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The Biochemical Journal Aug 1991We investigated the effects of glutamine and histidine analogues on glutamine transport processes in membrane vesicles prepared from rat liver (sinusoidal membrane) and...
We investigated the effects of glutamine and histidine analogues on glutamine transport processes in membrane vesicles prepared from rat liver (sinusoidal membrane) and skeletal muscle (sarcolemma). L-[14C]Glutamine is transported in these membranes predominantly by Systems N/Nm (liver and muscle respectively), and to a lesser extent by Systems A and L (e.g. about 60, 20 and 20% of total flux respectively via Systems N, A and L at 0.05 mM-glutamine in liver membrane vesicles). The glutamine anti-metabolites 6-diazo-5-oxo-L-norleucine and acivicin were relatively poor inhibitors of glutamine uptake into liver membrane vesicles (less than 25% inhibition at 20-fold excess) and appeared primarily to inhibit System A activity (i.e. N-methylaminoisobutyric acid-inhibitable glutamine uptake). In similar experiments azaserine (also a glutamine anti-metabolite) inhibited approx. 50% of glutamine uptake, apparently by inhibition of System A and also of System L (i.e. 2-amino-2-carboxybicyclo[2,2,1]heptane-inhibitable glutamine uptake). Glutamate gamma-hydroxamate, aspartate beta-hydroxamate, histidine and N'-methylhistidine were all strong inhibitors of glutamine uptake into liver membrane vesicles (greater than 65% inhibition at 20-fold excess), but neither homoglutamine nor N'-methylhistidine produced inhibition. L-Glutamate-gamma-hydroxamate was shown to be a competitive inhibitor of glutamine transport via System N (Ki approximately 0.6 mM). Glutamine uptake in sarcolemmal vesicles showed a similar general pattern of inhibition as in liver membrane vesicles. The results highlight limits on the substrate tolerance of System N; we suggest that the presence of both an L-alpha-amino acid group and a nitrogen group with a delocalized lone-pair of electrons (amide or pyrrole type), separated by a specific intramolecular distance (C2-C4 chain equivalent), is important for substrate recognition by this transporter.
Topics: Amino Acids; Amino Acids, Cyclic; Aminoisobutyric Acids; Animals; Azaserine; Biological Transport; Carrier Proteins; Cell Membrane; Diazooxonorleucine; Female; Glutamine; Kinetics; Lithium; Liver; Muscles; Rats; Rats, Inbred Strains; Sarcolemma; Sodium
PubMed: 1883322
DOI: 10.1042/bj2780105 -
The Biochemical Journal Jan 1991Incorporation of [35S]sulphate by cultures of matrix-free cells from chick embryo sterna in the presence of the glutamine analogue 6-diazo-5-oxo-L-norleucine (0.58 mM)...
Post-translational alterations in newly synthesized cartilage proteoglycans induced by the glutamine analogue 6-diazo-5-oxo-L-norleucine. Time course of inhibition and recovery.
Incorporation of [35S]sulphate by cultures of matrix-free cells from chick embryo sterna in the presence of the glutamine analogue 6-diazo-5-oxo-L-norleucine (0.58 mM) was inhibited in a time-dependent manner to less than 15% of that in control cultures after 2 h. Characterization of the major cartilage proteoglycan synthesized under these conditions showed that it contained few, if any, normal-sized chondroitin sulphate chains and only about half of the normal complement of substituted serine residues. Subsequent addition of D-glucosamine hydrochloride (final concn. 2 mM) resulted in a time-dependent recovery of [35S]sulphate incorporation to 90% of control cultures after 2 h, but restored the chondroitin sulphate chains to normal size within 15 min. On the basis of these results, it is concluded that a 2 h preincubation is necessary to deplete the chondrocytes of the endogenous supply of UDP-N-acetyl-D-glucosamine required for optimal glycoconjugate synthesis, and that this situation results in the synthesis of a chondroitin sulphate proteoglycan with significantly altered properties, owing to the paucity of glycosaminoglycan chains; however, this condition is completely reversible if the D-glucosamine pool is repleted.
Topics: Aggrecans; Animals; Cartilage; Cells, Cultured; Chick Embryo; Chondroitin Sulfate Proteoglycans; Chromatography, Gel; Diazooxonorleucine; Extracellular Matrix Proteins; Glucosamine; Glutamine; Glycoproteins; Kinetics; Lectins, C-Type; Protein Processing, Post-Translational; Proteoglycans; Sulfur Radioisotopes; Uridine Diphosphate N-Acetylglucosamine
PubMed: 1991027
DOI: 10.1042/bj2730283 -
The Journal of Biological Chemistry Jul 1990The glutamine antagonists, acivicin (NSC 163501), azaserine (NSC 742), and 6-diazo-5-oxo-L-norleucine (DON) (NSC 7365), are potent inhibitors of many glutamine-dependent...
