The major
nucleoside transporter of the human T
leukemia cell line CEM has been identified by photoaffinity labeling with the transport inhibitor nitrobenzylmercaptopurine riboside (
NBMPR). The photolabeled
protein migrates on SDS-PAGE
gels as a broad band with a mean apparent molecular weight (75,000 +/- 3000) significantly higher than that reported for the
nucleoside transporter in human erythrocytes (55,000) (Young et al. (1983) J. Biol. Chem. 258, 2202-2208). However,
after treatment with
endoglycosidase F to remove
carbohydrate, the
NBMPR-
binding protein in CEM cells migrates as a sharp peak with an apparent molecular weight (47,000 +/- 3000) identical to that reported for the deglycosylated
protein in human erythrocytes (Kwong et al. (1986) Biochem. J. 240, 349-356). It therefore appears that the difference in the apparent molecular weight of the
NBMPR-sensitive
nucleoside transporter between the CEM cell line and human erythrocytes is a result of differences in glycosylation. The
NBMPR-
binding protein from CEM cells has been solubilized with 1%
octyl glucoside and reconstituted into
phospholipid vesicles by a freeze-thaw sonication technique. Optimal reconstitution of
uridine transport activity was achieved using a sonication interval of 5 to 10 s and
lipid to
protein ratios of 60:1 or greater. Under these conditions transport activity in the reconstituted vesicles was proportional to the
protein concentration and was inhibited by
NBMPR. Omission of
lipid or
protein, or substitution of a
protein extract prepared from a
nucleoside transport deficient mutant of the CEM cell line resulted in vesicles with no
uridine transport activity. The initial rate of
uridine transport, in the vesicles prepared with CEM
protein, was saturable with a Km of 103 +/- 11 microM and was inhibited by
adenosine,
thymidine and
cytidine. The Km for
uridine and the potency of the other
nucleosides as inhibitors of
uridine transport (
adenosine greater than
thymidine greater than
cytidine) were similar to intact cells. Thus, although the
nucleoside transporter of CEM cells has a higher molecular weight than the human erythrocyte transporter, it exhibits typical
NBMPR-sensitive
nucleoside transport activity both in the intact cell and when reconstituted into
phospholipid vesicles.