Docosahexaenoic acid (DHA) alters the phospholipid molecular species composition of membranous vesicles exfoliated from the surface of a murine leukemia cell line

Previously, we presented evidence that the vesicles routinely exfoliated from the surface of T27A tumor cells arise from vesicle-forming regions of the plasma membrane and possess a set of lateral microdomains distinct from those of the plasma membrane as a whole. We also showed that docosahexaenoic acid (DHA, or 22:6n-3), a fatty acyl chain known to alter microdomain structure in model membranes, also alters the structure and composition of exfoliated vesicles, implying a DHA-induced change in microdomain structure on the cell surface. In this report we show that enrichment of the cells with DHA reverses some of the characteristic differences in composition between the parent plasma membrane and shed microdomain vesicles, but does not alter their phospholipid class composition. In untreated cells, DHA-containing species were found to be a much greater proportion of the total phosphatidylethanolamine (PE) pool than the total phosphatidylcholine (PC) pool in both the plasma membrane and the shed vesicles. After DHA treatment, the proportion of DHA-containing species in the PE and PC pools of the plasma membrane were elevated, and unlike in untreated cells, their proportions were equal in the two pools. In the vesicles shed from DHA-loaded cells, the proportion of DHA-containing species of PE was the same as in the plasma membrane. However, the proportion of DHA-containing species of PC in the vesicles (0.089) was much lower than that found in the plasma membrane (0.194), and was relatively devoid of species with 16-carbon acyl components. These data suggested that DHA-containing species of PC, particularly those having a 16-carbon chain in the sn-1 position, were preferentially retained in the plasma membrane. The data can be interpreted as indicating that DHA induces a restructuring of lateral microdomains on the surface of living cells similar to that predicted by its behavior in model membranes.     

The yeast vacuolar Rab GTPase Ypt7p has an activity beyond membrane recruitment of the homotypic fusion and protein sorting-Class C Vps complex

A previous report described lipid mixing of reconstituted proteoliposomes made using lipid mixtures that mimic the composition of yeast vacuoles. This lipid mixing required SNARE {SNAP [soluble NSF (N-ethylmaleimide-sensitive factor)-attachment protein] receptor} proteins, Sec18p and Sec17p (yeast NSF and α-SNAP) and the HOPS (homotypic fusion and protein sorting)-Class C Vps (vacuole protein sorting) complex, but not the vacuolar Rab GTPase Ypt7p. The present study investigates the activity of Ypt7p in proteoliposome lipid mixing. Ypt7p is required for the lipid mixing of proteoliposomes lacking cardiolipin [1,3-bis-(sn-3'-phosphatidyl)-sn-glycerol]. Omission of other lipids with negatively charged and/or small head groups does not cause Ypt7p dependence for lipid mixing. Yeast vacuoles made from strains disrupted for CRD1 (cardiolipin synthase) fuse to the same extent as vacuoles from strains with functional CRD1. Disruption of CRD1 does not alter dependence on Rab GTPases for vacuole fusion. It has been proposed that the recruitment of the HOPS complex to membranes is the main function of Ypt7p. However, Ypt7p is still required for lipid mixing even when the concentration of HOPS complex in lipid-mixing reactions is adjusted such that cardiolipin-free proteoliposomes with or without Ypt7p bind to equal amounts of HOPS. Ypt7p therefore must stimulate membrane fusion by a mechanism that is in addition to recruitment of HOPS to the membrane. This is the first demonstration of such a stimulatory activity--that is, beyond bulk effector recruitment--for a Rab GTPase.     

N-methyl-D-aspartate receptor function observed by rate of ligand dialysis from proteoliposome solution

This study reports rate-of-dialysis of an iodinated N-methyl-D-aspartate antagonist drug, [125-I] MK-801. from solutions of lipid vesicles and from proteoliposomes containing purified membrane proteins. A 170 kd protein precipitated from proteoliposomes cross reacts with monoclonal antibodies against cloned NMDA-NR2(A) and NR2(B) subunits. Drug binding in proteoliposomes includes contributions from lipid and from protein, in addition to lipid. A significant change in drug binding was observed in proteoliposomes in response to 10 uM agonist, NMDA. Rate-of-dialysis from agonist-stimulated proteoliposomes was sensitive to perturbation by decreased aqueous ligand concentration in a manner consistent with a lipid-mediated receptor/antagonist equilibrium.     

