Biomaterial Database

Identification and reconstitution of the nucleoside transporter of CEM human leukemia cells. 1990

C R Crawford, and C Y Ng, and B Ullman, and J A Belt

Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38101.

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.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008565	Membrane Proteins	Proteins which are found in membranes including cellular and intracellular membranes. They consist of two types, peripheral and integral proteins. They include most membrane-associated enzymes, antigenic proteins, transport proteins, and drug, hormone, and lectin receptors.	Cell Membrane Protein,Cell Membrane Proteins,Cell Surface Protein,Cell Surface Proteins,Integral Membrane Proteins,Membrane-Associated Protein,Surface Protein,Surface Proteins,Integral Membrane Protein,Membrane Protein,Membrane-Associated Proteins,Membrane Associated Protein,Membrane Associated Proteins,Membrane Protein, Cell,Membrane Protein, Integral,Membrane Proteins, Integral,Protein, Cell Membrane,Protein, Cell Surface,Protein, Integral Membrane,Protein, Membrane,Protein, Membrane-Associated,Protein, Surface,Proteins, Cell Membrane,Proteins, Cell Surface,Proteins, Integral Membrane,Proteins, Membrane,Proteins, Membrane-Associated,Proteins, Surface,Surface Protein, Cell
D008970	Molecular Weight	The sum of the weight of all the atoms in a molecule.	Molecular Weights,Weight, Molecular,Weights, Molecular
D010743	Phospholipids	Lipids containing one or more phosphate groups, particularly those derived from either glycerol (phosphoglycerides see GLYCEROPHOSPHOLIPIDS) or sphingosine (SPHINGOLIPIDS). They are polar lipids that are of great importance for the structure and function of cell membranes and are the most abundant of membrane lipids, although not stored in large amounts in the system.	Phosphatides,Phospholipid
D001798	Blood Proteins	Proteins that are present in blood serum, including SERUM ALBUMIN; BLOOD COAGULATION FACTORS; and many other types of proteins.	Blood Protein,Plasma Protein,Plasma Proteins,Serum Protein,Serum Proteins,Protein, Blood,Protein, Plasma,Protein, Serum,Proteins, Blood,Proteins, Plasma,Proteins, Serum
D002352	Carrier Proteins	Proteins that bind or transport specific substances in the blood, within the cell, or across cell membranes.	Binding Proteins,Carrier Protein,Transport Protein,Transport Proteins,Binding Protein,Protein, Carrier,Proteins, Carrier
D004910	Erythrocyte Membrane	The semi-permeable outer structure of a red blood cell. It is known as a red cell 'ghost' after HEMOLYSIS.	Erythrocyte Ghost,Red Cell Cytoskeleton,Red Cell Ghost,Erythrocyte Cytoskeleton,Cytoskeleton, Erythrocyte,Cytoskeleton, Red Cell,Erythrocyte Cytoskeletons,Erythrocyte Ghosts,Erythrocyte Membranes,Ghost, Erythrocyte,Ghost, Red Cell,Membrane, Erythrocyte,Red Cell Cytoskeletons,Red Cell Ghosts
D006026	Glycoside Hydrolases	Any member of the class of enzymes that catalyze the cleavage of the glycosidic linkage of glycosides and the addition of water to the resulting molecules.	Endoglycosidase,Exoglycosidase,Glycohydrolase,Glycosidase,Glycosidases,Glycoside Hydrolase,Endoglycosidases,Exoglycosidases,Glycohydrolases,Hydrolase, Glycoside,Hydrolases, Glycoside
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000345	Affinity Labels	Analogs of those substrates or compounds which bind naturally at the active sites of proteins, enzymes, antibodies, steroids, or physiological receptors. These analogs form a stable covalent bond at the binding site, thereby acting as inhibitors of the proteins or steroids.	Affinity Labeling Reagents,Labeling Reagents, Affinity,Labels, Affinity,Reagents, Affinity Labeling