Identification of hydrophobic interactions between proteins and lipids: free fatty acids activate phospholipase C delta1 via allosterism.
Lipids are well recognized ligands that bind to proteins in a specific manner and regulate their function. Most attention has been placed on the headgroup of phospholipids, and little is known about the role of the acyl chains in mediating any effects of lipids on proteins. In this report, free fatty acids (FFA) were found to bind and activate phospholipase C delta1(PLC delta1). The unsaturated FFA arachidonic acid (AA) and oleic acid were able to stimulate PLC delta1 up to 30-fold in a dose-dependent manner. The saturated FFA stearic acid and palmitic acid were less efficacious than unsaturated FFA, activating the enzyme up to 8-fold. The mechanism of activation appears to be due to a change in K(m) for substrate; 50 microM arachidonate reduced the K(m) for the soluble PLC substrate diC(4)PI from 1.7 +/- 0.6 mM to 0.24 +/- 0.04 mM (7-fold reduction). V(max) was not significantly altered. PLC delta1 bound to sucrose-loaded vesicles that contained AA in a concentration-dependent manner. A fragment of PLC delta1 that encompasses the EF-hand domain also bound to micelles containing AA using nondenaturing PAGE. This same fragment also inhibited AA activation of PLC delta1 in a competition assay. These results suggest that the function of the EF-hand domain of PLC delta1 is to bind lipid and to allosterically regulate catalysis. These results also suggest that esterified and nonesterified fatty acids can bind to and regulate protein function, identifying a functional role for hydrophobic interactions between lipids and proteins.
Kobayashi
Minae
M
Department of Pathology, Northwestern University, The Feinberg School of Medicine, 303 East Chicago Avenue, Chicago, Illinios 60611, USA.
Mutharasan
R Kannan
RK
Feng
Jianwen
J
Roberts
Mary F
MF
Lomasney
Jon W
JW
D000438:Alcohols / Q000737:chemistry / Q000494:pharmacology; D000494:Allosteric Regulation / Q000187:drug effects; D000495:Allosteric Site; D020832:EF Hand Motifs / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D005230:Fatty Acids, Nonesterified / Q000494:pharmacology*; D005960:Glucosides / Q000494:pharmacology; D057927:Hydrophobic and Hydrophilic Interactions; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D050356:Lipid Metabolism; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000494:pharmacology; D054803:Phospholipase C delta; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D000438:Alcohols; D005230:Fatty Acids, Nonesterified; D005960:Glucosides; D007527:Isoenzymes; D010446:Peptide Fragments; C040358:dodecyl maltoside; D010738:Type C Phospholipases; D054803:Phospholipase C delta