Sn-2 Acyl Research Articles

Structure and dynamics of plasmalogen model membranes containing cholesterol: a deuterium NMR study

Deuterium nuclear magnetic resonance ( 2H-NMR) was used to investigate the structure and dynamics of the sn-2 hydrocarbon chain of semi-synthetical choline and ethanolamine plasmalogens in bilayers containing 0, 30, and 50 mol% cholesterol. The deuterium NMR spectra of the choline plasmalogen yielded well-resolved quadrupolar splittings which could be assigned to the corresponding hydrocarbon chain deuterons. The sn-2 acyl chain was found to adopt a similar conformation as observed in the corresponding diacyl phospholipid, however, the flexibility at the level of the C-2 methylene segment of the plasmalogen was increased. Deuterium NMR spectra of bilayers composed of the ethanolamine plasmalogen yielded quadrupolar splittings of the C-2 segment much larger than those of the corresponding diacyl lipids, suggesting that the sn-2 chain is oriented perpendicular to the membrane surface at all segments. Cholesterol increased the ordering of the choline plasmalogen acyl chain to the same extent as in diacyl lipid bilayers. T 1 relaxation time measurements demonstrated only minor dynamical differences between choline plasmalogen and diacyl lipids in model membranes.

Motion and surface accessibility of spin-labeled lipids in a model lipoprotein containing cholesteryl oleate, dimyristoylphosphatidylcholine, and apolipoprotein E.

A series of spin-labeled phosphatidylcholines (PCs) and cholesteryl esters (CEs) bearing the paramagnetic 2,2-dimethyloxazolidinyl-1-oxy (doxyl) group at fatty acyl carbon C5', C12', or C16' were used to study acyl chain motions in the polar surface shell and hydrophobic core domains of microemulsion (ME) particles containing cholesteryl oleate and dimyristoylphosphatidylcholine (DMPC), and of particles with apolipoprotein E (apoE) bound to their surfaces. Electron paramagnetic resonance data obtained with the doxyl-labeled PCs indicated a gradient of motion in the ME surface monolayer similar to that observed with the same probes in a bilayer. The 5- and 12-doxyl-CEs clearly demonstrated a higher degree of order for the cholesteryl ester rich core than the corresponding doxyl-PCs showed for the phospholipid-rich surface over the entire range 10-60 degrees C. The temperature dependencies of spectra of the 16-doxyl-CE in the core and PC in the surface of the ME were almost identical, suggesting that there was no sharp boundary between core and surface domains. None of the probes detected either the surface phospholipid transition (31 degrees C) or the cholesteryl ester core transition (46 degrees C) measured previously by differential scanning calorimetry and 13C nuclear magnetic resonance. Binding of apoE to spin-labeled DMPC vesicles increased the order of the 5'-position of the sn-2 acyl chain over the range 15-33 degrees C; the thermal transition was broadened and its midpoint elevated. The effect of protein binding was not as striking for the ME particles.(ABSTRACT TRUNCATED AT 250 WORDS)

Fatty acyl chain specificity of phosphatidylcholine hydrolysis catalyzed by lipoprotein lipase. Effect of apolipoprotein C-II and its (56–79) synthetic fragment

Mixed acyl chain phosphatidylcholine molecules in Triton N-101 micelles were employed as substrates for lipoprotein lipase to test which substrate acyl chain has the greatest effect on activation of the enzyme by apolipoprotein C-II. The phospholipase A 1 activity of lipoprotein lipase was measured by pH-stat. The activation factor (lipoprotein lipase activity plus apolipoprotein C-II / activity minus apolipoprotein C-II) increased monotonically with apolipoprotein C-II concentration up to 1 μM apolipoprotein C-II at an enzyme concentration of 0.01 μM. The maximal activation factor for phosphatidylcholine substrate molecules with sn-2 acyl chain lengths of 14 averages 14.8. By contrast, for sn-2 acyl chain lengths of 16 the activation factor was 29.2. Varying the sn-1 acyl chain length had no significant effect on the activation factor. The chain-length dependence of the activation factor is similar with the apolipoprotein C-II peptide fragment comprising residues 56–79, which does not include the lipid-binding region of apolipoprotein C-II. These data are consistent with a model for activation of lipoprotein lipase in which residues 56–79 bind to lipoprotein lipase and alter the interaction of the sn-2 acyl chain of the phosphatidylcholine (PC) substrate or the lysoPC product within the activated state complex.

