peptide transport systems, and at least
one peptide transport system (PepT1, see
below) is capable of distinguishing
between cis and trans conformers (Table
1.2; Brandsch et al., 1997, 1998). The
similarities and differences in the affinities
of peptide transport systems have been of
practical importance to the pharmaco-
logical industry in the development of
peptidomimetic drugs. Whether these
differences can be exploited to enhance the
absorption of specific amino acids as pep-
tides, in nutritionally significant quantities,
remains to be determined.
Molecular Characterization of
Transport Proteins
The interpretation of biochemical studies
designed to characterize transporter activity
is complicated by the recognition of multi-
ple amino acids by transporter systems.
The recent generation of a number of com-
plementary DNA (cDNA) clones that
encode proteins with specific transport
activities, and the functional expression of
their corresponding mRNAs in various
expression models (Malandro and Kilberg,
1996), has clarified a number of questions
regarding the specificity, function and
expression of transporter activities. A list
of the cloned -amino acid and peptide
transport proteins is presented in Table 1.3,
except for the family of brain-specific
neurotransmitter transporters. Knowledge
of the molecular structure of proteins
capable of -amino acid transport has
allowed evolutionary and taxonomic rela-
tionships to be established based on
primary amino acid sequence homologies
and predicted membrane topologies (see
below). In addition, the gene structure of
several of the amino acid transporters has
been determined. An important under-
standing gained from knowing the gene
structure is that different mRNAs can be
transcribed from a single gene. The tran-
scription of mRNAs that encode different
proteins from a single gene occurs by
alternative gene promoters or alternative
splicing of transcripts and results in a
greater diversity of transporter isoforms
and functional characteristics. A good
example is the production of CAT2 (high-
affinity system y+ activity) and CAT2a
(low-affinity system y+ activity) proteins
from the CAT2gene (MacLeod et al., 1994).
The identification of cDNAs has also
allowed the amount of mRNA expressed to
be quantified (Northern analysis) and the
site of mRNA expression to be determined
(in situhybridization analysis). Based on
the sequence of the cDNA, the amino acid
sequence of the protein can be predicted,
thus facilitating the generation of anti-
bodies to transport proteins. With anti-
bodies, the amount (immunoblot/Western
blot) and site-specific expression (immuno-
histochemistry) of proteins can be deter-
mined. Based on the known cDNA
sequence of one species, the cDNA isoform
from another species can be identified by
hybridizing oligonucleotides that are
predicted to bind to regions of shared
homology. If oligonucleotides are designed
that encompass the whole protein-coding
sequence of the cDNA, then the region
can be amplified by polymerase chain reac-
tion (PCR), resulting in the cloning of
species-specific ‘full-length’ cDNA. These
techniques also can be combined to
identify the mRNA of transporters that
share homologous regions.CAT family of cationic amino acid
transportersThe Na+-independent transport of the
cationic amino acids arginine, lysine,
ornithine and that portion of histidine
molecules that is positively charged is
known as system y+activity. Even though
system y+transport is not coupled directly
to transmembrane-driving ion gradients,
system y+-mediated substrates can be
accumulated against their concentration
gradients because of the difference between
their positive charge and the relatively
negative charge on the cytosolic side of the
membrane. Currently, four cDNAs have
been identified that encode system y+
activity (cationic amino acid transport;Amino Acid and Peptide Transport Systems 9