Melittin-induced inhibition and aggregation of Ca-ATPase in skeletal muscle sarcoplasmic reticulum: a comparative study.
Incubation of melittin with sarcoplasmic reticulum membranes at pH 7. 0 and different melittin:Ca-ATPase molar ratios results in the progressive loss of enzyme activity. At high melittin:Ca-ATPase molar ratios (10:1 and 30:1), enzyme inhibition may be described by a biexponential curve. At pH 7.0, the values of the pseudo-first-order rate constants are 1.0 and 0.1 min-1 for the fast and slow phases of inhibition, respectively, at a melittin:Ca-ATPase molar ratio of 30:1. At pH 6.0 and a melittin:Ca-ATPase molar ratio of 30:1, melittin does not inhibit Ca-ATPase. Melittin-induced aggregation of Ca-ATPase molecules was studied using cupric phenanthroline as a chemical cross-linking agent. At a melittin:Ca-ATPase molar ratio of 5:1, aggregation of Ca-ATPase protein was not observed; however, the loss of enzyme activity was about 30% after 30 min. At melittin:Ca-ATPase molar ratios of 10:1 and 30:1, significant aggregation of Ca-ATPase protein takes place. The rate of Ca-ATPase aggregation is much lower than the rate of enzyme inhibition. At melittin:Ca-ATPase molar ratios of 10:1 and 30:1, the rate of Ca-ATPase protein aggregation is close to that for the slow phase of enzyme inhibition. At pH 6.0 and a melittin:Ca-ATPase molar ratio of 30:1, significant aggregation of Ca-ATPase occurs. It is concluded that melittin induces both Ca-ATPase inhibition and aggregation. These two processes may occur simultaneously, but under some conditions either inhibition or aggregation takes place independently of each other. Therefore, the aggregation of Ca-ATPase induced by melittin is not necessary for enzyme inhibition.
Shorina
E A
EA
Department of Biochemistry, School of Biology, Lomonosov Moscow State University, Russia.
Mast
N V
NV
Lopina
O D
OD
Rubtsov
A M
AM
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D003432:Cross-Linking Reagents; D007425:Intracellular Membranes / Q000187:drug effects / Q000201:enzymology; D008555:Melitten / Q000494:pharmacology*; D010618:Phenanthrolines / Q000494:pharmacology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology*; D013997:Time Factors
D003432:Cross-Linking Reagents; D010618:Phenanthrolines; C014741:bis(1,10-phenanthroline)copper(2+) ion; D008555:Melitten; D000252:Calcium-Transporting ATPases