Swelling of Phaseolus Mitochondria Induced by the Action of Phospholipase A

Abstract

Phaseolus vulgaris mitochondria incubated in sucrose swell rapidly upon the addition of phospholipase A. Bovine serum albumin inhibits the swelling. The release of free fatty acids as a result of phospholipase A action on the mitochondria is detected only in the presence of bovine serum albumin, which promotes the hydrolysis of both mitochondrial phospholipids and purified lecithin. Either free fatty acid or lysolecithin is able to initiate an extensive mitochondrial swelling in sucrose. It is suggested that phospholipase A-induced swelling results from the release of lysophosphatides plus free fatty acids and their subsequent detergent action on the membranes rather than phospholipid loss per se.Phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4) of snake venom catalyzes the hydrolysis of diacylated nitrogencontaining phosphoglycerides with the liberation of the fatty acid esterified at the ,3-position and formation of the lysophosphoglyceride. The metabolic results of adding snake venom or a phospholipase A preparation to animal mitochondria are well documented. Petrushka et al. (22) observed, following the addition of heated snake venom to rat mitochondria, an initial stimulation of the respiratory rate followed by a rapid inhibition. Phospholipase A has been shown to inhibit NADH oxidase, succinate-and NADH-cytochrome c reductase (28) and succinoxidase activities (13). Several workers have shown a decrease in the P/O ratio of isolated mitochondria as a result of phospholipase A action in vitro (23, 25) and also in vivo (2).In addition to the effects of phospholipase A on metabolism, there are several reports indicating that structural disorganization also occurs. The treatment of an NADH oxidase preparation resulted in extensive solubilization of the NADH dehydrogenase activity (24), and cytochrome c release occurred upon incubation of electron transport particles with phospholipase A (1). lipase A to provide a clear separation of the inner and outer membrane systems of beef heart mitochondria. It has also been reported that the addition of venom to rat liver mitochondria produced a marked swelling response (26).As part of a detailed study of volume changes exhibited by Phaseolus mitochondria (11, 12), it was of interest to determine whether phospholipase A was able to induce swelling in plant mitochondria as well as in animal. Phaseolus mitochondria show rapid spontaneous swelling in tris-buffered KC1 in a process strongly inhibited by bovine serum albumin but do not swell appreciably in tris-buffered sucrose (12). We wished to determine whether phospholipsase A would induce swelling in sucrose and whether BSA4 affected the system. MATERIALS AND METHODSIsolation ofMitochondria. Seed of dwarf French bean, Phaseolus vulgaris (var. Canadian wonder) was sown and germinated as described by Opik and Simon (21). Mitochondria were isolated from the etiolated hypocotyls on the 6th day following planting by a method similar to the procedure of Kenefick and Hanson (17) as described previously (11). Mitoch...

Defunctionalizing intracellular organelles such as mitochondria and peroxisomes with engineered phospholipase A/acyltransferases

“…1a ). Since a previous in vitro study showed that PLA deforms isolated mitochondria 15 , we chose mitochondria as a model organelle. To induce translocation of PLAAT3 from the cytosol to the mitochondria surface in living cells, we used the FKBP-FRB dimerizing unit which rapidly and strongly interacts with each other in the presence of rapamycin 21 .…”

“…Purified PLA proteins such as PLA 1 , PLA2G4A, PLA 2 from bee venom and transmembrane PLA from bacteria, induced membrane bending, budding, tubulation and pearling in model membranes such as giant vesicles and supported phospholipid bilayers [11][12][13][14] . Furthermore, PLA 1 from snake venom not only swelled isolated mitochondria but also perturbed their functions 15 , while inhibition of PLA 2 was reported to associate with tubulation of Golgi and trans Golgi network 16 . Of note, these PLAs need a supplementary condition such as non-physiologically high Ca 2+ concentration for the full catalytic activity.…”

Defunctionalizing Intracellular Organelles with Genetically-Encoded Molecular Tools Based on Engineered Phospholipase A/Acyltransferases (PLAATs)

“…1a ). Since a previous in vitro study showed that PLA deforms isolated mitochondria 15 , we chose mitochondria as a model organelle. To induce translocation of PLAAT3 from the cytosol to the mitochondria surface in living cells, we used the FKBP-FRB dimerizing unit which rapidly and strongly interacts with each other upon rapamycin treatment 18 .…”

“…Purified PLA proteins such as PLA 1 , PLA 2 G4A, PLA 2 from bee venom and transmembrane PLA from bacteria, induced membrane bending, budding, tubulation and pearling in model membranes such as giant vesicles and supported phospholipid bilayers [11][12][13][14] . Furthermore, PLA 1 from snake venom not only swelled isolated mitochondria but also perturbed their functions 15 , while inhibition of PLA 2 was reported to associate with tubulation of Golgi and trans Golgi network 16 . Of note, these PLAs need a supplementary condition such as non-physiologically high Ca 2+ concentration for the full catalytic activity.…”