Transverse Distribution Of Phospholipids Research Articles

Regulation of secretory type-II phospholipase A 2 and of lysophosphatidic acid synthesis

Secretory non-pancreatic phospholipase A2 (sPLA2), also called type II-PLA2, is produced in large amounts under inflammatory conditions, thus accumulating in inflammatory fluids. Since the enzyme is virtually inactive on phospholipids from intact cells, we have searched for conditions allowing the action of sPLA2 on membrane phospholipids. Based on an in vitro model, our studies suggest that only those membranes where the transverse distribution of phospholipids has been disturbed offer a convenient surface able to interact with the enzyme, which then achieves significant degradation of all glycerophospholipids. This results in the accumulation of various lysophospholipids such as lysophosphatidylcholine, lysophosphatidylethanolamine and lysophosphatidylserine. However, lysophosphatidic acid (LPA) can also be generated under these conditions involving accumulation of phosphatidic acid in the cytoplasmic leaflet of the membrane, followed by its transfer to the outer monolayer. Since LPA is now considered as a novel phospholipid mediator, this pathway deserves further studies concerning mainly platelets, the main source of LPA identified so far.

Relationship between the transverse distribution of phospholipids in plasma membrane and shape change of human platelets.

ESR spectroscopy was used to investigate the distribution of spin-labeled analogues of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in the presence of human platelets. Three rates were determined: hydrolysis of the ester bond at position 2, reduction of labels by cytoplasm, and internalization of labels situated in the outer leaflet of the plasma membrane. We found that the half-time for transverse diffusion of added phospholipids was shorter for aminophospholipids (40 min and less than 10 min for PE and PS, respectively) than for the choline derivatives (greater than 120 min for PC, not measurable for SM). Addition of any of the phospholipids led to a considerable change in the initial platelet shape (assessed by electron microscopy) from a discoid form to a smaller body with very long pseudopods. When aminophospholipids were used, the platelets quickly returned to the initial shape [half-time of 20 min and less than 5 min for (0.2)PE and (0.2)PS, respectively]. Conversely, there was no relaxation after (0.2)PC or (0.2)SM was added. We conclude that there is a relationship between the excess of phospholipids in the outer leaflet of the plasma membrane and cytoskeletal organization presumably via actin polymerization, which is responsible for platelet shape.

The transverse distribution of phospholipids in the membranes of Golgi subfractions of rat hepatocytes.

The transverse distribution of phospholipids in the membranes of subfractions of the Golgi complex was investigated by using phospholipase C and 2,4,6-trinitrobenzenesulphonic acid as probes. In trans-enriched Golgi membranes, 26% of the phosphatidylethanolamine is available for reaction with trinitrobenzenesulphonate or for hydrolysis by phospholipase C, and 72% of the phosphatidylcholine is hydrolysed by phospholipase C. In cis-enriched Golgi membranes, 45% of the phosphatidylethanolamine is available for reaction with trinitrobenzenesulphonate and for hydrolysis by phospholipase C, and 95% of the phosphatidylcholine is hydrolysed by phospholipase C. Under the conditions used with either probe the contents of the Golgi vesicles labelled with either [3H]palmitic acid or [14C]leucine were retained. Galactosyltransferase activity of the membrane vesicles was partially inhibited by the experimental procedures used to investigate the transverse distribution of phospholipids. However, the residual activity was latent, suggesting that the vesicles remained closed. Trinitrobenzenesulphonic acid caused no detectable morphological change in either Golgi fraction. Phospholipase C treatment caused morphological changes, including fusion of vesicles and the appearance of 'signet-ring' profiles in some vesicles; however, the vesicles remained closed and the bilayer was retained. It appears, therefore, that neither probe causes major disruption of the Golgi vesicles nor gains access to the inner surface of the membrane bilayer. These observations suggest that phospholipids have a transverse asymmetry in Golgi membranes, that this distribution differs in trans and cis membranes, and that the phospholipid structure of Golgi membranes is inconsistent with a simple flow of membrane bilayer from endoplasmic reticulum to Golgi membranes to plasma membrane.

48] Phospholipases as tools for studying structure and function of photoreceptor membranes

Publisher Summary This chapter discusses the phospholipases as tools for studying structure and function of photoreceptor membranes. Phospholipases have been shown to be useful tools in biomembrane research as moderate agents to remove or to modify phospholipids in the membrane. They have been used to study the influence of phospholipids on membrane-bound enzymatic activities and the transverse distribution of phospholipids over the two faces of biomembranes. Also, in photoreceptor membranes lipid–protein interactions play an important role, but the use of phospholipases to study these aspects has so far been limited. Interest in the topology of the photoreceptor membrane phospholipids, as studied by chemical modification, has led to similar investigations using phospholipases. Most complete hydrolysis of the phospholipids of photoreceptor membranes is achieved with phospholipase A 2 . The hydrolysis products, lysophospholipids and fatty acids, can be removed by washing with serum albumin.

Asymmetrical orientation of phospholipids and their interactions with marker enzymes in pig heart mitochondrial inner membrane

The transverse distribution of phospholipids and their interactions with marker enzymes were investigated in pig heart mitoplasts and inverted vesicles, using phospholipase A 2 from N. naja venom and chemical labeling with TNBS and FDNB. Morphological integrity was checked by freeze-fracturing. Fifty percent of phosphatidylcholine was hydrolyzed in mitoplasts as well as in inverted vesicles, suggesting an even distribution of this phospholipid on the two halves of the inner membrane; however, the fatty acid distribution did not appear the same in the two membrane fractions. Cardiolipin is exclusively hydrolyzed in inverted vesicles proving its location on the inner face of the inner membrane. The results obtained from phospholipase hydrolysis and TNBS labeling suggest that three different pools of phosphatidylethanolamine occur in the membrane: a first pool—about 50–60% of the total membrane phosphatidylethanolamine–is quickly accessible from the two sides of the membrane, a second pool—about 20–30% is slowly available, and finally 20–30% are buried within the membrane and inaccessible to the phospholipase and the probe. The cytochrome c oxidase activity increased in mitoplasts with the phospholipase attack suggesting a better accessibility of added cytochrome c after the attack. The rotenone-sensitive NADH-cytochrome c reductase was activated in mitoplasts but completely inactivated in inverted vesicles by the attack; the addition of cardiolipin liposomes restored the latter activity. The soluble matricial malate dehydrogenase was released, but the particulate form of this enzyme, strongly associated to the membrane, was detached only after attack of inverted vesicles.

Transverse distribution of phospholipids in the vertebrate photoreceptor membrane

A study has been made of the distribution of the three major phospholipids of the bovine photoreceptor membrane over the two faces of the lipid bilayer. Three different approaches have been used: a . Treatment of isolated intact outer segments and disk vesicles (lysed outer segments) with three different phospholipases and determination of the degradation pattern. As randomized control preparations a retinal lipid suspension and detergent solubilized disks were used. b . Treatment of intact and lysed outer segments with the amino group reagent trinitrobenzenesulfonic acid (with and without treatment by phospholipase D) and determination of modification of phosphatidylethanolamine and phosphatidylserine. c . Determination of the fatty acid composition of the membrane phospholipids during treatment by phospholipase D or TNBS. The results consistently indicate a nearly symmetric distribution of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine and their fatty acids over the two membrane faces, with a slight preponderance of the first phospholipid on the inner side and possibly a slight excess of the second one on the outer side.