Disaturated and dipolyunsaturated phospholipids in the bovine retinal rod outer segment disk membrane

Thin-layer chromatography was used to separate the major phospholipid headgroup classes of the rod outer segment disk membrane into subfractions which differ markedly in fatty acid composition. At least 18% of the rod outer segment phosphatidylcholine must contain two saturated fatty acids. Furthermore, two unsaturated fatty acids are found in at least 43% of the phosphatidylserine, 24% of the phosphatidylcholine, and 24% of the phosphatidylethanolamine. The unsaturated acids are predominantly polyunsaturated in all cases. A similar separation, but with less resolution, was achieved with silicic acid column chromatography.The temperature dependence of the polarization of the fluorescence of trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid) showed that the thermal behavior of aqueous dispersions of the phosphatidylcholine subfractions was consistent with their fatty acid compositions.     

Lipid composition of photoreceptor membranes from goldfish retinas

Isolation and biochemical characterization of goldfish retinal photoreceptor outer segment membranes are described. The lipid fraction is composed primarily of phospholipids (68 mol%) with substantial amounts of neutral lipids (32 mol%). Sterols account for only about 2 wt% of the membranes (about 9 mol% of the total lipids). The phospholipid class composition and fatty acid composition are similar to those of other vertebrate photoreceptor membranes. Two novel findings were the high levels of free fatty acids (21 mol% of the total lipids, primarily palmitic and docosahexaenoic acids) and the presence of relatively significant amounts of a C-32 diacylglycerol molecular species.     

Identification and Immunolocalization of Phospholipase C in Bovine Rod Outer Segments

Bovine rod outer segments (ROS) contain a phospholipase C (PLC) that hydrolyzes phosphatidylinositol 4,5-bisphosphate. Approximately 60-70% of PLC activity is recovered in soluble extracts of ROS. Moreover, the specific activity of this soluble PLC is approximately 10-fold higher than that of resealed ROS enzyme activity. Peptide-specific antiserum (Ab 1109) directed against a highly conserved sequence of the Y-region found in several PLC isozymes was used to detect any PLC belonging to this family. This antibody specifically recognized a protein of apparent molecular mass of approximately 140 kDa present in immunoblots of soluble extracts of both ROS and whole retina. The elution profile of this 140-kDa antigen from a Sephadex G-150 column coincided with the peak of PLC activity, suggesting PLC activity is associated with the 140-kDa protein. Immunocytochemical studies of bovine retina using Ab 1109 showed pronounced immunoreactive labeling in the photoreceptor layer. In resealed ROS and washed ROS membranes, Ab 1109 recognized an additional protein of apparent molecular mass of 70 kDa not usually detectable in soluble extracts of ROS, suggesting the presence of at least two isozymes of PLC in ROS.     

Influence of enzymatic phospholipid cleavage on the permeability of the erythrocyte membrane. I. Transport of non-electrolytes via the lipid domain

In order to further elucidate the influence of membrane lipids on transport via the lipid domain of the erythrocyte membrane, simple non-electrolyte diffusion was investigated by tracer flux measurements in whole cells after cleavage of up to 65% of phosphatidylcholine or sphingomyelin by phospholipase A₂ from Naja naja, or by sphingomyelinase.A new type of labelled model non-electrolyte was used in this study, readily available by reacting a non-labelled thiol with a labelled alkylating SH-reagent.In spite of the marked enzymatic alterations of the membrane, which lead to the occurrence of large quantities of lysophosphatidylcholine and long chain fatty acids, or of ceramide, the permeability of the lipid domain remained unaffected.This finding is very surprising, since the physical properties of the lipid phase (microviscosity, structure of the membrane interface) are likely to be perturbed in the enzyme-treated membranes.Sphingomyelinase-treated cells undergo stomatocytic shape changes followed by deep invaginations of the membrane and finally endocytosis, while phospholipase A₂-treated cells essentially maintain their normal shape.     