The glutamine antagonists, acivicin (NSC 163501), azaserine (NSC 742), and 6-diazo-5-oxo-L-norleucine (DON) (NSC 7365), are potent inhibitors of many glutamine-dependent amidotransferases in vitro. Experiments performed with mouse L1210 leukemia growing in culture show that each antagonist has different sites of inhibition in nucleotide biosynthesis. Acivicin is a potent inhibitor of CTP and GMP synthetases and partially inhibits N-formylglycineamidine ribotide (FGAM) synthetase of purine biosynthesis. DON inhibits FGAM synthetase, CTP synthetase, and glucosamine-6-phosphate isomerase. Azaserine inhibits FGAM synthetase and glucosamine-6-phosphate isomerase. Large accumulations of FGAR and its di- and triphosphate derivatives were observed for all three antagonists which could interfere with the biosynthesis of nucleic acids, providing another mechanism of cytotoxicity. Acivicin, azaserine, and DON are not potent inhibitors of carbamyl phosphate synthetase II (glutamine-hydrolyzing) and amidophosphoribosyltransferase in leukemia cells growing in culture although there are reports of such inhibitions in vitro. Blockade of de novo purine biosynthesis by these three antagonists results in a "complementary stimulation" of de novo pyrimidine biosynthesis.
Topics: Animals; Azaserine; Azo Compounds; Carbon-Nitrogen Ligases; Cell Line; Chromatography, High Pressure Liquid; Diazooxonorleucine; Glutamine; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); Isoxazoles; Leukemia L1210; Ligases; Mice; Nucleotides; Oxazoles; Spectrophotometry, Ultraviolet
PubMed: 2358467
DOI: No ID Found -
The American Journal of Anatomy Jun 1990Hyaluronate (HA) distribution patterns were examined in the cranial mesenchyme underlying the mesencephalic neural folds of mouse embryos maintained in roller tube...
Hyaluronate (HA) distribution patterns were examined in the cranial mesenchyme underlying the mesencephalic neural folds of mouse embryos maintained in roller tube culture. Using standard image-processing techniques, the digitized images of Alcian blue-stained or 3H-glucosamine-labeled sections digested with an enzyme specific for HA, were subtracted from adjacent, undigested sections. The resultant difference picture images (DPI) accurately depicted the distribution of stained or labeled HA within the cranial mesenchyme. 3H-glucosamine-labeled HA was distributed uniformly throughout the cranial mesenchyme as 12, 18, and 24 hr of culture. By contrast, the mesenchyme was uniformly stained with Alcian blue at 12 hr, but stain intensity decreased in the central regions of the mesenchyme at 18 and 24 hr. HA distribution patterns were also examined in the cranial mesenchyme of embryos cultured in the presence of diazo-oxo-norleucine (DON), a glutamine analogue that inhibits glycosaminoglycan and glycoprotein synthesis. In DON-treated mesenchyme, Alcian blue staining of HA was decreased from that in controls at 12, 18, and 24 hr. However, incorporation of 3H-glucosamine into HA was increased. The distribution of labeled HA within treated mesenchyme as 12, 18, and 24 hr resembled that in controls at 12 hr. These results indicate that the distribution of HA within the cranial mesenchyme of normal mouse embryos during neural fold elevation and convergence is not determined solely by regional differences in HA synthesis. We propose that HA distribution patterns result from the expansion of the HA-rich extracellular matrix of the central mesenchyme regions. This expansion may play a major role in fold elevation. These results also suggest that DON treatment reversibly inhibits HA synthesis, since treated mesenchymal cells retain the capability of synthesizing HA when provided with a glucosamine substrate. Patterns of 3H-glucosamine incorporation by DON-treated mesenchyme are similar to those observed in control mesenchyme prior to mesenchymal expansion at 12 hr.
Topics: Alcian Blue; Animals; Diazooxonorleucine; Glucosamine; Glycoproteins; Glycosaminoglycans; Hyaluronic Acid; Image Processing, Computer-Assisted; In Vitro Techniques; Mesencephalon; Mice; Neural Crest; Tritium
PubMed: 2375279
DOI: 10.1002/aja.1001880204 -
The American Journal of Anatomy Jun 1990Using the computer-assisted method of smoothed spatial averaging, spatial and temporal patterns of cell distribution and mitotic activity were analyzed in the cranial...
