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N-Acylethanolamine acid amidase (NAAA) is an N-terminal cysteine hydrolase primarily found in the endosomal-lysosomal compartment of innate and adaptive immune cells. NAAA catalyzes the hydrolytic deactivation of palmitoylethanolamide (PEA), a lipid-derived peroxisome proliferator-activated receptor-α (PPAR-α) agonist that exerts profound anti-inflammatory effects in animal models. Emerging evidence points to NAAA-regulated PEA signaling at PPAR-α as a critical control point for the induction and the resolution of inflammation and to NAAA itself as a target for anti-inflammatory medicines. The present Perspective discusses three key aspects of this hypothesis: the role of NAAA in controlling the signaling activity of PEA; the structural bases for NAAA function and inhibition by covalent and noncovalent agents; and finally, the potential value of NAAA-targeting drugs in the treatment of human inflammatory disorders.

Protein lipidation, including cysteine prenylation, N-terminal glycine myristoylation, cysteine palmitoylation, and serine and lysine fatty acylation, occurs in many proteins in eukaryotic cells and regulates numerous biological pathways, such as membrane trafficking, protein secretion, signal transduction, and apoptosis. We provide a comprehensive review of protein lipidation, including descriptions of proteins known to be modified and the functions of the modifications, the enzymes that control them, and the tools and technologies developed to study them. We also highlight key questions about protein lipidation that remain to be answered, the challenges associated with answering such questions, and possible solutions to overcome these challenges.

We have investigated the interaction of graphene oxide (GO) sheets with supported lipid membranes with focus on how the interaction depends on GO sheet size (three samples in the range of 90-5000 nm) and how it differs between small and large liposomes. The layer-by-layer assembly of these materials into multilamellar structures, as discovered in our previous research, is now further explored. The interaction processes were monitored by two complementary, real time, surface-sensitive analytical techniques: quartz crystal microbalance with dissipation monitoring (QCM-D, electroacoustic sensing) and indirect nanoplasmonic sensing (INPS, optical sensing). The results show that the sizes of each of the two components, graphene oxide and liposomes, are important parameters affecting the resulting multilayer structures. Spontaneous liposome rupture onto graphene oxide is obtained for large lateral dimensions of the graphene oxide sheets.

To achieve spatial control of fibroblast cell attachment and spreading on a biocompatible polymer coating, the effect of poly(acrylamide) (PAAm) brushes with varied grafting density was investigated. The synthesis of the brushes was performed by surface-initiated atom transfer radical polymerization (SI-ATRP). Gold substrates were modified with binary self-assembled monolayers (SAMs) of an initiator and 16-mercaptohexadecanoic acid (MHDA) as an "inert" thiol to initiate the ATRP of AAm. By using different mixtures for the binary SAMs, a series of polymer brushes with varied grafting densities were prepared. The fractional coverage of surface bound initiator was determined by grazing incidence Fourier transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and contact angle measurements. A linear relationship between the Br/S ratio determined by XPS and ToF-SIMS versus the fraction of initiator on the surface determined by water contact angle measurements was observed. The varied initiation concentration on the gold substrates yielded PAAm brushes with different thicknesses, indicating a transition from mushroom to brush regimes with increasing grafting density. Thereby we achieved exquisite control of the degree of cell adhesion. Cell attachment experiments with NIH 3T3 fibroblast cells revealed cell spreading on PAAm brushes with low grafting densities (initiator fractional coverage

Marine bacteria produce an abundance of suites of acylated siderophores characterized by a unique, species-dependent headgroup that binds iron(III) and one of a series of fatty acid appendages. Marinobacter sp. DS40M6 produces a suite of seven acylated marinobactins, with fatty acids ranging from saturated and unsaturated C12-C18 fatty acids. In the present study, we report that in the late log phase of growth, the fatty acids are hydrolyzed by an amide hydrolase producing the peptidic marinobactin headgroup. Halomonas aquamarina str. DS40M3, another marine bacterium isolated originally from the same sample of open ocean water as Marinobacter sp. DS40M6, produces the acyl aquachelins, also as a suite composed of a peptidic headgroup distinct from that of the marinobactins. In contrast to the acyl marinobactins, hydrolysis of the suite of acyl aquachelins is not detected, even when H. aquamarina str. DS40M3 is grown into the stationary phase. The Marinobacter cell-free extract containing the acyl amide hydrolase is active toward exogenous acyl-peptidic siderophores (e.g., aquachelin C, loihichelin C, as well as octanoyl homoserine lactone used in quorum sensing). Further, when H. aquamarina str. DS40M3 is cultured together with Marinobacter sp. DS40M6, the fatty acids of both suites of siderophores are hydrolyzed, and the aquachelin headgroup is also produced. The present study demonstrates that coculturing bacteria leads to metabolically tailored metabolites compared to growth in a single pure culture, which is interesting given the importance of siderophore-mediated iron acquisition for bacterial growth and that Marinobacter sp. DS40M6 and H. aquamarina str. DS40M3 were isolated from the same sample of seawater.

In this paper, we describe a procedure for the phase transfer of silver nanoprisms (AgNPrs) from aqueous solution to chloroform via an amide coupling reaction. AgNPrs are first modified with 16-mercaptohexadecanoic acid (MHA), and then primary or secondary amines are attached to the carboxylic acid end of the MHA ligand through a carbodiimide-mediated amide coupling step. Secondary amines, such as dicyclohexylamine and diphenylamine, are found to solubilize the nanoparticles in chloroform, whereas primary amines (e.g., butylamine and hexadecylamine) do not result in phase transfer. It is found that the AgNPrs functionalized with dicyclohexylamine show the highest stability and the least aggregation after undergoing phase transfer; in contrast, with a less nucleophilic amine, such as diphenylamine, the amide coupling reaction does not go to completion and the resultant AgNPrs are less stable and more prone to aggregation.

This study examines the lithographic capacity of tips in dip-pen nanolithography (DPN). The dependence of the transport rate (R) decay on the area of lithography (A(lith)), the dependence of A(lith) on the lithographic time (t), and the effect of piranha cleaning on the lithographic capacity are considered herein. The dependencies in the line-drawing lithography process are studied using 16-mercaptohexadecanoic acid (MHA) ink. On the basis of the linear decay dependence discovered in the R-A(lith) dependence, piranha treatment can increase the lithographic capacity by up to 35.5-fold, an improvement that may originate from a change in the tip's surface chemistry. Moreover, a theoretical model is derived to describe the A(lith)-t dependence accurately and to predict the tips' lifetime. Furthermore, an experiment involving DPN-based nanostructure fabrication demonstrates the importance of monitoring the tips' transport rate and lifetime. In addition to shedding light on the physical and chemical principles behind DPN, this study provides a comprehensive model for a quantitative analysis of the tips' behavior.

We present a detailed analysis of the behavior of the highly flexible post- translational lipid modifications of rhodopsin from multiple-microsecond all-atom molecular dynamics simulations. Rhodopsin was studied in a realistic membrane environment that includes cholesterol, as well as saturated and polyunsaturated lipids with phosphocholine and phosphoethanolamine headgroups. The simulation reveals striking differences between the palmitoylations at Cys322 and Cys323 as well as between the palmitoyl chains and the neighboring lipids. Notably the palmitoyl group at Cys322 shows considerably greater contact with helix H1 of rhodopsin, yielding frequent chain upturns with longer reorientational correlation times, and relatively low order parameters. While the palmitoylation at Cys323 makes fewer protein contacts and has increased order compared to Cys322, it nevertheless exhibits greater flexibility with smaller order parameters than the stearoyl chains of the surrounding lipids. The dynamical structure of the palmitoylationsas well as their extensive fluctuationssuggests a complex function for the post-translational modifications in rhodopsin and potentially other G protein-coupled receptors, going beyond their role as membrane anchoring elements. Rather, we propose that the palmitoylation at Cys323 has a potential role as a lipid anchor, whereas the palmitoyl−protein interaction observed for Cys322 suggests a more specific interaction that affects the stability of the dark state of rhodopsin.

