Abstract
Preconfluent or confluent fibroblasts grown in 5% serum medium yielded, without cell lysis, all the glucose-binding protein and most of the transport-stimulating activity in the cell wash fluid obtained with a 10 mM sodium octanoate-containing solution. For assay, octanoate was removed, and after the binding protein was labeled with [14C]glucose, the factors were chromatographed on Sephadex G-200 and the transport-stimulating and factor-bound [14C]glucose activities were measured. Three peaks were separated, which more or less overlapped for both functions; upon chromatography on DEAE-cellulose, these peaks yielded overlapping or separate peaks for the two functions, presumably indicating their separability. Serum, when similarly chromatographed, showed only peaks for transport which, with the exception of one major peak with both functions, more or less overlapped with those from the wash fluid. Glucose transport rates, when compared in fibroblasts grown in glucose and in fructose and in Rous sarcoma virus-transformed cells grown in glucose, were in the proportion of 1:3.7:6.3. Addition of extracted transport protein stimulated the transport of both the glucose-grown and fructose-grown normal cells but showed no effect on the transport of transformed cells. Addition of transport protein induced the formation of [14C]deoxyglucose 6-phosphate in amounts proportional to the increased transport of [14C]deoxyglucose into fibroblasts. On sodium dodecyl sulfate electrophoresis, using the tightly bound [14C]glucose for assay, purified binding protein yielded large fractions of 36,000 and 18,000 and small ones of 55,000 and 73,000 daltons; the 18,000-dalton fraction is supposedly the monomeric form of the binding protein.
Keywords: serum, uptake, phosphorylation, purification
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