Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei

Gundersen, L. E.; Dunlap, R. Bruce; Harding, Nigel G. L.; Freisheim, James H.; Ötting, Fritz; Huennekens, F.M.

doi:10.1021/bi00756a011

Cited by 88 publications

(27 citation statements)

References 23 publications

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“…As has been observed for dihydrofolate reductase from other sources (see Blakley, 1969), the Km values for the natural substrates are very low [Gundersen et al (1972) (Mathews & Huennekens, 1963). Fig.…”

Section: Kinetic Propertiessupporting

confidence: 67%

“…A notable feature is the absence of cysteine, which is also absent from the L. casei dihydrofolate reductase of Gundersen et al (1972). The presence of six histidine and five tyrosine residues per mol has been confirmed by n.m.r.…”

Section: Amino Acid Analysismentioning

confidence: 80%

“…of approximately 18000. Gundersen et al (1972) have reported the purification of dihydrofolate reductase from a methotrexateresistant strain of L. casei A.T.C.C. 7469 (Dunlap et al, 1971a), which has a mol.wt.…”

Section: Enzyme Assaymentioning

confidence: 99%

“…at a salt concentration of 0.1 M; a single maximum is observed at about pH7.3. Other bacterial dihydrofolate reductases also show a single pH optimum (Blakley, 1969;D'Souza et al, 1972;Gundersen et al, 1972;Poe et al, 1972), though usually in the range pH 6.0-6.5. By contrast, the enzyme from mammalian sources, when examined at moderate ionic strength, usually shows optimum activity at pH4.5-5.5, with a shoulder or subsidiary peak in the activity curve at neutral pH (Blakley, 1969;Rowe & Russel, 1973;Nakamura & Littlefield, 1972;Jarabak & Bachur, 1971 Fig.…”

Section: Kinetic Propertiesmentioning

confidence: 99%

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Large-scale purification and characterization of dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei

Dann¹,

Ostler²,

Bjur³

et al. 1976

Biochemical Journal

150

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Dihydrofolate reductase has been purified from a methotrexate-resistant strain of Lactobacillus casei NCB 6375. By careful attention to growth conditions, up to 2.5g of enzyme is obtained from a 400 litre culture. The purification procedure, involving polyethyleneimine treatment, DEAE-cellulose chromatography and affinity chromatography on methotrexate-aminohexyl-Sepharose, operates on the gram scale, with overall yields of 50-60%. Elution of the affinity column by reverse (upward) flow was used, as it led to recovery of the enzyme in a much smaller volume. The enzyme obtained appears to be more than 98% pure, as judged by gel electrophoresis, isoelectric focusing, and gel filtration. It has a mol.wt. ofapprox. 17900 and a turnover number of4s-(5OmM-triethanolamine/400mM-KCI, pH7.2, 25°C) with dihydrofolate and NADPH as substrates.The turnover number for folate is 0.02s-1. Michaelis constants for a variety of substrates have been measured by using a new fluorimetric assay (0.361uM-dihydrofolate; 0.78 uM-NADPH), and binding constants determined by using the quenching of protein fluorescence (dihydrofolate, 2.25 x 106M-1; NADPH, >108M-1). The pH/activity profile shows a single maximum at pH 7.3; at this pH, marked activation by 0.5M-NaCl is observed.Dihydrofolate reductase (tetrahydrofolate-NADP+ oxidoreductase, EC 1.5.1.3) is responsible for maintaining the intracellular pool of tetrahydrofolate by reducing dihydrofolate (arising either by biosynthesis de novo or by the action of thymidylate synthetase on 5,10-methylenetetrahydrofolate) to tetrahydrofolate (Blakley, 1969). This enzyme is of considerable pharmacological interest as the site of action of a group of powerful chemotherapeutic agents, the 'anti-folates', which includes methotrexate, trimethoprim and pyrimethamine (Blakley, 1969;Baker, 1967).We are undertaking a detailed study of the binding of substrates, coenzymes and inhibitors to the dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei, principally by highresolution n.m.r. (nuclear-magnetic-resonance) spec-* Present address:

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Section: Kinetic Propertiessupporting

confidence: 67%

Section: Amino Acid Analysismentioning

confidence: 80%

Section: Enzyme Assaymentioning

confidence: 99%

Section: Kinetic Propertiesmentioning

confidence: 99%

See 2 more Smart Citations

Large-scale purification and characterization of dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei

Dann¹,

Ostler²,

Bjur³

et al. 1976

Biochemical Journal

150

View full text Add to dashboard Cite

show abstract

“…This property was reported for Streptococcus faecium (2,3,14,15), Lactobacillus casei (5), and Diplococcus pneumoniae (17) as well as for mammalian (6) and tumor cells (4,16).…”

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confidence: 61%