Identification and purification of a stress associated nuclear carbohydrate binding protein (M(r) 33,000) from rat liver by application of a new photoreactive carbohydrate probe.

A photoreactive alpha-D-glucose probe has been designed for the specific detection of carbohydrate binding proteins (CBPs). The probe consists of four parts: (i) an alpha-D-glucose moiety; (ii) the digoxigenin tag; (iii) the photoreactive cross-linker; and (iv) the lysyl-lysine backbone. After incubation with lectins in the dark, the probe is activated and cross-linked to the CBPs after being treated by several flashes. Using this method we have identified a new alpha-D-glucose CBP of M(r) = 33,000, termed CBP33, in the nuclei of rats exposed to transient immobilization stress. Monoclonal antibodies were raised against the partially purified protein and subsequently used to enrich CBP33. It was purified (> 2400-fold) to apparent homogeneity from a 0.6 M nuclear salt extract by two subsequent affinity chromatography steps (antibody-affinity as well as alpha-D-glucose affinity column).