T4 bacteriophage-specific dihydrofolate reductase: Purification to homogeneity by affinity chromatography

Erickson, John S.; Mathews, Christopher K.

doi:10.1016/0006-291x(71)90585-7

Cited by 42 publications

(16 citation statements)

References 13 publications

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“…Analysis of the purified enzyme on a calibrated gel with several protein standards indicated an approximate molecular weight of 22,000. This is considerably lower than our earlier estimate of 29,000 (5), which was also based FIG. 2.…”

Section: Resultscontrasting

confidence: 79%

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Bacteriophage T4-coded dihydrofolate reductase: synthesis, turnover, and location of the virion protein

Mosher¹,

Mathews²

1979

J Virol

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Dihydrofolate reductase plays a dual role in bacteriophage T4, first, as an enzyme of thymidylate metabolism, and second, as a protein component of the tail baseplate. Antibody to the purified enzyme has been used to study its synthesis and intracellular turnover. The antibody specifically precipitates one protein from T4D-infected cell extracts. This has been identified as dihydrofolate reductase, although the polypeptide molecular weight (22,000) is lower than that earlier determined for this enzyme. The protein comigrates on gels with pY, a genetically undefined protein component of the baseplate. However, it is not pY, for pY is synthesized late in infection, whereas virtually no dihydrofolate reductase synthesis occurs later than 10 min after infection at 37°C. Dihydrofolate reductase, once formed, is neither degraded nor converted to proteins of higher or lower molecular weight. Thus, it is probably incorporated into virions at the same molecular weight as that of the soluble enzyme. 125I-radiolabeled antibody binds to the wedge substructure of the baseplate, and this binding is blocked by preincubation with purified T4 dihydrofolate reductase. Thus, the enzyme protein seems to be a component of the wedge. tect the reductase protein as a component of the wedge subassembly of the baseplate. (These results were taken from a Ph.D. thesis presented by R.A.M. to the University of Arizona.) MATERIALS AND METHODS Cell and bacteriophage 8trains. The wild-type phage strain T4D has been maintained in this laboratory for some time, as have Escherichia coli strains 94

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Section: Resultscontrasting

confidence: 79%

“…The purification of DFR was accomplished as described previously (5), with the one exception that the protein was passed twice through a Sephadex G-100 column (2.6 by 60 cm) in 0.04 M potassium phosphate-0.2 M KCl, pH 7.9.…”

mentioning

confidence: 99%

Bacteriophage T4-coded dihydrofolate reductase: synthesis, turnover, and location of the virion protein

Mosher¹,

Mathews²

1979

J Virol

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“…The ligands, nafenopin, ciprofibrate, and clofibric acid, were individually immobilized on AH-Sepharose 4B by carbodiimide reaction, coupling the -COOH group of the ligands and -NH2 groups on Sepharose beads (12,13 fTo whom reprint requests should be addressed.…”

Section: Methodsmentioning

confidence: 99%

Peroxisome proliferator-binding protein: identification and partial characterization of nafenopin-, clofibric acid-, and ciprofibrate-binding proteins from rat liver.

Lalwani¹,

Alvares²,

Reddy³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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“…In common with Whiteley et al (1972) and Kaufman (1974), we have used an aminohexyl 'spacer', since we find that this gives significantly better recoveries of the L. casei enzyme (Dann et al, 1972). Pastore et al, (1974) used a pteroyl-lysine-Sepharose, and Erickson & Mathews (1971) an N'0-formylaminopterin-aminoethyl-BioGel P-10 column, since the weaker binding of the enzyme by these ligands makes for easier elution. In earlier attempts to devise an affinity column from which the enzyme could be eluted without the use of a competing ligand, we prepared N10-formylaminopterin-aminohexyl-Sepharose.…”

Section: Enzyme Assaymentioning

confidence: 99%

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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T4 bacteriophage-specific dihydrofolate reductase: Purification to homogeneity by affinity chromatography

Cited by 42 publications

References 13 publications

Bacteriophage T4-coded dihydrofolate reductase: synthesis, turnover, and location of the virion protein

Bacteriophage T4-coded dihydrofolate reductase: synthesis, turnover, and location of the virion protein

Peroxisome proliferator-binding protein: identification and partial characterization of nafenopin-, clofibric acid-, and ciprofibrate-binding proteins from rat liver.

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

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