Mechanism of inhibition of E1-E2 ATPases by melittin.
The inhibition of Na,K-ATPase from duck salt gland and Ca-ATPase from rabbit skeletal muscle sarcoplasmic reticulum by melittin, a 26-residue peptide from bee venom, was studied. Melittin irreversibly inhibits both enzymes. At melittin/ATPase molar ratio (30-50):1, the time dependence of the inhibition is described by the sum of two exponential curves. At pH 7.0, the fast phase of the inhibition provides for about 50% of total loss of activity with pseudo-first order rate constants of 1.52 +/- 0.17 and 1.20 +/- 0.21 min-1 for Na,K- and Ca-ATPase, respectively. The corresponding pseudo-first order rate constants for the slow phase were 0.12 +/- 0.02 and 0.09 +/- 0.02 min-1. The inhibition of both enzymes by melittin depends upon pH; the inhibition increases when the pH is increased from 6.0 to 8.5. The enhancement of the inhibition concomitant to increase in pH is mainly due to an increase in the rate constant of the fast phase. ATP protects both enzymes from the inhibition by melittin; however, the character of protection is different for Ca-versus Na,K-ATPase. The protection of Ca-ATPase activity by ATP is due to an increase in melittin-insensitive activity. The protective effect of ATP on Na,K-ATPase is due to a decrease in the rate constant of fast phase as well as an increase in melittin-insensitive activity. The data suggest that the inhibition of Ca- and Na,K-ATPases by melittin results from the interaction of the peptide with two different sites. One of the sites may be located on the catalytic subunit of the enzymes, the other can be related to the lipid bilayer of the membrane.
Murtazina
D A
DA
Department of Biochemistry, School of Biology, Lomonosov Moscow State University, Russia.
Mast
N V
NV
Rubtsov
A M
AM
Lopina
O D
OD
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers; D008555:Melitten / Q000494:pharmacology*; D018482:Muscle, Skeletal / Q000201:enzymology; D011817:Rabbits; D012491:Salt Gland / Q000201:enzymology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*
D004791:Enzyme Inhibitors; D008051:Lipid Bilayers; D008555:Melitten; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase