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Purification and characterization of homogeneous protein synthesis initiation factor M1 from rabbit reticulocytes



Purification and characterization of homogeneous protein synthesis initiation factor M1 from rabbit reticulocytes



Journal of Biological Chemistry 250(4): 1197-1206



An eight-step procedure has been devised for the preparation of homogeneous rabbit reticulocyte IF-M1. Molecular weight determinations based on IF-M1 activity (gel filtration and sucrose density gradient sedimentation) and based on IF-M1 protein (low speed equilibrium sedimentation and sodium dodecyl sulfate gel electrophoresis) indicate that IF-M1 is active as a single polypeptide chain of 65,000 molecular weight. The amino acid composition of IF-M1 has been determined. There appears to be no unique features in the amino acid composition of IF-M1, except perhaps an elevated proline content (6.9 mol %). The catalytic properties of purified IF-M1 were similar to those previously reported by this laboratory for crude preparations of IF-M1. The sensitivity of IF-M1 activity to N-ethylmaleimide and heat (45 degrees) inactivation was tested in two model reactions requiring minimal complementary factors: (a) AUG-directed fMet-tRNAf binding to ribosomes; and (b) poly(U)-directed polyphenylalanine synthesis at 4 mM Mg2+ (IF-M2A, IF-M2B, EF-1, and EF-2 also required). IF-M1 activity proved to be sensitive to both N-ethylmaleimide and temperature (45 degrees). In addition, a contaminant of partially purified IF-M1 preparations has been found which is capable of fMet-tRNAf binding but is inactive in poly(U)-directed polyphenylalanine synthesis at low Mg2+ concentration.

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Accession: 041135131

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PMID: 1112800


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