Photoaffinity labeling and characterization of the cloned purine-cytosine transport system in Saccharomyces cerevisiae.

Abstract:

:8-Azido[2-3H]adenine was used as a photoaffinity label for the purine-cytosine transport system. After irradiation in the presence of the photoaffinity label, the cells were converted into protoplasts, their plasma membranes were purified, and the membrane proteins were extracted and separated by NaDodSO4/PAGE. The radioactivity was specifically incorporated into a protein with a molecular weight of 120,000. Photoaffinity labeling of this protein could be blocked by irradiation in the presence of natural substrates for the transport system. The molecular weight as determined by NaDod-SO4/PAGE was found to be twice the value calculated from mRNA analysis of the cloned gene. Incubation of exponentially growing cells with tunicamycin, an antibiotic that inhibits glycosylation of proteins, resulted in a 40% decrease in the overall initial uptake rate, which correlates with the reduction of the labeled Mr 120,000 protein. Treatment of the extracted labeled plasma membrane proteins with glycosidic enzymes resulted in disappearance of the Mr 120,000 peak and the appearance of new peaks at Mr 60,000 and Mr 73,000. These findings indicate that the purine-cytosine transport protein is a glycoprotein.

authors

Schmidt R,Manolson MF,Chevallier MR

doi

10.1073/pnas.81.20.6276

subject

Has Abstract

pub_date

1984-10-01 00:00:00

pages

6276-80

issue

20

eissn

0027-8424

issn

1091-6490

journal_volume

81

pub_type

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