Langmuir films from human placental membranes: preparation, rheology, transfer to alkylated glasses, and sigmoidal kinetics of alkaline phosphatase in the resultant Langmuir-Blodgett film.
In the present study, we studied the activity of human placental alkaline phosphatase (PLAP) constraint in a planar surface in controlled molecular packing conditions. For the first time, Langmuir films (LFs) were prepared by the spreading of purified placental membranes (PPM) on the air-water interface and their stability and rheological properties were studied. LFs exhibited a collapse pressure pi(C) = 48 mN/m, hysteresis during the compression-decompression cycle (C-D), indicating a plastic deformation, and a compressibility modulus (K) compatible with liquid-expanded phases. A phase transition point appeared at pi(T) = 28 mN/m and, following successive C-D, it moved toward lower surface areas and higher K, suggesting the lost of some non-PLAP proteins as components of vesicles that might protrude from the monolayer (confirmed by combining lipid/protein molar ratio analysis, PAGE-SDS and V(max)). LFs were transferred at 35 mN/m to alkylated glasses to obtain Langmuir-Blodgett films (LB(35)) the stability of which was confirmed by AFM. The kinetics of p-nitrophenyl phosphate (pNPP) hydrolysis at 37 degrees C catalyzed by PPM was Michaelian and exhibited the thermostability at 60 degrees C typical of PLAP. In LB(35), PLAP exhibited a sigmoidal kinetics which resembled the behavior of the partially metalated enzyme but might become from a cross-talk between protein and membrane structures.
Clop
Eduardo M
EM
Química Biológica-Biofísica Química, Dpto. Química, Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, Av. Vélez Sarsfield 1611, Córdoba, Argentina.
Perillo
María A
MA
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism*; D000478:Alkylation; D055162:Biocatalysis; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D005260:Female; D005898:Glass / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010920:Placenta / Q000737:chemistry / Q000166:cytology* / Q000201:enzymology*; D011247:Pregnancy; D012212:Rheology; D013499:Surface Properties
D004800:Enzymes, Immobilized; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D000469:Alkaline Phosphatase