Physical properties of liposomes and proteoliposomes prepared from Escherichia coli polar lipids

Reconstituted proteoliposomes serve as experimental systems for the study of membrane enzymes. Osmotic shifts and other changes in the solution environment may influence the structures and membrane properties of phospholipid vesicles (including liposomes, proteoliposomes and biological membrane vesicles) and hence the activities of membrane-associated proteins. Polar lipid extracts from Escherichia coli are commonly used in membrane protein reconstitution. The solution environment influenced the phase transition temperature and the diameter of liposomes and proteoliposomes prepared from E. coli polar lipid by extrusion. Liposomes prepared from E. coli polar lipids differed from dioleoylphosphatidylglycerol liposomes in Young's elastic modulus, yield point for solute leakage and structural response to osmotic shifts, the latter indicated by static light scattering spectroscopy. At high concentrations, NaCl caused aggregation of E. coli lipid liposomes that precluded detailed interpretation of light scattering data. Proteoliposomes and liposomes prepared from E. coli polar lipids were similar in size, yield point for solute leakage and structural response to osmotic shifts imposed with sucrose as osmolyte. These results will facilitate studies of bacterial enzymes implicated in osmosensing and of other enzymes that are reconstituted in E. coli lipid vesicles.     

N-Methyl-D-Aspartate Receptor Function Observed by Rate of Ligand Dialysis From Proteoliposome Solution

Abstract

This study reports rate-of-dialysis of an iodinated N-methyl-D-aspartate antagonist drug, [125-1] MK-801, from solutions of lipid vesicles and from proteoliposomes containing purified membrane proteins. A 170 kd protein precipitated from proteoliposomes cross reacts with monoclonal antibodies against cloned NMDA-NR2(A) and NR2(B) subunits. Drug binding in proteoliposomes includes contributions from lipid and from protein, in addition to lipid. A significant change in drug binding was observed in proteoliposomes in response to 10 uM agonist, NMDA. Rate-of-dialysis from agonist-stimulated proteoliposomes was sensitive to perturbation by decreased aqueous ligand concentration in a manner consistent with a lipid-mediated receptor/antagonist equilibrium.     

Lateral mobility of class I histocompatibility antigens in B lymphoblastoid cell membranes: modulation by cross-linking and effect of cell density

We have studied the lateral mobility of class 1 major histocompatibility complex (MHC) proteins in the membranes of human Epstein-Barr virus-transformed B cells using fluorescence photobleaching recovery. Class I MHC antigens were labeled with either W6/32 monoclonal antibody or its Fab fragment directly conjugated to fluorescein isothiocyanate. The diffusion coefficient of class I antigens labeled with Fab fragments of W6/32 was identical to that of a lipid analogue, fluorescein phosphatidylethanolamine, and was 10-fold greater than that of antigens labeled with intact W6/32. Furthermore, antigens labeled with Fab fragments but not with intact W6/32 had fractional mobilities identical to that of the lipid probe. The lateral mobility of class I antigens was dependent on the time of incubation with fluorescent antibody and on the presence of antibody microaggregates. Finally, class I MHC proteins labeled with intact W6/32 but not with Fab fragments were immobilized in the membranes of most cells grown in suspension at high cell density. These results suggest that, in the unperturbed state, class I MHC antigens diffuse as rapidly as membrane lipid, i.e., without cytoskeletal constraint. Cross-linking with bivalent ligand and growth to high cell density may trigger membrane events leading to slowing and immobilization of these proteins.     

