1-Butanol interferes with phospholipase D1 and protein kinase Calpha association and inhibits phospholipase D1 basal activity.

1-Butanol is commonly used as a substrate for phospholipase D (PLD) activity measurement. Surprisingly we found that, in the presence of 30mM 1-butanol (standard PLD assay conditions), PLD1 activity in COS-7 cells was lost after incubation for 2min. In contrast, in the presence of the protein kinase C (PKC) inhibitor staurosporine or dominant negative PKCα D481E, the activity was sustained for at least 30min. The binding between PLD1 and PKCα was also lost after 2min incubation with 30mM 1-butanol while staurosporine and D481E maintained the binding. 1-Butanol at 2mM did not inhibit PLD1 basal activity or PLD1 binding to PKCα, and staurosporine and PKCα D481E produced a constant increase in PLD1 basal activity of 2-fold. These results indicate that 1-butanol is inhibitory to PLD1 activity by reducing its association with PKCα, and that the concentration of 1-butanol is an important consideration in assaying basal PLD1 activity.