EVIDENCE THAT LACTOSE BINDING TO CBP35 DISRUPTS ITS INTERACTION WITH CBP70 IN ISOLATED HL-60 CELL-NUCLEI

We have previously reported that two carbohydrate-binding proteins (CB P35 and CBP70) can, under appropriate conditions of affinity chromatog raphy, be isolated from HL60 cell nuclear extracts as a complex. Moreo ver, we have demonstrated that, during affinity chromatography, the CB P70-CBP35 association can be modified by the binding of lactose to CBP 35. To determine whether the CBP70-CBP35 association could be disrupte d in the nucleus upon lactose binding to CBP35, the behaviors of CBP70 and CBP35 were analyzed in membrane-depleted nuclei of HL60 cells, in cubated with or without lactose. This study was performed by using an antiserum that cross-reacts with CBP35 and CBP70, an antiserum that wa s specifically raised against CBP70, and a monoclonal antibody (Mac 2) reactive against CBP35. Taken together, the results of indirect immun ofluorescent staining, immunoblotting experiments, and quantitative fl ow-cytofluorometric analysis show that (i) CBP7O and CBP35 are present and colocalized in the nuclei incubated without lactose, (ii) all the CBP70 molecules left the nuclei incubated in the presence of lactose, while CBP35 molecules, probably bound to RNA, remained inside the nuc lei, and (iii) glucose failed to have the same effect as lactose. Thes e results strongly suggest that, in membrane-depleted nuclei, CBP35 an d CBP70 interactions can be altered by a conformational change of CBP3 5 induced by the binding of lactose to its carbohydrate-recognition do main. (C) 1994 Academic Press, Inc.