Structural characterization of oxidized phospholipid products derived from arachidonate-containing plasmenyl glycerophosphocholine

Plasmenyl phospholipids are a structurally unique class of lipids that contain a vinyl ether substituent at the sn-1 position of the glycerol backbone, imparting unique susceptibility to oxidative reactions that may take place at the cell membrane lipid bilayer. Several studies have supported the hypothesis that plasmalogens may be antioxidant molecules that protect cells from oxidative stress. Because the molecular mechanism for the antioxidant properties of plasmenyl phospholipids is not fully understood, the oxidation of arachidonate-containing plasmalogen-glycerophosphocholine (GPC) was studied using electrospray tandem mass spectrometry after exposure to the free radical initiator 2, 2'-azobis(2-amidinopropane)hydrochloride (AAPH). Various oxidized GPC products involving the sn-1 position alone (1-formyl-2-arachidonyl lipids and lysophospholipid), oxidation products involving the sn-2 position alone (chain-shortened omega-aldehyde radyl substituents at sn-2) as well as products oxidized both at the sn-1 and sn-2 positions were observed and structurally identified.The results of these experiments suggest that oxidation of plasmenyl phospholipids esterified with polyunsaturated fatty acid groups at sn-2 likely undergo unique and specific free radical oxidation at the 1'-alkenyl position as well as oxidation of the double bond closest to the ester moiety at sn-2.     

StarD10, a START domain protein overexpressed in breast cancer, functions as a phospholipid transfer protein

We originally identified StarD10 as a protein overexpressed in breast cancer that cooperates with the ErbB family of receptor tyrosine kinases in cellular transformation. StarD10 contains a steroidogenic acute regulatory protein (StAR/StarD1)-related lipid transfer (START) domain that is thought to mediate binding of lipids. We now provide evidence that StarD10 interacts with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by electron spin resonance measurement. Interaction with these phospholipids was verified in a fluorescence resonance energy transfer-based assay with 7-nitro-2,1,3-benzoxadiazol-4-yl-labeled lipids. Binding was not restricted to lipid analogs since StarD10 selectively extracted PC and PE from small unilamellar vesicles prepared with endogenous radiolabeled lipids from Vero monkey kidney cells. Mass spectrometry revealed that StarD10 preferentially selects lipid species containing a palmitoyl or stearoyl chain on the sn-1 and an unsaturated fatty acyl chain (18:1 or 18:2) on the sn-2 position. StarD10 was further shown to bind lipids in vivo by cross-linking of protein expressed in transfected HEK-293T cells with photoactivable phosphatidylcholine. In addition to a lipid binding function, StarD10 transferred PC and PE between membranes. Interestingly, these lipid binding and transfer specificities distinguish StarD10 from the related START domain proteins Pctp and CERT, suggesting a distinct biological function.     

Oxidized phospholipids, linked to apolipoprotein B of oxidized LDL, are ligands for macrophage scavenger receptors

Previous studies have shown that macrophage receptors for oxidized LDL (OxLDL) recognize both the lipid and protein moieties, and that a monoclonal antibody against OxLDL, EO6, also recognizes both species. The present studies show directly that during LDL oxidation phospholipids become covalently attached to apolipoprotein B (apoB). After exhaustive extraction of lipids, apoB of native LDL contained 4 +/- 3 moles of phosphorus/mole protein. In contrast, apoB of OxLDL contained approximately 75 moles of phosphorus/mole protein. Saponification of this apoB released phosphorus, choline, and saturated fatty acids in a molar ratio of 1.0:0.98:0.84. When LDL was reductively methylated prior to oxidation, the amount of phospholipid covalently bound was reduced by about 80%, indicating that the phospholipids attach at lysine epsilon amino groups. Progressive decreases in the phospholipid associated with apoB of OxLDL decreased the ability of the protein to compete for binding to macrophage scavenger receptors and decreased its reactivity with antibody EO6.We postulate that some oxidized phospholipids containing fatty acid aldehydes at the sn-2 position bind to lysine residues of apoB while others remain unreacted within the lipid phase. This would account for the interchangeability of lipid and apolipoprotein of OxLDL with respect to receptor binding and antibody recognition.     

