Julieta M. Sánchez

Membrane topology modulates β-galactosidase activity against soluble substrates

Abstract The effect of bio-surfaces of contrasting curvature, on the kinetic parameters of ortho-nitrophenyl-β- d -galactopiranoside hydrolysis catalyzed by E. coli β-galactosidase, was investigated. The self-aggregating state and structure of the amphiphiles (Phosphatidylcholine, Lubrol-PX, Triton X-100, DocNa, SDS and CTAB) were inferred from their c.m.c. values and light-scattering measurements. Low curvature phosphatidylcholine or mixed phosphatidylcholine-detergent vesicles increased V max without affecting K M . High curvature micellar structures containing ionic detergents modulated negatively the enzyme activity (decreased or abolished V max and increased K M ). Neither micelles containing non-ionic detergents nor the amphiphiles in a monomeric form, affected enzyme activity. CTAB at a concentration bellow its c.m.c but incorporated into a bilayer, became an activator ( K M decreased respect to the control). Non-enzymatic interfacial hydrolysis of the substrate was discarded. Enzyme–membrane interaction and membrane elasticity, were evaluated using monomolecular layers at the air–water interface. Beyond particular molecular structures, topology affected the direction of the modulatory effects exerted by these amphiphiles on β-galactosidase activity.

Membrane adsorption or penetration differentially modulates β-galactosidase activity against soluble substrates

We investigated if in complex environments, like those where β-galactosidase activity is usually assayed, the kinetics of hydrolysis against soluble substrates could be modulated through enzyme-surface interactions. Kinetic parameters were determined using ortho-nitrophenyl-β-d-galactopiranoside (ONPG) as substrate, in the absence or presence of multilamellar vesicles (MLVs) of pure phosphatidyl cholines (PCs) or PCs:cholesterol mixtures, by visible spectroscopy. Light scattering was carefully corrected by three different methods obtaining similar results. The spectroscopic behavior of the reaction product in the presence of liposomes was also taken into account in order to avoid overestimating the reaction rate calculated from absorbance data. At low [PC] (<0.0024 mM) KM and Vmax decreased compared with the control in the absence of lipids. At high [PC] (1.2 mM), enzyme interaction with highly packed bilayers of dpPC induced an increment in both kinetic parameters. Both kinetic parameters decreased upon the interaction with low packed bilayers (soybean PC) at very low concentration (24 μM) but at higher concentration (1.2 mM) only an increment in Vmax was observed. The dpPC MLVs samples used were four times bigger than those of PCsoybean (approximately 1 μm mean diameter) as measured by quasi elastic light scattering. The increments in Vmax were due to a modulation of the kinetics of the enzymatic reaction and not to non-enzymatic hydrolysis of ONPG at the vesicle–water interface. Enzyme–membrane interaction was confirmed using monomolecular-layers at the air–water-interface. Interestingly, β-galactosidase showed a higher tendency to be localized at a lipid–water interface compared with the free air–water interface; membrane penetration was favored in lower packed membranes. Differences in surface curvature, and thus in surface molecular packing and hydration, might account for the effects observed as the main modulating factor. Our results suggest that β-galactosidase activity was differentially modulated according to the enzyme possibility to penetrate or just be adsorbed to a dimensionality restricted space.

α-Amylase kinetic parameters modulation by lecithin vesicles: binding versus entrapment

The effect of α-amylase–lipid interaction and entrapment inside lipidic soybean lecithin (PC) vesicles on the kinetic parameters of the enzyme was investigated. Human saliva was used as enzyme source and starch as substrate. Reaction kinetics were followed by measuring the consumption of starch as revealed by the decrease in the absorbance at 600 nm of the I2–starch complex. The presence of PC induced a decrease in A600 at low concentrations; above 0.1 g PC/l light scattering became important and an increase in absorbance was observed as a function of lipid concentration. These effects were taking into account in the interpretation of results. Multilamellar vesicles (MLVs) and giant unilamellar vesicles (GUVs) were prepared by established procedures; enzyme was ‘entrapped’ by the inclusion of saliva, conveniently diluted, in the buffer used for lipid dispersion during the preparation of vesicles. The kinetic parameters KM and Vmax were determined by adjusting saturation curves to the Michaelis–Menten equation by a computer assisted nonlinear regression analysis by the least squares method. The presence of PC in MLVs induced an increment of the value of KM of α-amylase for starch as the substrate both in the condition we designated as ‘entrapped’ and when the enzyme was applied to the already formed liposomes (‘untrapped’). The effects exerted by PCs MLVs on KM and Vmax were statistically significant only at the highest PC/protein mass ratio used (28 g/g). GUVs affected amylase KM only in some of the ‘entrapped’ samples at random suggesting certain aggresiveness of the GUVs preparation method. Vmax decreased significantly only in the ‘entrapped’ samples. The higher effect of MLVs in reducing the affinity of the enzyme for starch compared with that of GUVs is probably due to a higher protein adsorbing capacity of the lower molecular packing, higher surface tension and more curved MLV surface compared with the GUV surface. The binding of the enzyme to the lipidic surface is reversible and can be modulated by the presence of high salt concentrations or with the pH of the media which affect the surface electrostatics of both, the lipidic and the protein surfaces. The conclusions drown from the present experiments represent the average behavior of all the isozymes that represent α-amylase from human saliva and may be useful for understanding α-amylase activity in heterogenous media of physiological and technological importance.

