Biomedical Engineering Reference
In-Depth Information
a plateau value which represents a steady state. This steady state is a result of two
competing processes: active transport of drug by Pgp into the vesicle lumen (up a con-
centration gradient) and passive diffusion of the hydrophobic drug out of the vesicle
(down a concentration gradient). Addition of excess unlabeled drug to the vesicle
exterior once the steady state has been reached results in very rapid exchange with
labeled drug in the vesicle interior.
54
Ruetz and Gros expressed all three mouse Pgps
in the yeast mutant strain sec 6-4, which accumulates large numbers of secretory
vesicles because of a trafficking defect.
55
These vesicles contained sufficient Pgp for
characterization of the drug transport process using a rapid filtration approach.
Caution should be taken when using the fixed time-point rapid filtration approach
since transport can become nonlinear within 1 minute, making estimation of the initial
rates of Pgp-mediated transport difficult. In these situations, maximal uptake of drug
is measured instead, however, steady-state uptake values cannot be treated as kinetic
data and do not allow, for example, determination of
K
m
or
V
max
for the drug transport
process. In addition, this approach consumes relatively large amounts of membrane
vesicles and radiolabeled drug. Fluorescence approaches have been developed that cir-
cumvent these problems and allow continuous real-time monitoring of Pgp-mediated
drug transport in native membrane vesicle systems. For example, fluorescence quench-
ing of daunorubicin transported into the interior of DNA-loaded plasma membrane
vesicles allowed kinetic characterization of Pgp-mediated drug transport.
56
Biochemical characterization of Pgp requires purification of the protein in a func-
tional state. This has been accomplished by several research groups, using a variety of
drug-selected MDR cell lines and cells transfected with the MDR1 gene, as the source
of protein.
57-61
In general, expression of Pgp in heterologous systems (
Escherichia
coli
, baculovirus-infected insect cells, and yeast) has been fraught with difficulties
and has not led to the widespread use of this approach. The use of
E. coli
as a host cell
for expression was shown to lead to misfolding of the protein.
32
Overexpression in
the yeast
Pichia pastoris
is the exception and has led to the purification of milligram
amounts of both wild-type and mutant Pgps.
62
This system has also proved very useful
for overexpression of other ABC transport proteins.
63
Purified Pgp has been charac-
terized with respect to both its ATPase and drug transport activities (see below), and
various biophysical studies have been carried out to assess its structure and confor-
mation, using circular dichroism (CD) spectroscopy,
64
fluorescence spectroscopy,
24
and EM.
44
Pgp has been reconstituted successfully into proteoliposomes, so that both
its ATPase and drug transport functions are retained.
25
,
26
,
58
,
65-68
10.7. ATP BINDING AND HYDROLYSIS BY P-GLYCOPROTEIN
ATP hydrolysis supplies the energy for active drug transport. In most ATP-driven
transporters, ATP hydrolysis is tightly coupled to substrate transport, so that it is
hydrolyzed only when substrate is transported concurrently. However, Pgp is unusual
in displaying a high level of constitutive (basal) ATPase activity, which is observed in
the absence of added drugs for plasma membrane vesicles from MDR cells
49
,
50
and
insect cells overexpressing recombinant Pgp,
69
,
70
and purified Pgp.
57-59
Constitutive
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