Phosphatidylglucoside: a new marker for lipid rafts

Lipid rafts are functional microdomains enriched with sphingolipids and cholesterol. The fatty acyl chain composition of sphingolipids is a critical factor in the localization of lipids in lipid rafts. The recent studies suggest that lipid rafts are more heterogeneous than previously thought. In addition, our discovery of a new glycolipid, phosphatidylglucoside (PtdGlc), also supports the notion of raft heterogeneity. The complete structural characterization of PtdGlc shows that it consists solely of saturated fatty acyl chains: C18:0 at the sn-1 and C20:0 at the sn-2 positions of the glycerol backbone. This unique fatty acyl composition comprising a single molecular species rarely occurs in known mammalian lipids. Although the structure of PtdGlc is similar to that of phosphatidylinositol, PtdGlc localizes to the outer leaflet of the plasma membrane and is possibly involved in cell-cell interaction signaling in the central nervous system.     

Computational analysis of the membrane association of group IIA secreted phospholipases A2: a differential role for electrostatics

Secreted phospholipases A2 (sPLA2's) are enzymes that hydrolyze glycerophospholipids at the sn-2 position, which leads to the production of lipid mediators of many cellular processes. These interfacial enzymes are regulated by their lipid specificity at two levels: membrane binding and substrate recognition. Different sPLA2's utilize different combinations of electrostatic and hydrophobic interactions to adsorb to membrane surfaces, which results in the wide range of membrane binding behaviors observed. Here, the finite difference Poisson Boltzmann (FDPB) method is used to quantitatively analyze the contribution of electrostatic interactions to the membrane association of two highly basic group II sPLA2's: Agkistrodon piscivorus piscivorus (AppD49) sPLA2 and nonpancreatic human group IIA (hGIIA) sPLA2. The calculations predict how membrane binding is affected by ionic strength, membrane composition, substitutions of residues in the enzymes, and the presence of calcium in the active site. In addition, the results provide molecular models for the membrane-associated forms of the enzymes. Furthermore, these models account for (1) changes in orientation and protonation state of both the native and charge reversal forms of the enzymes at the membrane surface and (2) the effect of protein/vesicle aggregation, as observed for hGIIA sPLA2. Importantly, the modeling quantitatively describes the complex membrane binding behaviors of these interfacial enzymes in terms of simple physical forces and provides structural information that is difficult to obtain experimentally. The computational analysis shows that nonspecific electrostatic interactions not only play a major role in recruiting these enzymes to membrane surfaces but also orient the enzymes for productive catalysis at the membrane interface.     

Coupled changes between lipid order and polypeptide conformation at the membrane surface. A 2H NMR and Raman study of polylysine-phosphatidic acid systems

Thermotropism and segmental chain order parameters of sn-2-perdeuteriated dimyristoyl-phosphatidic acid (DMPA)-water dispersions, with and without poly(L-lysine) (PLL) of different molecular weights, have been investigated by solid-state deuterium NMR spectroscopy. The segmental chain order parameter profile of this negatively charged lipid is similar to that already found for other lipids. Addition of long PLL (MW = 200,000) increases the temperature, Tc, of the lipid gel-to-fluid phase transition, whereas short PLL (MW = 4000) has practically no effect on Tc. In the fluid phase both varieties of PLL increase the "plateau" character of segmental order parameters up to carbon position 10. At the same reduced temperature, long PLL more significantly increases the segmental ordering, especially at the methyl terminal position. This leads to the conclusion that polar head-group capping and charge neutralization by PLL induce severe changes in lipid chain ordering, even down to the bilayer core. The structure of PLL bound to the lipid bilayer surface was monitored by Raman spectroscopy, following the amide I bands. Results show that the lipid gel-to-fluid phase transition triggers a conformational transition from ordered beta-sheet to random structure of short PLL, while it does not affect the strongly stabilized beta-sheet structure of long PLL. It is concluded that both short and long PLL can efficiently cap and neutralize lipid head groups, whatever their structure, and that peptide length is a key parameter in whether lipids or peptides are the driving force in conformationally coupled changes of both partners in the membrane.     