Phase behavior of isolated photoreceptor membrane lipids is modulated by bivalent cations

The phase behavior of isolated photoreceptor membrane lipids is further investigated by ³¹P-NMR, in view of earlier discrepant results [(1979) Biochim. Biophys. Acta 558, 330–337; (1982) FEBS Lett. 124, 93–99]. We present evidence that the discrepancy is due to bivalent cations. When resuspended in aqueous media at neutral pH in the absence of bivalent cations, the isolated photoreceptor membrane lipids largely adopt the bilayer configuration. However, upon addition of such cations (Ca²⁺ Mg²⁺) or when resuspended in their presence, the formation of other phases (hexagonal H_II, lipidic particles) results. The rate of this transition depends on cation concentration and temperature. The transition is not easily reversed by addition of EDTA. Implications with regard to photoreceptor membrane structure and function need further study.     

Asymmetry of arachidonic acid metabolism in the phospholipids of the human platelet membrane as studied with purified phospholipases

(1) Human platelets were incubated with high density lipoproteins (HDL) doubly labelled with either free [¹⁴C]arachidonate/[³H]arachidonoylphosphatidylcholine or free [¹⁴C]oleate/[³H]oleoylphosphatidylcholine. Whereas [¹⁴C]arachidonate was incorporated at a 10–15 times higher rate than [¹⁴C]oleic acid, the exchange of both species of phosphatidylcholine occurred to the same extent. In both cases, free ³H-labelled fatty acids were generated during the labelling procedure, indicating phospholipase A₂ hydrolysis. A redistribution of radioactivity to other phospholipids was noted after exchange of [³H]arachidonoylphosphatidylcholine only. (2) The exchange of phosphatidylcholine to platelets was confirmed using [¹⁴C]choline-labelled dipalmitoyl- and 1-palmitoyl-2-arachidonoylphosphatidylcholines. (3) Non-lytic degradation of platelet phospholipids by phospholipases revealed that free fatty acids were incorporated at the inside of the cells, whereas exchange was taking place on the platelet outer surface. However, 2-arachidonoylphosphatidylcholine displayed a more rapid movement towards the cell inside. The above findings suggest a topological asymmetry for the two pathways (acylation and exchange) of fatty acid renewal in platelets. The possible mechanisms and physiological relevance of the translocation of the external arachidonic acid pool across the membrane are discussed.     

Phosphorylation of rod outer segment proteins modulates phosphatidylethanolamine N-methyltransferase and phospholipase A2 activities in photoreceptor membranes

The activities of enzymes involved in lipid metabolism—phospholipase A₂ (PLA₂) and phosphatidylethanolamine N-methyltransferase (PE N-MTase)—were found to be differently affected by pre-incubation of rod outer segments (ROS) under protein phosphorylating or dephosphorylating conditions. Exposure to cAMP-dependent protein kinase (PKA), under dark or light conditions, produced a significant increase in PE N-MTase activity, whereas PLA₂ activity decreased. Under standard protein kinase C (PKC) phosphorylating conditions in light, PE N-MTase activity was stimulated and PLA₂ activity was not affected. When the assays were performed in the dark, both enzymatic activities were unaffected when compared to the corresponding controls. Incubation of ROS membranes in light in the presence of PKC activators phorbol 12,13-dibutyrate (PDBu) and dioctanoylglycerol (DOG) resulted in the same pattern of changes in enzyme activities as described for standard PKC phosphorylating condition. Pre-incubation of membranes with the PKC inhibitor H-7 reduced the stimulation of PDBu on PE N-MTase activity, and had no effect on PLA₂ activity in ROS membranes incubated with the phorbol ester. Pre-treatment of isolated ROS with alkaline phosphatase resulted in decreased PE N-MTase activity and produced a significant stimulation of PLA₂ activity under dark as well as under light conditions when compared to the corresponding controls. These findings suggest that ROS protein phosphorylation and dephosphorylation modulates PE N-MTase and PLA₂ activities in isolated ROS, and that these activities are independently and specifically modulated by particular kinases. Furthermore, dephosphorylation of ROS proteins has the opposite effect to that produced by protein phosphorylation on the enzymes studied.     

