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 30 mM 1-butanol (standard PLD assay conditions), PLD1 activity in COS-7 cells was lost after incubation for 2 min. In contrast, in the presence of the protein kinase C (PKC) inhibitor staurosporine or dominant negative PKCalpha D481E, the activity was sustained for at least 30min. The binding between PLD1 and PKCalpha was also lost after 2 min incubation with 30 mM 1-butanol while staurosporine and D481E maintained the binding. 1-Butanol at 2 mM did not inhibit PLD1 basal activity or PLD1 binding to PKCalpha, and staurosporine and PKCalpha 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 PKCalpha, and that the concentration of 1-butanol is an important consideration in assaying basal PLD1 activity.
Hu
Tianhui
T
Department of Molecular Physiology and Biophysics, Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Exton
John H
JH
D020001:1-Butanol / Q000494:pharmacology*; D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010739:Phospholipase D / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051381:Rats; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors
D004791:Enzyme Inhibitors; D001224:Aspartic Acid; D020001:1-Butanol; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D010739:Phospholipase D; C106801:phospholipase D1; D019311:Staurosporine