Mini-Symposium - Abstracts
10 STRUCTURE/FUNCTION RELATIONSHIPS OF K49-PHOSPHOLIPASE A2(PLA2) HOMOLOGUES - FLUORESCENCE SPECTROSCOPY STUDIES USING BOTHROPSTOXIN I FROM Bothrops jararacussu AS A MODEL SYSTEM
R.J. WARD
Department of Physics, Institute of Biosciences, Humanitie\s and Physical Sciences, São Paulo State University, São José do Rio Preto, Brazil.
Fluorescence spectroscopy is a versatile and sensitive technique that has significantly contributed to the understanding of the structure/function relationships of a diverse range of proteins. The interaction of proteins with lipid membranes often results in significant conformation changes in protein structure, a process that is readily detected by spectrofluorimetric methods. Various fluorescence techniques have been applied to investigate the unusual membrane damaging activity of bothropstoxin I (BthTx-I), a K49-PLA2 homologue isolated from the venom of Bothrops jararacussu. Although a D49K substitution virtually eliminates the Ca
dependent hydrolytic activity, K49-PLA2s continue to disrupt membranes by a poorly understood mechanism of action even in the presence of Ca chelating agents. The hypothesis that BthTx-I dimer identified by x-ray crystallography is the membrane active form has been tested by measuring the release of liposome entrapped calcein, a self-quenching fluorophore, on addition of BthTx-I. Release kinetics measured by an increased fluorescence signal as the calcein dequenches due to liposomal damage, show second order kinetics that suggests a dimeric active species. In addition, variations in the lipid composition of the model membrane system allow differences in the marker release to be correlated with changes in the intrinsic tryptophan fluorescence emission (ITFE) of the BthTx-I on membrane association. Changing the negatively charged lipid component of the liposome from DMPA (dimyristroil phosphatidic acid) to DMPG (dimyristroil phosphatidylglycerol) abolishes marker release. Changes in the ITFE spectra of BthTx-I show a blue-shift and increased quantum yield on association with DMPA liposomes, yet little change on interaction with DMPG liposomes. This suggests that the membrane damaging activity involves an inactive membrane associated intermediate, perhaps analogous to the E* S (enzyme/substrate) intermediate complex proposed for catalytically active D49 PLA2s (Verheij et al., 1981, Rev. Physiol. Biochem. Pharmacol. 91, 91-203; Jain et al., 1993, Biochemistry 32, 11319-11329).
CORRESPONDENCE TO:
Dr. Richar J. Ward - Departamento de Física, IBILCE, UNESP, Rua Cristóvão Colombo, 2265, CEP 15054-000, São José do Rio Preto, SP, Brasil. email: richard@thor.ibilce.unesp.br
Publication Dates
-
Publication in this collection
08 Jan 1999 -
Date of issue
1997