Role of membrane integrity on G protein-coupled receptors: Rhodopsin stability and function

Rhodopsin is a prototypical G protein-coupled receptor (GPCR) - a member of the superfamily that shares a similar structural architecture consisting of seven-transmembrane helices and propagates various signals across biological membranes. Rhodopsin is embedded in the lipid bilayer of specialized disk membranes in the outer segments of retinal rod photoreceptor cells where it transmits a light-stimulated signal. Photoactivated rhodopsin then activates a visual signaling cascade through its cognate G protein, transducin or Gt, that results in a neuronal response in the brain. Interestingly, the lipid composition of ROS membranes not only differs from that of the photoreceptor plasma membrane but is critical for visual transduction. Specifically, lipids can modulate structural changes in rhodopsin that occur after photoactivation and influence binding of transducin. Thus, altering the lipid organization of ROS membranes can result in visual dysfunction and blindness.     

Transmembrane distribution of phospholipids and their involvement in electron transport,as revealed by phospholipase A2 treatment of spinach thylakoids

Thylakoid membranes were treated with either pancreatic or snake venom phospholipase A₂, and the residual phospholipid content of these membranes was determined and compared to the rates of Photosystem II and/or Photosystem I electron transports. The hydrolysis curves of both phosphatidylglycerol and phosphatidylcholine displayed a first, rapid phase which was almost temperature-insensitive, followed by a second, slower phase which depended strongly on the temperature. When pancreatic phospholipase A₂ had access either to the outer face or to both faces of the thylakoid membrane, either only part of or all the phospholipids, respectively, could be hydrolysed. These results were interpreted as indicating an asymmetric distribution of phospholipids across the thylakoid membrane, phosphatidylglycerol and phosphatidylcholine being preferentially located in the outer and the inner layer, respectively. When acting on uncoupled thylakoid membranes, phospholipase A₂ exerted an inhibitory effect on Photosystem II activity and a stimulatory effect on Photosystem I activity. The involvement of phosphatidylcholine and of phosphatidylglycerol in electron transport activities of Photosystem II and of Photosystem I are discussed with special reference to the role of the external and internal pools of these phospholipids.     

The regulation of phospholipase D by inositol phospholipids and small GTPases

Phospholipase D1 and D2 (PLD1, PLD2) both have PX and PH domains in their N-terminal regions with these inositol lipid binding domains playing key roles in regulating PLD activity and localisation. The activity of PLD1 is also regulated by protein kinase C and members of the Rho and Arf families of GTPases. Each of these proteins binds to unique sites; however, there appears to be little in vitro discrimination between individual family members. In agonist-stimulated cells, however, there is specificity, with, for example in RBL-2H3 cells, antigen stimulating the activation of PLD1 by association with Arf6, Rac1 and protein kinase Calpha. PLD2 appears to be less directly regulated by GTPases and rather is primarily controlled through interaction with phosphatidylinositol 4-phosphate 5-kinase that generates the activating phosphatidylinositol 4,5-bisphosphate.     

