Identification of the monkey lens glucose transporter by photoaffinity labelling with cytochalasin B

V. A. Lucas, J. S. Zigler

Research output: Contribution to journalArticlepeer-review

Abstract

Polypeptide constituents of the lens glucose transporter have been identified by photoaffinity labelling with cytochalasin B. The urea-insoluble fraction of monkey lens was irradiated at 280 nm for 30 min in the presence of 5 x 10-7 M 3H-cytochalasin B. After extensive washing, the membranes wer solubilized and their polypeptide composition determined by SDS-PAGE. Radioactivity was extracted from gel slices to determine the position of photoincorporated label. 3H-cytochalasin B was irreversibly incorporated into a broad molecular weight region from M(r) > 94,000 to 43,000 with the peak of activity occurring at M(r) 53,000. Photoincorporation was inhibited by D-glucose (500 mM) and phloretin (1 x 10-5) but was unaffected by L-glucose (500 mM), cytochalasin E (1 x 10-5) and phloridzin (1 x 10-5 M). Cortex and nucleus membrane preparations contained the same range of labelled polypeptides after photoaffinity labelling but nuclear membranes contained approximately twice the activity of cortical membranes indicating an enrichment of glucose transporters in the nucleus. Treatment of labelled membranes with endoglycosidase F converted the broad band of labelling to a sharp band of M(r) 45,000. The lens glucose transporter is therefore a glycoprotein and the broadness of the photoaffinity labelled peak is due to heterogeneous N-linked glycosylation of a core polypeptide. From these studies it appears that the monkey lens glucose transporter closely resembles that of the human erythrocyte.

Original languageEnglish (US)
Pages (from-to)630-635
Number of pages6
JournalInvestigative Ophthalmology and Visual Science
Volume29
Issue number4
StatePublished - 1988

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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