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.     

Lipid-protein and protein-protein interactions in double recombinants of myelin proteolipid apoprotein and myelin basic protein with dimyristoylphosphatidylglycerol.

The integral proteolipid apoprotein (PLP) from bovine spinal cord has been reconstituted in dimyristoylphosphatidylglycerol (DMPG) bilayers, and the mutual interactions on binding the peripheral myelin basic protein (MBP) have been studied. Quantitation of protein and lipid contents in the MBP-PLP-DMPG double recombinants at different PLP:DMPG ratios led to the conclusion that MBP binds only to the DMPG lipid headgroups and is hindered from interaction with the first shell of lipids surrounding the PLP. No specific PLP-MBP association could be detected. Electron spin resonance (ESR) spectra of phosphatidylglycerol spin-labeled at position n = 5 in the sn-2 chain showed that complexation of MBP with the PLP-DMPG recombinants leads to a decrease in lipid chain mobility to an extent which correlates with the degree of MBP binding. At low DMPG:PLP ratios, the perturbations of lipid mobility by both proteins are mutually enhanced. In single recombinants of PLP with DMPG, the ESR spectra of phosphatidylglycerol spin-labeled at position n = 14 in the sn-2 chain indicated that approximately 10 lipids/protein are motionally restricted by direct contact with the intramembranous surface of the protein. This number is in agreement with earlier results for reconstitutions of PLP in dimyristoylphosphatidylcholine (DMPC) [Brophy, P. J., Horváth, L. I., & Marsh, D. (1984) Biochemistry 23, 860-865] and is consistent with a hexameric arrangement of the PLP molecules in DMPG bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrospray ionization mass spectrometric analyses of phospholipids from INS-1 insulinoma cells: comparison to pancreatic islets and effects of fatty acid supplementation on phospholipid composition and insulin secretion

Insulin secretion by pancreatic islet beta-cells is impaired in diabetes mellitus, and normal beta-cells are enriched in phospholipids with arachidonate as sn-2 substituent. Such molecules may play structural roles in exocytotic membrane fusion or serve as substrates for phospholipases activated by insulin secretagogues. INS-1 insulinoma cells respond to secretagogues and permit the study of effects of culture with free fatty acids on phospholipid composition and secretion. INS-1 cell glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) lipids are demonstrated here by electrospray ionization mass spectrometry to contain a lower fraction of molecules with arachidonate and a higher fraction with oleate as sn-2 substituent than native islets. Palmitic acid supplementation induces little change in these INS-1 cell lipids, but supplementation with linoleate or arachidonate induces a large rise in the fraction of INS-1 cell GPC species with polyunsaturated sn-2 substituents and a fall in oleate-containing species to yield a GPC profile similar to native islets. The fraction of GPE lipids comprised of plasmenylethanolamine species with polyunsaturated sn-2 substituents in early-passage INS-1 cells is similar to that of islets, but declines on serial passage. Such molecules might participate in exocytotic membrane fusion, and late-passage INS-1 cells have reduced insulin secretory responses. Arachidonate supplementation induces a rise in the fraction of INS-1 cell GPE lipids with polyunsaturated sn-2 substituents and partially restores responses to insulin secretagogues by late-passage INS-1 cells, but does not further amplify secretion by early-passage cells. Effects of extracellular free fatty acids on beta-cell phospholipid composition and secretory responses could be involved in changes in beta-cell function during the period of hyper-free fatty acidemia that precedes diabetes mellitus.     

Protein-induced vertical lipid dislocation in a model membrane system: spin-label relaxation studies on avidin-biotinylphosphatidylethanolamine interactions.

