Abstract
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.
Original language | English (US) |
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Pages (from-to) | 373-382 |
Number of pages | 10 |
Journal | Archives of Biochemistry and Biophysics |
Volume | 342 |
Issue number | 2 |
DOIs | |
State | Published - Jun 15 1997 |
Keywords
- Baculovirus expression system
- Deglycosylation
- Glycoproteins
- Heroin
- p67
- p67- DG
ASJC Scopus subject areas
- Biophysics
- Biochemistry
- Molecular Biology