Calmodulin is a nonessential activator of secretory phospholipase A(2).
Ammodytoxins are presynaptically neurotoxic snake venom group IIA secreted phospholipase A(2) enzymes that interact specifically with calmodulin in the cytosol of nerve cells. We show that calmodulin behaves as an activator of ammodytoxin under both nonreducing and reducing (cytosol-like) conditions by stimulating its enzymatic activity up to 21-fold. Kinetic analysis, using a general modifier mechanism, and surface plasmon resonance measurements reveal that calmodulin influences both the catalytic and the vesicle binding properties of the enzyme without affecting its calcium binding properties. The equilibrium dissociation constant of the ammodytoxin-calmodulin complex under cytosol-like conditions is in the low nanomolar range (3 nM), while under nonreducing conditions, the binding affinity is in the subnanomolar range (0.07-0.18 nM). Upon exposure to cytosol-like conditions, ammodytoxin undergoes a slow hysteretic transition to a less active state. Calmodulin stabilizes the conformation of ammodytoxin and thereby restores its activity. These results provide insights into the neurotoxic action of ammodytoxins and the mechanisms involved in the regulation of secreted phospholipase A(2) activity within the cytosol.
Kovacic
Lidija
L
Department of Molecular and Biomedical Sciences, Jozef Stefan Institute, Ljubljana, Slovenia.
Novinec
Marko
M
Petan
Toni
T
Baici
Antonio
A
Krizaj
Igor
I
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D002413:Cations, Divalent; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D008958:Models, Molecular; D009474:Neurons / Q000737:chemistry / Q000378:metabolism; D054467:Phospholipases A2 / Q000737:chemistry / Q000378:metabolism*; D012910:Snake Venoms / Q000201:enzymology*; D013552:Swine
D002147:Calmodulin; D002413:Cations, Divalent; D012910:Snake Venoms; D054467:Phospholipases A2; D002118:Calcium