Molecular Packing Tunes the Activity of Kluyveromyces lactis β-Galactosidase Incorporated in Langmuir−Blodgett Films

Functional consequences of constraining beta-Gal in bidimensional space were studied at defined molecular packing densities and constant topology. Langmuir-Blodgett films, LB15 and LB35 composed of dipalmitoyl phosphatidylcholine and K. lactis beta-Gal, were obtained by transferring Langmuir films (L) initially packed at 15 and 35 mN/m, respectively, to alkylated glasses. The beta-Gal-monolayer binding equilibrium, mainly the adsorption rate and affinity, depended on the initial monolayers surface pressure (lower for higher pi i). At pi i = 15 and 35 mN/m, the surface excess (Gamma) followed downward parabolic and power-law tendencies, respectively, as a function of subphase protein concentration. Gamma values in L roughly reflected the protein surface density chemically determined in LBs (0-7.5 ng/mm2 at pi i = 0-35 mN/m and [beta-Gal] subphase = 0-100 microg/mL). The beta-Gal-catalyzed hydrolysis of o-nitrophenyl-galactopyranoside showed a Michaelian kinetics in solution as well as in LB15. KM, KM,LB15, Vmax, and Vmax,LB15 were 5.15 +/- 2.2 and 9.25 +/- 6 mM and 39.63 and 0.0096 +/- 0.0027 micromol/min/mg protein, respectively. The sigmoidal kinetics observed with LB35 was evaluated by Hills model (K0.5 = 9.55 +/- 0.4 mM, Vmax,35 = 0.0021 micromol/min/mg protein, Hill coefficient n = 9) and Savageaus fractal model (fractal constant K f = 9.84 mM; reaction order for the substrate gs = 9.06 and for the enzyme ge = 0.62). Fractal reaction orders would reflect the fractal organization of the environment, demonstrated by AFM images, more than the molecularity of the reaction. Particular dynamics of the protein-lipid structural coupling in each molecular packing condition would have led to the different kinetic responses.

Surface Behavior and Peptide-Lipid Interactions of the Cyclic Neuropeptide Melanin Concentrating Hormone

The kinetics and the thermodynamics of melanin concentrating hormone (MCH) adsorption, penetration, and mixing with membrane components are reported. MCH behaved as a surface active peptide, forming stable monolayers at a lipid-free air-water interface, with an equilibrium spreading pressure, a collapse pressure, and a minimal molecular area of 11 mN/m, 13 mN/m, and 140 A (2), respectively. Additional peptide interfacial stabilization was achieved in the presence of lipids, as evidenced by the expansion observed at pi > pi sp in monolayers containing premixtures of MCH with zwitterionic or charged lipids. The MCH-monolayer association and dissociation rate constants were 9.52 x 10 (-4) microM (-1) min (-1) and 8.83 x 10 (-4) min (-1), respectively. The binding of MCH to the dpPC-water interface had a K d = 930 nM at 10 mN/m. MCH penetration in lipid monolayers occurred even up to pi cutoff = 29-32 mN/m. The interaction stability, binding orientation, and miscibility of MCH in monolayers depended on the lipid type, the MCH molar fraction in the mixture, and the molecular packing of the monolayer. This predicted its heterogeneous distribution between different self-separated membrane domains. Our results demonstrated the ability of MCH to incorporate itself into biomembranes and supports the possibility that MCH affects the activity of mechanosensitive membrane proteins through mechanisms unrelated with binding to specific receptors.

PEG-induced molecular crowding leads to a relaxed conformation, higher thermal stability and lower catalytic efficiency of Escherichia coli β-galactosidase.