Here, we present a simple platform for the use of the enhanced emission of 16-mercaptohexadecanoic acid (16-MHA) capped CdSe quantum dots (QDs) as a probe for ultrasensitive copper(II) detection. In this study, the photoluminescence (PL) of the QDs was first enhanced by Ag nanoprisms which were self-assembled on Si surfaces and then further increased by photobrightening. Using this approach, the control and different analytes could be readily probed all on a single platform using fluorescence microscopy. The enhanced PL intensity of CdSe QDs was selectively quenched in the presence of Cu(2+), accompanied by the emergence of a new red-shifted luminescence band. The quenching mechanism was found to be due to a cation exchange mechanism as confirmed by X-ray photoelectron spectroscopy (XPS) measurements. Herein, we have demonstrated that this simple methodology can offer a rapid and reliable detection of Cu(2+) with a detection limit as low as 5 nM and a dynamic range up to 100 muM in a fixed fast reaction time of 5 min. The potential applications of this technique were tested in two ways, for mixed-ion solutions and in physiological fluids, and both experiments exhibited good selectivity toward Cu(2+).

The coordination of iron(III) to the marine amphiphilic marinobactin and aquachelin siderophores, as well as to petrobactin, an unusual 3,4-dihydroxybenzoyl siderophore is reported. Potentiometric titrations were performed on the apo siderophore to determine the ligand pK(a) values, as well as the complex formed with addition of 1 equiv of Fe(III). The log K(ML) values for Fe(III)-marinobactin-E and Fe(III)-aquachelin-C are 31.80 and 31.4, respectively, consistent with the similar coordination environment in each complex, while log K(ML) for Fe(III)-petrobactin is estimated to be about 43. The pK(a) of the beta-hydroxyaspartyl hydroxyl group was determined to be 10.8 by (1)H NMR titration. (13)C NMR and IR spectroscopy were used to investigate Ga(III) coordination to the marinobactins. The coordination-induced shifts (CIS) in the (13)C NMR spectrum of Ga(III)-marinobactin-C compared to apo-marinobactin-C indicates that the hydroxamate groups are coordinated to Ga(III); however, the lack of CISs for the carbons of the beta-hydroxyamide group suggests this moiety is not coordinated in the Ga(III) complex. Differences in the IR spectrum of Ga(III)-marinobactin-C and Fe(III)-marinobactin-C in the 1600-1700 cm(-1) region also corroborates Fe(III) is coordinated to the beta-hydroxyamide moiety, whereas Ga(III) is not coordinated.

The ability to organize nanomaterials, e.g., Au nanoparticles (NPs) and graphene oxide (GO) sheets, into ordered structures with high accuracy and resolution on a substrate is crucially important for fundamental studies and applications. In this letter, we developed a simple and efficient method to generate positively charged 11-amino-1-undecanethiol (AUT) templates on Au substrates, which were successfully used for controlled assembly of negatively charged Au NPs or GO sheets from aqueous solution. The templates were obtained by passivation of the exposed Au area with AUT after 16-mercaptohexadecanoic acid (MHA) patterns were generated by dip-pen nanolithography (DPN) on Au. The electrostatic interaction ensures that the Au NPs and GO sheets only adsorb on the designed AUT areas. Importantly, by using this method, the number of Au NPs adsorbed on patterned areas can be controlled, and a single Au NP array was successfully achieved.

Ceramides are particularly abundant in the stratum corneum lipid matrix, where they determine its unusual mesostructure, are involved in the lateral segregation of lipid domains in biological cell membranes, and are also known to act as signaling agents in cells. The importance attributed to ceramides in several biological processes has heightened in recent years, demanding a better understanding of their interaction with other membrane components, namely, cholesterol. Structural data concerning pure ceramides in water are relatively scarce, and this is even more the case for mixtures of ceramides with other lipids commonly associated with them in biological systems. We have derived the thermotropic binary phase diagram of mixtures of N-palmitoyl- D-erythro-sphingosine, C16:0-ceramide, and cholesterol in excess water, using differential scanning calorimetry and small- and wide-angle X-ray diffraction. These mixtures are self-organized in lamellar mesostructures that, between other particularities, show two ceramide to cholesterol crystalline phases with molar proportions that approach 2:3 and 1:3. The 2:3 phase crystallizes in a tetragonal arrangement with a lamellar repeat distance of 3.50 nm, which indicates an unusual lipid stacking, probably unilamellar. The uncommon mesostructures formed by ceramides with cholesterol should be considered in the rationalization of their singular structural role in biological systems.

Monolayers prepared with polar or ionic amino acids with short side chains have a reduced nonspecific adsorption of serum proteins compared to that of hydrophobic amino acids and organic monolayers immobilized on the gold surface of surface plasmon resonance (SPR) biosensors. Proteins contained in biological samples adsorb on most surfaces, which in the case of biosensors causes a nonspecific response that hinders the quantification of biomarkers in these biological samples. To circumvent this problem, self-assembled monolayers (SAM) of N-3-mercaptopropyl-amino acids (3-MPA-amino acids) were prepared from 19 natural amino acids. These SAM were investigated to limit the nonspecific adsorption of proteins contained in biological fluids and to immobilize molecular receptors (i.e., antibodies) that are necessary in the construction of biosensors. SPR and Ge attenuated total reflection (GATR) FTIR spectroscopy were employed to characterize the formation of the amino acid SAMs. Monolayers of 3-MPA-amino acids densely packed on the surface of the SPR biosensors result in a surface concentration of approximately 10 (15) molecules/cm (2). SPR also quantifies the surface concentration of serum proteins nonspecifically adsorbed on 3-MPA-amino acids following the exposure of the biosensor to undiluted bovine serum. The concentration of nonspecifically bound proteins ranged from approximately 400 ng/cm (2) with polar and ionic amino acids to approximately 800 ng/cm (2) with amino acids of increased hydrophobicity. The nonspecific adsorption of serum proteins on the 3-MPA-amino acids increases in the following order: AspAsnSerMetGluGlnThrGlyHisCysArgPheTrpValProIleLeuAlaTyr. The analysis of the adsorption and desorption curves for serum proteins on the SPR sensorgram has demonstrated the strong irreversibility of the protein adsorption on each surface. The effective hydrophilicity of the SAMs was measured from the contact angle with a saline buffer and has demonstrated that surfaces minimizing the contact angle with PBS performed better in serum. The antibody for beta-lactamase was immobilized on a 3-MPA-glycine SAM, and beta-lactamase was detected in the nanomolar range. The presence of beta-lactamase is an indicator of antibiotic resistance.

The metal-induced micelle-to-vesicle phase change that the ferric complex of the microbially produced amphiphile, marinobactin E (M(E)), undergoes has been investigated by X-ray diffraction (XRD) and X-ray absorption spectroscopy (XAS). Marinobactin E is one member of the suite of siderophores, marinobactins A-E, that are used by the source bacterium to facilitate iron acquisition. Fe(III)-M(E) undergoes a micelle-to-multilamellar vesicle transition in the presence of Cd(II) and Zn(II). XRD measurements indicate the interlamellar repeat distance of the Cd(II)- and Zn(II)-induced multilamellar vesicles is approximately 5.3 nm. XAS spectra of the sedimented Cd(II)- and Zn(II)-induced multilamellar vesicles suggests hexadentate coordination of Cd(II) and Zn(II) consisting of two monodentate carboxylate ligands and four water ligands. This coordination environment supports the hypothesis that Cd(II) and Zn(II) bridge the terminal carboxylate moiety of two Fe(III)-M(E) headgroups, pulling the headgroups together in an arrangement that favors vesicle formation over the formation of micelles. XAS spectra of the Fe(III) center in the sedimented Cd(II)- and Zn(II)-induced vesicles confirm the anticipated six-coordinate geometry of Fe(III) by the M(E) headgroup via the two hydroxamate groups and the alpha-hydroxy amide moiety.

