Purification and Properties of a Phospholipase A2/Lipase Preferring Phosphatidic Acid, Bis(monoacylglycerol) Phosphate, and Monoacylglycerol from Rat Testis

Abstract

Phospholipase A 2 (PLA 2 ) was purified to homogeneity from the supernatant fraction of rat testis homogenate. The purified 63-kDa enzyme did not require Ca 2؉ ions for activity and exhibited both phosphatidic acid-preferring PLA 2 and monoacylglycerol lipase activities with a modest specificity toward unsaturated acyl chains. Anionic detergents enhanced these activities. Serine-modifying irreversible inhibitors, (p-amidinophenyl) methanesulfonyl fluoride and methylarachidonyl fluorophosphonate, inhibited both activities to a similar extent, indicating a single active site is involved in PLA 2 and lipase activities. The sequence of NH 2 -terminal 12 amino acids of purified enzyme was identical to that of a carboxylesterase from rat liver. The optimal pH for PLA 2 activity (around 5.5) differed from that for lipase activity (around 8.0). At pH 5.5 the enzyme also hydrolyzed bis(monoacylglycerol) phosphate, or lysobisphosphatidic acid (LBPA), that has been hitherto known as a secretory PLA 2 -resistant phospholipid and a late endosome marker. LBPA-enriched fractions were prepared from liver lysosome fractions of chloroquinetreated rats, treated with excess of pancreatic PLA 2 , and then used for assaying LBPA-hydrolyzing activity. LBPA and the reaction products were identified by microbore normal phase high performance liquid chromatography/electrospray ionization ion-trap mass spectrometry. These enzymatic properties suggest that the enzyme can metabolize phosphatidic and lysobisphosphatidic acids in cellular acidic compartments.

Selective decrease of bis(monoacylglycero)phosphate content in macrophages by high supplementation with docosahexaenoic acid

“…Another explanation for the decrease of BMP in the absence of vitamin E is that DHA and 22:6/22:6-PG could promote the activation of BMPhydrolyzing enzyme, possibly through the generation of oxidant species/lipid peroxides. As a matter of fact, lysosomal phospholipase A 2 activity has been reported to exert hydrolytic activity on BMP, including DHA containing BMP (49). It is yet totally unknown whether this enzyme is sensitive to lipids or lipid peroxides.…”

Alteration of the 4-sphingenine scaffolds of ceramides in keratinocyte-specific Arnt-deficient mice affects skin barrier function

“…Epidermal ceramide molecular species were identified by electrospray ionization (ESI)-ion-trap mass spectrometry connected online with normal-phase HPLC (23). The HPLC system consisted of two Gilson model 302 liquid-delivery modules, a Gilson model 811 dynamic mixer (Gilson SAS, Villiers Le Bel, France), and a trap column (1 × 20 mm), and two Valco switching valves (Valco Instruments Co., Houston, Texas, USA) were used to remove components eluting earlier and later than the ceramides.…”

Differential Membrane Packing of Stereoisomers of Bis(monoacylglycero)phosphate

“…However, recent work has revealed the existence of phospholipase A 2 that catalyzes the hydrolysis of naturally occurring BMP (22). Considering the high level of accumulation of BMP in late endosomes, it is suggested that the specific stereoconfiguration of BMP plays an important role in the function of degradative organelles which are forming characteristic multilamellar/multivesicular structures.…”