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RADIOACTIVE LABELING OF PROTEINS OF SACCHAROMYCES CARLSBERGENSIS PROTOPLAST MEMBRANE
Author(s) -
PATEL PURUSHOTTAM C.,
LEWIS MICHAEL J.
Publication year - 1980
Publication title -
journal of food biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.507
H-Index - 47
eISSN - 1745-4514
pISSN - 0145-8884
DOI - 10.1111/j.1745-4514.1980.tb00637.x
Subject(s) - protoplast , chromatography , elution , membrane , chemistry , size exclusion chromatography , mannitol , yeast , saccharomyces , biochemistry , sucrose , sodium , saccharomyces cerevisiae , enzyme , organic chemistry
Yeast (Saccharomyces carlsbergensis) protoplasts were suspended in 0.8 M mannitol‐phosphate buffer (pH 9.2) and were labeled with dansyl chloride (GJH). When the protoplasts were washed and then ruptured in non isotonic media, and subsequently separated in a discontinuous sucrose gradient, over 92% of the radioactivity present was associated with the membrane fraction. The isolated labeled cytoplasmic membrane was solubilized in sodium dodecyl sulfate and 2‐mercaptoethanol and separated by gel filtration chromatography on Bio gel P‐100 and P‐150 columns. The elution pattern showed three peaks of radioactivity, but the bulk of protein, which eluted in the void volume, was found to have a comparatively low level of radioactivity. This suggests that there was selectivity in the labeling reaction, i.e. only certain proteins are exposed in such a way as to be accessible to labeling by dansylation. We suggest these proteins are at the protoplast surface. It is suggested that there are three or possibly four polypeptides which have an N‐terminal group exposed on the outer surface of the yeast membrane.