Off-campus UNL users: To download campus access dissertations, please use the following link to log into our proxy server with your NU ID and password. When you are done browsing please remember to return to this page and log out.
Non-UNL users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
PURIFICATION AND PROPERTIES OF AN M(R) 80,000 POLYPEPTIDE (CO-EIF-2A(80)) WITH CO-EIF-2A ACTIVITY FROM RABBIT RETICULOCYTES AND ISOLATION OF CO-EIF-2A(80) MESSENGER RNA FROM ANIMAL CELLS
Abstract
A high molecular weight rabbit reticulocyte protein factor Co-eIF-2 regulates eIF-2 activity during initiation of protein synthesis. Co-eIF-2 contains Co-eIF-2A and Co-eIF-2C activities. Co-eIF-2A stimulates Met-tRNA(,f) binding to eIF-2 and stabilizes the ternary complex. Co-eIF-2C stimulates Met-tRNA(,f) binding to eIF-2 in the presence of Mg('2+) by relieving Mg('2+) inhibition of ternary complex formation from eIF-2. Co-eIF-2 protein complex contains several polypeptides including M(,r) 80,000 and 50,000 polypeptides. Three polypeptides (M(,r) 80,000, 50,000 and 25,000) are present in 0.5M KCl ribosomal salt wash and each possesses Co-eIF-2A activity. M(,r) 80,000 polypeptide (Co-eIF-2A('80)) has been purified to homogeneity and its properties studied: (1) Co-eIF-2A('80) stimulated Met-tRNA(,f) binding to eIF-2 and the complex formed was resistant to aurintricarboxylic acid. (2) Co-eIF-2A('80) activity was NEM resistant and heat-labile. (3) Antibodies prepared against homogenous Co-eIF-2A('80) strongly inhibited protein synthesis in reticulocyte lysates and, also, eIF-2 and Co-eIF-2 promoted Met-tRNA(,f) binding to 40S ribosomes. Inhibition of protein synthesis in reticulocyte lysates was overcome by preincubation of anti-Co-eIF-2A('80) with homogenous Co-eIF-2A('80) and was partially overcome by similar preincubation with Co-eIF-2. (4) Upon limited digestion with Staphylococcus aureus V8 protease, the homogenous Co-eIF-2A('80) gave two major polypeptide fragments (M(,r) 50,000 and 25,000). Upon similar treatment, an M(,r) 80,000 polypeptide band isolated from the SDS-gel of the Co-eIF-2 complex gave four major polypeptide fragments, and two of these fragments (M(,r) 50,000 and 25,000) were similar to those given by Co-eIF-2A('8-), indicating that this M(,r) 80,000 polypeptide band contains the Co-eIF-2A('80) component. In another study, we isolated the messenger RNA corresponding to Co-eIF-2A('80) from erythroleukemia cells. The procedure was: (1) Screening of human liver cDNA library with Co-eIF-2A('80) antibody to isolate a clone expressing Co-eIF-2A. (2) The insert DNA corresponding to Co-eIF-2A was isolated and nick translated to form the radioactive probe. (3) A northern hybridization was carried out with the total polyA RNA from erythroleukemia cells and the radioactive Co-eIF-2A DNA probe. (4) The hybridization studies show a mRNA of size 4.5 kb corresponding to Co-eIF-2A('80).
Subject Area
Biochemistry
Recommended Citation
CHAKRAVARTY, INDRANI, "PURIFICATION AND PROPERTIES OF AN M(R) 80,000 POLYPEPTIDE (CO-EIF-2A(80)) WITH CO-EIF-2A ACTIVITY FROM RABBIT RETICULOCYTES AND ISOLATION OF CO-EIF-2A(80) MESSENGER RNA FROM ANIMAL CELLS" (1985). ETD collection for University of Nebraska-Lincoln. AAI8602926.
https://digitalcommons.unl.edu/dissertations/AAI8602926