PHOSPHOLIPASE A(2) FROM PLASMA OF PATIENTS WITH SEPTIC SHOCK IS ASSOCIATED WITH HIGH-DENSITY-LIPOPROTEINS AND C3 ANAPHYLATOXIN - SOME IMPLICATIONS FOR ITS FUNCTIONAL-ROLE

被引:35
作者
GIJON, MA
PEREZ, C
MENDEZ, E
CRESPO, MS
机构
[1] CSIC, DEPT BIOQUIM & FISIOL, E-47005 VALLADOLID, SPAIN
[2] FDN JIMENEZ DIAZ, SERV MED INTENS, E-28040 MADRID, SPAIN
[3] HOSP RAMON Y CAJAL, SERV ENDOCRINOL, E-28034 MADRID, SPAIN
关键词
D O I
10.1042/bj3060167
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
Phospholipase A(2) (PLA(2)) activity was purified 12 544-fold with a 13% yield from the plasma of patients diagnosed of septic shock by the sequential use of heparin-agarose affinity chromatography, gel filtration, and reverse-phase f.p.l.c. Gel-filtration chromatography of plasma omitting high-ionic-strength buffer revealed a molecular mass different from that of purified PLA(2) and co-elution with apolipoprotein A-I peaks, which suggests its association with high-density lipoproteins (HDL). N-terminal analysis of the enzyme activity protein band, electroblotted from a SDS-acrylamide gel and with an assessed molecular mass of 19 kDa, showed an identical sequence to that of alpha-chain of human C3 complement component, suggesting the presence in this band of a complex formed by a complement C3-derived anaphylatoxin (C3a)-related fragment and the PLA(2) linked side-by-side. Because the preparation of plasma enzyme showed lower activity than the enzyme obtained from fibroblasts transfected with the coding sequence of human group-II PLA(2), and because the addition of C3-derived anaphylatoxins from human serum inhibited the activity of this recombinant PLA(2), it was considered that C3a-related peptides behave as inhibitors of group-II PLA(2). The enzyme showed optimal activity on [C-14]oleate-labelled autoclaved E. coli, on synthetic phosphatidylethanolamine, and on [H-3]arachidonate-labelled membranes of the monoblast cell line U937, but it did not show any activity on the release of [H-3]arachidonate from pre-labelled human polymorphonuclear leukocytes (PMNs). In short, PLA(2) from plasma of sepsis patients shows unique associations with other plasma proteins which may influence its functional properties. The association with C3-related peptides shows an inhibitory effect on the enzyme activity, whereas the association with HDL might influence its environment and/or its interaction with cells. The study of the catalytic properties shows a prominent effect on bacterial phospholipids, synthetic phosphatidylethanolamine, and membranes from U937 monoblasts, but not on synthetic phosphatidylcholine or on PMNs, even when these cells were maintained in culture to allow spontaneous apoptosis and became a good substrate for pancreatic type PLA(2).
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页码:167 / 175
页数:9
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