Using the computer-assisted method of smoothed spatial averaging, spatial and temporal patterns of cell distribution and mitotic activity were analyzed in the cranial mesenchyme underlying the mesencephalic neural folds of mouse embryos maintained in roller tube culture. Total cell density increased in central and medial mesenchymal regions after 12 hr in culture, decreased after 18 hr, and showed a further decrease after 24 hr when the neural folds of the embryos had elevated, converged, and were fusing or fused. Mitotic activity, as measured by the ratio of 3H-thymidine-labeled cells to unlabeled cells, was highest in the central mesenchyme at all culture times. Embryos were also cultured in the presence of diazo-oxo-norleucine (DON), which inhibits glycosaminoglycan and glycoprotein synthesis. After 24 hr in culture, neural folds of DON-treated embryos had failed to elevate. Total cell density increased in central and medial regions of the mesenchyme of DON-treated folds at 12 hr but showed no significant decrease in these regions with further culture. Mitotic activity was highest in the central mesenchyme of these treated embryos. These results suggest that cell distribution patterns observed in the cranial mesenchyme during neural fold elevation in normal cultured embryos are not produced by regional differences in mitotic activity. Rather, we propose that cell distribution patterns in the central and medial regions of the mesenchyme result from expansion of a glycosaminoglycan-rich extracellular matrix that disperses cells from these regions and decreases their density. In DON-treated embryos, in which expansion of the mesenchyme is prohibited by the decreased glycosaminoglycan and glycoprotein content of the extracellular matrix, mitotic activity apparently determines these patterns.
Topics: Animals; Cell Count; Cell Division; Diazooxonorleucine; Epithelium; Glycoproteins; Glycosaminoglycans; Hyaluronic Acid; Image Processing, Computer-Assisted; In Vitro Techniques; Mesencephalon; Mice; Mitosis; Neural Crest
PubMed: 2375278
DOI: 10.1002/aja.1001880203 -
Journal of Bacteriology Feb 1988In Bacillus subtilis, the formation of glutaminyl-tRNA is accomplished by first charging tRNA(Gln) with glutamate, which is then amidated. Glutamine was preferred over...
In Bacillus subtilis, the formation of glutaminyl-tRNA is accomplished by first charging tRNA(Gln) with glutamate, which is then amidated. Glutamine was preferred over asparagine and ammonia as the amide donor in vitro. There is a functional analogy of this reaction to that catalyzed by glutamine synthetase. Homogeneous glutamine synthetase, from either B. subtilis or Escherichia coli, catalyzed the amidotransferase reaction but only about 3 to 5% as well as a partially purified preparation from B. subtilis. Several classes of glutamine synthetase mutants of B. subtilis, however, were unaltered in the amidotransferase reaction. In addition, there was no inhibition by inhibitors of either glutamine synthetase or other amidotransferases. A unique, rather labile activity seems to be required for this reaction.
Topics: Ammonia; Asparagine; Bacillus subtilis; Diazooxonorleucine; Glutamate-Ammonia Ligase; Glutamine; Hot Temperature; Methionine Sulfoximine; Mutation; Nitrogenous Group Transferases; RNA, Transfer, Amino Acyl; RNA, Transfer, Gln; Transferases
PubMed: 2892827
DOI: 10.1128/jb.170.2.916-920.1988 -
Journal of Bacteriology Sep 1987The specific activities of glutamine synthetase (GS) and glutamate synthase (GOGAT) were 4.2- and 2.2-fold higher, respectively, in cells of Azospirillum brasilense...
The specific activities of glutamine synthetase (GS) and glutamate synthase (GOGAT) were 4.2- and 2.2-fold higher, respectively, in cells of Azospirillum brasilense grown with N2 than with 43 mM NH4+ as the source of nitrogen. Conversely, the specific activity of glutamate dehydrogenase (GDH) was 2.7-fold higher in 43 mM NH4+-grown cells than in N2-grown cells. These results indicate that NH4+ could be assimilated and that glutamate could be formed by either the GS-GOGAT or GDH pathway or both, depending on the cellular concentration of NH4+. The routes of in vivo synthesis of glutamate were identified by using 13N as a metabolic tracer. The products of assimilation of 13NH4+ were, in order of decreasing radioactivity, glutamine, glutamate, and alanine. The formation of [13N]glutamine and [13N]glutamate by NH4+-grown cells was inhibited in the additional presence of methionine sulfoximine (an inhibitor of GS) and diazooxonorleucine (an inhibitor of GOGAT). Incorporation of 13N into glutamine, glutamate, and alanine decreased in parallel in the presence of carrier NH4+. These results imply that the GS-GOGAT pathway is the primary route of NH4+ assimilation by A. brasilense grown with excess or limiting nitrogen and that GDH has, at best, a minor role in the synthesis of glutamate.
Topics: Alanine; Ammonia; Glutamate Dehydrogenase; Glutamate Synthase; Glutamate-Ammonia Ligase; Glutamates; Glutamine; Gram-Negative Bacteria; Nitrogen; Transaminases
PubMed: 2887545
DOI: 10.1128/jb.169.9.4211-4214.1987 -
The Journal of Biological Chemistry Aug 1987We have investigated the biosynthesis of heparan sulfate proteoglycan in human colon carcinoma cells cultured in either the absence of L-glutamine or the presence of...
