Protein synthesis in rabbit reticulocytes. Purification and properties of an Mr 80,000 polypeptide (Co-eIF-2A80) with Co-eIF-2A activity.

I Chakravarty,M K Bagchi,R Roy,A C Banerjee,N K Gupta

doi:10.1016/s0021-9258(18)88871-8

Abstract

The high molecular weight protein complex, Co-eIF-2, contains both Co-eIF-2A and Co-eIF-2C activities (Bagchi, M. K., Banerjee, A. C., Roy, R., Chakravarty, I., and Gupta, N. K. (1982) Nucleic Acids Res. 10, 6501-6510). Co-eIF-2A stimulated Met-tRNAf binding to eukaryotic initiation factor-2 (eIF-2) both in the presence and absence of Mg2+. Co-eIF-2C stimulates Met-tRNAf binding to eIF-2 in the presence of Mg2+ by relieving Mg2+ inhibition of ternary complex formation from eIF-2. Co-eIF-2 protein complex contains several polypeptides including Mr 80,000 and 50,000 polypeptides. Three polypeptides (Mr 80,000, 50,000 and 25,000) are present in 0.5 M KCl ribosomal salt wash and each possesses Co-eIF-2A activity. Mr 80,000 polypeptide (Co-eIF-2A80) has been purified to homogeneity and its properties studied. 1) Co-eIF-2A80 stimulated Met-tRNAf binding to eIF-2 and the complex formed was resistant to aurintricarboxylic acid. 2) Co-eIF-2A80 activity was N-ethylmaleimide-resistant and heat-labile; it was destroyed by heating at 55 degrees C for 4 min. 3) Antibodies prepared against homogeneous Co-eIF-2A80 strongly inhibited protein synthesis in reticulocyte lysates and, also, eIF-2 and Co-eIF-2 promoted Met-tRNAf binding to 40 S ribosomes. Inhibition of protein synthesis in reticulocyte lysates was overcome by preincubation of anti-Co-eIF-2A80 with homogeneous Co-eIF-2A80 and was partially overcome by similar preincubation with Co-eIF-2. 4) Upon limited digestion with Staphylococcus aureus V8 protease, the homogeneous Co-eIF-2A80 gave two major polypeptide fragments (Mr 50,000 and 25,000). Upon similar treatment, an Mr 80,000 polypeptide band isolated from the sodium dodecyl sulfate-gel of the Co-eIF-2 protein complex gave four major polypeptide fragments, and two of these fragments (Mr 50,000 and 25,000) were similar to those given by Co-eIF-2A80, indicating that this Mr 80,000 polypeptide band contains the Co-eIF-2A80 component. We suggest that Co-eIF-2A80 is a component of Co-eIF-2 and is also essential for Co-eIF-2 activity and overall peptide chain initiation.

Highlights

2, contains both Co-eIF-2A and Co-eIF-2C activities
It was recently claimed that wheat neous Co-eIF-2Aa0 gave two major polypeptide frag- germ Co-eIF-2A activity isrequired for protein synthesis ina ments (Mr 50,000 and 25,000)
We have reported that two high molecular weight protein complexes, Co-eukaryotic initiation factor-2 (eIF-2) and RF,contain Co-eIF-2A activity

Summary

PURIFICATIONANDPROPERTIESOF ACTIVITY*

AN M , 80,000 POLYPEPTIDE (Co-eIF-2Am) WITH Co-eIF-2A (Received for publication, August 14, 1984). Complex gave four major polypeptide fragments, and ( termed Co-eIF-2C (16S),P [17], andSF [18]).A similar two of these fragments (Mr50,000 and 25,000) were factor activity has been reported to be present inmouse similar to those given byCo-eIF-2ASo,indicating that this M, 80,000 polypeptide band contains the Co-eiF2AS0component. We have observed that reticulocyte high salt wash preparations contain, besides the M , 25,000 polypeptide reportedearlier [3], two other polypeptides (Mr 80,000 and 50,000) which possess Co-eIF-2A activity. We report the results of our studies on the roles of homogeneous Co-eIF-2Amin peptide chain initiation and thepossible relationship of Co-eIF-2Asoto Co-eIF-2 protein complex and to the otherisolated polypeptides with Co-eIF-2A activity

DISCUSSION

TABLE IV