Purification of the human erythrocyte nucleoside transporter by immunoaffinity chromatography

F. Y P Kwong, A. Davies, Chung Ming Tse, J. D. Young, P. J F Henderson, S. A. Baldwin

Research output: Contribution to journalArticle

Abstract

The nucleoside transporter has been purified by passage of a preparation of human erythrocyte-membrane band-4.5 proteins through a column of immobilized antibodies specific for the glucose transporter. This procedure removed > 99.8% of the glucose transporters and achieved an approx. 18-fold purification of the nucleoside transporter, constituting a 478-fold purification from erythrocyte membranes. The isolated protein migrated as a single broad band of average apparent M(r) 55000 on SDS/polyacrylamide gels and bound approx. 0.6 mol of nitrobenzylthioinosine/mol of polypeptide, with a K(d) of 1.1 ± 0.14 (S.E.M.) nM. Upon reconstitution into large unilamellar phospholipid vesicles it catalysed the uptake of uridine with an apparent specific activity 6-fold greater than that of the unfractionated band-4.5 proteins. Furthermore, the purified nucleoside transporter was not labelled on Western blots by monoclonal antibody raised against the glucose transporter. It is concluded that the nucleoside transporter has been purified to near homogeneity.

Original languageEnglish (US)
Pages (from-to)243-249
Number of pages7
JournalBiochemical Journal
Volume255
Issue number1
StatePublished - 1988
Externally publishedYes

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Nucleoside Transport Proteins
Chromatography
Purification
Facilitative Glucose Transport Proteins
Erythrocytes
Erythrocyte Membrane
Monosaccharide Transport Proteins
Immobilized Antibodies
Membranes
Unilamellar Liposomes
Proteins
Uridine
Phospholipids
Western Blotting
Monoclonal Antibodies
Peptides
Scanning electron microscopy

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kwong, F. Y. P., Davies, A., Tse, C. M., Young, J. D., Henderson, P. J. F., & Baldwin, S. A. (1988). Purification of the human erythrocyte nucleoside transporter by immunoaffinity chromatography. Biochemical Journal, 255(1), 243-249.

Purification of the human erythrocyte nucleoside transporter by immunoaffinity chromatography. / Kwong, F. Y P; Davies, A.; Tse, Chung Ming; Young, J. D.; Henderson, P. J F; Baldwin, S. A.

In: Biochemical Journal, Vol. 255, No. 1, 1988, p. 243-249.

Research output: Contribution to journalArticle

Kwong, FYP, Davies, A, Tse, CM, Young, JD, Henderson, PJF & Baldwin, SA 1988, 'Purification of the human erythrocyte nucleoside transporter by immunoaffinity chromatography', Biochemical Journal, vol. 255, no. 1, pp. 243-249.
Kwong FYP, Davies A, Tse CM, Young JD, Henderson PJF, Baldwin SA. Purification of the human erythrocyte nucleoside transporter by immunoaffinity chromatography. Biochemical Journal. 1988;255(1):243-249.
Kwong, F. Y P ; Davies, A. ; Tse, Chung Ming ; Young, J. D. ; Henderson, P. J F ; Baldwin, S. A. / Purification of the human erythrocyte nucleoside transporter by immunoaffinity chromatography. In: Biochemical Journal. 1988 ; Vol. 255, No. 1. pp. 243-249.
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