Biomaterial Database

Multiple routes and regulation by tyrosine phosphorylation characterize the ATP-dependent transport of 2,4-dinitrophenyl S-glutathione in inside-out vesicles from human erythrocytes. 1997

M Saxena, and G B Henderson

Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California, 92037, USA.

ATP-dependent efflux routes for 2,4-dinitrophenyl S-glutathione (DNP-SG) were investigated using inside-out vesicles from human erythrocytes. Nonlinear double-reciprocal plots of transport at substrate concentrations ranging from 0.07 to 500 micro;m indicated that multiple transport routes were operative. Sensitivity to naphthyl glucuronide separated [3H]DNP-SG transport into two low-affinity components which by computer analysis exhibited Km values of 330 and 1400 micro;m, respectively. At low substrate concentrations, two high-affinity routes were observed. The predominant activity (hMOAT3a) exhibited a Km of 0.18 micro;m (Vmax = 22 pmol/min/mg protein), whereas the second activity (MOAT3b) had a Km of 0.58 micro;m (Vmax = 16 pmol/min/mg protein). High-affinity transport of DNP-SG increased substantially (2.5-fold) in vesicles preincubated with genistein or other tyrosine kinase inhibitors. Kinetic analyses in vesicles pretreated with 50 micro;m genistein showed that increased transport resulted from the appearance of a new activity (hMOAT3c) with a Km of 0.85 micro;m and a substantially elevated Vmax (80 pmol/min/mg protein). At varying concentrations of genistein, a progression was observed that was consistent with the conversion of hMOAT3b to hMOAT3a and hMOAT3a to hMOAT3c. Phenylarsine oxide, a phosphotyrosine phosphatase inhibitor, produced an opposite progression. Specificity studies showed that hMOAT3a exhibited the highest affinity for various anionic conjugates, and had a notable binding preference for glutathione disulfide. The relative effectiveness of the various inhibitors was similar for hMOAT3a, hMOAT3b, and hMOAT3c, as well as for a corresponding mMOAT3 activity from L1210 mouse cells. The results show that human erythrocytes contain multiple ATP-dependent efflux systems for DNP-SG and that separation of these systems can be achieved on the basis of substrate Km value and inhibitor and activator specificity. High-affinity transport can proceed via three activities which appear to be subforms of a single system with differing levels of tyrosine phosphorylation. Multiple hMOAT3 subforms provide flexibility for extruding various anionic conjugates and may have evolved in erythrocytes to expedite the efflux of GS-SG.

UI	MeSH Term	Description	Entries
D007529	Isoflavones	3-Phenylchromones. Isomeric form of FLAVONOIDS in which the benzene group is attached to the 3 position of the benzopyran ring instead of the 2 position.	3-Benzylchroman-4-One,3-Benzylidene-4-Chromanone,Homoisoflavone,Homoisoflavones,Isoflavone,Isoflavone Derivative,3-Benzylchroman-4-Ones,3-Benzylidene-4-Chromanones,Isoflavone Derivatives,3 Benzylchroman 4 One,3 Benzylchroman 4 Ones,3 Benzylidene 4 Chromanone,3 Benzylidene 4 Chromanones,Derivative, Isoflavone,Derivatives, Isoflavone
D007939	Leukemia L1210	An experimental LYMPHOCYTIC LEUKEMIA of mice.	Leukemia L 1210,L 1210, Leukemia,L1210, Leukemia
D011505	Protein-Tyrosine Kinases	Protein kinases that catalyze the PHOSPHORYLATION of TYROSINE residues in proteins with ATP or other nucleotides as phosphate donors.	Tyrosine Protein Kinase,Tyrosine-Specific Protein Kinase,Protein-Tyrosine Kinase,Tyrosine Kinase,Tyrosine Protein Kinases,Tyrosine-Specific Protein Kinases,Tyrosylprotein Kinase,Kinase, Protein-Tyrosine,Kinase, Tyrosine,Kinase, Tyrosine Protein,Kinase, Tyrosine-Specific Protein,Kinase, Tyrosylprotein,Kinases, Protein-Tyrosine,Kinases, Tyrosine Protein,Kinases, Tyrosine-Specific Protein,Protein Kinase, Tyrosine-Specific,Protein Kinases, Tyrosine,Protein Kinases, Tyrosine-Specific,Protein Tyrosine Kinase,Protein Tyrosine Kinases,Tyrosine Specific Protein Kinase,Tyrosine Specific Protein Kinases
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
D002474	Cell-Free System	A fractionated cell extract that maintains a biological function. A subcellular fraction isolated by ultracentrifugation or other separation techniques must first be isolated so that a process can be studied free from all of the complex side reactions that occur in a cell. The cell-free system is therefore widely used in cell biology. (From Alberts et al., Molecular Biology of the Cell, 2d ed, p166)	Cellfree System,Cell Free System,Cell-Free Systems,Cellfree Systems,System, Cell-Free,System, Cellfree,Systems, Cell-Free,Systems, Cellfree
D004791	Enzyme Inhibitors	Compounds or agents that combine with an enzyme in such a manner as to prevent the normal substrate-enzyme combination and the catalytic reaction.	Enzyme Inhibitor,Inhibitor, Enzyme,Inhibitors, Enzyme
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
D005965	Glucuronates	Derivatives of GLUCURONIC ACID. Included under this heading are a broad variety of acid forms, salts, esters, and amides that include the 6-carboxy glucose structure.	Glucosiduronates,Glucuronic Acids,Acids, Glucuronic
D005978	Glutathione	A tripeptide with many roles in cells. It conjugates to drugs to make them more soluble for excretion, is a cofactor for some enzymes, is involved in protein disulfide bond rearrangement and reduces peroxides.	Reduced Glutathione,gamma-L-Glu-L-Cys-Gly,gamma-L-Glutamyl-L-Cysteinylglycine,Glutathione, Reduced,gamma L Glu L Cys Gly,gamma L Glutamyl L Cysteinylglycine
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man