Atomic force microscope visualization of lipid bilayer degradation due to action of phospholipase A2 and Humicola lanuginosa lipase.
An important application of liquid cell Atomic Force Microscopy (AFM) is the study of enzyme structure and behaviour in organized molecular media that mimic in-vivo systems. In this study we demonstrate the use of AFM as a tool to study the kinetics of lipolytic enzyme reactions occurring at the surface of a supported lipid bilayer. In particular, the time course of the degradation of lipid bilayers by Phospholipase A(2) (PLA(2)) and Humicola Lanuginosa Lipase (HLL) has been investigated. Contact mode imaging allows visualization of enzyme activity on the substrate with high lateral resolution. Lipid bilayers were prepared by the Langmuir-Blodgett technique and transferred to an AFM liquid cell. Following injection of the enzyme into the liquid cell, a sequence of images was acquired at regular time intervals to allow the identification of substrate structure, preferred sites of enzyme activation, and enzyme reaction rates.
Balashev
Konstantin
K
Sofia University, Department of Physical Chemistry, Lab. of Biophysical Chemistry, Sofia 1164, 1, James Bourchier Ave., Bulgaria. fhkb@chem.uni-sofia.bg
John DiNardo
N
N
Callisen
Thomas H
TH
Svendsen
Allan
A
Bjørnholm
Thomas
T
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000378:metabolism; D003435:Crotalid Venoms / Q000201:enzymology; D004075:Diglycerides / Q000378:metabolism; D005658:Fungi / Q000201:enzymology; D006868:Hydrolysis; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000378:metabolism*; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008066:Lipolysis; D018625:Microscopy, Atomic Force; D008968:Molecular Conformation; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D054467:Phospholipases A2
C031442:Agkistrodon venoms; D003435:Crotalid Venoms; D004075:Diglycerides; D008051:Lipid Bilayers; D015060:1,2-Dipalmitoylphosphatidylcholine; C005071:dipalmitin; D008049:Lipase; D010741:Phospholipases A; D054467:Phospholipases A2