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.
Regulation of a 67 kDa glycoprotein (p67) and ap67-deglycosylase during the protein synthesis initiation in eukaryotes
Abstract
A 67 kDa glycoprotein (p67) protects eIF-2 $\alpha$ subunit from kinase catalyzed phosphorylation and thus promotes protein synthesis in the presence of active kinases. In this present work I have studied the roles of p67-deglycosylase in regulation of protein synthesis in baculovirus infected insect cells. Like vaccinia viral infection baculovirus infection of insect cells also induced appearance of a p67-deglycosylase. However, p67-deglycosylase activity could not be detected as these cells do not contain detectable level of p67. The baculovirus expression vector system (BEVS), however, promotes significant expression of cloned p67-cDNA. The expression of p67 was significantly enhanced by the addition of hemin to the growth medium. Maximum enhancement was observed at 5 $\mu$M hemin. This p67 was purified to homogeneity and was identical to native p67 in several reactions tested. Data suggest that hemin prevents the activation of latent p67-deglycosylase inside the cell and does not have any effect on p67 gene transcription. To gain a better understanding of the mechanism of p67-deglycosylase activation and hemin stimulation of p67 synthesis we have now purified p67-deglycosylase from baculovirus infected insect cells. We prepared antibodies against this protein. These antibodies reacted with a 105 kDa in cell extracts from the uninfected insect cells (Sf9), KRC-7 and L929 (animal cells). In addition these antibodies reacted with an additional 60 kDa protein in the cell extracts of baculovirus infected Sf9 cells and vaccinia virus infected KRC-7 and L929 cells. Data is also presented to show that the antibodies against p67-deglycosylase reacted more efficiently (40%) with the 60 kDa protein in both hemin deficient reticulocyte lysate and hemin deficient baculovirus infected cells. We suggest that hemin prevents the conversion of an inactive p67-deglycosylase into an active form possibly by covalent modification such as protein phosphorylation or protein glycosylation. The active form is more efficiently recognized by the p67-deglycosylase antibodies since these antibodies were prepared against the active form of p67-deglycosylase.
Subject Area
Biochemistry|Molecular biology
Recommended Citation
Saha, Debabrata, "Regulation of a 67 kDa glycoprotein (p67) and ap67-deglycosylase during the protein synthesis initiation in eukaryotes" (1997). ETD collection for University of Nebraska-Lincoln. AAI9730281.
https://digitalcommons.unl.edu/dissertations/AAI9730281