A series of nanoscale chemical patterning methods based on soft and hybrid nanolithographies have been characterized using scanning electron microscopy with corroborating evidence from scanning tunneling microscopy and lateral force microscopy. We demonstrate and discuss the unique advantages of the scanning electron microscope as an analytical tool to image chemical patterns of molecules highly diluted within a host self-assembled monolayer and to distinguish regions of differential mass coverage in patterned self-assembled monolayers. We show that the relative contrast of self-assembled monolayer patterns in scanning electron micrographs depends on the operating primary electron beam voltage, monolayer composition, and monolayer order, suggesting that secondary electron emission and scattering can be used to elucidate chemical patterns.

5 pages, 3 figures.-- PMID: 17253792 [PubMed].-- Supporting information (Additional methods and procedures, 63 pages) available at: http://pubs.acs.org/doi/suppl/10.1021/jo061592s/suppl_file/jo061592ssi20061124_060211.pdf, The synthesis of two hexadeuterated palmitic acids differing in the position of the diagnostic labels, and their use to decipher the cryptoregiochemistry of a Δ(13) desaturation are described. A dithiane and a triple bond functionalities were used to introduce the diagnostic (C13 or C14) and tagging (C8 and C9) labels, respectively, in the palmitic acid skeleton. Using these probes, the cryptoregiochemistry of the Δ(13) desaturation involved in the biosynthesis of Thaumetopoea pityocampa sex pheromone was studied by means of kinetic isotope effect determinations. Transformation of both (Z)-11-hexadecenoic and 11-hexadecynoic acids into (Z, Z)-11,13-hexadecadienoic and (Z)-13-hexadecen-11-ynoic acids, respectively, is initiated by abstraction of the hydrogen atom at the C13 position, followed by the fast elimination of the C14 hydrogen to give the double bond., We gratefully acknowledge CICYT and FEDER (Grant AGL2001-0585) and Generalitat de Catalunya (Grant 2005SGR00726) for financial support. J.-L.A. thanks Spanish MEC for a Ramón y Cajal contract. The authors also acknowledge Rodolfo Hernández from Laboratorio de Sanidad Forestal (Gobierno de Aragón Mora de Rubielos, Teruel) for providing T. pityocampa female pupae.

Rings of single-walled carbon nanotubes (SWNTs) were assembled by dip-pen nanolithography (DPN) generated molecular templates consisting of COOH-terminated monolayers in circular patterns surrounded by passivating CH3-terminated SAMs. Experimental data and atomic-level Monte Carlo simulations show that SWNTs assemble into rings with radii as small as 100 nm at the edge of the COOH templates. This directed assembly is strongly length-dependent; only when the length of a SWNT is longer than half of the circumference of the circle does the SWNT bend to precisely follow the interface of the COOH-terminated monolayer. The theoretical modeling shows that the strain energy of each SWNT is balanced by the energy difference between the van der Waals interactions of the tube with COOH and CH3 templates to produce the resulting ring structure.

This report describes the reactivity of acid fluoride (AF)-terminated self-assembled monolayers (SAMs) on gold toward amine and alcohol compounds and the potentiality of AF as a reactive intermediate for surface functionalizations. The AF group was generated in situ on a gold surface by reacting the terminal carboxylic acid group in the SAM of 16-mercaptohexadecanoic acid with cyanuric fluoride and pyridine under the optimized conditions. AF was found to be highly reactive toward various amine groups, such as primary and secondary amines, but it did not react effectively with alcohol. In addition, the amide coupling reaction by microcontact printing (microCP) was compared with the solution-based reaction: when amine-derivatized ferrocene compound was used for 1-min microCP on the AF-activated surface, the surface coverage of the reaction product was about 83% of 3.45 x 1014 cm-2, the coverage obtained in the solution-based reaction. On the basis of the high reaction efficiency of microCP, the AF-activated surface was also used as a platform for patterning a biological ligand, biotin.

Serum and plasma are the major sources of human material for clinical molecular diagnostics and drug discovery. However, due to the high abundance of some proteins, of which serum albumin (SA) is most prominent, lower-abundance proteins often remain undetectable in proteomic analysis of these body fluids. We have used hexadecanedionic acid (HDA) immobilized to Sepharose 4B to develop an affinity resin that is effective in the removal of SA from plasma. Two-dimensional gel analysis of the SA-depleted samples shows a significant enhancement of the low-abundance proteins and highly specific capture of serum albumin. The HDA resin shows better performance in terms of specificity than dye-based resins.

UV-vis absorbance measurements and associated studies of cytochrome P450 BM-3 in complex with N-palmitoylglycine (NPG) indicate that a conformational change occurs in the active site of the complex where the terminal atoms of the ligand move from a site distant from the heme iron, as seen in the low temperature crystal structure to a site proximal to the heme iron at biological temperatures. We employ replica exchange molecular dynamics simulations to study this conformational change. The population of the proximal state is found to increase with temperature in agreement with UV-vis absorbance and NMR measurements. In addition to the conformations characterized by X-ray crystallography and computer modeling, this study shows that a new conformational state is significantly populated at room temperature. The observed increase in the population of conformations where the terminal atoms of NPG are proximal to the heme iron with increasing temperature indicates that the proximal state is stabilized by conformational entropy. A proposal for the origin of this entropic stabilization is provided on the basis of the structure of the newly identified state. We use the temperature weighted histogram (T-WHAM) method to characterize the transition state regions of the conformational ensemble and propose a mechanism of interconversion between these low free energy conformational states.

Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) techniques have been applied to study the self-assembly processes of a microbially produced siderophore, marinobactin E (ME). ME is one of a series of marinobactins A-E that facilitate Fe(III) acquisition by the source bacterium through coordination of Fe(III) by the marinobactin headgroup. ME is a six-amino-acid peptide amphiphile appended by palmitic acid (C16), and differs only in the nature of the fatty acid moiety from the other marinobactins. Apo-ME (uncoordinated ME) assembles to form micelles with an average diameter of 4.0 nm. Upon coordination of one equivalent of Fe(III), the mean micellar diameter of Fe(III)-ME shrinks to approximately 2.8 nm. However, in the presence of excess Fe(III), Fe(III)-ME undergoes a micelle-to-vesicle transition (MVT). At a small excess of Fe(III) over Fe(III)-ME (i.e.

In this paper, we report surface-initiated d(A-T) polymerization by Taq DNA polymerase as a method for constructing DNA-tethered surfaces using an enzyme. The enzymatic polymerization was conducted successfully via two steps: tethering of oligo d(A-T)s onto the surface presenting carboxylic acids by amide coupling and surface-initiated polymerization using Taq DNA polymerase. In this enzymatic polymerization process, the design and construction of carboxylic acid-presenting surfaces were found to be an important factor: DNA growth did not occur on the gold surface coated only with the self-assembled monolayer (SAM) of 16-mercaptohexadecanoic acid (MHDA), but effectively proceeded on the surfaces presenting mixed SAMs of MHDA and 1-pentadecanethiol. The coupling of oligo d(A-T)s and the subsequent DNA polymerization reaction were characterized by polarized infrared external reflectance spectroscopy, ellipsometry, X-ray photoelectron spectroscopy, and atomic force microscopy.

Posttranslational modifications (PTMs) of the beta-2 adrenoceptor (B2AR) play a fundamental role in receptor regulation by agonists. We have examined the effects of several agonists on net levels of B2AR palmitoylation and phosphorylation using epitope tagging in stably transfected human embryonal kidney (HEK) 293 cells, immunoaffinity purification, and mass spectrometry combined with the method of stable isotope labeling by amino acids in cell culture (SILAC). Palmitoylation of Cys341 was confirmed and did not change detectably after 30 min exposure of cells to saturating concentrations of dopamine, epinephrine, or isoproterenol. However, all of these agonists produced a marked increase in net phosphorylation. Phosphorylation of the third cytoplasmic loop was increased to a similar degree by all three agonists, whereas differences between agonists were observed in net phosphorylation of the carboxyl-terminal cytoplasmic domain (isoproterenol approximately epinephrine >> dopamine). Interestingly, agonist-induced phosphorylation of the carboxyl-terminal cytoplasmic domain was observed exclusively in a proximal portion (between residues 339-369). None of the agonists produced detectable phosphorylation in a distal portion of the cytoplasmic tail, which contains all sites of agonist-induced phosphorylation identified previously by in vitro reconstitution. These results provide insight to agonist-dependent regulation of the B2AR in intact cells, suggest the existence of significant differences in regulatory phosphorylation events occurring between in vitro and in vivo conditions, and outline a general analytical approach to investigate regulated PTM of receptors in mammalian cells.