Modulation of heparan sulfate biosynthesis. Effects of 6-diazo-5-oxo-L-norleucine and low glutamine on the synthesis of heparan sulfate proteoglycan by human colon carcinoma cells.
We have investigated the biosynthesis of heparan sulfate proteoglycan in human colon carcinoma cells cultured in either the absence of L-glutamine or the presence of 6-diazo-5-oxo-L-norleucine (DON), a glutamine analogue. Following a 24-h incubation with 100 micrograms/ml DON (0.58 mM) or without L-glutamine, the incorporation of [35S]sulfate was maximally inhibited to about 50%, whereas the incorporation of [3H]leucine or [3H]serine and their specific activity were not significantly affected. Several lines of evidence indicate that the inhibition of [35S]sulfate incorporation was mediated by a reduction in the intracellular pool of UDP-N-acetylhexosamine: the intracellular hexosamine levels were reduced by about 50%; taking into account the changes in specific activity, the incorporation of [3H]glucosamine was also significantly inhibited; and exogenous D-glucosamine (2.3 mM) was capable of substantially reversing the inhibitory effects of DON. This decrease in endogenous sugar supply resulted in the generation of an underglycosylated, lower buoyant density proteoglycan which contained significantly fewer heparan sulfate chains of otherwise normal size and sulfation and even fewer O-linked oligosaccharides. These biochemical changes were corroborated ultrastructurally by the appearance of smaller ruthenium red-stained proteoglycans on the surface of the cells. DON also caused a marked inhibition of cell proliferation and profound morphological changes, both of which were reversible upon culturing in DON-free medium. These results demonstrate that perturbations in glutamine metabolism have profound effects on the structure of heparan sulfate proteoglycan and on the phenotype of human colon carcinoma cells and indicate that DON treatment could be useful for studying post-translational modifications of proteoglycans in various cell systems.
Topics: Azo Compounds; Cell Division; Cells, Cultured; Centrifugation, Density Gradient; Chemical Phenomena; Chemistry; Chondroitin Sulfate Proteoglycans; Colonic Neoplasms; Diazooxonorleucine; Electrophoresis, Polyacrylamide Gel; Glucosamine; Glutamine; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Hexosamines; Microscopy, Electron; Oligosaccharides; Protein Biosynthesis; Proteoglycans; Sulfates
PubMed: 2956263
DOI: No ID Found -
The Journal of Biological Chemistry Jul 1987Incubation of embryonic chick chondrocytes with 6-diazo-5-oxo-L-norleucine (DON), a glutamine analogue, led to a dose-dependent inhibition of [35S]sulfate incorporation...
Incubation of embryonic chick chondrocytes with 6-diazo-5-oxo-L-norleucine (DON), a glutamine analogue, led to a dose-dependent inhibition of [35S]sulfate incorporation into proteoglycan. In the absence of exogenous L-glutamine, a maximal inhibition of 50-60% was achieved with DON concentrations greater than or equal to 1 microgram/ml (6 microM); the ED50 was approximately 0.2 microM. This inhibitory effect could be partially restored by the addition of 100-fold molar excess of either exogenous L-glutamine or M-glucosamine. The quantitative changes were due neither to inhibition of protein core synthesis nor to undersulfation of glycosaminoglycan chains. Rather, the proteoglycan synthesized in the presence of DON contained substantially fewer (approximately 50% of control) and smaller (10-15% of control, on the average) chondroitin sulfate chains as well as a paucity of keratan sulfate chains. The result of these structural changes was a proteoglycan with significantly lower molecular weight, buoyant density, and anionic charge. In spite of these modifications, the altered proteoglycan synthesized in the presence of DON was secreted normally and retained the ability to interact with exogenous hyaluronic acid and link proteins. The results of our experiments also indicate that DON substantially diminished the pool of hexosamine precursors required for glycosaminoglycan synthesis. We conclude that this decrease was responsible for the molecular alterations described above; and these, in turn, can account for the morphological changes previously seen in cartilage matrix synthesized in the presence of DON.
Topics: Animals; Azo Compounds; Cartilage; Chick Embryo; Chondroitin Lyases; Chromatography, Gel; Diazooxonorleucine; Glycoside Hydrolases; Molecular Weight; Protein Conformation; Proteoglycans; beta-Galactosidase
PubMed: 3112139
DOI: No ID Found -
British Journal of Cancer Jun 1987
Topics: Antimetabolites; Azo Compounds; Cell Cycle; Cell Line; Colonic Neoplasms; Diazooxonorleucine; Drug Evaluation, Preclinical; Glutaminase; Glutamine; Humans; Isoxazoles; Lung Neoplasms; Neoplasms, Experimental; Oxazoles
PubMed: 3620309
DOI: 10.1038/bjc.1987.133