Dihydrofolate reductase. Purification and characterization of the enzyme from an amethopterin-resistant mutant of Escherichia coli

Poe, Martin; Greenfield, Norma J.; Hirshfield, J.; Williams, Myra N.; Hoogsteen, Karst

doi:10.1021/bi00756a012

Cited by 115 publications

(76 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In addition, the two enzymes differ in their activity towards folate (see below). It seems likely that these differences can be attributed to the different strains of bacteria used; similar differences have been noted for the dihydrofolate reductases from different strains of E. coli (Mathews & Sutherland, 1965;Burchall & Hitchings, 1965;Burchall & Chan, 1969;Poe et al, 1972;Erickson & Mathews, 1973) and S. faecalis (see, for example, Albrecht & Hutchinson, 1969;D'Souza et al, 1972).…”

Section: Enzyme Assaymentioning

confidence: 68%

“…In many of the previous reports of purification of dihydrofolate reductase by affinity chromatography, the column material used was methotrexate-aminoethyl-Sepharose (Nakamura & Littlefield, 1972;Poe et al, 1972;Chello et al, 1972;Gauldie & Hillcoat, 1972;Gauldie et al, 1973). 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).…”

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%

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

149

View full text Add to dashboard Cite

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:

show abstract

Section: Enzyme Assaymentioning

confidence: 68%

Section: Enzyme Assaymentioning

confidence: 99%

Section: Kinetic Propertiesmentioning

confidence: 99%

See 1 more Smart Citation

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

Dann¹,

Ostler²,

Bjur³

et al. 1976

Biochemical Journal

149

View full text Add to dashboard Cite

show abstract

“…11 ) To measure the turnover number of DHFR synthesized in cell-free system, enzymatic activities with various concentrations of methotrexate were measured by the method of Poe et 01. 12 ) The translated product was also confirmed by SDS polyacrylamide gel followed by immunoblotting using anti-DHFR serum as described previously.13)…”

Section: Methodsmentioning

confidence: 99%

An Increased Rate of Cell-free Protein Synthesis by Condensing Wheat-germ Extract with Ultrafiltration Membranes

Nakano

Tanaka

Kawarasaki

et al. 1994

Bioscience, Biotechnology, and Biochemistry

View full text Add to dashboard Cite

“…Since, as shown later, our symmetry-related reflection intensities agree with each other to within 5 % on the average and since some reflection intensity deviation can be attributed to quantum statistics, we can conclude that the average nonuniformity of the chamber is less than 5 %. The new multiwire area detector is used routinely to collect data from crystals of a complex of dihydrofolate reductase and metho-trexate (a cancer drug) (Poe, Greenfield, Hirshfield, Williams & Hoogsteen, 1972). This work is done in collaboration with Drs D. Matthews, R. Alden, S. Freer and J. Kraut of the UCSD Chemistry Department.…”

mentioning

confidence: 99%

A xenon-filled multiwire arc detector for X-ray diffraction

Cork¹,

Hamlin²,

Vernon³

et al. 1975

Acta Cryst Sect A

View full text Add to dashboard Cite

A xenon-filled multiwire proportional chamber with high and uniform photon-detection efficiency has been built and incorporated into a high-speed data-collection system for protein crystallography. This system has been used to collect more than 200 000 reflection intensities from one parent and four heavy-atom derivative crystals of a complex of dihydrofolate reductase and metho-trexate (a cancer drug). Its data collection rate is about 30 times faster than the rate of a standard diffractometer (1400 reflection intensities per hour versus 50 reflection intensities per hour). The precision of the chamber data is also better with an intensity reliability R of 5 % as compared with 6"7 % from the diffractometer data. The difference Patterson maps between parent and heavy-atom derivative data show very clearly the positions of the heavy atoms.We have reported previously the use of an argon-filled multiwire proportional chamber as an area X-ray detector for protein crystallography (Cork, Fehr, Hamlin, Vernon, Xuong & Perez-Mendez, 1973. Since then we have successfully built a xenon-filled chamber with much higher photon-detection efficiency, better spatial resolution and more uniform quantum-detection efficiency. This chamber is now incorporated into a high-speed data-collection system for protein crystallography. The system collects reflection intensities at a rate about 30 times faster than a standard diffractometer. The precision of the new data is better than that obtained with the diffractometer.The advantage of a xenon chamber over an argon-filled chamber is higher photon-detection efficiency. Xenon is, however, much more expensive than argon so the chamber enclosure must be carefully built to reduce gas leakage. The new chamber has three electrodes: one beryllium window and two wire detection planes. The window, made of a flat beryllium plate (1 mm thick), is mounted in electrical contact with the aluminum enclosure. The two detection planes are similar to those in the previously described argon chamber. The center electrode consists of 150 horizontal wires (20 pm stainless steel with 2 mm spacing). The back electrode consists of 300 vertical wires (50 pm of stainless steel with 1 mm spacing). The two wire planes are mounted on 4 mm thick fiber-glass frames and stacked behind the beryllium window. The aluminum enclosure is composed of three parts: a picture frame holding the front window, another picture frame containing the detection planes and a back plate. The three parts are mounted together with O-rings to eliminate gas leakage. There is enough extra room inside the container for the two delay lines used in the read-out process . All electrical feedthrough connections are standard B.N.C. seated in a leaktight adhesive sealant. The chamber is filled with a gas mixture of 90 % xenon and 10 % CO2 at a pressure of about 1 atm. The center electrode is maintained at 2.8 kV, the front window and the aluminum container are grounded. The back electrode is held at -80 V. The front window has to be kept flat to withi...

show abstract

Dihydrofolate reductase. Purification and characterization of the enzyme from an amethopterin-resistant mutant of Escherichia coli

Cited by 115 publications

References 22 publications

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

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

An Increased Rate of Cell-free Protein Synthesis by Condensing Wheat-germ Extract with Ultrafiltration Membranes

A xenon-filled multiwire arc detector for X-ray diffraction

Contact Info

Product

Resources

About