The Mechanism of Inhibition of the Ca2+-ATPase by Mastoparan

Abstract

The amphiphilic peptide mastoparan, isolated from wasp venom, is a potent inhibitor of the sarcoplasmic reticulum Ca 2؉ -ATPase. At pH 7.2, ATPase activity is inhibited with an inhibitory constant (K i ) of 1 ؎ 0.13 M.

The widely utilized brominated flame retardant tetrabromobisphenol A (TBBPA) is a potent inhibitor of the SERCA Ca2+ pump

“…These results would suggest that TBBPA binds directly to the SERCA, without the need to partition first into the phospholipid bilayer. Figure 6(A) shows the effect of 3.5 µM TBBPA on the tryptophan fluorescence changes of SERCA upon the addition of a range of free Ca 2+ concentrations, which is often used to assess the affinity of Ca 2+ binding to the ATPase [7,8,31]. In the absence of TBBPA, the addition of Ca 2+ gave a F max of 9.1 + − 0.3 % and a K d (app) of 0.7 + − 0.1 µM, whereas in the presence of 3.5 µM TBBPA, there was a decrease in the maximal fluorescence change ( F max = 5.21 + − 0.04 %) and a substantial increase in K d (app).…”

“…The activities of SERCA Ca 2+ pumps have been found to be altered by a wide spectrum of lipophilic molecules, including a number of environmental endocrine disrupters [4][5][6]. Although a number of these molecules have non-specific effects on these pumps, some of these hydrophobic molecules interact specifically at distinct sites on the protein and, in some cases, these molecules have proved invaluable in elucidating mechanistic steps within the Ca 2+ transport processes [7][8][9][10]. TBBPA (tetrabromobisphenol A) is a highly lipophilic halogenated aromatic molecule and currently the most widely used type of BFR (brominated flame retardant) [11].…”

“…Rapid kinetic fluorescence measurements were performed using a stopped-flow fluorescence spectrophotometer as described in [7,8]. In the Ca 2+ -binding experiments, syringe A, containing the SERCA (1 µM) in 20 mM Hepes/Tris (pH 7.2), 100 mM KCl, 5 mM MgSO 4 and 50 µM EGTA, was rapidly mixed with syringe B, containing 1 mM Ca 2+ (final concentration).…”

“…• C as described in [4,7,8]. The SERCA was diluted to 0.1 mg/ml in 20 mM Hepes/Tris (pH 7.2) containing 100 mM KCl, 5 mM MgSO 4 and 1 mM CaCl 2 in a total volume of 1 ml.…”

“…Excitation was at 342 nm and emission at 395 nm. to the SERCA was monitored by observing the change in intrinsic tryptophan fluorescence as described in [4,7,8]. SERCA (50 µg/ml in the absence and presence of 3.5 µM TBBPA) was added to a buffer containing 20 mM Hepes/Tris, 100 mM MgSO 4 (pH 7.0) and any conformational changes were measured as a percentage change in total tryptophan fluorescence over a range of free Ca 2+ concentrations (10-100 µM) at 25…”