Abstract
Serum contains a factor that co-purifies with albumin and causes neurite retraction in PC12 cells, inhibits the proliferation of tumor cells in vitro, and activates the phosphatidylinositol/Ca2+ second messenger system in Xenopus oocytes and other cells. The activity of serum albumin depends on several lysophospholipids bound to albumin. Thin layer chromatographic analysis of the lipids extracted by methanol from serum albumin revealed over a dozen components, several of which evoked oscillatory currents in oocytes. In contrast to serum albumin, most of these lipids were absent in plasma, which lacks the biological activity. The most abundant naturally occurring active component was identified as stearoyl-lysophosphatidic acid. Synthetically prepared lysophosphatidates reproduced the biological activities of the natural serum factor. Adding synthetic lysophosphatidates to inactive fatty acid-free albumin restored activity to the albumin, making the active factor nondialyzable against aqueous solvents and protecting against digestion by various lipases. Since the biologically active lysophosphatidates were produced during blood clotting, in the presence of platelets, and lysophosphatidates have been shown previously to activate platelets, we propose that lysophosphatidates may play an important role in linking platelet activation to receptor-mediated tissue regeneration.
Highlights
Serum contains a factor that co-purifies with albu- potent mitogen for cultured fibroblasts.’ Responses to active min and causes neurite retraction in PC12 cells, inhib- serum albumin in these bioassays were accompanied by the its the proliferation of tumor cells in vitro, and acti- concomitant activation of the phosphatidylinositol/Ca*+ sigvates the phosphatidylinositol/Ca2+second messenger naling system
Rents in Xenopus oocytes through a receptor-mediated activation of the phosphatidylinositol second messenger system (Tigyi et al, 1990).The serum factorresponsible forthe effect is produced during blood clotting and co-purifies with a particularsubfraction of serumalbumin (Tigyi et d., 1991a, (100 PM dissolved with 100 PM FAF-HSA) was dialyzed against 1,000fold excess distilled water or methanol (HPLC grade) using Spectra Por 2 membrane for 16 h
In all subsequent ledi, 1982).Lipids were dissolved in minimal volume of methanol, experiments, methanol was used to extract the active comdiluted further (>lOO-fold) in frog Ringer's solution, and applied by ponent from serum albumin
Summary
Thin layer chromatographic analy- 1991a) and because it regulates cell proliferation and differsis of the lipids extracted by methanol from serum albumin revealed over a dozen components, several of which evoked oscillatory currents in oocytes. Exceeding over a millionfold, elicit oscillatory chloride cur- Human serumalbumin (100 PM in distilled water) or LPA 18:O rents in Xenopus oocytes through a receptor-mediated activation of the phosphatidylinositol second messenger system (Tigyi et al, 1990).The serum factorresponsible forthe effect is produced during blood clotting and co-purifies with a particularsubfraction of serumalbumin Enzymatic Digestion--100 PM HSA, 1 mg of Bond Elut purified phospholipids, or 200 PM LPA 181 was used as substrate in the different enzymatic cleavage reactions. (Alabaster, under nitrogen, and the digests were stored at -80 "C until further AL) and Sigma The purity of those components that were found to analysis.
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