ISSN:
0091-7419
Keywords:
membrane proteins
;
transport proteins
;
glucose transport
;
reconstitution of glucose transport
;
purification of glucose transporter
;
cytochalasin B
;
Life Sciences
;
Molecular Cell Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
Notes:
The D-glucose transporter from human erythrocytes has been purified and reconstituted by Kasahara and Hinkle (J Biol Chem 252:7394-7390). Using a similar purification scheme, we have isolated the protein with 65% of the extracted phospholipid at a lipid-protein ratio of 14:1 by weight. The KD (0.14 μM) and extent (11 nmoles/mg protein) for binding of 3H-cytochalasin B was determined by equilibrium dialysis. Glucose was a linear competitive inhibitor of binding of cytochalasin B, with an inhibition constant of 30 mM. To further characterize the protein, samples were filtered in the presence of sodium dodecyl sulfate (SDS) through Sepharose 6B to remove 95% of the lipid followed by filtration of Sephadex G150 to remove the remaining lipid and a contaminating amount of a minor, lower-molecular-weight protein. This preparation contains only 24% acidic and basic amino acids. The protein also contains 5% neutral sugars (of which 3% is galactose), 7% glucosamine, and 5% sialic acid.
Additional Material:
3 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/jss.400080407
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