Dihydrofolate reductase from soybean seedlings

Reddy, V.Anthony; Rao, N. Appaji

doi:10.1016/0003-9861(76)90398-2

Cited by 37 publications

(17 citation statements)

References 35 publications

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“…The DHFR activity was eluted with a molecular mass corresponding to ϳ140 kDa. Similar molecular masses were previously reported for DHFR originating from soybean seedling (41) and carrot cell suspension cultures (42). In protozoa, the apparent molecular mass of the enzyme was also estimated at 150 kDa by gel filtration (43).…”

Section: Distribution Of Folate-synthesizing Enzymes In Leaf Tissue-supporting

confidence: 67%

Mitochondria Are a Major Site for Folate and Thymidylate Synthesis in Plants

Neuburger

Rébeillé

Jourdain

et al. 1996

Journal of Biological Chemistry

132

128

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The subcellular distributions of folate and folate-synthesizing enzymes were investigated in pea leaves. It was observed that the mitochondrial folate pool (ϳ400 M) represented ϳ50% of the total pool. Furthermore, all the enzymes involved in tetrahydrofolate polyglutamate synthesis were present in the mitochondria. In marked contrast, we failed to detect any significant activity of these enzymes in chloroplasts, cytosol, and nuclei. The presence of the tetrahydrofolate synthesis pathway in mitochondria is apparently a general feature in plants since potato tuber mitochondria also contained a high folate concentration (ϳ200 M) and all the enzymes required for tetrahydrofolate polyglutamate synthesis.The specific activities of tetrahydrofolate-synthesizing enzymes were rather low (1.5-15 nmol h ؊1 mg ؊1 matrix protein), except for dihydrofolate reductase (180 -500 nmol h ؊1 mg ؊1 matrix protein). Dihydrofolate reductase was purified to homogeneity. The enzyme had a native molecular mass of ϳ140 kDa and was constituted of two identical 62-kDa subunits. Interestingly, this mitochondrial protein appeared to be a bifunctional enzyme, also supporting thymidylate synthesis. The cell distribution of thymidylate synthase was also investigated. No significant activity was observed in cell fractions other than mitochondria, indicating that plant cell mitochondria are also a major site for thymidylate synthesis.

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Section: Distribution Of Folate-synthesizing Enzymes In Leaf Tissue-supporting

confidence: 67%

Mitochondria Are a Major Site for Folate and Thymidylate Synthesis in Plants

Neuburger

Rébeillé

Jourdain

et al. 1996

Journal of Biological Chemistry

132

128

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“…The anitfolate compounds bind very strongly to dihydrofolate reductase from mammalian sources (10) and also from soybean (19). It was, therefore, of interest to examine the effect of these compounds on mung bean serine hydroxymethyltransferase activity.…”

Section: Discussionmentioning

confidence: 99%

Purification and Regulatory Properties of Mung Bean (Vigna Radiata L.) Serine Hydroxymethyltransferase

Rao¹,

Rao²

1982

Plant Physiol.

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Serine hydroxymethyltransferase, the rirst enzyme in the pathway for interconversion of C1 fragments, was purified to homogeneity for the first time from any plant source. The enzyme from 72-h mung bean (Vigna radiata L.) seedlings was isolated using Blue Sepharose CL-6B and folate-AH-Sepharose4B affinity matrices and had the highest specific activity (1. The sigmoid saturation pattern of H4folate (nH = 2.0), one of the substrates, the abolition of sigmoidicity by NADH, an allosteric positive effector (nH = 1.0) and the increase in sigmoidicity by NAD+ and adenine nucleotides, negative allosteric effectors (nH = 2.4) clearly established that this key enzyme in the folate metabolism was an allosteric protein. Further support for this conclusion were the observations that (a) seine saturation exhibited an intermediary plateau region; (b) partial inhibition by methotrexate, aminopterin, O-phosphoserine, DL-a-methylserine and DL-O-methylserine; (c) subunit nature of the enzyme; and (d) decrease in the nH value from 2.0 for Hfolate to 1.5 in presence of L-serine.These results highlight the regulatory nature of mung bean serine hydroxymethyltransferase and its possible involvement in the modulation of the interconversion of folate coenzymes.The major storage form of folate coenzymes in plants is N5-methyltetrahydrofolate (21). Although this compound is present in abundant quantities the metabolism of C1 compounds and the 'This investigation was supported from a research

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“…MTX inhibits growth and blocks DHFR activity in plants (4,8,19), as well as in plant tissue cultures (4,24). Variability in MTX tolerance among plant species has been described (4).…”

Section: Introductionmentioning

confidence: 99%

Isolation of methotrexate-resistant cell lines in Petunia hybrida upon stepwise selection procedure

et al. 1984

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Cell suspensions of Petunia hybrida were subjected to a selection procedure in which the concentration of the selective agent, methotrexate (MTX), was gradually elevated. In mammalian cells, this procedure frequently results in MTX-resistant mutants due to amplification of the gene coding for dihydrofolate reductase (DHFR), the target protein of MTX.Five suspension lines were isolated, with degrees of resistance ranging from 10 to 500 μM MTX (in wild type the LD99.9 is 0.2 μM). MTX(R) phenotypes were unstable, as manifested by the loss of resistance upon prolonged growth in the absence of drug. All of the mutants also exhibited high values of MTX-binding protein (60- to 400-fold higher than that of the wild type), which declined to intermediate values upon MTX withdrawal. Finally, cellular extracts from all of the mutants also showed high specific staining of DHFR-activity in gels.The results suggest that the resistance of MTX in these plant cell-lines is mediated by the elevation of the amounts of DHFR, probably as a consequence of gene amplification.

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Dihydrofolate reductase from soybean seedlings

Cited by 37 publications

References 35 publications

Mitochondria Are a Major Site for Folate and Thymidylate Synthesis in Plants

Mitochondria Are a Major Site for Folate and Thymidylate Synthesis in Plants

Purification and Regulatory Properties of Mung Bean (Vigna Radiata L.) Serine Hydroxymethyltransferase

Isolation of methotrexate-resistant cell lines in Petunia hybrida upon stepwise selection procedure

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