Biomedical Engineering Reference
In-Depth Information
very stable; V
i
and ADP dissociate slowly from the catalytic site, and ATPase activity
is regained.
72
Nucleotide binding to Pgp is of relatively low affinity, making it difficult to measure
by classical techniques. Binding of both unmodified nucleotides
74
,
81
and fluorescent
TNP-labeled nucleotides
71
to purified Pgp has been quantitated using fluorescence
spectroscopic approaches.
24
Recently, an electron paramagnetic resonance (EPR)
spectroscopy study also examined binding of a spin-labeled ATP analog.
82
These
studies were consistent in showing a
K
d
value for ATP or ADP binding in the range 0.2
to 0.4 mM. TNP-labeled nucleotides bind with higher affinity (
K
d
of 30 to 40
M),
probably because the nitrophenyl ring engages in additional interactions with hy-
drophobic residues in the nucleotide-binding site.
71
The stoichiometry of ATP binding
is normally 2 (i.e., both catalytic sites are occupied)
82
,
83
In the V
i
-trapped complex,
the second untrapped catalytic site can still bind ATP with the same affinity.
83
10.8. DRUG BINDING TO P-GLYCOPROTEIN
Several different approaches have been used to characterize the binding of drugs and
modulators to Pgp. Photoaffinity labeling by analogs of substrates and modulators,
such as [
3
H]azidopine and [
125
I]iodoarylazidoprazosin, has been used widely to study
the drug-binding properties of Pgp.
84
,
85
Competition experiments with photoactive
substrate analogs have given an indication of binding affinity and demonstrated inter-
actions between substrates and modulators. However, labeling stoichiometry is often
very low, complicating interpretation of the results. In addition, kinetic analysis of
binding, and quantitation of dissociation constants, is not possible.
Direct binding studies using radioactive drugs and modulators have been carried
out using native plasma membrane vesicles containing Pgp.
53
,
86-89
Such an approach
is technically difficult because of the high levels of nonspecific background binding
obtained with hydrophobic drugs, which arises from nonspecific partitioning into the
membrane. Detailed kinetic analysis led to the estimation of
K
d
values for binding,
and rates of association and dissociation could also be quantitated. Complex allosteric
interactions were found between multiple drug-binding sites.
53
Fluorescence quenching approaches have been developed to monitor binding and
obtain quantitative estimates of
K
d
for binding of drugs, and modulators to purified
Pgp.
24
,
90
These techniques can measure equilibrium binding without the need to
separate Pgp-bound drug from free drug. The first approach used Pgp labeled at
the two Walker A motif Cys residues with 2-(4
-maleimidylanilino)naphthalene-6-
sulfonic acid (MIANS).
74
Saturable quenching of MIANS fluorescence was obtained
with nucleotides, drugs, and modulators, and fitting of the data led to an estimate
of the
K
d
value for binding. More recently, saturable quenching of the intrinsic Trp
fluorescence of purified Pgp was observed with nucleotides, drugs, and modulators,
and again led to quantitation of the binding affinity.
81
Values of
K
d
for a large number
of different drugs and modulators range from 37 nM for paclitaxel (a high-affinity
substrate) to 158
M for colchicine (a low-affinity substrate).
24
,
91
Thus, the substrate-
binding affinity of Pgp covers a range of 10.
4
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