Biochemical and Molecular Characterization of Two Phosphatidic Acid-selective Phospholipase A1s, mPA-PLA1α and mPA-PLA1β

Abstract

We have identified a novel phospholipase A 1 , named mPA-PLA 1 ␤, which is specifically expressed in human testis and characterized it biochemically together with previously identified mPA-PLA 1 ␣. The sequence of mPA-PLA 1 ␤ encodes a 460-amino acid protein containing a lipase domain with significant homology to the previously identified phosphatidic acid (PA)-selective PLA 1 , mPA-PLA 1 ␣. mPA-PLA 1 ␤ contains a short lid and deleted ␤9 loop, which are characteristics of PLA 1 molecules in the lipase family, and is a member of a subfamily in the lipase family that includes mPA-PLA 1 ␣ and phosphatidylserine-specific PLA 1 . Both mPA-PLA 1 ␤ and mPA-PLA 1 ␣ recombinant proteins exhibited PA-specific PLA 1 activity and were vanadate-sensitive. When mPA-PLA 1 ␤-expressing cells were treated with bacterial phospholipase D, the cells produced lysophosphatidic acid (LPA). In both mPA-PLA 1 ␣ and ␤-expressing cells, most of the PA generated by the phospholipase D (PLD) treatment was converted to LPA, whereas in control cells it was converted to diacylglycerol. When expressed in HeLa cells most mPA-PLA 1 ␣ protein was recovered from the cell supernatant. By contrast, mPA-PLA 1 ␤ was recovered almost exclusively from cells. Consistent with this observation, we found that mPA-PLA 1 ␤ has higher affinity to heparin than mPA-PLA 1 ␣. We also found that the membrane-associated mPA-PLA 1 s were insoluble in solubilization by 1%

Phosphatidic acid metabolism in rat liver cell nuclei

“…In this respect, our results showed that under PLA assay conditions LPA is formed from PA and that it could be rapidly converted into MAG and glycerol-3-phosphate by PLA or LPP and by LPA-phospholipase, respectively. This also agrees with other observations [18] according to which most of the PA generated by treatment with PLD in cells expressing PA-PLA forms is converted into LPA. Some of the PLA activities which hydrolyze either PC or PE have been identified in nuclei but they do not hydrolyze PA [36,38].…”

“…PA-PLA activity was determined as described previously [18]. Lipids were extracted with chloroform:methanol (2:1, v/v) and washed with 0.2 volume of CaCl 2 (0.05%) [19].…”

ABCA3 as a Lipid Transporter in Pulmonary Surfactant Biogenesis

“…The content of choline phospholipids in the interstitial fraction of lung tissues was analyzed by mass spectrometry. MS analysis was performed according to previous reports (23,24) by using a Quattro Micro tandem quadrupole mass spectrometer (Waters) equipped with an ESI source. Phospholipid molecular species were separated and analyzed using normal-phase Cap-liquid chromatography ESI-MS with a silica column (Deverosil Si60, 150 ϫ 0.3 mm inner diameter, 5 m particle size, Nomura Chemicals, Nagoya, Japan).…”

Bile salt–dependent efflux of cellular phospholipids mediated by ATP binding cassette protein B4

“…Mass spectrometric analyses were performed with a triple quadrupole instrument model Quattro micro (Waters, Milford, MA) equipped with an electrospray source as described. 22 The samples were provided by the UltiMate high-performance liquid chromatography system (Dionex Corp., Sunnyvale, CA) into the electrospray interface at a flow rate of 4 l/minute in a solvent system of acetonitrile-methanol-water (2:3:1) containing 0.1% ammonium formate (pH 6.4). The mass spectrometer was operated in the positive and negative scan modes.…”