Multiple forms of dihydrofolate reductase

Mell, Galen P.; Martelli, Massimo F.; Kirchner, Jeffrey E.; Huennekens, F.M.

doi:10.1016/0006-291x(68)90257-x

Cited by 36 publications

(9 citation statements)

References 9 publications

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“…As found for the same enzyme from hamster cells [24], LM 4 mouse lymphoma cells [25], Lactobacillus casei [26], and chicken liver [27] polyacrylamide gel electrophoresis of the purified reductase indicated two protein components both possessing enzymatic activity (Fig. 1 A).…”

Section: Separation Of the Two Forms Of The Enzymementioning

confidence: 95%

Dihydrofolate Reductase from Bovine Liver

Wilson

1975

European Journal of Biochemistry

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Dihydrofolate reductase from bovine liver has been purified 5000-fold employing conventional techniques and methotrexate/aminohexyl/Sepharose affinity chromatography. Electrophoresis of the isolated enzyme on polyacrylamide gels resulted in the separation of two enzymatically active protein components which were not interconvertible by treatment with dihydrofolate and/or the coenzyme. The two forms, present in a ratio of 20: 1, were found by isoelectric focusing to have isoelectric points of 7.15 and 5.94. They had identical specific activities towards dihydrofolate (26.1 -27.0 Ujmg) and folate (1.3 -2.2 Ujmg), and h identical molecular weights (23 500) and amino acid compositions.Due to the small quantity of the acidic form and the similarity of the two forms, the amino-terminal sequence (19 residues) was determined on a mixture of carboxymethylated reductase.The single sulfhydryl group of the enzyme can be modified by several sulfhydryl reagents in the native enzyme without loss of activity. Modification of the same residue occurs in the denaturated state and partially inhibits renaturation to the fully active enzyme. One disulfide bridge was detected by reduction and alkylation. The cleavage of this bond did not effect the enzymatic activity.Dihydrofolate reductase catalyzes the reversible NADPH-dependent reduction of dihydrofolate to tetrahydrofolate which is required at various stages in DNA, RNA, and amino acid biosynthesis [1]. Due to the central role of this enzyme in cellular metabolism during growth various substrate analogs were developed which have been successfully employed as cancer chemotherapeutic agents [2,3]. At the same time numerous investigations of the enzymatic and physical properties of dihydrofolate reductases isolated from both procaryotes and eucaryotes have been reported (see [4] for a review). However to date all detailed structural investigations have been restricted to the dihydrofolate reductases isolated from the inhibitor-resistant strains of Esclzevichia coli [5] and Streptococcusfuecium [6]. With the recently developed preparative methods based on affinity chromatography [7] sufficient amounts of vertebrate enzymes can now also be easily isolated for such structural studies.Utilizing this method we have purified the dihydrofolate reductase from bovine liver. In this communication we extend the structural characterization of the enzyme, first investigated by Rowe and Russel reporting its amino acid composition, isoelectric properties and amino terminal sequence. The effects of sulfhydryl reagents on the enzymatic activity and stability of this enzyme are also reported. MATERIALS AND METHODSBovine liver dihydrofolate reductase was isolated employing a combination of the methods previously outlined by Rowe and Russel [8] and Kaufman and Pierce [9]. Table 1 illustrates the isolation procedure. Steps 1 -4 are identical to those used by Rowe and Russel in their isolation of dihydrofolate reductase by convential chromatographic methods. The preparation of the methotrexate/aminoh...

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Section: Separation Of the Two Forms Of The Enzymementioning

confidence: 95%

Dihydrofolate Reductase from Bovine Liver

Wilson

1975

European Journal of Biochemistry

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“…A current of 1 mA/tube was applied for 30min then increased to 2mA/tube for a further lih. The gels were then removed and incubated in the dark at 25°C in 2.Oml of assay mixture to localize the enzyme as blue bands formed on reduction of a tetrazolium salt [3-(4,5dimethylthiazol -2 -yl) -2,5 -diphenyltetrazolium bromide] by the tetrahydrofolate formed (Gunlack et al, 1968;Mell et al, 1968a;Hiebert et al, 1972). The position of the bands was recorded as previously described (Hiebert et al, 1972).…”

Section: Trimethoprimmentioning

confidence: 99%

Purification and properties of dihydrofolate reductase from cultured mammalian cells

Gauldie

Marshall

Hillcoat

1973

Biochemical Journal

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Dihydrofolate reductase was purified quickly and simply from small quantities of cultured mammalian cells by affinity chromatography. On gel electrophoresis of the purified enzyme, multiple bands of activity resulted from enzyme-buffer interaction at low but not high buffer concentration. A Ferguson plot (Ferguson, 1964) showed that this heterogeneity was due to a charge difference with no alteration in the size of the enzyme. Stimulation of enzyme activity by KCl, urea and p-hydroxymercuribenzoate, and inhibition by methotrexate and trimethoprim, showed only minor differences between the various enzymes.

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“…We have not been able to activate this nonfunctional big species of DHFR from the CML cytoplasm with KC1, urea, guanidine hydrochloride, or excess NADPH, compounds that have been reported to increase the activity or alter the electrophoretic mobility of DHFR (28)(29)(30)(31)(32). However, physiologic activation might require, in addition to NADPH and substrate, some other intracellular factor(s) that are only present in mitotically active cells.…”

Section: Resultsmentioning

confidence: 99%

Human leukemia and normal leukocytes contain a species of immunoreactive but nonfunctional dihydrofolate reductase.

Rothenberg¹,

Iqbal²

1982

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

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A quantitative radioimmunoassay has been developed for human dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) by using antiserum raised in rabbits against the active enzyme purified from calf liver. An immunoreactive protein could be identified in the cytoplasm of chronic myelogenous leukemia cells, which contained no functional dihydrofolate reductase activity. Its concentration was stoichiometric to the volume of cytoplasm assayed and paralleled the standard curve obtained with purified enzyme, indicating that this protein in the human cells is antigenically similar to the homologous antigen. The concentration of this immunoreactive protein in the cytoplasm of human leukemia and normal leukocytes in all instances greatly exceeded the concentration of functional dihydrofolate reductase, which was measured by the binding of [3H]methotrexate. This nonfunctional immunoreactive protein in the cytoplasm and cytosol from two different samples of chronic myelogenous leukemia cells analyzed by gel filtration had an apparent molecular weight of 41,000, which is twice the molecular weight of the functional enzyme.

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Multiple forms of dihydrofolate reductase

Cited by 36 publications

References 9 publications

Dihydrofolate Reductase from Bovine Liver

Dihydrofolate Reductase from Bovine Liver

Purification and properties of dihydrofolate reductase from cultured mammalian cells

Human leukemia and normal leukocytes contain a species of immunoreactive but nonfunctional dihydrofolate reductase.

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