Enzymatic activities were historically assayed in dilute solutions where molecular crowding, molecular confinement and their consequences were not taken into account. Here we report how macromolecular crowding tunes catalytic parameters for the tetrameric β-Galactosidase from Escherichia coli, β-Gal. We detected increases in KM (weaker substrate binding) and a nonlinear variation in Vmax, with a minimum at 25% W/P of the crowding agent (polyethyleneglycol molecular mass 6000, PEG(6000)) resulting in a linear decrease in the catalytic efficiency (kcat/KM) within the whole [PEG(6000)] range tested). Presence of crowding agent affected β-Gal structural content and increased its thermal resistance. Steady state fluorescence and Fourier transformed infrared spectroscopic observations are compatible with crowding-induced disordering and restricted internal dynamics as a result of excluded volume and solvent structuring effects. This leads to a non-optimal substrate-binding site and a less conformationally strained protein.

β-Galactosidase at the membrane–water interface: A case of an active enzyme with non-native conformation

Previously we demonstrated that Escherichia coli beta-galactosidase (β-Gal) binds to zwitterionic lipid membranes improving its catalytic activity. To understand the activation mechanism from the protein perspective, here the thermal dependence of the catalytic activity was evaluated in conjunction with parameters derived from spectroscopy and calorimetry, in the presence and absence of egg-yolk phosphatidylcholine vesicles. In solution, the native state of β-Gal exhibits a loose conformation according to the λmax of fluorescence emission, which is in the upper end of the emission range for most proteins. A non-two state thermal unfolding mechanism was derived from DSC experiments and supported by the sequential unfolding temperatures exhibited by fluorescence (55°C) and CD (60°C) spectroscopies. Quenching of β-Gals intrinsic fluorescence, provided evidence for a novel and even looser folding for the lipid-bound protein. However, DSC data showed that the thermal unfolding in the presence of lipids occurred with a significant decrease in ΔH compared to what happened in solution, suggesting that only the population of non-bound protein molecules were involved in this process. Concluding, upon binding to a lipid-water interface β-Gal becomes trapped in a partially unfolded state, more active than that of the native protein in solution.

StarD7 behaves as a fusogenic protein in model and cell membrane bilayers.

StarD7 is a surface active protein, structurally related with the START lipid transport family. So, the present work was aimed at elucidating a potential mechanism of action for StarD7 that could be related to its interaction with a lipid-membrane interface. We applied an assay based on the fluorescence de-quenching of BD-HPC-labeled DMPC-DMPS 4:1 mol/mol SUVs (donor liposomes) induced by the dilution with non-labeled DMPC-DMPS 4:1 mol/mol LUVs (acceptor liposomes). Recombinant StarD7 accelerated the dilution of BD-HPC in a concentration-dependent manner. This result could have been explained by either a bilayer fusion or monomeric transport of the labeled lipid between donor and acceptor liposomes. Further experiments (fluorescence energy transfer between DPH-HPC/BD-HPC, liposome size distribution analysis by dynamic light scattering, and the multinuclear giant cell formation induced by recombinant StarD7) strongly indicated that bilayer fusion was the mechanism responsible for the StarD7-induced lipid dilution. The efficiency of lipid dilution was dependent on StarD7 electrostatic interactions with the lipid-water interface, as shown by the pH- and salt-induced modulation. Moreover, this process was favored by phosphatidylethanolamine which is known to stabilize non-lamellar phases considered as intermediary in the fusion process. Altogether these findings allow postulate StarD7 as a fusogenic protein.

Superactive β-Galactosidase inclusion bodies

Bacterial inclusion bodies (IBs) were historically considered one of the major obstacles in protein production through recombinant DNA techniques and conceived as amorphous deposits formed by passive and rather unspecific structures of unfolded proteins aggregates. Subsequent studies demonstrated that IBs contained an important quantity of active protein. In this work, we proved that recombinant β-galactosidase inclusion bodies (IBβ-Gal) are functional aggregates. Moreover, they exhibit particular features distinct to the soluble version of the enzyme. The particulate enzyme was highly active against lactose in physiological and in acid pH and also retained its activity upon a pre-incubation at high temperature. IBβ-Gal washing or dilution induced the spontaneous release of active enzymes from the supramolecular aggregates. Along this process, we observed a continuous change in the values of several kinetic parameters, including specific activity and Michaelis-Menten constant, measured in the IBβ-Gal suspensions. Simultaneously, IBβ-Gal turned into a more heterogeneous population where smaller particles appeared. The released protein exhibited secondary structure features more similar to those of the soluble species than to the aggregated enzyme. Concluding, IBβ-Gal represents a reservoir and packed source of highly active and stable enzyme.