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

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Abstract

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 πC = 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 πT = 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 (LB35) the stability of which was confirmed by AFM. The kinetics of p-nitrophenyl phosphate (pNPP) hydrolysis at 37°C catalyzed by PPM was Michaelian and exhibited the thermostability at 60°C typical of PLAP. In LB35, 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.

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Abbreviations

A1, A2, and A3 :

Monolayer aspirated at 35 mN/m during the 1st, 2nd, and 3rd C–D cycles, respectively

AFM:

Atomic force microscopy

AP:

Alkaline phosphatase

C–D:

Compression–decompression cycle

% CV:

Percent variation coefficient

EDTA:

Ethylendiamintetraacetic acid

GPI:

Glycophostatidyl-inositol

K M :

Michaelis–Menten constant

LB35 :

Langmuir-Blodgett film packed at 35 mN/m

LF:

Langmuir film

2-ME:

2-Mercaptoethanol

M r :

Relative mobility

MWM:

Molecular weight markers

PAGE:

Polyacrylamide gel electrophoresis

PLAP:

Placental alkaline phosphatase

pNPP:

para-nitrophenylphosphate

pNPx:

para-nitro phenoxide

PPM:

Purified placental membrane

SDS:

Sodium dodecyl-sulfate

s.e.m.:

Standard error of the mean

V max :

Maximal velocity

V 0 :

Initial velocity

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Acknowledgments

Authors gratefully acknowledge the Dr M.J.Sartori for helping in placentas obtaining, dissection and fractionation. This work was supported by grants from CONICET, SeCyT-UNC, Agencia Córdoba Ciencia and Foncyt. EMC is a graduate students from the Doctorado en Ciencias Biológicas, FCEFyN, UNC and a fellowships holder from CONICET. MAP is a career member of the later institution.

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  1. Química Biológica-Biofísica Química, Dpto. Química, Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, Av. Vélez Sarsfield 1611, X 5016GCA, Córdoba, Argentina

    Eduardo M. Clop & María A. Perillo

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Clop, E.M., Perillo, M.A. Langmuir Films from Human Placental Membranes: Preparation, Rheology, Transfer to Alkylated Glasses, and Sigmoidal Kinetics of Alkaline Phosphatase in the Resultant Langmuir-Blodgett Film. Cell Biochem Biophys 56, 91–107 (2010). https://doi.org/10.1007/s12013-009-9073-4

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