Effects of peptide toxins on the Ca2+-ATPase of sarcoplasmic reticulum

Baker, Keren Julie (1994) Effects of peptide toxins on the Ca2+-ATPase of sarcoplasmic reticulum. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

The effects of the peptide toxin melittin, from honey bee venom and peptide fragments corresponding the N- and C-terminal region of myotoxin a from Crotalus viridis viridis snake venom have been investigated, on the function of the Ca²⁺-ATPase. Melittin and the C-terminal region of myotoxin a were shown to inhibit Ca²⁺-dependent ATPase activity by causing a reduction in the rate of the dephosphorylation step from E2P to E2. In addition the myotoxin a C-terminal peptide reduced the Ca²⁺ affinity of both the inner and outer Ca²⁺ binding sites on the Ca²⁺-ATPase. The effects of various melittin analogues on the inhibition of ATPase activity have shown that the C-terminal tetrapeptide of melittin is necessary for inhibition of ATPase activity. Melittin and its analogues shift the E1/E2 equilibrium towards E1. This effect correlated better with effects on Ca²⁺ accumulation than on ATPase activity. It is suggested that these effects follow from binding at the membrane-water interface whereas effects on the rate of dephosphorylation and ATP hydrolysis follow from binding to the cytoplasmic domain of the Ca²⁺-ATPase.

The three dimensional structure of the Ca²⁺-ATPase has been studied using the technique of resonance energy transfer. The Ca²⁺-ATPase has been labelled with 5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulphonic acid (IAEDANS) at Cys-670/674 and the distance between Cys-670/674 and Glu-439 labelled with 5-(bromomethyl)fluorescein (BrF) or Lys-515 labelled with fluorescein isothiocyanate (FITC) estimated as 40 and 54 Å respectively. The distance between Glu-439 labelled with BrF and Lys-515 labelled with eosin isothiocyanate (EITC) has been estimated as 41 Å. The height of Cys-670/674 above the phospholipid/water interface has been measured by resonance energy transfer between IAEDANS-labelled ATPase and fluorescein-labelled phosphatidylethanolamine (PE) as 54 Å. Conformational changes on the Ca²⁺-ATPase or toxin binding have been shown to result in no significant changes in the measured distances.

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