An amphitropic cAMP-binding protein in yeast mitochondria

ABSTRACT: We describe the first example of a mitochondrial protein with a covalently attached phos-phatidylinositol moiety acting as a membrane anchor. The protein can be metabolically labeled with both stearic acid and inositol. The stearic acid label is removed by phospholipase D whereupon the protein with the retained inositol label is released from the membrane. This protein is a cAMP receptor of the yeast Saccharomyces cereuisiae and tightly associated with the inner mitochondrial membrane. However, it is converted into a soluble form during incubation of isolated mitochondria with Ca2+ and phospholipid (or lipid derivatives). This transition requires the action of a proteinaceous, N-ethylmaleimide-sensitive component of the intermembrane space and is accompanied by a decrease in the lipophilicity of the cAMP receptor. We propose that the component of the intermembrane space triggers the amphitropic behavior of the mitochondrial lipid-modified CAMP-binding protein through a phospholipase activity. Only in recent years specific fatty acids have been recog-nized to play important roles in the association of proteins with membranes. Both noncovalent and covalent interactions be-tween fatty acids and proteins have been reported. Among the latter are GTP-binding proteins (Molenaar et al., 1988)

The phospholipidomic signatures of human blood microparticles, platelets and platelet-derived microparticles: A comparative HILIC-ESI-MS investigation

The phospholipidomic signatures of human blood microparticles and platelets, evaluated by hydrophilic interaction liquid chromatography coupled to electrospray ionization - mass spectrometry, were compared. The phospholipidome of platelet-derived microparticles, obtained by platelets stimulation with a mixture of Ca(II), thrombin and collagen, was also considered for the comparison. Platelets, blood microparticles and platelet-derived microparticles displayed qualitatively similar phospholipidomes, all based on eight major phospholipid classes, namely: phosphatidylcholines, diacyl- and plasme(a)nyl-phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, sphingomyelins and lyso forms of phosphatidylcholines and phosphatidylethanolamines. However, while the phospholipidomes of platelets and platelet-derived microparticles were found to be generally similar also from a quantitative point of view, a higher relative incidence of species bearing polyunsaturated side chains, especially in phospholipid classes sharing the choline head (i.e. phosphatidylcholines and lyso-phosphatidylcholines), was observed in the case of blood microparticles. As a further peculiar feature, never reported before, the relative abundance of lyso-phosphatidylcholines among the eight identified phospholipid classes was found to be significantly higher in the lipid extracts of blood microparticles

Hydrophilic interaction liquid chromatography – electrospray ionization - tandem mass spectrometry of a complex mixture of native and oxidized phospholipids.

A mixture of native and oxidized phospholipids (PLs), generated by the soybean lipoxygenase type V-catalyzed partial oxidation of a lipid extract obtained from human platelets, was analyzed by HydrophilicInteraction Liquid Chromatography-ElectroSpray Ionization-Tandem Mass Spectrometry (HILIC–ESI-MS/MS). The complexity of the resulting mixture was remarkable, considering that the starting lipid extract, containing (as demonstrated in a previous study) about 130 native PLs, was enriched with enzy-matically generated hydroperoxylated derivatives and chemically generated hydroxylated forms of PLsbearing polyunsaturated side chains. Nonetheless, the described analytical approach proved to be verypowerful; indeed, focusing on phosphatidylcolines (PCs), the most abundant PL class in human platelets,about fifty different native/oxidized species could be identified in a single HILIC–ESI-MS/MS run. Low-energy collision induced dissociation tandem MS (CID-MS/MS) experiments on chromatographicallyseparated species showed single neutral losses of H2O2 and H2O to be typical fragmentation pathways of hydroperoxylated PCs, whereas a single H2O loss was observed for hydroxylated ones. Moreover, diag-nostic losses of n-hexanal or n-pentanol were exploited to recognize PCs hydroperoxylated on the last butfive carbon atom of a -6 polyunsaturated side chain. Despite the low resolution of the 3D ion trap mass analyzer used, the described HILIC–ESI-MS/MS approach appears very promising for the identification ofoxidized lipids in oxidatively stressed complex biological systems