Effect of Molecular Surface Packing on the Enzymatic Activity Modulation of an Anchored Protein on Phospholipid Langmuir Monolayers

Abstract

The catalytic activity of a glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase has been studied in Langmuir phospholipid monolayers at different surface pressures. The enzyme substrate, p-nitrophenyl phosphate, was injected into the subphase of mixed enzyme/lipid Langmuir monolayers. Its hydrolysis product was followed by monitoring the absorbance at 410 nm in situ in the monolayer subphase of the Langmuir trough. Several surface pressures, corresponding to different molecular surface densities, were attained by lateral compression of the monolayers. The morphology of the monolayers, observed by fluorescence microscopy, showed three different types of domains owing to the heterogeneous partition of the enzyme within the mixed enzyme/lipid film. The catalytic activity was modulated by the enzyme surface density, and it increased until a pressure of 18 mN/m was reached, but it decreased significantly when the equilibrium in-plane elasticity (surface compressional modulus) increased more noticeably, resulting in alterations in the interface morphology. A model for the modulation of the enzyme orientation and catalytic activity by lipid/enzyme surface morphology and enzyme surface packing at the air/liquid interface is proposed. The results might have an important impact on the comprehension of the enzymatic activity regulation of GPI-anchored proteins in biomembranes.

Langmuir Films from Human Placental Membranes: Preparation, Rheology, Transfer to Alkylated Glasses, and Sigmoidal Kinetics of Alkaline Phosphatase in the Resultant Langmuir-Blodgett Film

“…This contact would allow the transmission of information between the protein and the interface that could lead to structural changes which in turn may affect its catalytic activity. This is supported by results obtained with the alkaline phosphatase purified from rat osseous plate which incorporated in a self assembled lipid monolayer showed a molecular packing-dependent activity [19].…”

Adsorption kinetics and dilatational rheological studies for the soluble and anchored forms of alkaline phosphatase at the air/water interface

“…On the other hand, considering the absence of the hydrophobic anchor in PLSAP and, therefore, its high hydrophilic character, it is very likely that, during its air/ water interface adsorption, a certain denaturation takes 5,2005 place at the interface due to the exposition of the hydrophobic groups. [27][28][29][30][31] Figure 3B shows the kinetics for DSAP adsorption in short times (less than 30s).…”

“…It has been recently demonstrated in our laboratory that there is a correlation between the equilibrium surface compressibility of a planar mixed lipid/enzyme monolayer and the catalytic activity of a glicosylphosphatidil-inositol (GPI) anchored enzyme, 5 with the enzyme reaction J. Braz. Chem.…”

Protein-mediated surface structuring in biomembranes

“…This was specifically shown for the cross-talk between the PLA 2 -and sphingomyelinase-driven reactions whereby they can become mutually amplified or dampened by fluctuations of the lateral surface pressure controlling the lateral lipid packing and dipolar organization (21). Recently, it was shown that the lateral packing and inplane elasticity of the lipid-protein interface can also regulate the activity of the GPI-anchored protein alkaline phosphatase and modulate information exchange between the interface and the aqueous environment (50).…”