Tear lipocalin (TL), a major component of human tears, shows pH-dependent endogenous ligand binding. The structural and conformational changes associated with ligand release in the pH range of 7.5-3.0 are monitored by circular dichroism spectroscopy and site-directed tryptophan fluorescence. In the transition from pH 7.5 to pH 5.5, the ligand affinity for 16-(9-anthroyloxy)palmitic acid (16AP) and 8-anilino-1-naphthalenesulfonic acid is reduced. At pH 4.0 these ligands no longer bind within the TL calyx. From pH 7.3 to pH 3.0, the residues on loops CD and EF, which overhang the calyx entrance, show reduced accessibility to acrylamide. In addition resonance energy transfer is enhanced between residues on the two loops; the distance between the loops narrows. These findings suggest that apposition of the loops at low pH excludes the ligand from the intracavitary binding site. The conformational changes observed in transition from pH 7.3 to pH 3.0 for loops CD and EF are quite different. The CD loop shows less population reshuffling than the EF loop with an acidic environment, probably because backbone motion is restrained by the adjacent disulfide bond. The Trp fluorescence wavelength maximum (lambda(max)) reflects internal electrostatic interactions for positions on loops CD and EF. The titration curves of lambda(max) for mutants on the EF loop fit the Hendersen-Hasselbalch equation for two apparent pK(a) values, while the CD loop positions fit satisfactorily with one pK(a) value. Midpoints of transition for the binding affinity of TL tryptophan mutants to 16AP occur at pH 5.5-6.1. Replacement of each amino acid on either loop by single tryptophan mutation does not disrupt the pH-dependent binding affinity to 16AP. Taken together the data suggest that pH-driven ligand release involves ionization changes in several titratable residues associated with CD and EF loop apposition and occlusion of the calyx.

Anodic electrogenerated chemiluminescence (ECL) with tri-n-propylamine (TPrA) as a coreactant was used to determine DNA and C-reactive protein (CRP) by immobilizations on Au(111) electrodes using tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)(3)(2+)) labels. A 23-mer synthetic single-stranded (ss) DNA derived from the Bacillus anthracis with an amino-modified group at the 5' end position was covalently attached to the Au(111) substrate precoated with a self-assembled thiol monolayer of 3-mercaptopropanoic acid (3-MPA) in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and then hybridized with a target ssDNA tagged with Ru(bpy)(3)(2+) ECL labels. Similarly, biotinylated anti-CRP species were immobilized effectively onto the Au(111) substrate precovered with a layer of avidin linked covalently via the reaction between avidin and a mixed thiol monolayer of 3-MPA and 16-mercaptohexadecanoic acid on Au(111) in the presence of EDAC and N-hydroxysuccinimide. CRP and anti-CRP tagged with Ru(bpy)(3)(2+) labels were then conjugated to the surface layer. ECL responses were generated from the modified electrodes described above by immersing them in a TPrA-containing electrolyte solution. A series of electrode treatments, including blocking free -COOH groups with ethanol amine, pinhole blocking with bovine serum albumin, washing with EDTA/NaCl/Tris buffer, and spraying with inert gases, were used to reduce the nonspecific adsorption of the labeled species. The ECL peak intensity was linearly proportional to the analyte CRP concentration over the range 1-24 microg/mL. CRP concentrations of two unknown human plasma/serum specimens were measured by the standard addition method based on this technique.

The endogenous fatty acid amide anandamide (AEA) has, as a result of its actions on cannabinoid and vanilloid receptors, a number of important pharmacological properties including effects on nociception, memory processes, spasticity, and cell proliferation. Inhibition of the metabolism of AEA, catalyzed by fatty acid amide hydrolase (FAAH), potentiates the actions of AEA in vivo and therefore may be a useful target for drug development. In the present study, we have investigated whether substitution of the headgroup of the endogenous alternative FAAH substrate palmitoylethanolamide (PEA) can result in the identification of novel compounds preventing AEA metabolism. Thirty-seven derivatives of PEA were synthesized, with the C16 long chain of palmitic acid kept intact, and comprising 20 alkylated, 12 aromatic, and 4 halogenated amides. The ability of the PEA derivatives to inhibit FAAH-catalyzed hydrolysis of [(3)H]AEA was investigated using rat brain homogenates as a source of FAAH. Inhibition curves were analyzed to determine the potency of the inhibitable fraction (pI(50) values) and the maximal attained inhibition for the compound, given that solubility in an aqueous environment is a major issue for these compounds. In the alkylamide family, palmitoylethylamide and palmitoylallylamide were inhibitors of AEA metabolism with pI(50) values of 5.45 and 5.47, respectively. Halogenated derivatives (Cl and Br) exhibit pI(50) values of approximately 5.5 but rather low percentages of maximal inhibition. The -OH group of the ethyl head chain of N-palmitoylethanolamine was not necessary for interaction with FAAH. Amides containing aromatic moieties were less potent inhibitors of AEA metabolism. Compounds containing amide and ester bonds, 13 and 37, showed pI(50) values of 4.99 and 5.08, respectively. None of the compounds showed obvious affinity for CB(1) or CB(2) receptors expressed on Chinese hamster ovary (CHO) cells. It is concluded that although none of the compounds were dramatically more potent than PEA itself at reducing the metabolism of AEA, the lack of effect of the compounds at CB(1) and CB(2) receptors makes them useful templates for development of possible therapeutic FAAH inhibitors.

We describe a new combinatorial method for studying the exchange between solution adsorbates and nanoscale features within libraries generated via dip-pen nanolithography. Four different compounds, 1-octadecanethiol, 16-mercaptohexadecanoic acid, ferrocene (11-mercaptoundecyl), and ferrocene (11-mercapto-1-oxoundecyl), are studied on amorphous and single-crystal gold substrates. This series of adsorbates allows us to compare the exchange properties of patterns of nanoscale features as a function of composition, feature size, and type of underlying substrate. Moreover, these properties can be compared and contrasted with bulk SAM properties. The novel strategy provides not only a method for initiating site-specific exchange processes but also a way of extracting kinetic information about the rate of such processes in situ.

Three different classes of small lipid-binding protein (LBP) are found in helminth parasites. Although of similar size, the ABA-1A1 (also designated As-NPA-A1) and Ov-FAR-1 (formerly known as Ov20) proteins of nematodes are mainly alpha-helical and have no known structural counterparts in mammals, whereas Sj-FABPc of schistosomes is predicted to form a beta-barrel structure similar to the mammalian family of intracellular fatty acid binding proteins. The parasites that produce these proteins are unable to synthesize their own complex lipids and, instead, rely entirely upon their hosts for supply. As a first step in elucidating whether these helminth proteins are involved in the acquisition of host lipid, the process by which these LBPs deliver their ligands to acceptor membranes was examined, by comparing the rates and mechanisms of ligand transfer from the proteins to artificial phospholipid vesicles using a fluorescence resonance energy transfer assay. All three proteins bound the fluorescent fatty acid 2-(9-anthroyloxy)palmitic acid (2AP) similarly, but there were clear differences in the rates and mechanisms of fatty acid transfer. Sj-FABPc displayed a collisional mechanism; 2AP transfer rates increased with acceptor membrane concentration, were modulated by acceptor membrane charge, and were not diminished in the presence of increasing salt concentrations. In contrast, transfer of ligand from Ov-FAR-1 and ABA-1A1 involved an aqueous diffusion step; transfer rates from these proteins were not modulated by acceptor membrane concentration or charge but did decrease with the ionic strength of the buffer. Despite these differences, all of the proteins interacted directly with membranes, as determined using a cytochrome c competition assay, although Sj-FABPc interacted to a greater extent than did Ov-FAR-1 or rABA-1A1. Together, these results suggest that Sj-FABPc is most likely to be involved in the intracellular targeted transport and metabolism of fatty acids, whereas Ov-FAR-1 and ABA-1A1 may behave in a manner analogous to that of extracellular LBPs such as serum albumin and plasma retinol binding protein.

