The roles of p67-deglycosylase (p67-DG) in the regulation of protein synthesis in baculovirus-infected insect cells were studied. Like vaccinia vital infection, baculovirus infection of insect cells also induced the appearance of a p67-DG. However, p67-DG activity could not be detected because these cells do not contain a 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 heroin to the growth medium. Maximum enhancement was observed at 5 μM heroin. Data suggest that heroin prevents the activation of latent p67- DG inside the cell and does not have any effect on p67 gene transcription. To gain a better understanding of the mechanism of p67-DG activation and hemin stimulation of p67 synthesis, we have now purified p67-DG from baculovirus- infected insect cells. We prepared antibodies against this protein. These antibodies reacted with a 105-kDa protein 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 are also presented to show that the antibodies against p67-DG 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-DG 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-DG antibodies since these antibodies were prepared against the active form of p67-DG.
Bibliographical noteFunding Information:
This work was supported by NIGMS Grant GM22079, an American Heart Association (Nebraska Chapter) grant, and a Nebraska State grant for Cancer and Smoking Diseases. We also thank Professor John W. B. Hershey (University of California, Davis) for critical reading and many valuable suggestions during the preparation of the manuscript.
- Baculovirus expression system
- p67- DG