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Journal of Biochemistry Advance Access originally published online on December 5, 2006
Journal of Biochemistry 2007 141(1):77-84; doi:10.1093/jb/mvm007
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© 2006 The Japanese Biochemical Society.

Cytosolic PLA2 in Zymogen Granule Fusion and Amylase Release: Inhibition of GTP-induced Fusion by Arachidonyl Trifluoromethyl Ketone Points to cPLA2 in G-Protein-mediated Secretory Vesicle Fusion

Akm A. Sattar* and Reazul Haque

Division of Endocrinology, Department of Internal Medicine, Wayne State University School of Medicine, 421 East Canfield Avenue, Detroit, Michigan 48201, USA

*To whom correspondence should be addressed. Tel: (313) 577-9405, Fax: (313) 577-8615, E-mail: asattar{at}med.wayne.edu

Received October 17, 2006; Accepted November 14, 2006


  Abstract

Previously we reported that the G-protein G{alpha}i3 localized in pancreatic zymogen granule (ZG) membrane participates in vesicular fusion at the cell plasma membrane (PM). In the present study, the presence of cytosolic phosholipase A2 (cPLA2) in rat ZGs was demonstrated and its potential role in G-protein-mediated ZG–PM fusion was investigated. In vitro fusion assays utilizing both enzymatic and fluorimetric techniques demonstrate that ZGs fuse with PM with a greater potency in the presence of GTP. Arachidonyl trifluoromethyl ketone (AACOCF3) at 40 µM reduces GTP-induced ZG–PM fusion by 25–50%. Anti-cPLA2 antibody reduces ZG–PM fusion in a dose-dependent manner and a 50% reduction of the fusion takes place in the range of 0.48–0.64 ratios of cPLA2 antibody to ZG proteins. PLAP, a cPLA2 activator synthetic peptide increases ZG–PM fusion in a limited dose-dependent manner and tends to inhibit at higher concentrations. Exogenous arachidonic acid inhibits GTP-induced ZG–PM fusion in a dose-dependent manner. Furthermore, a non-hydrolysable GTP analogue, Gpp(NH)p, reduces PLAP effect in ZG–PM fusion; and the net effect of Gpp(NH)p and PLAP differs significantly from the net effect of GTP and PLAP on ZG–PM fusion suggesting that cPLA2 is involved in G-protein-mediated secretory vesicle fusion.

Key Words: phospholipase A2, phospholipase A2 inhibitor, G-protein, membrane fusion, pancreas, zymogen granule

Abbreviations: ZG, zymogen granule; PM, plasma membrane; GTP, guanosine 5'-triphoshate; Gpp(NH)p, guanosine 5'-[ß, {gamma}-imido]triphosphate; G{alpha}, G protein alpha subunit; HELSS, haloenol lactone suicide substrate; PMSF, phenylmethanesulfonyl fluoride; PBS, phosphate-buffered saline; AACOCF3, arachidonyl trifluoromethyl ketone; PLA2, phospholipase A2; cPLA2, cytosolic PLA2; iPLA2, calcium-independent PLA2; sPLA2, secretory PLA2; PLAP, PLA2 activator synthetic peptide,; ESPLIAKVLTTEPPIITPVR, that spans a region of homology between natural PLAP and melittin


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