Cytochrome P450s constitute a superfamily of enzymes that catalyze the oxidation of a vast number of structurally and chemically diverse hydrophobic substrates. Herein, we describe the crystal structure of a complex between the bacterial P450BM-3 and the novel substrate N-palmitoylglycine at a resolution of 1.65 A, which reveals previously unrecognizable features of active site reorganization upon substrate binding. N-palmitoylglycine binds with higher affinity than any other known substrate and reacts with a higher turnover number than palmitic acid but with unaltered regiospecificity along the fatty acid moiety. Substrate binding induces conformational changes in distinct regions of the enzyme including part of the I-helix adjacent to the active site. These changes cause the displacement by about 1 A of the pivotal water molecule that ligands the heme iron, resulting in the low-spin to high-spin conversion of the iron. The water molecule is trapped close to the heme group, which allows it to partition between the iron and the new binding site. This partitioning explains the existence of a high-spin-low-spin equilibrium after substrate binding. The close proximity of the water molecule to the heme iron indicates that it may also participate in the proton-transfer cascade that leads to heterolytic bond scission of oxygen in P450BM-3.

The tertiary structure of murine adipocyte fatty acid-binding protein (AFABP) is a flattened 10-stranded beta-barrel capped by a helix-turn-helix segment. This helical domain is hypothesized to behave as a "lid" or portal for ligand entry into and exit from the binding cavity. Previously, we demonstrated that anthroyloxy-labeled fatty acid (AOFA) transfer from AFABP to phospholipid membranes occurs by a collisional process, in which ionic interactions between positively charged lysine residues on the protein surface and negatively charged phospholipid headgroups are involved. In the present study, the role of specific lysine residues located in the portal and other regions of AFABP was directly examined using site-directed mutagenesis. The results showed that isoleucine replacement for lysine in the portal region, including the alphaI- and alphaII-helices and the beta C-D turn, resulted in much slower 2-(9-anthroyloxy)palmitate (2AP) transfer rates to acidic membranes than those of native AFABP. An additive effect was found for mutant K22,59I, displaying the slowest rates of FA transfer. Rates of 2AP transfer from "nonportal" mutants on the beta-G and I strands were affected only moderately; however, a lysine --isoleucine mutation in the nonportal beta-A strand decreased the 2AP transfer rate. These studies suggest that lysines in the helical cap domain are important for governing ionic interactions between AFABP and membranes. Furthermore, it appears that more than one distinct region, including the alphaI-helix, alphaII-helix, beta C-D turn, and the beta-A strand, is involved in these charge-charge interactions.

N-Terminal myristoylation and thiopalmitoylation of the endothelial isoform of nitric oxide synthase (eNOS) are required for targeting the enzyme to specialized signal-transducing microdomains of plasma membrane termed caveolae. We have previously documented that the subcellular localization of eNOS is dynamically regulated by agonists such as bradykinin, which promotes enzyme depalmitoylation and translocation from caveolae. More recently, we have shown that association of eNOS with caveolin, the principal structural protein in caveolae, leads to enzyme inhibition, in a reversible process modulated by Ca2+-calmodulin (CaM). We now report studies of the respective roles of acylation and caveolin interaction for regulating eNOS activity. Using eNOS truncation and deletion mutants expressed in COS-7 cells, we have identified an obligatory role for the N-terminal half of eNOS in stabilizing its association with caveolin. By exploring the differential effects of detergents (CHAPS vs octyl glucoside), we have shown that this direct interaction between both proteins is facilitated by, but does not require, eNOS acylation, and, importantly, that treatment of intact aortic endothelial cells with the calcium ionophore A23187 leads to the rapid disruption of the eNOS-caveolin complexes. Finally, using transiently transfected COS-7 cells, we have observed that the myristoylation-deficient cytosol-restricted eNOS mutant (myr-) as well as the cytosolic fraction of the palmitoylation-deficient eNOS mutant (palm-) may both interact with caveolin; this association also leads to a marked inhibition of enzyme activity, which is completely reversed by addition of calmodulin. We conclude that the regulatory eNOS-caveolin association is independent of the state of eNOS acylation, indicating that agonist-evoked Ca2+/CaM-dependent disruption of the caveolin-eNOS complex, rather than agonist-promoted depalmitoylation of eNOS, relieves caveolin's tonic inhibition of enzyme activity. We therefore propose that caveolin may serve as an eNOS chaperone regulating NO production independently of the enzyme's residence within caveolae or its state of acylation.

Endothelins regulate blood pressure in mammals through G protein-coupled receptors. Two receptor subtypes, ETA and ETB, exist which differ by their agonist profiles. Here we show subtype-specific differences in the inactivation of these endothelin receptors. Using a modified inositol phosphate accumulation assay, we found that stimulation of ETA by endothelin-1 results in sustained activation of the subtype, retaining >30% of its initial activity even 20 min after agonist administration, whereas the ETB rapidly deactivated after agonist stimulation, losing >80% of its initial activity within 5 min after endothelin application. The discrepancy in receptor inactivation is reflected by subtype-specific differences in receptor phosphorylation. Whereas ETA failed to undergo ligand-induced phosphorylation, the ETB was rapidly phosphorylated in response to agonist stimulation. By contrast, the kinetics of ligand-induced internalization were essentially identical for the receptor subtypes, suggesting endothelin receptor internalization being independent of ligand-induced receptor phosphorylation. Interestingly, a strong correlation was observed between the time course of ETA inactivation and ETA internalization. Therefore, our data suggest a subtype-specific inactivation of human endothelin receptors: fast receptor phosphorylation in the case of ETB and slow receptor internalization in the case of ETA. Subtype-specific modulation of endothelin receptors may account for the short-term hypotensive effects of endothelins via rapidly down-regulating ETB receptors and the long-lasting hypertensive effects due to sustained ETA activation.

Bilayers composed of phosphatidylcholine initially resist catalysis by phospholipase A2. However, after a latency period, they become susceptible when sufficient reaction products (lysolecithin and fatty acid) accumulate in the membrane. Temperatures near the main bilayer phase transition and saturated long-chain diacylglycerol in the bilayer modulate the effectiveness of the reaction products. The purpose of this study was to identify possible mechanisms for these effects of temperature and diacylglycerol. Various fluorescent probes were used to asses changes in the ability of the reaction products to perturb the bilayer and promote enzyme binding to he membrane surface. Temperature appeared to cause three effects. First, the degree of binding of enzyme at the end of the latency period was greatest near the phase transition temperature where the latency was shortest. Second, the bilayer was more sensitive to perturbation by reaction products near the transition. Third, the disturbance provoked by the products was confined to the membrane surface below the transition but affected deeper regions at higher temperature where the latency period was greater. The latter two effects of temperature required the presence of calcium. Diacylglycerol promoted lateral segregation of reaction products in the bilayer. This effect corresponded with the tendency of diacylglycerol to reduce the length of the latency period at temperature below the phase transition. Therefore, it appeared that temperature affects the latency period by alternating the binding of the enzyme and the depth and magnitude of the bilayer perturbation caused by reaction products. Alternatively, diacylglycerol may enhance the effectiveness of reaction products by inducing them to segregate in the bilayer and thus create local regions of increased impact on the bilayer surface.