The change in vertical location of spin-labeled N-biotinyl phosphatidylethanolamine in fluid-phase dimyristoyl phosphatidylcholine bilayer membranes, on binding avidin to the biotinyl headgroup, has been investigated by progressive saturation electron spin resonance measurements. Spin-labeled phospholipids were present at a concentration of 1 mol%, relative to total membrane lipids. For avidin-bound N-biotinyl phosphatidylethanolamine spin-labeled on the 8 C atom of the sn-2 chain, the relaxation enhancement induced by 30 mM Ni2+ ions confined to the aqueous phase was 2.5 times that induced by saturating molecular oxygen, which is preferentially concentrated in the hydrophobic core of the membrane. For phosphatidylcholine also spin-labeled at the 8 position of the sn-2 chain, this ratio was reversed: the relaxation enhancement by Ni2+ ions was half that induced by molecular oxygen. In the absence of avidin, the enhancement by either relaxant was the same for both spin-labeled phospholipids. For a double-labeled system, in which both N-biotinyl phosphatidylethanolamine and phosphatidylcholine were spin-labeled on the 12 C atom of the sn-2 chain, the relaxation rate in the absence of avidin was greater than that predicted from linear additivity of the corresponding singly labeled systems, because of mutual spin-spin interactions between the two labeled lipid species. On binding of avidin to the N-biotinyl phosphatidylethanolamine, this relaxation enhancement by mutual spin-spin interaction was very much decreased. These results indicate that, on binding of avidin to the lipid headgroup, N-biotinyl phosphatidylethanolamine is lifted vertically within the membrane, relative to the phosphatidylcholine host lipids. The specific binding of avidin to N-biotinyl phosphatidylethanolamine parallels the liftase activity proposed for activator proteins associated with the action of certain gangliosidases.     

Constitutive internalization of murine MHC class I molecules

The total number of cell surface glycoprotein molecules at the plasma membrane results from a balance between their constitutive internalization and their egress to the cell surface from intracellular pools and/or biosynthetic pathway. Constitutive internalization is net result of constitutive endocytosis and endocytic recycling. In this study we have compared spontaneous internalization of murine major histocompatibility complex (MHC) class I molecules (K(d), D(d), full L(d), and empty L(d)) after depletion of their egress to the cell surface (Cycloheximide [CHX], brefeldin A [BFA]) and internalization after external binding of monoclonal antibody (mAb). MHC class I alleles differ regarding their cell surface stability, kinetics, and in the way of internalization and degradation. K(d) and D(d) molecules are more stable at the cell surface than L(d) molecules and, thus, constitutively internalized more slowly. Although the binding of mAbs to cell surface MHC class I molecules results in faster internalization than depletion of their egress, it is still slow and, thereby, can serve as a model for tracking of MHC class I endocytosis. Internalization of fully conformed MHC class I molecules (K(d), D(d), and L(d)) was neither inhibited by chlorpromazine (CP) (inhibitor of clathrin endocytosis), nor with filipin (inhibitor of lipid raft dependent endocytosis), indicating that fully conformed MHC class I molecules are internalized via the bulk pathway. In contrast, internalization of empty L(d) molecules was inhibited by filipin, indicating that non-conformed MHC class I molecules require intact cholesterol-rich membrane microdomains for their constitutive internalization. Thus, conformed and non-conformed MHC class I molecules use different endocytic pathways for constitutive internalization.     

Ladderane lipid distribution in four genera of anammox bacteria

Intact ladderane phospholipids and core lipids were studied in four species of anaerobic ammonium oxidizing (anammox) bacteria, each representing one of the four known genera. Each species of anammox bacteria contained C(18) and C(20) ladderane fatty acids with either 3 or 5 linearly condensed cyclobutane rings and a ladderane monoether containing a C(20) alkyl moiety with 3 cyclobutane rings. The presence of ladderane lipids in all four anammox species is consistent with their putative physiological role to provide a dense membrane around the anammoxosome, the postulated site of anammox catabolism. In contrast to the core lipids, large variations were observed in the distribution of ladderane phospholipids, i.e. different combinations of hydrophobic tail (ladderane, straight chain and methyl branched fatty acid) types attached to the glycerol backbone sn-1 position, in combination with different types of polar headgroup (phosphocholine, phosphoethanolamine or phosphoglycerol) attached to the sn-3 position. Intact ladderane lipids made up a high percentage of the lipid content in the cells of "Candidatus Kuenenia stuttgartiensis", suggesting that ladderane lipids are also present in membranes other than the anammoxosome. Finally, all four investigated species contained a C(27) hopanoid ketone and bacteriohopanetetrol, which, indicates that hopanoids are anaerobically synthesised by anammox bacteria.     