Purification of lysosomal phospholipase A and demonstration of proteins that inhibit phospholipase A in a lysosomal fraction from rat kidney cortex.

Phospholipase A has been isolated from a crude lysosomal fraction from rat kidney cortex and purified 7600-fold with a recovery of 9.8% of the starting activity. The purified enzyme is a glycoprotein having an isoelectric point of pH 5.4 and an apparent molecular weight of 30,000 by high-pressure liquid chromatography gel permeation. Naturally occurring inhibitors of lysosomal phospholipase A are present in two of the lysosomal-soluble protein fractions obtained in the purification. They inhibit hydrolysis of 1,2-di[1-14C]oleoylphosphatidylcholine by purified phospholipase A1 with IC50 values of 7-11 micrograms. The inhibition is abolished by preincubation with trypsin at 37 degrees C, but preincubation with trypsin at 4 degrees C has no effect, providing evidence that the inhibitors are proteins. The results suggest that the activity of lysosomal phospholipase A may be regulated in part by inhibitory proteins. Lysosomal phospholipase A from rat kidney hydrolyzes the sn-1 acyl group of phosphatidylcholine, does not require divalent cations for full activity, and is not inhibited by ethylenediaminetetraacetic acid. It has an acid pH optimum of 3.6-3.8. Neither p-bromophenacyl bromide, diisopropyl fluorophosphate, nor mercuric ion inhibits phospholipase A1. In contrast to rat liver, which has two major isoenzymes of acid phospholipase A1, kidney cortex has only one isoenzyme of lysosomal phospholipase A1.

Fluorescence quenching of cytochrome b5 in vesicles with an asymmetric transbilayer distribution of brominated phosphatidylcholine.

Several fluorescence techniques have been used to estimate the depth, in the membrane, of the endogenous tryptophans of membrane-bound proteins. We reported recently the use of phosphatidylcholines specifically brominated at different positions of the sn-2 acyl chain for this purpose (Markello, T., Zlotnick, A., Everett, J., Tennyson, J., and Holloway, P. W. (1985) Biochemistry 24, 2895-2901). The membranes made from these brominated lipids will have the brominated lipid in both monolayers, and so the estimated depth of the fluorophore will be relative to either the inner or outer surface of the membrane, but will not distinguish between these two extremes. To differentiate between these two models vesicles have now been made with an asymmetric distribution of brominated lipid, by use of phosphatidylcholine exchange protein. The asymmetric vesicles were isolated by virtue of their density, and their asymmetry was established by addition of an amphipathic fluorescent carbazole compound. With these vesicles it was shown that the tryptophan in the membrane-binding domain of cytochrome b5 which is quenched by bromolipid is located 0.7 nm below the outer surface of the membrane vesicles, rather than 0.7 nm from the inner surface.

Substrate specificity and partial characterization of the PAF-acylhydrolase in human serum that rapidly inactivates platelet-activating factor.

The structure of the potent inflammatory mediator, platelet-activating factor, is 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC, PAF-acether). Human sera contain an acid labile factor (ALF) that is a Ca+2-independent 2-acylhydrolase-specific for AGEPC and AGEPC-like molecules. The enzyme functions by catalytically removing the sn-2 acetyl moiety from AGEPC, producing the biologically inactive sn-2 hydroxy form or 2-lyso-GEPC. Incubation of ALF with sn-2 acyl PAF analogs indicated that the enzyme hydrolyzes the sn-2 fatty acid only if the chain length is five carbons or less, the sn-1 position fatty acid length is greater than 10 carbon units, and at least one methyl group is present on the terminal amine of the choline group. The enzyme was active with either an ether or ester linkage at the sn-1 position. ALF is inactivated by heating to 65 degrees C for 30 min. It is pronase and trypsin sensitive but resistant to papain and papain with dithiothreitol. Further characteristics of human ALF indicated a broad pH range of activity with an optimum of pH 6.2 and an isoelectric point of 6.2 to 6.7. The specificity and Ca+2 independence of human ALF sets it apart from phospholipase A2. It is proposed that human ALF be called human serum PAF-acylhydrolase to distinguish it from other hydrolases currently known to exist.

