Purification and characterization of homogeneous protein synthesis initiation factor M1 from rabbit reticulocytes.
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.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004355:Drug Stability; D004589:Electrophoresis, Disc; D005033:Ethylmaleimide / Q000494:pharmacology; D006358:Hot Temperature; D008970:Molecular Weight; D010442:Peptide Chain Initiation, Translational / Q000187:drug effects; D010448:Peptide Initiation Factors / Q000302:isolation & purification; D011487:Protein Conformation; D011817:Rabbits; D012156:Reticulocytes / Q000187:drug effects / Q000201:enzymology*; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism
D000596:Amino Acids; D010448:Peptide Initiation Factors; D005033:Ethylmaleimide