The structure of heart fatty acid binding protein (HFABP) is a flattened beta-barrel comprising 10 antiparallel beta-sheets capped by two alpha-helical segments. The helical cap region is hypothesized to behave as a portal "lid" for the entry and release of ligand from the binding pocket. The transfer of fatty acid from HFABP is thought to occur via effective collisional interactions with membranes, and these interactions are enhanced when transfer is to membranes of net negative charge, thus implying that specific basic residues on the surface of HFABP may govern the transfer process [Wootan, M. G.,Storch, J. (1994) J. Biol. Chem. 269, 10517-10523]. To directly examine the role of charged lysine residues on the HFABP surface in specific interactions with membranes, chemical modification and selective mutagenesis of HFABP were used. All surface lysine residues were neutralized by acetylation of recombinant HFABP with acetic anhydride. In addition, seven mutant HFABPs were generated that resulted in charge alterations in five distinct sites of HFABP. Modification of the protein did not significantly alter the structural or ligand binding properties of HFABP, as assessed by circular dichroism, fluorescence quantum yield, and ligand binding analyses. By using a resonance energy transfer assay, transfer of 2-(9-anthroyloxy)palmitate (2AP) from acetylated HFABP to membranes was significantly slower than transfer from native HFABP. In addition, in distinct contrast to transfer from native protein, the 2AP transfer rate from acetylated HFABP was not increased to acceptor membranes of increased negative charge. Transfer of 2AP from HFABP mutants involving K22, located on alpha-helix I (alpha-I) of the helical cap region, was 3-fold slower than transfer from wild-type protein, whereas rates from a mutant involving the K59 residue, located on the beta 2-turn of the barrel near the helical cap, were 2-fold faster than those of wild type. A double mutant involving K22 and K59 resulted in transfer rates identical to those of wild type, indicating that at least two domains are involved in determining the overall rate of ligand transfer. In addition, 2AP transfer rates from HFABP mutated at position 22 were totally unaffected by the charge characteristics of acceptor membranes, in marked contrast to wild type and other members of the mutant series. Further, by introducing a positive charge to alpha-helix II (alpha-II) of the helical cap region, 2AP transfer rates increased by 4-fold and properties of HFABP transfer began to approach those seen for AFABP, another member of the FABP family thought to transfer ligand via collisional interactions with membranes, which has a lysine residue in the alpha-II helix. These studies demonstrate that the helical cap region of HFABP may play an important role in governing ionic interactions between binding protein and membranes.

It has been proposed that each of the lipids from the Acholeplasma laidlawii membrane prefers to form either a lamellar or a reversed cubic or hexagonal phase when dispersed in excess water at physiologically relevant temperatures and ionic strengths. In this study, we have reinvestigated the thermotropic phase behavior of all the major membrane lipids from A. laidlawii B membranes derived from cells grown in equimolar palmitic and elaidic acids. We confirm that phosphatidylglycerol (PG) and diglucosyldiacylglycerol (DGDG) from such membranes do indeed form only lamellar phases over the temperature range 5-80 degrees C. We also confirm that the monoglucosyldiacylglycerol (MGDG) and acyl polyprenyl glucoside (APG) exist in lamellar phases at lower temperatures but do form reversed phases at higher temperatures. However, we present here optical, differential scanning calorimetric, quasielastic light scattering, and 2H- and 31P-nuclear magnetic resonance spectroscopic evidence indicating that one lipid component of the A. laidlawii B membrane, namely, glycerylphosphoryldiglucosyldiacylglycerol (GPDGDG), actually forms normal micellar rather than lamellar or reversed phases when dispersed in excess water at physiological temperatures. To the best of our knowledge, this is the first demonstration of the existence of a micellar phase-preferring lipid in a prokaryotic cell membrane, and only the second demonstration of the existence of a micellar phase-forming lipid in any biological membrane. We also show that GPDGDG levels change greatly depending on the fatty acid composition of the membrane lipids of this organism.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of temperature and various glycerides to modulate the ability of lysolecithin and fatty acid to promote high phospholipase A2 activity was studied using dipalmitoylphosphatidylcholine large unilamellar vesicles as substrate. The length of the lag phase prior to the accumulation of sufficient hydrolysis products (lysolecithin and fatty acid) to support high phospholipase activity was shortest at temperatures near the thermotropic phase transition of the phospholipid substrate. A reduction in the lag phase correlated with a reduction in the requirement for hydrolysis products at the phase transition temperature, where the bilayer exists in a state of fluctuating domains of gel and liquid crystal. Dipalmitoylglycerol and tripalmitoylglycerol also reduced the length of the lag phase. This reduction was both concentration-dependent and temperature-dependent relative to the phase transition in the presence of the glycerides. As with the effect of temperature, the ability of di- and triglycerides to decrease the lag time correlated with a decrease in the amount of reaction products necessary to promote high phospholipase activity. This effect coincided with the tendency of the glycerides to form domains in the bilayer. Glycerides that did not form domains either had no effect (monopalmitoylglycerol) or increased the length of the lag phase (dicaprylglycerol). These data suggest that the effect of the reaction products to increase phospholipase A2 activity is aided by the presence of fluctuations in lipid domains within the bilayer.

To gain insight into the structural consequences of acylation for membrane proteins, we have covalently attached palmitic acid to the ethanolamine end of gramicidin A (gA), which functions as a well-characterized cation-selective membrane channel. Next, we investigated by NMR methods the effect of acylation on the side chains of Trp9, Leu10, and Trp11, which are expected to be close to the acyl chain, and of Val7, which is expected to be far from the acyl chain. Two-dimensional NMR spectroscopy in a sodium dodecyl sulfate (SDS) environment suggests that one of the beta-hydrogens of Leu10 of gA is severely shielded by a nearby aromatic ring. This shielding disappears upon acylation. Deuterium NMR spectra for labeled samples in hydrated dimyristoylphosphatidylcholine (DMPC) bilayers show that, for the major gA conformation, the (deuterated) side chains of Trp9 and Leu10 are markedly influenced by acylation, whereas the side chains of Val7 and Trp11 are essentially unaffected. The NMR results in both environments suggest that the indole ring of Trp9 is situated near the side chain of Leu10 and moves away upon acylation. We propose that acylation provides a subtle mechanism to modulate protein and lipid interactions and to regulate the stability and function of proteins within membranes.

The activity of phospholipase A2 from snake venom to hydrolyze bilayers of phosphatidylcholines is greatly enhanced by the presence of the hydrolysis products, lysolecithin and fatty acid, in the bilayer. The fluorescence of several probes of membrane structure was used to monitor changes in bilayer physical properties during vesicle hydrolysis. These changes were compared to emission spectra and fluorescence polarization results occurring upon direct addition of lysolecithin and/or fatty acid to the bilayer. The excimer to monomer ratio of 1,3-bis(1-pyrene)propane was insensitive to vesicle hydrolysis, suggesting that changes in the order of the phospholipid chains were not relevant to the effect of the hydrolysis products on phospholipase activity. The fluorescence of 6-propionyl-2-(dimethylamino)-naphthalene (Prodan) suggested that the polarity of the bilayer in the region of the phospholipid head groups increases as the hydrolysis products accumulate in the bilayer. The fluorescence of 6-dodecanoyl-2-(dimethylamino)naphthalene (Laurdan) confirmed that such effects were restricted to the bilayer surface. Furthermore, the lysolecithin appeared to be the product most responsible for these changes. These results suggested that lysolecithin increases the activity of phospholipase A2 during vesicle hydrolysis by disrupting the bilayer surface, making the phospholipid molecules more accessible to the enzyme active site.

