Biomedical Engineering Reference
In-Depth Information
ATPase activity has since been reported for other eukaryotic ABC proteins, including
MRP1 (ABCC1), CFTR (ABCC7), ABCA1, ABCR (ABCA4), and several bacterial
ABC transporters. Purified Pgp has a maximal basal ATPase activity as high as 3 to
5
mol/min per milligram of protein, depending on the presence of detergent, lipids,
and drugs.
62
,
71
The
K
m
for ATP hydrolysis by membrane-bound and purified Pgp reported by
several laboratories is quite high (in the range 0.4 to 0.8 mM), indicating that Pgp has
a relatively low nucleotide affinity compared to other transporters. A divalent cation is
necessary for ATP hydrolysis. Physiologically, this ion is Mg
2
+
, although both Mn
2
+
and Co
2
+
can support ATP hydrolysis at lower rates.
72
Several inhibitors of Pgp AT-
Pase activity have been identified, including
ortho
-vanadate and various sulfhydryl-
modifying agents, including maleimides, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole
(NBD-Cl),
p
-chloromercuribenzenesulfonate, HgCl
2
, and others. Sulfhydryl reagents
covalently modify two Cys residues, one in each Walker A motif (Cys 431 and 1074 in
human Pgp)
73
and thereby inhibit catalysis, although ATP binding still takes place.
74
These Cys are not required for ATPase activity, since a Cys-less Pgp protein is still
active,
30
and the loss of activity when they are modified probably results from steric
interference.
The basal ATPase activity of Pgp is modulated by drug substrates and modulators
in a complex and puzzling fashion. Three different patterns have been observed. Many
drugs display a biphasic pattern, with stimulation of ATPase activity at low concen-
trations and varying degrees of inhibition at higher concentrations. Some compounds
have been observed to only stimulate activity; for example, many linear peptides,
cyclic peptides, and ionophores stimulate Pgp ATPase activity up to 2.5-fold.
75
On
the other hand, some substrates appear to produce only inhibition of activity. The
molecular basis of these differences in ATPase modulation is not known. The bipha-
sic pattern might arise from the presence of a “stimulatory” drug-binding site and
an overlapping “inhibitory” drug-binding site,
76
but why such an arrangement would
be intrinsic to the mechanism of Pgp is not clear. To complicate matters further, ex-
tremely variable results have been seen from one research lab to another. For example,
vinblastine stimulated the ATPase activity of human Pgp
77
but inhibited the ATPase
activity of hamster Pgp.
57-59
Modulation of Pgp ATPase activity by drugs and mod-
ulators is also highly dependent on the detergent used to isolate the protein or the
surrounding lipid environment.
62
,
65
,
78
The ATPase activity of Pgp is inhibited rapidly and completely by the P
i
analog,
ortho
-vanadate (V
i
), in the presence of ATP. V
i
is trapped after a single catalytic
turnover in only one NB domain,
72
M
2
+
, where M
2
+
is a
divalent cation, usually Mg
2
+
. The trapped complex can also form from ADP and V
i
,
but at a lower rate. The V
i
-trapped complex displays no ATPase activity, suggesting
that both catalytic sites must be functional for ATP hydrolysis to take place. Based
on these observations, Senior et al. proposed that Pgp operates by an alternating sites
mechanism, whereby only one catalytic site can be in the transition-state conformation
at any time, and the two sites alternate in catalysis.
79
Based on studies of myosin and
other nucleotide-utilizing proteins, the V
i
-trapped complex is believed to resemble
the catalytic transition state structurally.
80
However, the V
i
-trapped complex of Pgp is
as the complex ADP
·
V
i
·
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