Autocatalytic acylation of phospholipase-like myotoxins.
Several snake venoms contain a phospholipase A2 in which position 49 in the active site is occupied by a lysine or a serine instead of the aspartate residue normally found. Although these proteins do not bind Ca2+ and are devoid of catalytic activity, they are still highly specific myotoxins and have recently been shown to induce membrane leakage by a new type of cytolytic mechanism. Three of these toxins, myotoxin II from Bothrops asper, ammodytin L from Vipera ammodytes, and the K49 protein from Agkistrodon piscivorus piscivorus, were examined for their interaction with fatty acids and were found to bind long-chain fatty acids covalently by a rapid, spontaneous, autocatalytic process. The fatty acids could be released by treatment with 1 M NH2OH or NaOH, but not with 1 M NaCl or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electron spin resonance studies using spin-labeled fatty acids showed that only the carboxyl headgroup of the fatty acid was linked to protein amino acid, the carbon chain had free mobility and did not bind tightly to the protein surface. Stearic acid methyl esters and short-chain fatty acids did not bind to the toxins. Acylated myotoxins bound to the surface of liposomes and isolated muscle membranes, with the fatty acid moiety inserted into the lipid bilayer and possibly acting as an anchor. The phospholipase-like myotoxins represent the first group of proteins able to undergo acylation by spontaneous reaction with free fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)
Pedersen
J Z
JZ
Department of Biology, University of Rome Tor Vergata, Italy.
Lomonte
B
B
Massoud
R
R
Gubensek
F
F
Gutiérrez
J M
JM
Rufini
S
S
D000215:Acylation; D001665:Binding Sites; D002384:Catalysis; D003435:Crotalid Venoms / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005227:Fatty Acids / Q000378:metabolism; D005454:Fluorescence Polarization; D054501:Group II Phospholipases A2; D009498:Neurotoxins / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D030162:Reptilian Proteins; D014757:Viper Venoms / Q000378:metabolism*
C031442:Agkistrodon venoms; D003435:Crotalid Venoms; D005227:Fatty Acids; D009498:Neurotoxins; D030162:Reptilian Proteins; D014757:Viper Venoms; C065419:ammodytin L; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054467:Phospholipases A2; C061056:myotoxin II, Bothrops asper