The unfolding of bovine beta-lactoglobulin, a small globular protein that unfolds reversibly at low pH in the presence of urea or guanidine hydrochloride, has been studied at pH 6.72 in phosphate buffer at 21 degrees C. The midpoint urea concentration for the loss of CD intensity at 220 nm, loss of CD intensity at 293 nm, quenching of intrinsic fluorescence, shift in the wavelength of the maximum of the intrinsic fluorescent emission, and loss of fluorescence intensity from 1-anilino-8-naphthalenesulfonate (ANS) (and probably the hydrophobic binding site) was close to 4.4 M. Addition of sodium dodecyl sulfate (SDS) at concentrations less than 100 microM to the beta-lactoglobulin solutions increased the midpoint urea concentration for the CD and intrinsic fluorescence parameters to about 5.8 M. Palmitic acid had a similar effect to that shown by SDS in altering the CD intensity at 293 nm, and both SDS and palmitic acid attained a maximum effect in altering the CD at 293 nm at a 1:1 molar ratio to beta-lactoglobulin. It seems likely that the beta-sheet structure of beta-lactoglobulin breaks down simultaneously with the loss of the hydrophobic binding site and exposure of tryptophan-19 to the external environment, supporting the view that the major hydrophobic binding site of beta-lactoglobulin is closely involved with the beta-sheet core of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphatidylethanolamine (PE) was isolated from Bacillus megaterium grown at 20 and 55 degrees C (PE-20 and PE-55). Iso and anteiso methyl-branched, saturated acyl chains are predominant in B. megaterium, and the value of the molar ratio of iso/anteiso acyl chains is more than 20-fold higher in PE-55 than in PE-20. Moreover, about 21 mol% of the acyl chains of PE-20 are monounsaturated. The phase equilibria differ between the two PE preparations: (1) PE-20 is more prone to form reversed nonlamellar phases than PE-55; (2) PE-20 forms both reversed cubic (I2) and reversed hexagonal (H(II)) phases while PE-55 forms only an HII phase; and (3) the lamellar liquid-crystalline (L alpha) phase of PE-20 takes up about 70% more water than the L alpha phase of PE-55. These differences can be explained by the differences in the acyl chain composition. When the growth temperature is raised, PE molecules with a reduced tendency to form nonlamellar phases are probably synthesized by B. megaterium in order to counteract the bilayer destabilizing effect of the temperature. The regulation of the acyl chain composition is not needed in order to regulate the temperature for the transition between gel/crystalline and L alpha phases of the membrane lipids. Acholeplasma laidlawii strain A-EF22 was grown at 37 degrees C on 15-(1,1,1(-2) H3)methylhexadecanoic acid, 14-(1,1,1(-2)H3)methylhexadecanoic acid or 13-(1,1,1(-2)H3)methylhexadecanoic acid, and these acids constituted 84-89 mol% of the acyl chains in the membrane lipids. The molar ratio between the two dominating lipids, monoglucosyldiacylglycerol (MGLcDAG) and diglucosyldiacylglycerol (DGlcDAG), decreased, and the molar fraction of the anionic lipids increased, when the methyl branch was moved from position 15 to position 13. Concomitantly, the order of the methyl branch increased in cells as well as in total lipid extracts. The phase equilibria of total lipid extracts (neutral lipids removed) were studied with 20 wt % of water, and HII and I2 phases were formed above 63-67 degrees C. These results indicate that the regulation of the polar head-group composition compensates for the difference in acyl chain packing introduced into the bilayer by the three branched-chain fatty acids. The regulation of the polar head-group composition of the A. laidlawii lipids cannot regulate the temperature for the transition between gel/crystalline and L alpha phases of the lipids, i.e. the transition to fluid acyl chains.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipocyte lipid-binding protein is a 14.6-kDa polypeptide that is responsible for the intracellular trafficking of fatty acids. Its structure previously has been solved in the apo and holo forms complexed with stearate and oleate. To examine the binding of lipids other than those with a carboxylate headgroup, we have determined the structure of ALBP in complex with a sulfonic acid, hexadecanesulfonic acid, and compared its structure with the natural fatty acid analog, palmitate. Crystallographic refinement led to similar models, both with R-factors of about 20% and a resolution of 1.6 A. results can be compared with earlier studies on C18 fatty acids, both saturated and unsaturated. The previously refined complexes with stearate and oleate in combination with the complexes of palmitate and hexadecanesulfonic acid demonstrate specific positions for water molecules bound in the internal cavity. Many of the water-binding sites are present in both the apo form and the holo forms of the protein. With ligand present, a network of 10 internalized water molecules appear to form a hydrophobic hydration region. In spite of the sp3 geometry of the sulfonic acid derivative, the headgroup occupies the same site as that of the planar carboxylate in natural fatty acids. These results demonstrate that intracellular lipid-binding proteins are capable of binding a wider variety of lipids than previously considered and reveal the importance of interior ordered water molecules in the binding cavity.

In an effort to determine the functionality of palmitoylation in rhodopsin, a number of physicochemical properties of depalmitoylated rhodopsin were monitored. Approximately 70% of the rhodopsin was depalmitoylated in rod outer segments by a mild hydroxylamine treatment that resulted in minimal bleaching of rhodopsin. Subsequent purification by affinity chromatography could be used to remove hydroxylamine-bleached rhodopsin. Parallel physical studies were performed on both purified, detergent-solubilized rhodopsin and rhodopsin in rod outer segments. No effect was seen on the rate of metarhodopsin II formation for depalmitoylated rhodopsin. A small effect was seen in the biphasic behavior of the rate of retinal regeneration. The circular dichroism spectrum of depalmitoylated, purified rhodopsin was virtually identical to that of the native protein. These results suggest that depalmitoylation does not greatly affect the conformational structure of rhodopsin. Circular dichroism at 222 nm was used to monitor the thermal denaturation of depalmitoylated and native rhodopsin. A small but significant decrease in the in rod outer segments. In both cases, the van't Hoff parameters showed an increase in positive enthalpy for denaturation relative to the native state. This is largely counterbalanced by an increase in positive entropy relative to the native states. The circular dichroism of the "denatured" state showed a high alpha-helix content. Depalmitoylated rhodopsin had a lower helix content than native protein in this high-temperature state. The changes in the thermodynamics upon depalmitoylation were attributed to structural changes in the denatured state.

Cytochrome P450BM-3 preferentially oxidized fatty acids with terminal double or triple bonds to the omega-2 hydroxylated fatty acids rather than, respectively, to the epoxide or diacid metabolites. The enzyme is inactivated during catalytic turnover of long, terminally unsaturated fatty acids but not by the analogous medium-length fatty acids. Enzyme inactivation by 17-octadecynoic acid and 16-hydroxy-17-octadecynoic acid is due to alkylation of the prosthetic heme group to given an adduct tentatively identified as N-(2-oxo-3-hydroxy-17-carboxyheptadecyl)protoporphyrin IX by its chromatographic and spectroscopic properties. Catalytic turnover of 17-octadecenoic acid also results in heme modification. Fatty diacid monoethyl thioesters are introduced as a new class of irreversible inhibitors that exploit the omega-2 oxidation specificity of cytochrome P450BM-3. Catalytic oxidation of the monoethyl thioesters of dodecanedioic and hexadecanedioic acids results in enzyme inactivation and formation of the parent diacids as metabolites. Limited tryptic digestion of the enzyme after incubation with the monoethyl thioester of [14C]hexadecanedioic acid shows that the inactivating agent binds covalently to both the heme and flavin domains. This finding, and the observation that glutathione prevents inactivation of the enzyme by the monoethyl thioesters, indicate that a diffusible metabolite, probably the sulfoxide, is responsible for enzyme inactivation. The strong preference for omega-2 allylic or propargylic hydroxylation over terminal pi-bond oxidation is opposite to the usual cytochrome P450 pattern and requires that the enzyme actively suppress terminal pi-bond oxidation. The inference that the enzyme binds and sequesters the terminal carbon in a lipophilic pocket is consistent with the crystal structure of the hemoprotein domain of cytochrome P450BM-3.

Pancreatic islets, when stimulated with D-glucose, secrete insulin by processes requiring glycolytic metabolism and generation of ATP. Hydrolysis of membrane phospholipids also occurs in glucose-stimulated islets, resulting in accumulation of nonesterified arachidonate, which facilitates Ca2+ entry and the rise in beta-cell [Ca2+] that triggers insulin secretion. Glucose-induced hydrolysis of arachidonate from islet phospholipids is mediated in part by an ATP-stimulated, Ca(2+)-independent (ASCI) phospholipase A2 (PLA2) which prefers plasmenyl over diacyl phospholipid substrates. Here we characterize the endogenous plasmalogen content of islet cells and subcellular membranes. Fast atom bombardment mass spectrometric analyses demonstrated that three of the most abundant molecular species of ethanolamine phospholipids in rat pancreatic islets were plasmalogens with sn-2 arachidonate residues and palmitic, oleic, or stearic aldehyde residues, respectively, in the sn-1 position. Purified populations of beta-cells prepared by fluorescence-activated cell sorting were also found to contain these plasmenylethanolamine molecular species in abundance similar to that in intact islets and greater than that in islet alpha-cells. Both islet plasma membranes (PM) and endoplasmic reticulum (ER) also contained these plasmenylethanolamine species, which accounted for 42% (PM) to 64% (ER) of the ethanolamine phospholipid arachidonate content of these membranes, as measured by stable isotope dilution mass spectrometry. Plasmenylethanolamine species were also abundant constituents of human pancreatic islets (accounting for 58% of their ethanolamine phospholipid arachidonate content) and were hydrolyzed more rapidly than diacyl ethanolamine phospholipid by human islet cytosolic ASCI-PLA2. Both secretagogue-induced eicosanoid release and insulin secretion from human islets were attenuated by an ASCI-PLA2 suicide substrate which sterically resembles plasmalogens. These observations are consistent with the hypotheses that islet beta-cell ASCI-PLA2-catalyzed hydrolysis of arachidonate from endogenous plasmenylethanolamine substrates may occur in membrane compartments which participate in regulation of the beta-cell cytosolic [Ca2+] and that this may be an intermediary biochemical event in the induction of insulin secretion.

