William E. Momsen

Physical and photophysical characterization of a BODIPY phosphatidylcholine as a membrane probe.

Lipids containing the dimethyl BODIPY fluorophore are used in cell biology because their fluorescence properties change with fluorophore concentration (C.-S. Chen, O. C. Martin, and R. E. Pagano. 1997. Biophys J. 72:37-50). The miscibility and steady-state fluorescence behavior of one such lipid, 1-palmitoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphocholine (PBPC), have been characterized in mixtures with 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC). PBPC packs similarly to phosphatidylcholines having a cis-unsaturated acyl chain and mixes nearly ideally with SOPC, apparently without fluorophore-fluorophore aggregation. Increasing PBPC mole fraction from 0.0 to 1.0 in SOPC membranes changes the emission characteristics of the probe in a continuous manner. Analysis of these changes shows that emission from the excited dimethyl BODIPY monomer self quenches with a critical radius of 25.9 A. Fluorophores sufficiently close (< or =13.7 A) at the time of excitation can form an excited dimer, emission from which depends strongly on total lipid packing density. Overall, the data show that PBPC is a reasonable physical substitute for other phosphatidylcholines in fluid membranes. Knowledge of PBPC fluorescence in lipid monolayers has been exploited to determine the two-dimensional concentration of SOPC in unilamellar, bilayer membranes.

Purification and characterization of cholesterol esterase from porcine pancreas

A protein catalyzing the hydrolysis of cholesterol esters and p-nitrophenyl acetate has been purified 200-fold from porcine pancreas. The enzyme is homogenous as judged by polyacrylamide gel electrophoresis and exhibits a molecular weight of 80 000 as determined by sodium dodecyl sulfate electrophoresis and gel filtration. Activity toward p-nitrophenylacetate exhibits a broad pH optimum and is influenced by a group with a pKa of 5.5--6.0. The enzyme is completely inhibited by diisopropylfluorophosphate at concentrations as low as 10(-5) M, suggesting that it is a serine esterase. Partial inhibition was observed with p-chloromercuribenzoate.

Lipid-lipid complexes: Properties and effects on lipase binding to surfaces

In the presence of bulk water, the lipase-catalyzed synthesis and hydrolysis of insoluble lipid esters occur at the lipid-water interface. For water-soluble lipases, a necessary step in this process is the partitioning of enzyme from the bulk aqueous phase to the surface phase. In surface phases of phospholipids and the substrates and products of lipolysis, physical studies have demonstrated the formation of preferred packing arrays or lipid-lipid “complexes.” Such interactions involve changes in both lipid molecular area and hydration. Binding of pancreatic carboxylester lipase (cholesterol esterase) and colipase to monomolecular films of a phosphatidylcholine and its complexes with fatty acid or diglyceride is negligible. In contrast, saturation of film of pure fatty acid or diglyceride correlates with formation of a protein monolayer. With mixtures of complex and uncomplexed fatty acid or diglyceride, binding to the uncomplexed lipid occurs, but only with colipase can saturation of available sites be achieved. The lower affinity of carboxylester lipase for surfaces containing complexes can be qualitatively explained by differences in the size of lipid and protein molecules. Because it involves no direct interaction between enzyme and complex, such “proxinhibition” of enzyme binding is potentially an important regulation of lipid-protein interactions.

Recovery of monomolecular films in studies of lipolysis.

Publisher Summary The collection of lipid monolayers provides a simple, but effective, method for the determination of reactant distribution or adsorbed lipase in monomolecular films. Monolayer collection has been applied in two variations. One is based on aspiration of the surface phase 9–12 and the second on the adsorption of the surface phase to a hydrophobic support. Although conceptually identical, there are practical differences in the data obtainable with each of these techniques. The first is sensitivity for determining net lipase adsorption. Sensitivity of the measurements is limited by the volume of the aqueous subphase that is adventitiously collected with the monolayer. When lipase adsorption is relatively low and subphase enzyme concentration is relatively high, the correction for enzyme carried over in the aqueous subphase can greatly exceed the quantity of adsorbed lipase, especially with the aspiration approach.

The suitability of nichrome for measurement of gas-liquid interfacial tension by the wilhelmy method

Abstract Determination of surface tension by the Wilhelmy method is facilitated by having a zero contact angle between the Wilhelmy plate and the liquid phase. Compared to filter paper or platinum, nichrome was shown to exhibit minimal hysteresis between advancing and receding contact angles under several experimental conditions. Calibration with a series of solvents of known surface tension indicates that the receding contact angle is zero. Comparison of equilibrium spreading pressures for several lipids measured by the Wilhelmy and Langmuir methods showed proportionality with a slope near 1.0. Thus, nichrome is superior to other commonly used materials for measurements of surface tension at the gas-liquid interface.

Regulation of lipases by lipid-lipid interactions: implications for lipid-mediated signaling in cells.

Lipases are extracellular peripheral proteins that act at the surface of lipid emulsions stabilized, typically, by phospholipids. At a critical composition lipase activity toward substrates in phospholipid monolayers is discontinuously switched on by a small increase in substrate mole fraction. This occurs in part because lipase binding is inhibited by phospholipids. Binding of the lipase cofactor, colipase, is also inhibited by phospholipids. The initial rate of colipase binding increases abruptly at a substrate mole fraction that is approximately half the critical composition for lipase activity and just above that in substrate-phospholipid complexes. Moreover, complex collapse areas show an approximately 1:1 correlation with phospholipid excluded areas determined from an analysis of colipase adsorption rates. Thus, complexes inhibit colipase binding rate. Additionally, the switching of lipase activity likely occurs when uncomplexed substrate becomes the majority species in the interface. Lipase substrates, e.g. diacylglycerols, are typically the same lipids generated in the cytoplasmic surface of the plasma membrane of stimulated cells. As colipase binding is nonspecific and complexes involving lipase substrates form on the basis of lipid-lipid interactions alone, complexes should form in the plasma membrane of stimulated cells and may regulate protein translocation to the membrane.

GLTP-fold interaction with planar phosphatidylcholine surfaces is synergistically stimulated by phosphatidic acid and phosphatidylethanolamine

Among amphitropic proteins, human glycolipid transfer protein (GLTP) forms a structurally-unique fold that translocates on/off membranes to specifically transfer glycolipids. Phosphatidylcholine (PC) bilayers with curvature-induced packing stress stimulate much faster glycolipid intervesicular transfer than nonstressed PC bilayers raising questions about planar cytosol-facing biomembranes being viable sites for GLTP interaction. Herein, GLTP-mediated desorption kinetics of fluorescent glycolipid (tetramethyl-boron dipyrromethene (BODIPY)-label) from lipid monolayers are assessed using a novel microfluidics-based surface balance that monitors lipid lateral packing while simultaneously acquiring surface fluorescence data. At biomembrane-like packing (30–35 mN/m), GLTP uptake of BODIPY-glycolipid from POPC monolayers was nearly nonexistent but could be induced by reducing surface pressure to mirror packing in curvature-stressed bilayers. In contrast, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) matrices supported robust BODIPY-glycolipid uptake by GLTP at both high and low surface pressures. Unexpectedly, negatively-charged cytosol-facing lipids, i.e., phosphatidic acid and phosphatidylserine, also supported BODIPY-glycolipid uptake by GLTP at high surface pressure. Remarkably, including both 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (5 mol%) and POPE (15 mol%) in POPC synergistically activated GLTP at high surface pressure. Our study shows that matrix lipid headgroup composition, rather than molecular packing per se, is a key regulator of GLTP-fold function while demonstrating the novel capabilities of the microfluidics-based film balance for investigating protein-membrane interfacial interactions.