磷酯酶D在胞吞与胞吐中的作用

磷脂酶D（phsopholipaseD,PLD)水解其主要底物磷脂酰胆碱是细胞信号转导的重要途径之一。大量研究表明PLD激活是受体介导的胞吞和胞吐过程中关键的一步。本文主要介绍PLD在受体介导的胞吞和胞吐过程中的作用及作用机制的研究发展。     

The erythrocyte membrane in essential hypertension characterization of the temperature dependence of lithium efflux

Erythrocytes of most patients with essential hypertension are distinguished by a typical pattern of temperature-dependence of Li efflux. In the present study we have attempted to characterize this unique temperature response. Measurements of Li efflux into Na medium and Li_i-Na_o countertransport were conducted simultaneously at finely spaced temperature intervals with increments of 1 to 2°C in the range of 10–40°C. The Arrhenius plots for the efflux in Na medium and for Li_i-Na_o countertransport in erythrocytes of both normotensives and hypertensives were biphasic with slopes representing apparent energies of activation of about 28 and 8 kcal/mol below and above the ‘break’, respectively. However, the ‘break’ in the Arrhenius plot appeared at distinctly different temperatures: 30°C for normotensives and 20°C for hypertensives. The Li efflux was resolved into N-ethylmaleimide-sensitive and -insensitive components. The sensitive component exhibited a typical biphasic temperature response, with the characteristic ‘break’: at 30°C for normotensives and at 20°C for hypertensives. In contrast, the N-ethylmaleimide-insensitive component was alike in normotensives and hypertensives. It is concluded that: (a) the unique temperature dependence of Li efflux in erythrocytes of hypertensives results from a localized modification in the membrane; (b) the N-ethylmaleimide-sensitive component represents a protein moiety which distinguishes between the erythrocyte membrane of normotensives and hypertensives; (c) the expression of the temperature dependence as judged by the sharp transition in slope (within 1 to 2°C), apparently reflects the cooperative involvement of membrane lipids, associated with the Li efflux system.     

A study on the topological distribution of phospholipids in microsomal membranes of chick brain using phospholipase C and trinitrobenzenesulfonic acid

The transbilayer distribution of phospholipids in chicken brain microsomal membranes has been investigated using trinitrobenzenesulfonic acid and phospholipase C from Clostridium weichii. The exposure of intact microsomes to trinitrobenzenesulfonic acid showed that the labelling of aminophospholipids followed biphasic kinetics, indicating that these membranes contain a fast- and a slow-reacting pool of aminophospholipids. Use of microsomes radioiodinated on their surface led to the conclusion that the fast-reacting pool may be located on the outer leaflet of the microsomal vesicles. It contains about 35% of the phosphatidylethanolamine, 29% of the ethanolamine plasmalogens and 18% of the phosphatidylserine. The treatment of intact microsomes with the phospholipase C Cl. welchii produced the hydrolysis of 50% of the phospholipids without any loss of their permeability properties, indicating that they are not permeable to the hydrolase. Phospholipids extracted from the microsomes were hydrolyzed rapidly by the phospholipase C with the exception of phosphatidylserine and phosphatidylinositol. In intact microsomes about 90% of phosphatidylcholine, 32% of ethanolamine phospholipids and 60% of sphingomyelin were accessible to the phospholipase. These results suggest that the phospholipids have an asymmetric distribution in chicken brain microsomes, the external leaflet containing about 75% of the choline phospholipids and 25% of the aminophospholipids, whereas an opposite distribution is observed in the inner leaflet.     

Identification of caspase 3 motifs and critical aspartate residues in human phospholipase D1b and phospholipase D2a

Stimulation of mammalian cells frequently initiates phospholipase D-catalyzed hydrolysis of phosphatidylcholine in the plasma membrane to yield phosphatidic acid (PA) a novel lipid messenger. PA plays a regulatory role in important cellular processes such as secretion, cellular shape change, and movement. A number of studies have highlighted that PLD-based signaling also plays a pro-mitogenic and pro-survival role in cells and therefore anti-apoptotic. We show that human PLD1b and PLD2a contain functional caspase 3 cleavage sites and identify the critical aspartate residues within PLD1b that affect its activation by phorbol esters and attenuate phosphatidylcholine hydrolysis during apoptosis.     

