Lipid-protein interactions in monolayers (original) (raw)
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Lysozyme Influence on Monolayers of Individual and Mixed Lipids
Colloids and Interfaces, 2022
Fatty acids, cholesterol, and phospholipids are amphiphilic compounds of biological interest, which form ordered monolayers mimicking biomembranes, and can be studied with the Langmuir technique using surface pressure-area isotherms and compressibility plots. Proteins are also components of biomembranes or are present in body fluids. In this study, the influence of lysozyme on different films of a fatty acid (stearic acid or oleic acid), cholesterol, a phospholipid (dipalmitoylphosphatidylcholine, DPPC, or palmitoyloleoylphosphatidylcholine, POPC), and mixtures of them is presented using a 0.9% saline solution as subphase. Results show that the presence of lysozyme alters the lipid monolayer formation in an important way at the beginning (low surface pressures) and the middle (intermediate surface pressures) parts of the isotherm. At high surface pressures, the phospholipids DPPC and POPC and the saturated fatty acid, stearic acid, expel lysozyme from the surface, while oleic acid a...
Enzymatic Modification of a Chemisorbed Lipid Monolayer †
Langmuir, 1996
The selectivity and specificity of enzymes may be exploited to create chemically complex surfaces which are difficult or impossible to achieve using classical synthetic chemistry. In this paper we discuss the preparation of a chemisorbed lipid film on a silicon wafer and explore the activity of free phospholipase C (PLC) on that film. A carboxylic acid derivative of the lipid dimyristoylphosphatidylcholine (DMPC) was attached to an amino-terminal silane (EDA) via amide bond formation to create an immobilized lipid layer (EDA-DMPC). Films were characterized using X-ray photoelectron spectroscopy (XPS), secondary-ion mass spectrometry (SIMS), atomic force microscopy (AFM), X-ray reflectivity, and ellipsometry. Following treatment with the enzyme phospholipase C (PLC), which catalyzes the cleavage of the lipid headgroup at the glycerol-phosphate ester bond, the lipid film was reanalyzed using the above techniques. Before analysis, nonspecifically adsorbed PLC was removed with a 25% trifluoroethanol rinse. XPS and SIMS results of the cleaned films show nearly complete removal of the phosphate from the lipid layer, indicating enzymatic activity of the PLC on the chemisorbed lipid layer.
Monolayers of Carbohydrate-Containing Lipids at the Water- Air Interface
Cell Culture
Glycolipids are important members of the glycoconjugate family that are distributed on cell surfaces and are important in aspects of cellular behavior including signal transduction, protein trafficking, cell surface recognition and cell adhesion. Errors in the synthesis or mutations of these glycoconjugates are often linked with various human pathological conditions. The complex nature of their molecular structures coupled with the complexity of cellular structure make their study a challenging process, which can be simplified by fabrication of model membrane systems. Liposomes and monolayers of lipids at the airwater interface are two of the most frequently used model membrane systems. Techniques for fabrication of monolayer models and methods used for their studies are discussed with a focus on glycolipids.
Journal of Colloid and Interface Science, 1999
The penetrant ability of the native glucose oxidase, GOx, and of the hydrophobically modified enzyme GO(mod) realized by grafting to its lysine residues alkyl C16 chains, into phosphatidylcholine dibehenoyl (DBPC), phosphatidylcholine dipalmitoyl (DPPC), phosphatidyl-ethanolamine dipalmitoyl (DPPE), phosphatidyl-serine dipalmitoyl (DPPS), and cholesterol (CHOL) monolayers was assessed by surface pressure measurements at constant area by enzyme injection to the aqueous phase beneath spread monolayers. As revealed by the magnitude of surface pressure increments (DeltaPi), both the quantities and the rates of penetration of the enzymes into these monolayers were lipid chemical nature and enzyme concentration dependent. When compared with GOx, GO(mod) displayed an enhanced penetrant ability into all the studied monolayers that resulted in rapidly attained DeltaPi plateau values, characteristic of stable systems. The influence of lipid hydrocarbon chain length and of the polar headgroup charge on the efficiency and effectiveness of GOx and GO(mod) penetration into these monolayers is discussed. Copyright 1999 Academic Press.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2015
Surface pressure Isochoric method Bothrops diporus sPLA 2 Bothrops asper myotoxin We present an analysis of lipid monolayer hydrolysis at a constant area to assess the optimal lateral surface pressure value (Π opt) and thus, the surface packing density of the lipid, at which the activity of a given lipolytic enzyme is maximal. This isochoric method consists of a measurement of the decrease down to zero of the Π opt of phospholipid substrate monolayer due to continuous hydrolysis using only one reaction compartment. We performed the comparison of both approaches using several commercially available and literature-evaluated sPLA 2 s. Also, we characterized for the first time the profile of hydrolysis of DLPC monolayers catalyzed by a sPLA 2 from Streptomyces violaceoruber and isoenzymes purified from Bothrops diporus venom. One of these viper venom enzymes is a new isoenzyme, partially sequenced by a mass spectrometry approach. We also included the basic myotoxin sPLA 2-III from Bothrops asper. Results obtained with the isochoric method and the standard isobaric one produced quite similar values of Π opt , validating the proposal. In addition, we propose a new classification parameter, a lipolytic ratio of hydrolysis at two lateral pressures, 20 mN•m −1 and 10 mN•m −1 , termed here as LR 20/10 index. This index differentiates quite well "high surface pressure" from "low surface pressure" sPLA 2 s and, by extension; it can be used as a functional criterion for the quality of a certain enzyme. Also, this index could be added to the grouping systematic criteria for the superfamily proposed for phospholipase A 2 .
