Purification and properties of glucose 1-phosphate binding protein in Agrobacterium tumefaciens

Abstract

The presence of two kinds of glucose 1-phosphate-binding protein in sonic extract of sucrose-grown cells of Agrobacterium tumefaciens was demonstrated by DEAE-cellulose column chromatography. Both of them showed high specificity for glucose 1-phosphate ( Glc-1-P ) and were purified as homogeneous proteins in polyacrylamide disc gel electrophoresis. The properties of the two binding proteins (I and II) are as follows: ratio of A 280 nm /A 260 nm , 1.82 and 1.70; molecular weight, 35 000 and 42 000; K D for Glc-1-P , 8 · 10 −7 M and 1.3 · 10 −6 M; substrate binding ( Glc-1-P mole/protein mole), 0.84 and 0.93; optimal pH, 8.0–8.4 (narrow range) and 5.4–8.4 (wide range). The binding protein II was exclusively released from cells by osmotic shock, and the restoration of Glc-1-P uptake activity of shocked cells occurred on addition of the binding protein II. On the other hand, by treatment with the binding protein I, the isolated cell envelope which was prepared from sonic extract of resting cells gained Glc-1-P- binding activity, while by treatment with the binding protein II the envelope gained no activity.