The wax ester n-dodecyl palmitate is shown to be synthesized by retinal pigment epithelial membranes. The biosynthesis of this ester is phospholipid dependent and occurs via the transfer of a palmitoyl group from the sn-1 position of lecithin to n-dodecanol. When retinal pigment epithelial membranes are used as the source of enzyme, the apparent Michaelis constant for n-dodecanol in this process is 65.8 microM, and the maximal velocity for n-dodecyl palmitate synthesis is 16.2 nmol/(h.mg of protein). The enzymatic activity is membrane associated and shows a maximum velocity between pH8 and pH9. This transesterification process appears to be similar to the lecithin retinol acyl transferase reaction and is a further example of acyl group transfer reactions from the sn-1 position of phospholipids.

Analysis of the primary protein structure of the human serotonin1B (5-HT1B) receptor reveals consensus sites for phosphorylation and a putative site for palmitoylation. To investigate these posttranslational modifications, we have expressed a c-myc epitope-tagged 5-HT1B (m5-HT1B) receptor in Sf9 cells. This strategy enabled receptors to be detected by immunoblot analysis and purified by immunoprecipitation using a monoclonal antibody, 9E10, specific for the c-myc epitope. Agonist radioligand [3H]5-HT binding studies showed that the expressed 5-HT1B and m5-HT1B receptors displayed the characteristic pharmacological profile of the neuronal 5-HT1B receptor. The expressed receptors displayed both high- and low-affinity states for [3H]5-HT, suggesting that the receptors were coupled to endogenous G-proteins. Indeed, agonist binding to the high-affinity receptor state was regulated in the presence of GTP gamma S, Gpp(NH)p, and pertussis toxin. [32P]ADP-ribosylation experiments identified a major approximately 41-kDa ADP-ribosylated protein present in Sf9 membranes that comigrated with partially purified bovine brain Gi alpha/G(o) alpha subunits. Measurements of adenylyl cyclase activity in membranes from cells expressing m5-HT1B receptors showed that serotonergic agonists mediated the inhibition of adenylyl cyclase activity with a rank order of potency comparable to their affinity constants. Immunoblot analysis of membranes prepared from cells expressing m5-HT1B receptors and photoaffinity labeling of the immunoprecipitated material revealed photolabeled species at approximately 95 and at approximately 42 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

We are investigating the mechanism of the activation of phosphocholine cytidylyltransferase by selective lipids. In the previous paper [Cornell, R. B. (1991) Biochemistry (preceding paper in this issue)] we found that the Triton X-100 present in the purified enzyme preparation interfered with activation of the enzyme by neutral lipid activators. The enzyme preparation was also contaminated with micromolar levels of phospholipids and fatty acids. To eliminate these interferences in our analysis, we removed 99.9% of the Triton and 99% of the endogenous lipid by DEAE-Sepharose chromatography. This preparation was inactive but could be reactivated in a lipid-specific manner up to 40-fold when added back to preformed liposomes. Small unilamellar vesicles were more effective than multilamellar vesicles of the same composition. The transferase showed a requirement for membrane acyl chains longer than 12 carbons and was poorly activated by lipids in the gel phase. Using PG-PC liposomes that undergo phase transitions within a useful temperature range (27 and 36 degrees C), we found an anomalous enhancement of enzyme activity around the phase transition temperature. These results support the idea that cytidyltransferase activation depends on intercalation into the membrane bilayer.

We performed scanning microbeam small-angle X-ray diffraction (micro-SAXD) experiments, differential scanning calorimetry (DSC) analysis, and optical microscopic observation of palm mid fraction (PMF) crystals in oil-in-water emulsion droplets. The scanning micro-SAXD experiment was performed by irradiating a synchrotron radiation X-ray microbeam having an area of 5 x 5 microm(2) onto different positions on a 50 microm diameter emulsion droplet after the crystallization of PMF by chilling the emulsion at 5 degrees C. The micro-SAXD patterns were recorded with a two-dimensional (2D) detector, which enabled spatial analysis of polymorphic structures and the orientation of lamella planes of PMF crystals at different positions inside the emulsion droplet. Particular attention was paid to compare the crystallization of PMF in two types of emulsion droplets, hydrophilic polyoxyethylene sorbitan mono-oleate (Tween 80) alone (Tween 80 emulsion) and Tween 80 and hydrophobic sucrose palmitic acid oligoester (P-170) (Tween 80+P-170 emulsion). The DSC study revealed that the PMF crystallization temperature in the Tween 80+P-170 emulsion droplets increased by 3 degrees C compared to that of the Tween 80 emulsion because of the effects of the P-170 additive in promoting PMF crystallization. The micro-SAXD studies revealed the following results. (1) The lamella planes of PMF crystals near the outer edges of the droplet in the Tween 80+P-170 emulsion were mostly parallel to an oil-water interface, whereas the lamella planes of PMF crystals were not always aligned with the oil-water interface in the Tween 80 emulsion droplet. (2) The degree of orientation of the lamellar planes of PMF crystals, which was evaluated from the values of full width at half-maximum of 2D micro-SAXD patterns with respect to azimuthal angle extension, was remarkably higher in the Tween 80+P-170 emulsion than in the Tween 80 emulsion. (3) Polymorphic transformation of PMF from alpha to beta' in the Tween 80+P-170 emulsion was retarded compared to that in the Tween 80 emulsion. These results confirmed that the P-170 additive caused interfacial heterogeneous nucleation through hydrophobic interactions at the oil-water interfaces in the emulsion, which subsequently influenced the arrangements of fat crystals so that the lamellar planes of fat crystals were parallel to the oil-water interface.

The enhancement of the fluorescence quantum yield of 1,8-anilinonaphthalenesulfonic acid (ANS) upon binding to intestinal fatty acid protein (I-FABP) was exploited to devise an assay for free I-FABP. With this assay, we monitored the competition for free I-FABP between ANS and fatty acids and thereby extracted values for the dissociation constants (K(FA)) of fatty acids for I-FABP. We obtained these constants for the I-FABP ligands oleic acid, arachidonic acid, and palmitic acid. In addition, we measured the dependence of K(FA) for oleic acid upon temperature and at two pH values. From these data, we calculate the van't Hoff enthalpy of oleic acid binding. This enthalpy is compared with the enthalpies of binding obtained directly from titration calorimetry. Our experiments with the fluorescence-based assay generate values of K(FA) which disagree with older values obtained from calorimetry and other methods. Our own calorimetric data were analyzed with a view to improving the technique involved in subtraction of a "reference" dilution of the ligand into solution in the absence of the protein. By this maneuver, we obtained "corrected" titrations which could be fitted to values of K(FA) more in agreement with the values we determined via the fluorescence-based assay than wer the older literature values. Our new values for K(FA) also agree substantially with values derived using a complementary assay technique, one measuring the concentration of free fatty acid, that has recently been developed by Richiere et al [Richiere et al. (1995) J. Biol. Chem. 270, 15076-15084]. We compare the values of delta H degrees, delta S degrees, and delta C(p)degrees for fatty acid binding we have obtained in this work with those we found in earlier work with ANS binding to I-FABP [Kirk et al. (1996) Biophys. J. 70, 69-83]. Our interpretation of the origin of the thermodynamic changes for ANS binding in our earlier work is here substantiated and extended to include an evaluation in physical terms of the interaction of I-FABP with fatty acids.