR'-'lacZ fusion strain showed that the expression of rocR is not induced by arginine and is negatively autoregulated.
J Mol Biol 1995 Jun 23
PMID:Expression of the rocDEF operon involved in arginine catabolism in Bacillus subtilis. 754 Jun 94

Ornithine decarboxylase from Lactobacillus 30a (L30a OrnDC) is representative of the large, pyridoxal-5'-phosphate-dependent decarboxylases that act on lysine, arginine or ornithine. The crystal structure of the L30a OrnDC has been solved to 3.0 A resolution using MIR phases in combination with density modification (space group P6; a = 195.6 A, c = 97.6 A; dimer of 1460 amino acid residues/asymmetric unit; VM = 3.26 A3/Da). The refined crystallographic R-value was 0.219 (Rfree = 0.268) using 2-fold restraints with a 4 sigma cutoff and 8.0 to 3.0 A resolution data. Six dimers related by C6 symmetry compose the enzymatically active dodecamer (approximately 10(6) Da). Each monomer of L30a OrnDC can be described in terms of five sequential folding domains. The amino-terminal domain, residues 1 to 107, consists of a five-stranded beta-sheet termed the "wing" domain. Two wing domains of each dimer project inward towards the center of the dodecamer and contribute to dodecamer stabilization. The "linker" domain, residues 108 to 160, consists of short alpha-helices separated by a loop that fills in the PLP pocket. The third domain, residues 161 to 413, is an alpha/beta domain containing a seven stranded beta-sheet that resembles the PLP-binding domain of the aspartate aminotransferases. The fourth domain, residues 414 to 569, resembles the "small" domain of the aspartate aminotransferases, but is significantly larger due to insertions. The remaining carboxy-terminal domain, residues 570 to 730, is organized into multiple antiparallel loops and seven alpha-helices that help form a deep channel leading to the PLP-binding site.
J Mol Biol 1995 Oct 06
PMID:Crystallographic structure of a PLP-dependent ornithine decarboxylase from Lactobacillus 30a to 3.0 A resolution. 756 80

The transport properties and growth-inhibitory potential of 37 classic and novel antifolate compounds have been tested in vitro against human and murine cell lines expressing different levels of the reduced folate carrier (RFC), the membrane-associated folate binding protein (mFBP), or both. The intracellular targets of these drugs were dihydrofolate reductase (DHFR), glycinamide ribonucleotide transformylase (GARTF), folylpolyglutamate synthetase (FPGS), and thymidylate synthase (TS). Parameters that were investigated included the affinity of both folate-transport systems for the antifolate drugs, their growth-inhibitory potential as a function of cellular RFC/mFBP expression, and the protective effect of either FA or leucovorin against growth inhibition. Methotrexate, aminopterin, N10-propargyl-5,8-dideazafolic acid (CB3717), ZD1694, 5,8-dideazaisofolic acid (IAHQ), 5,10-dideazatetrahydrofolic acid (DDATHF), and 5-deazafolic acid (efficient substrate for FPGS) were used as the basic structures in the present study, from which modifications were introduced in the pteridine/quinazoline ring, the C9-N10 bridge, the benzoyl ring, and the glutamate side chain. It was observed that RFC exhibited an efficient substrate affinity for all analogues except CB3717, 2-NH2-ZD1694, and glutamate side-chain-modified FPGS inhibitors. Substitutions at the 2-position (e.g., 2-CH3) improved the RFC substrate affinity for methotrexate and aminopterin. Other good substrates included PT523 (N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine), 10-ethyl-10-deazaaminopterin, and DDATHF. With respect to mFBP, modifications at the N-3 and 4-oxo positions resulted in a substantial loss of binding affinity. Modifications at other sites of the molecule were well tolerated. Growth-inhibition studies identified a series of drugs that were preferentially transported via RFC (2,4-diamino structures) or mFBP (CB3717, 2-NH-ZD1694, or 5,8-dideazaisofolic acid), whereas other drugs were efficiently transported via both transport pathways (e.g., DDATHF, ZD1694, BW1843U89, or LY231514). Given the fact that for an increasing number of normal and neoplastic cells and tissue, different expression levels of RFC and mFBP are being recognized, this folate antagonist structure-activity relationship can be of value for predicting drug sensitivity and resistance of tumor cells or drug-related toxicity to normal cells and for the rational design and development of novel antifolates.
Mol Pharmacol 1995 Sep
PMID:Carrier- and receptor-mediated transport of folate antagonists targeting folate-dependent enzymes: correlates of molecular-structure and biological activity. 756 26

N omega-Substituted analogues of L-arginine have proven useful as specific inhibitors of nitric oxide formation in various biological systems. In the present study we describe the characteristics of amino acid transporters that mediate uptake of N omega-methyl-L-arginine (L-NMA) and N omega-nitro-L-arginine (L-NNA) into cultured porcine aortic endothelial cells. The transport of L-[14C]NMA showed biphasic kinetics, with Km values of 4 and 368 microM, and was inhibited by L-arginine, L-homoarginine, L-lysine, and L-ornithine but not by L-leucine or L-isoleucine. Similar transport kinetics (Km values of 6 and 609 microM) and substrate specificities were obtained for L-[3H]arginine uptake, indicating that L-arginine and L-NMA are transported by the same system. In contrast to L-arginine and L-NMA transport, uptake of L-[3H]NNA was monophasic (Km = 617 microM) and was inhibited by L-leucine and L-isoleucine but not by L-arginine, L-homoarginine, L-NMA, L-lysine, or L-ornithine. Uptake studies with L-[3H]leucine revealed that the transport of this amino acid occurred in a manner very similar to that of L-[3H]NNA transport, suggesting that the uptake of both compounds may be mediated by the same system. In additional experiments, we determined the effects of L-NMA and L-NNA on the A23187-induced accumulation of intracellular cGMP, to establish to what extent these transport systems are involved in the actions of nitric oxide synthase inhibitors. L-Lysine and L-ornithine, which both inhibited L-NMA uptake, increased the IC50 of L-NMA from 7.8 microM to 57 microM but did not reduce the inhibitory effects of L-NNA. In the presence of L-leucine or L-isoleucine, however, which both inhibited L-NNA uptake, the IC50 of L-NNA was increased from 1.2 microM to 37 microM but the inhibitory actions of L-NMA remained unaffected. These data demonstrate that the endothelial transport systems for L-arginine and L-leucine mediate the biological effects of L-NMA and L-NNA, respectively.
Mol Pharmacol 1993 Sep
PMID:Characterization of endothelial cell amino acid transport systems involved in the actions of nitric oxide synthase inhibitors. 769 Apr 51


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