The effect of calcium on the bilayer stability of lipids from bovine rod outer segment disk membranes

The phase behavior of bovine rod outer segment disk lipids has been investigated using freeze-fracture and ³¹P nuclear magnetic resonance (NMR) techniques. ³¹P-NMR spectra of isolated disk membranes were taken as a function of temperature between 25°C and 45°C. The ³¹P-NMR spectrum characteristic of phospholipid bilayers was observed at all temperatures both in the absence of Ca²⁺ and in the presence of 10 mM and 50 mM Ca²⁺. A similar study was performed on lipids isolated from the disk membranes. In the absence of Ca²⁺ only lamellar phase behavior was observed. In the presence of less than 10 mM Ca²⁺, however, there was a change in morphology to non-lamellar structures. Removal of the Ca²⁺ caused the system to reassume the lamellar form.     

Conservation of Docosahexaenoic Acid in Rod Outer Segments of Rat Retina During n-3 and n-6 Fatty Acid Deficiency

We investigated the mechanism by which rat retina conserves docosahexaenoic acid during essential fatty acid deficiency. Weanling female albino rats were fed diets containing either 10% by weight hydrogenated coconut oil, safflower oil, or linseed oil for 15 weeks. Plasma and rod outer segment (ROS) membranes were prepared for fatty acid and phospholipid molecular species analysis. In addition, retinas were removed for morphometric analysis. We found the following: (1) Plasma phospholipids and cholesterol esters from coconut oil, safflower oil, and linseed oil diet groups were enriched in 20:3(n-9), 20:4(n-6), and 20:5(n-3), respectively. The levels of these 20-carbon fatty acids in the ROS, however, were only slightly affected by diet. (2) The fatty acids and molecular species of ROS phospholipids from the safflower oil and coconut oil groups showed a selective replacement of 22:6(n-3) with 22:5(n-6), as evidenced by a reduction of the 22:6(n-3)-22:6(n-3) molecular species and an increase in the 22:5(n-6)-22:6(n-3) species. (3) The renewal rate of ROS integral proteins, determined by autoradiography, was 10% per day for each diet group. (4) Morphometric analysis of retinas showed no differences in the outer nuclear layer area or in ROS length between the three groups. We conclude that the conservation of 22:6(n-3) in ROS is not accomplished through reductions in the rate of membrane turnover, the total amount of ROS membranes, or in the number of rod cells. The retina may conserve 22:6(n-3) through recycling within the retina or between the retina and the pigment epithelium, or through the selective uptake of 22-carbon polyunsaturated fatty acids from the circulation.     

Developmental changes and regional distribution of phospholipase D and base exchange enzyme activities in rat brain

Phospholipase D (PL-D) activity per mg protein of whole homogenate increased 5.1 fold between Embryonic (E) day 17 and Postpartum (P) day 14 and slightly decreased by P 30 days. This was due to the decrease of PL-D activity of the P₂ fraction. The PL-D activity of P₂ and P₃ fractions increased 11.2 and 6.1 fold respectively between E 17 and P 14. The 3 base exchange enzyme (BEE) activities per mg protein of whole homogenate increased up to P 14 or P 21 and then decreased. This decrease was greater in the P₂ fraction and the P₃ fraction increased after P14. Brains from 1 day to 25 month old rats were dissected into 7 separate regions and both PL-D and BEE activities were measured. In adult rats, the hippocampus and hypothalamus had the highest PL-D activities while medulla+pons and cerebellum had the lowest PL-D activities. The developmental patterns of 5 regions except for hippocampus and hypothalamus were similar. PL-D activity in the hippocampus was maximum at P 7 followed by a steep decrease till P30 suggesting that the PL-D activity of the hypothalamus develops later and that of the hippocampus develops earlier than any other region. The distributions of BEE activities were quite different from those of PL-D activities. In adult rats, the cerebellum had the highest activity while the striatum and medulla+pons had the lowest. The BEE activities of cerebellum were lowest at P 1 and showed steep increase during the next 2 weeks.To whom to address reprint request are to be sent.