Immunological characterization of sn-1,2-diacylglycerol and sn-2-monoacylglycerol kinase from pig brain.

Rabbit antisera were raised against diacylglycerol kinase purified from pig brain cytosol. Upon immunoblot analysis, the antibody was specifically reactive with the kinase (Mr = 79,000-80,000). Pig brain cytosol, microsomal, and synaptosomal fractions all contained the immunoreactive Mr = 80,000 polypeptide, thus showing that the same enzyme is present in the soluble as well as membrane fractions of the brain. The antibody could precipitate only 60% of the kinase activity present in the crude cytosol. Further, the antibody exhibited very little or no cross-reactivity toward liver cytosolic enzymes obtained from different animals including pigs. Immunostaining of brain tissues demonstrated that neurons, in particular, their nuclei, were positively stained, whereas glial cells were not stained. It is likely that there exists a tissue-and/or cell-dependent immunological multiplicity of diacylglycerol kinase. The enzyme activities phosphorylating sn-1 and sn-2 monoacylglycerols were co-precipitated by the antibody, indicating their identity with diacylglycerol kinase. The enzyme activity toward sn-1 monoolein was much lower than that obtained with sn-2 monoolein. Enzymic as well as chemical analyses of acyl isomers of the reaction products showed that even tested with pure (greater than 95%) sn-1 monoolein, about 70% of the formed lysophosphatidate was of the sn-2 acyl type. The results show that diacylglycerol kinase phosphorylates almost exclusively the sn-2 acyl type of monoacyl-glycerol.

Conformation of fatty acyl chains in α- and β-phosphatidylcholine and phosphatidylethanolamine derivatives in sonicated vesicles

Mono- and dimethylated derivatives constitute important intermediates in the conversion of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in eucaryote membranes. 1H-NMR techniques were utilized to examine the conformation of the region of the fatty acyl chains that is close to the polar group in the series of α-phospholipids: PE, N- methyl-PE, N,N- dimethyl-PE , and PC. The same series of polar groups, but on phospholipid containing sn-1 and/or sn-3 fatty acyl chains (β-phospholipids) were also examined. All of the phospholipids were in the form of small sonicated vesicles which are widely utilized as membrane models. The α-methylene group of the sn-1 and sn-2 fatty acyl chains of the α-phospholipids give rise to separate signals due to the non-equivalency of these chains with respect to the glycerol phosphate backbone on all α-phospholipids tested. Additionally, differences in the environment of the PC molecules as well as N- methyl-PE , and N,N- dimethyl-PE , but not PE itself on the inside and outside of the vesicles are reflected in the chemical shift of the α-methylene protons. On the other hand, all of the β-phospholipids (including β-PE) were found to reflect the inside/outside packing differences in their α-methylene groups. The bilayer packing does not induce any nonequivalence in the chemically equivalent acyl chains. In mixed micelles with detergents, β-phospholipids showed one α-CH 2 signal for all phospholipids. These results are consistent with a common conformational arrangement for the fatty acyl chains in all α-phospholipids that have been investigated no matter what aggregated form. The conformational arrangement in the β-phospholipids is different, but again is similar for all of the compounds tested in various aggregated forms.

Semi-synthetic approach for the preparation of homogeneous plasmenylethanolamine utilizing phospholipase D from Streptomyces chromofuscus.