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2013
Atomic force microscopy (AFM) is employed to reveal the morphological changes of the supported phospholipid bilayers hydrolyzed by a phospholipase A 2 (PLA 2 ) enzyme in a buffer solution at room temperature. Based on the high catalytic selectivity of PLA 2 toward L-enantiomer phospholipids, five kinds of supported bilayers made of L-and D-dipalmitoylphosphatidylcholines (DPPC), including L-DPPC (upper leaflet adjacent to solution)/L-DPPC (bottom leaflet) (or L/L in short), L/D, D/L, D/D, and racemic LD/LD, were prepared on a mica surface in gel-phase, to explicate the kinetics and mechanism of the enzyme-induced hydrolysis reaction in detail. AFM observations for the L/L bilayer show that the hydrolysis rate for L-DPPC is significantly increased by PLA 2 and most of the hydrolysis products desorb from substrate surface in 40 min. As D-enantiomers are included in the bilayer, the hydrolysis rate is largely decreased in comparison with the L/L bilayer. The time used to hydrolyze the as-prepared bilayers by PLA 2 increases in the sequence of L/L, L/D, LD/LD, and D/L (D/D is inert to the enzyme action). D-enantiomers in the enantiomer hybrid bilayers remain on the mica surface at the end of the hydrolysis reaction. It was confirmed that the hydrolysis reaction catalyzed by PLA 2 preferentially occurs at the edges of pits or defects on the bilayer surface. The bilayer structures are preserved during the hydrolysis process. Based on these observations, a novel kinetics model is proposed to quantitatively account for the PLA 2 -catalyzed hydrolysis of the supported phospholipid bilayers. The model simulation demonstrates that PLA 2 mainly binds with lipids at the perimeter of defects in the upper leaflet and leads to a hydrolysis reaction, yielding species soluble to the solution phase. The lipid molecules underneath subsequently flip up to the upper leaflet to maintain the hydrophilicity of the bilayer structure. Our analysis shows that D-enantiomers in the hybrid bilayers considerably reduce the hydrolysis rate by its ineffective binding with PLA 2 .
Organization, structure and activity of proteins in monolayers
Colloids and Surfaces B: Biointerfaces, 2007
Many different processes take place at the cell membrane interface. Indeed, for instance, ligands bind membrane proteins which in turn activate peripheral membrane proteins, some of which are enzymes whose action is also located at the membrane interface. Native cell membranes are difficult to use to gain information on the activity of individual proteins at the membrane interface because of the large number of different proteins involved in membranous processes. Model membrane systems, such as monolayers at the air-water interface, have thus been extensively used during the last 50 years to reconstitute proteins and to gain information on their organization, structure and activity in membranes. In the present paper, we review the recent work we have performed with membrane and peripheral proteins as well as enzymes in monolayers at the air-water interface. We show that the structure and orientation of gramicidin has been determined by combining different methods. Furthermore, we demonstrate that the secondary structure of rhodopsin and bacteriorhodopsin is indistinguishable from that in native membranes when appropriate conditions are used. We also show that the kinetics and extent of monolayer binding of myristoylated recoverin is much faster than that of the nonmyristoylated form and that this binding is highly favored by the presence polyunsaturated phospholipids. Moreover, we show that the use of fragments of RPE65 allow determine which region of this protein is most likely involved in membrane binding. Monomolecular films were also used to further understand the hydrolysis of organized phospholipids by phospholipases A2 and C.