Activity of Phospholipase A2 in the Inner Membrane of Rat-Liver Mitochondria

Abstract

The inner membrane of rat liver mitochondria contains a highly active phospholipase Az which has alkaline pH optimum and requires Ca2+ in the micromolar range. The phospholipase is particularly active on the endogenous phosphatidylethanolamine and release relatively high amounts of docosahexanoic acid. The phospholipase A2 of mitochondria or mitoplasts is not dependent on calmodulin. Using fluorescamine-labelled mitoplasts there are indications that the enzyme is localized on both sides of the inner membrane.Mitochondria are known to possess a Ca2+-dependent phospholipase A2 which, beside being involved in the turnover of membrane phospholipids, is responsible for the membrane damage observed in aged mitochondria [l -31 and might be involved in the process of Ca2+-release from mitochondria [4,5]. The mitochondrial phospholipase A2 has an alkaline pH optimum and it prefers phosphatidylethanolamine as endogenous substrate [6]. The enzyme seems to be primarly located on the outer membrane of the mitochondrion [7], however some activity has also been found associated with the inner membrane [7,8]. Presumably the outer membrane enzyme is mainly involved in the turnover of membrane phospholipids whereas the inner membrane enzyme might be more involved in determining the permeability properties and the functional integrity of the inner membrane. It is still unclear how the activity of the mitochondrial and the other intracellular phospholipase A2 is regulated [9]. Since very little has been done to characterize the inner membrane phospholipase A2 we have decided to carry out a detailed study of its functional properties. For this study we have used both mitochondria completely devoid of outer membrane (mitoplasts) and inner membrane vesicles. The results obtained are presented in this article and are discussed in the general context of the metabolic regulation and function of the mitochondrial phospholipase Az. MATERIALS A N D METHODS Preparation of Mitochondria and MitoplastsLiver mitochondria were prepared from Wistar rats by the standard sucrose procedure of Schneider [lo]. The homogenization medium was buffered with 20 mM Tris/Cl pH 7.4 and contained 0.2 mM EDTA. The mitochondrial pellets were washed three times with medium without EDTA to reduce microsomal contamination. The mitochondria which were directly used for phospholipase A2 assay were treated with 30 pg digitonin/mg of protein to eliminate most of the contaminant lysosomes. Mitoplasts were prepared from intact mitochondria with a procedure similar to that described by Abbreviations. PtdEtn, phosphatidylethanolamine; IysoPtdEtn, lysophosphatidylethanolamine; PtdCho, phosphatidylcholine; Hcpes, 4-(2-hydroxyethy1)-l -piperazineethane sulfonic acid.Chan et al. [I I ] using 130 pg digitonin/mg of mitochondrial protein. To obtain complete removal of the outer membrane, the mitoplasts were suspended in 250 mM sucrose, 10 mM Tris/Cl pH 7.5, 0.2 mM EDTA at a concentration of 5 mg/ml. Aliquots of 3.5 ml were then sonicated for 5 s at 0 "C using a Branson sonifier (...

Mechanism of Fatty-Acid-Dependent UCP1 Uncoupling in Brown Fat Mitochondria

“…These results suggest that an UCP1 activator, most likely LCFAs, is produced within the IMM. The smaller I UCP1 recovery at acidic pH correlates with decreased activity of mitochondrial PLA2 at a lower pH (Kinsey et al, 2007; Zurini et al, 1981). Because Ca 2+ in our solutions was in the low nM range, the putative PLA2 involved in UCP1 activation must be Ca 2+ independent.…”

Phospholipase A2: Its Usefulness in Laboratory Diagnostics

“…451 It has been purified to near homogeneity 452,453 and its partial sequence has been determined. 454 PLA 2 has also been detected in rat liver microsomes, 455 plasma membranes, 455 Golgi membranes, 456 nuclear membrane and matrix, 457 and lysosomes.…”

Inhibition of mitochondrial phospholipase A2 by mono- and dilysocardiolipin

“…liat liver mitochondria contain phospholipase A2 activity associated with their inner and outer membranes (Waite, 1969;Nachbaur et al, 1972;Zurini et al, 1981). Recently, highly purified preparations of this enzyme have been obtained (De Winter et al, 1982;Natori et al, 1983; Aarsman et al, 1984), and some properties of purified protein have been described (De Winter et al, 1984Winter et al, , 1987Lenting et al, 1987).…”