Phosphatidylethanolamine

Abstract: IAbbreviations used in this article are as follows: AraC= l -,B-d arabinofuranosyl cytosine, Chol=cholesterol, DNA=deoxyribonucleic acid, DMPA=dimyristoyl phos­ phatidic acid, DMPC = dimyristoyl phosphatidylcholine, DMPE = dimyristoyl phos­ phatidylethanolamine, DOPC = dioleoyl phosphatidylcholine, DOPE = dioleoyl phos­ phatidylethanolamine, DPPA=dipaJmitoyl phosphatidic acid, DPPC=dipaJmitoyl phos­ phatidylcholine, DPPG = dipaJmitoyl phosphatidylglycerol, DPPS;= dipalmitoyl phos­ phatidylserine, DSPC = distearoyl phosphatidylcholine, EPC = egg phosphatidylcholine, EDTA=ethylene diamine tetracetic acid, HDL=high density lipoprotein, HPLC=high performance liquid chromatography, LUV = large unilamellar vesicle, MLV = multilamellar vesicle, NT A = nitrilotriacetic acid, NMR = nuclear magnetic resonance, PA phosphatidic acid, PC = phosphatidylcholine, PE = phosphatidylethanolamine, PO = phosphatidylglycerol, PS = phosphatidylserine, REV = reverse-phase evaporation vesicle, RNA = ribonucleic acid, SUV=small unilameUar vesicle, Tc=transition temperature. 2Present address: Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.

Abstract: An assay for vesicle--vesicle fusion involving resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl), the energy donor, and rhodamine, the energy acceptor, has been developed. The two fluorophores are coupled to the free amino group of phosphatidylethanolamine to provide analogues which can be incorporated into a lipid vesicle bilayer. When both fluorescent lipids are in phosphatidylserine vesicles at appropriate surface densities (ratio of fluorescent lipid to total lipid), efficient energy transfer is observed. When such vesicles are fused with a population of pure phosphatidylserine vesicles by the addition of calcium, the two probes mix with the other lipids present to form a new membrane. This mixing reduces the surface density of the energy acceptor resulting in a decreased efficiency of resonance energy transfer which is measured experimentally. These changes in transfer efficiency allow kinetic and quantitative measurements of the fusion process. Using this system, we have studied the ability of phosphatidylcholine, phosphatidylserine, and phosphatidylcholine--phosphatidylserine (1:1) vesicles to fuse with cultured fibroblasts. Under the conditions employed, the majority of the cellular uptake of vesicle lipid could be attributed to the adsorption of intact vesicles to the cell surface regardless of the composition of the vesicle bilayer.

Abstract: The proenzyme of a Ca2+-dependent protease-activated protein kinase previously obtained from mammalian tissues (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616) was enzymatically fully active without limited proteolysis when Ca2+ and a membrane-associated factor were simultaneously present in the reaction mixture. The activation process was reversed by removing Ca2+ with ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid. An apparent Ka value for Ca2+ was less than 5 x 10(-5) M. Other divalent cations were inactive except for Sr2+, which was 5% as active as Ca2+. The factor was almost exclusively localized in membrane fractions of various tissues including brain, liver, kidney, skeletal muscle, blood cells, and adipose tissue. It was easily extractable with chloroform/methanol (2:1), and was recovered in the phospholipid fraction. In fact, this membrane factor could be replaced by chromatographically pure phosphatidylinositol, phosphatidylserine, phosphatidic acid, or diphosphatidylglycerol. Phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin were far less effective under the comparable conditions. Ca2+-dependent modulator protein was unable to support enzymatic activity. The enzyme thus activated showed an ability to phosphorylate five histone fractions and muscle phosphorylase kinase, and appeared to possess multifunctional catalytic activities.

Abstract: 1. 1. Phospholipase A2 (phosphatide acylhydrolase, EC 3.1.1.4) from Naja naja hydrolyses 68% of the lecithin of the intact human erythrocyt without changing the freeze fracture faces of the membrane. Phospholipase A2 (Naja naja) treatment of ghosts produces complete breakdown of the glycerophospholipids and induces aggregation of particles on the freeze-fracture faces of the membrane. 2. 2. Phospholipase C (phosphatidylcholine choline phosphohydrolase, EC 3.1.4.3) from Bacillus cereus does not attack intact cells and no change in freeze-etch morphology is observed. The glycerophospholipids of ghosts are almost completely degraded by this enzyme, which causes a reduction in tangentially-splitted membranes and a formation of large diglyceride droplets, which are also visible by phase-contrast microscopy. 3. 3. Sphingomyelinase (sphingomyelin choline phosphohydrolase) from Staphylococcus aureus, hydrolyses 80–85% of the sphingomyelin of the intact human red cel, and produces aggregation of the particles and the formation of small spheres (75 A and 200 A in diameter) on the outer fracture face with corresponding pits on the inner fracture face. Treatment of ghosts with this enzyme causes a complete degradation of the sphingomyelin and produces, in addition to aggregation of particles, the formation of droplets (1000–3000 A in diameters) whcih are adherent to the membrane and are not visible by phase-contrast microscopy. 4. 4. When the cells are treated successively with phospholipase A2 (Naja naja) and sphingomyelinase (Staphylococcus aureus) no lysis occurs although the osmotic fragility is markedly increased. By this treatment, up to 48% of the total phospholipids are degradd. It is concluded that this phospholipid fraction (which contains the majority of the choline-containing phospholipids and some phosphatidylethanolamine) forms the outer monolayer of the membrane.

Abstract: The regulation of the Ca(2+)- and phorbol ester-insensitive zeta isozyme of protein kinase C (PKC zeta) by phospholipids was studied. Phosphatidylserine (PS) stimulated the activity to the same extent as proteolysis by calpain. However, the PS stimulation was abolished by phosphatidylethanolamine (PE) or phosphatidylcholine. Phosphatidylinositol-3,4,5-P3 (PIP3) produced a large stimulation of PKC zeta in the absence or presence of PS plus PE that was equal to that seen with PS alone. In the presence of PS plus PE, PIP3 was half-maximally effective at 50 nM. Phosphatidylinositol-3,4-P2 also fully activated PKC zeta, but higher concentrations (0.5 microM) of phosphatidylinositol-3-P, phosphatidylinositol-4-P, and phosphatidylinositol-4,5-P2 produced only partial (11-30%) activation of the enzyme. In contrast, when tested with "conventional" PKC purified from rat brain, none of the inositol phospholipids produced more than one-third of the stimulation seen with PS plus Ca2+ plus phorbol ester, and there was little difference between the efficacy of PIP3 and that of the other phospholipids. PIP3 produced a marked stimulation of the autophosphorylation of PKC zeta, indicating that it interacted with the enzyme directly. These results suggest that PKC zeta may be a target for PIP3 and thus may be involved in the signaling mechanism(s) for growth factors and oncogenes that increase phosphatidylinositol 3-kinase activity.