title: Synergism between mellitin and phospholipase A2 from bee venom: apparent activation by intervesicle exchange of phospholipids.
abstract: Mellitin, a cationic amphiphilic peptide, has an apparent activating effect on interfacial catalysis by phospholipase A2 (PLA2) of bee venom on zwitterionic vesicles of 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine (POPC) and on anionic vesicles of 1,2-dimyristoylglycero-sn-3-phosphomethanol (DMPM), as well as on covesicles of POPC/DMPM (3:7). On the other hand, mellitin-induced increase in the rate of pig pancreatic PLA2 is seen only on anionic vesicles. Interfacial kinetic protocols and spectroscopic methods show that the activation is due to enhanced substrate replenishment resulting from intervesicle exchange of zwitterionic or anionic phospholipids through vesicle-vesicle contacts established by mellitin. It is shown that as the hydrolysis on POPC vesicles progresses, due to a high propensity of bee PLA2 for binding to the product containing zwitterionic vesicles, most of the enzyme in the reaction mixture is trapped on few vesicles that are initially hydrolyzed, and thus reaction ceases. Under these conditions, mellitin promotes substrate replenishment by direct exchange of the products of hydrolysis from the enzyme-containing vesicles with the substrate present in excess vesicles which have not been hydrolyzed. Pig PLA2 has poor affinity for POPC vesicles, and the affinity is only modestly higher in the presence of low mole fractions of the products of hydrolysis; therefore, the enzyme is not trapped on those vesicles. Biophysical studies confirm that the phospholipid exchange occurs through stable intervesicle contacts formed by low mole fractions of mellitin, without transbilayer movement of phospholipids or fusion of vesicles. At high mole fraction (> 1.5%) mellitin induces leakage in POPC vesicles and does not form additional contacts. In POPC/DMPM vesicles, the contacts are formed even at high mole fractions of mellitin. Changes in intrinsic tryptophan fluorescence of mellitin indicate that bound mellitin exists in at least two different functional forms depending on the lipid composition and on the lipid:peptide ratio. A model is proposed to accommodate amphiphilic mellitin as a transmembrane channel or an intervesicle contact.
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark 19716, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Cajal
authors.forename: M K
authors.initials: MK
authors.lastname: Jain
mesh_terms: D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001514:Bee Venoms / Q000201:enzymology*; D004227:Dithionite / Q000378:metabolism; D004357:Drug Synergism; D005227:Fatty Acids / Q000494:pharmacology; D005456:Fluorescent Dyes; D020404:Glycerophospholipids; D006868:Hydrolysis; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism*; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D008555:Melitten / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology; D010712:Phosphatidic Acids / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*; D011721:Pyrenes / Q000378:metabolism; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013552:Swine
chemical_list: D000178:Acrylamides; D001514:Bee Venoms; D005227:Fatty Acids; D005456:Fluorescent Dyes; D020404:Glycerophospholipids; D008081:Liposomes; D008244:Lysophosphatidylcholines; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D011721:Pyrenes; D004227:Dithionite; D008555:Melitten; D020106:Acrylamide; C029313:dimyristoylmethylphosphatidic acid; C042077:N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine; C030984:pyrene; D010741:Phospholipases A; D054467:Phospholipases A2; C028694:1-palmitoyl-2-oleoylphosphatidylcholine