Separation of Reticulocyte Initiation Factor M ₂ Activity into Two Components

Abstract

IF-M2, one of three initiation factors isolated by DEAE-cellulose chromatography from the 0.5 M KCl-wash fraction of rabbit reticulocyte ribosomes, has been separated by (3), reticulocyte elongation factors EF-1 and EF-2 (5), and the aminoacyltRNA species (8) were prepared as described.Preparation of IF-Ml. IF-Ml was purified from the 0.5 M KCl-wash fraction from ribosomes essentially as described previously (Table 3, (Fig. 1). 36 mg of IF-M2 protein in a 3-ml volume was chromatographed on a 1.5 X 86-cm Sephadex G-200 column equilibrated with 10 mM Tris * HCl (pH 7.5)-100 mM KC1-1 mM dithiothreitol-0.1 mM EDTA. Protein was eluted with the same buffer at a flow rate of 10 ml/hr; 2-ml fractions were collected. Activity

Polypeptide chain initiation in eukaryotes: initiation factor requirements for translation of natural messengers.

“…In Table 4 the effect of omitting single factors is shown (A) in the absence of further additions and (B) in the presence of ribosomal salt wash from undeveloped embryos. From (A) it is evident that the basic system is deficient (samples [4][5][6][7][8] in at least four of the rabbit reticulocyte initiation factors, (12). To determine whether this factor participates in mRNA translation we used antibody against A. salina IF-Mi.…”

“…In eukaryotes the situation is more complex. There are two (1, 4) IF-2-like factors (IF-MP and IF-Mi), a factor(s) (5-7) corresponding to IF-3 (IF-M3), and several additional factors whose function is less well defined (4,(8)(9)(10)(11). At least three of these additional factors (IF-M2A, IF-M2Ba, and IF-M2Bfl) are well characterized.…”

A Ribosome Dissociation Factor from Rabbit Reticulocytes Distinct from Initiation Factor M 3

“…Quantitative assays of crude fractions (steps [1][2][3] were not reproducible because the contaminating proteins caused aggregation of subunits and ribosomes (1); hence, only the 10-fold purification in steps 4-7 is reported.…”

“…At least four different initiation factors have also been separated from the same extract (2,3). Since initiation in eukaryotes (as well as in bacteria) involves subunits, and since the ribosome dissociation factor of Escherichia coli has been identified with initiation factor IF-3 (4,5), it seemed, important to determine whether or not the reticulocyte DF is identical with any of the reticulocyte initiation factors, particularly IF-M3.…”

Partial purification and characterization of two hen oviduct protein synthesis initiation factors capable of initiation complex formation

“…eIF-2A was, nevertheless, capable of binding the natural unformylated initiator tRNA to the AUG codon . The initiation factors eIF-5 and eIF-4C stimulated this reaction and GTP was then required (Shafritz et al, 1972). More recently, however, using a homogeneous preparation of eIF-2A the requirements for eIF-5 and GTP were lost while stimulation by eIF-4C was retained (Adams et al, 1975).…”