trans

202 PURINES AND PYRIMIDINES
activity that was distinct from the induced
activity at the erythrocyte membrane because
it was both stereo-selective and saturable.
Recently, the gene that likely encodes this
nucleoside transport activity was identified
in P. falciparum. PfNT1 (PfENT1) is a broad
specificity purine and pyrimidine nucleoside,
and possibly purine nucleobase, transporter,
although it appears to have a significantly
higher affinity for adenosine. The preference
of PfNT1 for adenosine is reasonable since
ADA activity is augmented in intraerythrocytic
P. falciparum and is likely the preferred salvageable
nucleoside. PfNT1 also recognizes
L-nucleosides at millimolar affinity. However,
PfNT1 is an unlikely candidate for the activity
induced at the erythrocyte membrane since
it is saturable and localizes primarily to the
parasite plasma membrane. During the intraerythrocytic
lifecycle, PfNT1 is constitutively
expressed, but expression is significantly upregulated
in the trophozoite and schizont stages
when the demand for nucleic acid precursors is
acute. Plasmodium has a V-type H -pump on
its plasma membrane, which is predicted to
result in a significant negative inward proton
gradient, but whether PfNT1 acts as a proton
symporter is not yet known. Thus far PfNT1 is
the only nucleoside transporter gene to be
cloned and functionally characterized from
Plasmodium. Another related nucleoside transporter
sequence (PfNT2) also has been identified
within The Institute for Genomic Research
database, although the protein has not been
functionally validated.
Toxoplasma
The actively dividing mammalian form of
T. gondii also is intracellular. Unlike Plasmodium,
it can invade and propagate in virtually
any human or avian nucleated cell type.
T. gondii resides within a parasitophorous
vacuole (PV) and is dependent upon exogenous
purines that are supplied by the host cell (see
Figure 9.3). Thus, preformed purines must
permeate into the parasitophorous vacuolar
space. The parasitophorous vacuolar membrane
(PVM) in T. gondii is strikingly similar
to that described for Plasmodium. It also is
thought to be derived from the host-cell membrane,
and appears to act as a molecular sieve
with a size exclusion limit similar to the PVM
of P. falciparum. Until recently, the hydrolysis
of adenine nucleotides to adenosine within the
PV by parasite-secreted NTP hydrolases was
thought to provide the primary purine source
for T. gondii. However, it now appears that the
activity for the terminal hydrolysis (the release
of adenosine from AMP) is absent within the
PV. From direct transport measurements into
tachyzoites and drug sensitivity profiles with
toxic purine analogs, it appears that T. gondii
transports the nucleobases hypoxanthine, xanthine,
and uracil, and the nucleosides adenosine,
inosine, and guanosine. A low-affinity
adenosine transporter, which also recognizes
inosine and guanosine, and possibly the
nucleobases hypoxanthine and adenine, has
been characterized in T. gondii tachyzoites.
There appears to be a second route of uptake
for inosine distinct from the adenosine transporter.
Recently the cDNA for an adenosine transporter
(TgAT) has been cloned by disruption
of the TgAT locus by insertional mutagenesis
to generate adenine arabinoside-refractory
parasites and subsequent identification of the
TgAT cDNA by hybridization with part of the
disrupted locus. TgAT encodes an ENT-type
protein of 462 amino acids, which shares
limited sequence identity with the human
ENTs 1 and 2. Functional expression of TgAT in
Xenopus oocytes indicates that it shares most
of the characteristics of the adenosine transporter,
although it does not appear to recognize
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA