Purification and characterization of homogeneous initiation factor M2A from rabbit reticulocytes.

作者: W C Merrick , W M Kemper , W F Anderson

DOI: 10.1016/S0021-9258(19)41216-7

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摘要: Abstract Rabbit reticulocyte initiation factor M2A has been prepared in homogeneous form. The final preparation was purified 2,300-fold and ran as a single band on polyacrylamide gel electrophoresis three different buffer systems: alkaline, sodium dodecyl sulfate, acidic 6.5 M urea. IF-M2A also isoelectric focusing experiments with an apparent pI of 6.45. molecular weight approximately 125,000 based determinations by low speed equilibrium centrifugation (118,000), sulfate (130,000) s20,w combined Stokes radius (124,000). amino acid composition revealed unusual features: a) the basic acids represented 19.4 mol %; b) glutamicacid (plus glutamine) constituted 18.8 mol%; c) tryptophan cysteine residues were only 0.4 0.7 mol%, respectively. Homogeneous tested several assays using either natural or artificial mRNAs. In each assay tested, fully substituted for cruder preparations at concentrations commensurate its increased purity. examined ribosome-dependent GTP hydrolysis, requiring ribosomes but no other factors. Analysis data yielded Km 10 muM Vmax hydrolysis 1.20pmol/mug IF-M2A/min. addition, mediated required both 40 S 60 subunits maximal activity. possibility that is joining discussed.

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