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Melittin-induced inhibition and aggregation of Ca-ATPase in skeletal muscle sarcoplasmic reticulum: a comparative
E A Shorina1, N V Mast, O D Lopina
1Department of Biochemistry, School of Biology, Lomonosov Moscow State University, Russia.
Abstract:
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.
Insights
Melittin inhibits Ca-ATPase enzyme activity and causes protein aggregation. This aggregation is not required for melittin
Area of Science:
- Biochemistry
- Membrane protein function
- Enzyme kinetics
Background:
- Sarcoplasmic reticulum Ca-ATPase (SERCA) is crucial for muscle calcium regulation.
- Melittin, a peptide toxin, is known to interact with cell membranes.
- The effect of melittin on Ca-ATPase activity and structure is not fully understood.
Purpose of the Study:
- To investigate the impact of melittin on Ca-ATPase activity and aggregation.
- To determine the relationship between melittin-induced Ca-ATPase inhibition and aggregation.
- To elucidate the role of pH in melittin's effects on Ca-ATPase.
Main Methods:
- Incubation of melittin with sarcoplasmic reticulum membranes at varying pH and molar ratios.
- Assay of Ca-ATPase enzyme activity.
- Chemical cross-linking with cupric phenanthroline to study protein aggregation.
Main Results:
- Melittin progressively inhibits Ca-ATPase activity at pH 7.0, with inhibition kinetics described by a biexponential curve at high melittin:Ca-ATPase ratios.
- At pH 6.0, melittin does not inhibit Ca-ATPase but induces significant aggregation.
- Ca-ATPase aggregation occurs at higher melittin:Ca-ATPase ratios and proceeds slower than enzyme inhibition.
Conclusions:
- Melittin induces both Ca-ATPase inhibition and aggregation.
- These processes can occur independently, suggesting aggregation is not a prerequisite for inhibition.
- pH significantly influences the effects of melittin on Ca-ATPase.