Membrane interactions of amphiphilic polypeptides mastoparan, melittin, polymyxin B, and cardiotoxin. Differential inhibition of protein kinase C, Ca2+/calmodulin-dependent protein kinase II and synaptosomal membrane Na,K-ATPase, and Na+ pump and differentiation of HL60 cells.
Interactions of certain naturally occurring, amphiphilic polypeptides with membranes were investigated. Mastoparan (wasp venom toxin), melittin (bee venom toxin), cardiotoxin (cobra venom toxin), and polymyxin B (antibacterial antibiotic) inhibited protein kinase C stimulated by phosphatidylserine bilayer or arachidonate monomer and blocked binding of [3H] phorbol 12,13-dibutyrate to protein kinase C in the presence of phosphatidylserine bilayer, with IC50 values (concentrations causing 50% inhibition) of 1-8 microM. Mastoparan and polymyxin B were much less inhibitory (IC50, 10-20 microM), whereas melittin and cardiotoxin were similarly inhibitory (IC50, 1-4 microM), when protein kinase C was activated instead by synaptosomal membrane. Kinetic analysis indicate that mastoparan inhibited protein kinase C, assayed using phosphatidylserine or synaptosomal membrane as the phospholipid cofactor, competitively with the phospholipid cofactor, in a mixed manner with CaCl2 or diacylglycerol, noncompetitively with histone, and uncompetitively with ATP, with apparent Ki values of 1.6-18.7 microM. Inhibition of Na,K-ATPase in the membrane by these polypeptides had relative potencies different from those for their inhibition of protein kinase C activated by the same membrane preparation; mastoparan and melittin inhibited the two activities with comparable potencies, but polymyxin B and cardiotoxin were far less effective in inhibiting Na,K-ATPase. The same relative inhibitory potencies of the polypeptides (melittin greater than mastoparan greater than polymyxin B) for inhibition of Na,K-ATPase were also noted for their inhibition of Ca2+/calmodulin-dependent protein kinase II, 86Rb uptake (Na+ pump) by HL60 cells and the phorbol ester-induced differentiation of the leukemia cells. These findings were consistent with discrete interactions of the polypeptides with functionally distinct sites on the membrane, leading to differential inhibition of biological activities associated with the membrane. Actions of certain polypeptides appeared to be more specific compared to those of lipid second messengers such as lyso-phosphatidylcholine and sphingosine, and the antineoplastic ether lipid analogs such as 1-O-octadecyl-2-methyl-rac-glycero-3-ophosphocholine.
Raynor
R L
RL
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322.
Zheng
B
B
Kuo
J F
JF
D000818:Animals; D001692:Biological Transport; D002454:Cell Differentiation; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004179:Cobra Cardiotoxin Proteins / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008555:Melitten / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011112:Polymyxin B / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*; D013552:Swine; D013574:Synaptosomes / Q000201:enzymology*; D014862:Wasp Venoms / Q000378:metabolism
D004179:Cobra Cardiotoxin Proteins; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D047428:Protein Kinase Inhibitors; D014862:Wasp Venoms; D008555:Melitten; C037500:mastoparan; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011112:Polymyxin B