Dihydrofolate Reductase from a Resistant Subline of the L1210 Lymphoma
1967; Elsevier BV; Volume: 242; Issue: 20 Linguagem: Inglês
10.1016/s0021-9258(18)99522-0
ISSN1083-351X
AutoresJohn P. Perkins, Brian L. Hillcoat, Joseph R. Bertino,
Tópico(s)Protein purification and stability
ResumoAbstract Dihydrofolate reductase has been purified 1000-fold from a methotrexate-resistant subline of the L1210 lymphoma with 49% yield and purity up to 100%. The procedure involves ammonium sulfate and acid precipitation, gel filtration on Sephadex G-100, and chromatography on hydroxylapatite. Purity has been determined by titrating enzymic activity and protein fluorescence with a stoichiometric inhibitor, methotrexate, and confirmed by electrophoresis and immunodiffusion. The isoelectric point in phosphate buffer is approximately pH 6.0, but in tris(hydroxymethyl)aminomethane buffer, the isoelectric point is above pH 8.5. The protein contains one reactive sulfhydryl group and is stabilized to heat and to urea denaturarion by each of its substrates.
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