Biomedical Engineering Reference
In-Depth Information
of two-dimensional crystals of purified protein in a lipid bilayer.
44
The resulting
low-resolution projection structure (22 A) was compact and suggested that the two
cytoplasmic NB domains interact closely.
More recently, a higher-resolution EM structure was obtained for human Pgp which
shows close association of the NB domains
45
and bears a much greater resemblance
to the mouse Pgp structure (Figure 10.1
c
), so it seems likely that the NB domains
indeed form the sandwich dimer observed for other ABC proteins. This structure also
clearly showed the existence of 12 TM segments, supporting the proposed topology
of the protein, but the resolution was not high enough to discern further details. The
packing arrangement of the TM helices of Pgp has been explored systematically by
Clarke and co-workers, who introduced Cys residues into defined positions within
a Cys-less Pgp construct and then carried out cross-linking studies.
46
The pattern
observed suggested that TM6 is close to TM10, 11, and 12, whereas TM12 is close
to TM4, 5, and 6. Recent work showed that the ends of TM2 and TM11 are close
together on the cytoplasmic side of the membrane,
47
as are the cytoplasmic ends of
TM5 and TM8.
48
10.6. SUBCELLULAR SYSTEMS FOR STUDYING P-GLYCOPROTEIN
Much early work on the molecular basis of MDR was carried out on intact cells se-
lected for MDR by growth in high concentrations of drugs, such as colchicine and vin-
blastine. However, the difficulties involved in dissecting such a complex system soon
led to attempts to use simpler subcellular systems to study the MDR phenomenon.
Native plasma membrane vesicles isolated from MDR cells expressing high levels
of Pgp have proved to be very useful. Compared to membrane preparations from
the drug-sensitive parent cell line, they often display much higher levels of ATPase
activity, which are attributable to the presence of large amounts of Pgp in the plasma
membrane.
49
,
50
In addition, membrane vesicles were found to be labeled by pho-
toaffinity analogs of both MDR drugs
51
and nucleotides,
52
providing some of the
first biochemical evidence that Pgp binds these molecules. Since then, membrane
vesicles have been used for sophisticated kinetic studies of substrate binding using
radiolabeled drugs.
53
Plasma membrane vesicles have also proved useful in studies of Pgp-mediated drug
transport. Most vesicle preparations consist of a mixture of right-side-out and inside-
out vesicles,
54
and if they are well sealed, the latter population can transport drug from
the external medium into the vesicle lumen when provided with ATP. When using a
vesicle system where other membrane-bound ATPases are present, it is often necessary
to add an ATP-regenerating system, such as creatine kinase and creatine phosphate,
to prevent rapid depletion of ATP in the external solution. Substrate uptake into the
vesicle interior can be measured in one of two ways. If drug is available in radioactive
form (e.g., [
3
H]colchicine, [
3
H]vinblastine, [
125
I]peptide), it is added to the vesicle
preparation at time zero, together with ATP and a regenerating system, and vesicles
are removed at various times (typically, ranging up to 30 minutes) and collected by
rapid filtration.
54
Drug uptake into the vesicles increases with time, usually reaching
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