Enhancement of phospholipase D activity following baculovirus and adenovirus infection in Sf9 and COS-7 cells.
In order to purify the human phospholipase D1 (hPLD1) for analysis of its functional properties, we applied a baculovirus-based high-expression system. As expected, Sf9 cells infected with a baculovirus encoding for the hPLD1 displayed a 7.5-fold increase in PLD activity compared to uninfected cells. Sf9 cells infected with the wild-type (WT) and other recombinant baculoviruses were used as an expression control. Surprisingly, all baculoviruses tested led to a 3-5 fold increase in basal PLD activity when compared to uninfected cells. To further characterize the nature of the increased PLD activity, the influence of ADP-ribosylation factor (ARF) and phorbol 12-myristate 13-acetate (PMA) was studied. In contrast to membranes containing the hPLD1, the PLD activity in membranes from uninfected and WT-infected Sf9 cells was not stimulated by ARF. PMA did not affect the increase in PLD activity in any case. To further study whether the virus-mediated increase in PLD activity is a more general phenomenon, we infected COS-7 cells with recombinant and WT adenoviruses. Only the infection with the WT adenovirus resulted in an approx. 2-fold increase in PLD activity. Our results demonstrate for the first time that a viral infection elevates the PLD activity in insect and mammalian cells.
Höer
A
A
Institut für Pharmakologie, Fachbereich Humanmedizin der Freien Universität Berlin, Germany. adhoer@zedat.fu-berlin.de
Schöneberg
T
T
Harteneck
C
C
Cetindag
C
C
Oberdisse
E
E
D020727:ADP-Ribosylation Factors; D000256:Adenoviridae / Q000201:enzymology / Q000235:genetics*; D000257:Adenoviridae Infections / Q000201:enzymology; D000818:Animals; D016367:Baculoviridae / Q000201:enzymology / Q000235:genetics*; D019556:COS Cells; D002460:Cell Line; D003474:Curcumin / Q000494:pharmacology; D019204:GTP-Binding Proteins; D007313:Insecta; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D010739:Phospholipase D; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D003474:Curcumin; D013755:Tetradecanoylphorbol Acetate