Gossypol modification of Ala-1 of secreted phospholipase A(2): A probe for the kinetic effects of sulfate glycoconjugates

被引:21
作者
Yu, BZ
Rogers, J
Ranadive, G
Baker, S
Wilton, DC
ApitzCastro, R
Jain, MK
机构
[1] UNIV DELAWARE, DEPT CHEM & BIOCHEM, NEWARK, DE 19716 USA
[2] UNIV SOUTHAMPTON, DEPT BIOCHEM, SOUTHAMPTON SO16 7PX, HANTS, ENGLAND
[3] IVIC, THROMBOSIS LAB, CARACAS, VENEZUELA
关键词
D O I
10.1021/bi962972i
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Gossypol is shown to covalently modify secreted phospholipase A(2) (PLA2) in the aqueous phase, but not at the interface. A rapid initial noncovalent binding of gossypol is followed by a slow covalent modification of the alpha-amino group of Ala-l by stoichiometric amounts of gossypol, The rate of modification increases in the presence of calcium, but occupancy of the substrate binding site does not alter the rate. Pancreatic PLA2 is modified at the alpha-amino group of the N terminus to form a Schiff base, which can be stabilized by reduction with borohydride. Residual activity of the gossypol-modified PLA2 from several different sources is about 10%, indicative of impaired catalytic turnover. The halftime for the inactivation is about 10 min, and it is more than 100-fold longer for PLA2 at the interface. Gossypol promotes binding of PLA2 to the interface, and the binding of PLA2 to the interface promotes only the noncovalent binding of gossypol, but not the covalent modification. Gossypol, in conjunction with spectroscopic and kinetic protocols, is used to characterize the kinetic effects of sulfated glycoconjugates, heparin and artery wall proteoglycans, with human inflammatory and pancreatic PLA2(1) The conjugates do not interfere with the binding of PLA2 to the interface or with the catalytic cycle at the interface. The conjugates do not influence the kinetics of modification of PLA2 by gossypol in the aqueous phase, and the enzyme at the interface is not modified in the presence of the conjugates, The conjugates bind to PLA2 at the interface with only a modest effect on the interfacial catalytic turnover without dislodging the bound enzyme, Complex kinetic effects induced by the conjugates are shown to be due to sequestration of PLA2 in the aqueous phase as a high-molecular mass complex, which dissociates with added NaCl.
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页码:12400 / 12411
页数:12
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