A simple method for the preparation of homogeneous molecular species of plasmenylcholine and plasmenylethanolamine was developed. The method utilized reverse phase high performance liquid chromatography to isolate homogeneous molecular species of plasmenylcholine prepared by acylation of lysoplasmenylcholine. Plasmenylcholine was directly converted to plasmenylethanolamine by transphosphatidylation utilizing phospholipase D from Streptomyces chromofuscus. This method permits the facile labeling of homogeneous molecular species of plasmalogens in the polar head group, the sn-2 acyl chain, or both, for the first time.

Comparison of the hydrolysis of phosphatidylethanolamine and phosphatidyl ( N-acyl) ethanolamine in Dictyostelium discoideum amoebae

In Dictyostelium discoideum, ( N-acyl)ethanolamine glycerophospholipids disappear as the amoebae aggregate, whereas the amount of ethanolamine glycerophospholipids remains relatively constant, suggesting that each type of ethanolamine-containing phospholipid might have a separate metabolic pathway. To study their metabolism, phosphatidylethanolamine and phosphatidyI( N-acyl)ethanolamine containing either [ 14C]ethanolamine or a 14C-labeled sn-2 fatty acyl group were incubated with D. discoideum homogenates, and the conversion of the substrates into radioactive products was monitored. At pH values 3.8 and 4.5, phosphatidyl( N-acyl)ethanolamine was hydrolyzed by a phospholipase A 1 to form the sn-2 acyl form of the lipid. Only minor hydrolysis occurred at pH values of 5.2 or higher. ( N-acyl)Ethanolamine was also released by a phospholipase D type activity at 0.1 the rate of the lysophospholipid formation. Phosphatidyl ( N-acyl)ethanolamine was not hydrolyzed to form phosphatidylethanolamine or water soluble components. At pH 7.2 and at the low pH range of 3.8–4.5, phosphatidylethanolamine was hydrolyzed to lysophosphatidylethanolamine, which was then further degraded to water-soluble components. At pH 7.2, a phospholipase A 2 initially hydrolyzed the phosphatidylethanolamine, whereas at the low pH range a phospholipase A 1 was the most active enzyme. Although both types of ethanolamine-containing phospholipid were hydrolyzed by a phospholipase A 1 at the low pH range, phosphatidylethanolamine hydrolysis was more sensitive to inhibition by Trition X-100, and phosphatidylethanolamine was hydrolyzed to water-soluble components, whereas phosphatidyl( N-acyl)ethanolamine was not. At pH 7.2, phosphatidylethanolamine was hydrolyzed, but phosphatidyl( N-acyl)ethanolamine was not hydrolyzed at all. These results indicate that there are separate routes of degradation for the two types of ethanolamine-containing phospholipid in D. discoideum.

Packing of ether and ester phospholipids in monolayers. Evidence for hydrogen-bonded water at the sn-1 acyl group of phosphatidylcholines

Packing of ether and ester phospholipids in monolayers. Evidence for hydrogen-bonded water at the sn-1 acyl group of phosphatidylcholines

Phosphatidylcholine and cholesterol interactions in model membranes

Various phosphatidylcholines differing either in the stereochemistry around their chiral center or in the position of a cis double bond along the acyl chains were synthesized in order to study critical contact regions in the phospholipid molecule with adjacent cholesterol in model membranes. Microviscosities calculated from fluorescence depolarization of diphenylhexatriene and chain order from spin label studies were measured to monitor physical membrane properties. The enhancing effect of cholesterol on the microviscosity of membranes containing phosphatidylcholines with comparable acyl chain length was largest when the two acyl chains were saturated and smallest when both were unsaturated. Membranes prepared from phosphatidylcholines having a single cis double bond at different positions along the sn-2 acyl chain showed roughly the same changes of microviscosity or chain order upon incorporation of cholesterol. No discrimination was evident in the interaction between cholesterol and enantiomeric phosphatidylcholines or between the enantiomeric phosphatidylcholine molecules themselves. We conclude that the rigidifying effect of cholesterol in membranes does not depend on specific sites of interactino and that with respect to physical membrane properties phosphatidylcholine behaves as an achiral molecule.

Calorimetric investigations of saturated mixed-chain phosphatidylcholine bilayer dispersions.

