Journal of Lipid Research, Vol 28, 1434-1443, Copyright © 1987 by Lipid Research, Inc.
Nonspecific high affinity binding of bile salts to carboxylester lipases [published erratum appears in J Lipid Res 1988 Mar;29(3):396]
T Tsujita, NK Mizuno and HL Brockman
Hormel Institute, University of Minnesota, Austin 55912.
The interactions with bile salts of carboxylester lipases (EC 3.1.1.13)
from human pancreatic juice and pig pancreas were characterized by physical
methods. Bile salts cause a decrease in the fluorescence intensity of the
proteins at the emission maximum of 333-335 nm. The concentration
dependence of this decrease shows saturation behavior, is relatively
nonspecific with respect to bile salt conjugation or the presence of the 7
alpha-hydroxyl group, and is consistent with a 1:1 interaction between
enzyme and bile salt. Direct measurement of the binding of [3H]cholate by
equilibrium dialysis supports the stoichiometry. Other detergents also
bind, causing fluorescence changes, but with much lower affinities. Binding
of taurocholate to the monomeric pig enzyme is enhanced by increasing ionic
strength, indicating the predominance of hydrophobic interactions. In the
range of pH 5.5-6.8, binding is pH-independent with dissociation constants
of 3-20 microM. At higher pH, affinity is greatly reduced and the
fluorescence spectrum changes, indicating the importance of a protonated
group for efficient interaction. Occupancy of the bile salt binding site
partially stabilizes the enzyme against inactivation by heat but not
trypsin. However, circular dichroism spectra do not indicate that bile salt
binding is accompanied by any change in secondary structure. The monomeric
pig enzyme binds to the argon/water interface in the presence of bile salts
and binding of taurocholate to diisopropylphosphoryl-enzyme is similar to
that measured with native enzyme. These results suggest that surface
binding and catalysis occur at sites distinct from the bile salt binding
site of the enzyme. Stabilization of the monomeric pig enzyme against
denaturation at high energy surfaces occurs concomitantly with occupancy of
the bile salt binding site. Overall, the data suggest that an important
role of bile salts in vivo is to stabilize these enzymes at lipid-water
interfaces.
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