Annexin I-mediated vesicular aggregation: mechanism and role in human neutrophils.

Whole cytosol isolated from human neutrophils was found to accelerate the Ca(2+)-dependent fusion of phospholipid vesicles with neutrophil plasma membranes as measured by several fluorescence resonance energy transfer lipid dilution assays or by the fate of an encapsulated aqueous soluble fluorophore. The Ca2+ (threshold of 2-10 microM) and protein concentration dependencies for fusion mediated by purified human neutrophil annexin I (lipocortin I), recombinant annexin I and des(1-9)annexin I showed behavior similar to that of whole cytosol. A monoclonal antibody against the N-terminal region of annexin I strongly inhibited the action of isolated annexins as well as whole cytosol, indicating that annexin I is the major activity of this type in whole neutrophil cytosol and that it functions even in this complex mixture of proteins. Residual Ca(2+)-dependent fusion activity in the absence of cytosol or annexin I was not inhibited by several antibodies against annexin I, implicating an as yet unknown protein. Kinetic analysis of liposomal fusion showed that annexin I, as in the case of synexin, accelerates aggregation of vesicles but not the actual fusion event per se. The disposition of annexin I in liposomal aggregates was studied by monitoring binding of the protein with a pyrene-phospholipid and by simultaneously monitoring vesicular aggregation by turbidity. An antibody to the N-terminus of annexin I inhibited vesicular aggregation but not binding, suggesting that initial binding of annexin I is similar to that of annexin V. A relatively small proportion of the bound annexin was involved in intervesicular linkage, and no exchange of bound annexin to subsequently added vesicles was observed. The lack of extensive contact between lipids of aggregated vesicles was supported by a lack of energy transfer between phospholipid probes on separate aggregating vesicles. Covalent linkage of maleimidyl or photoaffinity phospholipid derivatives with annexin I in vesicular aggregates did not allow complete disaggregation of vesicles by EDTA, suggesting that monomers of annexin I can contact two membranes simultaneously at the point of intervesicular linkage. These data are discussed in terms of possible models for the structure of this site.     

Ultrastructure of human erythrocyte GLUT1

This study was undertaken to examine GLUT1 quaternary structure. Independent but complementary methodologies were used to investigate the influence of membrane-solubilizing detergents on GLUT1/lipid/detergent micelle hydrodynamic radii. Hydrodynamic size analysis and electron microscopy of GLUT1/lipid/detergent micelles and freeze-fracture electron microscopy of GLUT1 proteoliposomes support the hypothesis that the glucose transporter is a multimeric (probably tetrameric) complex of GLUT1 proteins. GLUT1 forms a multimeric complex in octyl glucoside that dissociates upon addition of reductant. Some detergents (e.g., CHAPS and dodecyl maltoside) promote the dissociation of GLUT1 oligomers into smaller aggregation states (dimers or monomers). These complexes do not reassemble as larger oligomers when dissociating detergents are subsequently replaced with nondissociating detergents such as octyl glucoside or cholic acid. When dissociating detergents are replaced with lipids, the resulting proteoliposomes catalyze protein-mediated sugar transport, and the subsequent addition of solubilizing, nondissociating detergents generates higher (tetrameric) GLUT1 aggregation states. These findings suggest that some detergents stabilize while others destabilize GLUT1 quaternary structure. GLUT1 does not appear to exchange rapidly between protein/lipid/detergent micelles but is able to self-associate in the plane of the lipid bilayer.