A series of saturated mixed-chain phosphatidylcholines were prepared whose sn-2 acyl chains are two, four, six, and eight carbon atoms shorter than the sn-1 acyl chain. The calorimetric behavior of multilamellar bilayers of these phosphatidylcholines in excess water is investigated. The phosphatidylcholines display cooperative phase transitions which are dependent upon both the difference in chain length and the position of the acyl chains on the glycerol backbone of the phospholipid. A model is proposed which suggests that the thermotropic behavior of the mixed-chain phosphatidylcholines results from progressively greater interdigitation of the acyl chains of the phospholipid across the bilayer center, in the gel state, as the chain-length difference is increased beyond a minimum value. The disruptive effect of the terminal methyl groups of the fatty acyl chains upon the bilayer packing stability is also stressed. Dispersions of some of the mixed-chain phosphatidylcholines display transition endotherms which appear to be composites of two or more individual transition peaks. The dependence of this behavior on the thermal history of the dispersions is investigated. It is proposed that these peaks arise from the ability of the phosphatidylcholines' acyl chains to pack in more than one interdigitated conformation in the gel state.

Specific positional distribution of acyl moieties in phospholipids is not generally deleted in neoplastic cells.

The distribution of acyl moieties at sn-1 and sn-2 positions of cholinphosphoglycerides (CPG) and ethanolaminephosphoglycerides (EPG) has been determined for neurosarcoma, sarcoma 180 and leukemia L 1210. In all the three samples, the positional distribution of acyl moieties in the two major classes of phospholipids is found to be similar to that in cellular phospholipids of most mammalian tissues. The saturated acyl moieties are located predominantly at sn-1 and polyunsaturated acyl moieties at sn-2, whereas the monounsaturated acyl moieties are randomly distributed between these two positions. Apparently, a disruption of specific positioning of acyl moieties in phospholipids, which hitherto has been considered to be a general metabolic deletion in neoplasia, does not exist in an all neoplastic cells.

Proton and carbon-13 nuclear magnetic resonance studies of rhodopsin-phospholipid interactions.

Proton and carbon-13 nuclear magnetic resonance (1H and 13C NMR) spectra of rhodopsin-phospholipid membrane vesicles and sonicated disk membranes are presented and discussed. The presence of rhodopsin in egg phosphatidylcholine vesicles results in homogeneous broadening of the methylene and methyl resonances. This effect is enhanced with increasing rhodopsin content and decreased by increasing temperature. The proton NMR data indicate the phospholipid molecules exchange rapidly (less than 10(-3) s) between the bulk membrane lipid and the lipid in the immediate proximity of the rhodopsin. These interactions result in a reduction in either or both the frequency and amplitude of the tilting motion of the acyl chains. The 13C NMR spectra identify the acyl chains and the glycerol backbone as the major sites of protein lipid interaction. In the disk membranes the saturated sn-1 acyl chain is significantly more strongly immobilized than the polyunsaturated sn-2 acyl chain. This suggest a membrane model in which the lipid molecules preferentially solvate the protein with the sn-1 chain, which we term an edge-on orientation. The NMR data on rhodopsin-asolectin membrane vesicles demonstrate that the lipid composition is not altered during reconstitution of the membranes from purified rhodopsin and lipids in detergent.

Purified phospholipase A 2 from sheep erythrocyte membrane : Preferential hydrolysis according to polar groups and 2-acyl chains

Hydrolysis of natural phospholipids by pure erythrocyte membrane phospholipase A 2 was compared to the reaction catalyzed by the soluble pancreatic enzyme. Fatty acids liberated during both types of reaction were quantitatively analyzed by gas liquid chromatography. We confirm for the pancreatic enzyme lack of specificity with respect to the sn-2 acyl chain of the phospholipids and preference for negatively charged polar head groups. Conversely, the membranous enzyme preferentially attacks uncharged phospholipids and within one class of phospholipid preferentially splits long chain unsaturated fatty acids in the sn-2 position. The significance of such differences between pancreatic and sheep erythrocyte enzyme is discussed in relation to the possible physiological role of the latter enzyme.