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Identification and purification of a stress associated nuclear carbohydrate binding protein (Mr 33000) from rat liver by application of a new photoreactive carbohydrate probe

Abstract

A photoreactive α-D-glucose probe has been designed for the specific detection of carbohydrate binding proteins (CBPs). The probe consists of four parts: (i) an α-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 α-D-glucose CBP ofM r=33000, 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 0.6M nuclear salt extract by two subsequent affinity chromatography steps (antibody-affinity as well as α-D-glucose affinity column).

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Abbreviations

BSA:

bovine serum albumin

CBP:

carbohydrate binding protein

DIG:

digoxigenin

Gal:

galactose

Glc:

glucose

Lys:

lysine

PAGE:

polyacrylamide gel electrophoresis

SDS:

sodium dodecyl sulphate.

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Correspondence to Werner E. G. Müller.

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Lauc, G., Flögel, M., Diehl-seifert, B. et al. Identification and purification of a stress associated nuclear carbohydrate binding protein (Mr 33000) from rat liver by application of a new photoreactive carbohydrate probe. Glycoconjugate J 11, 541–549 (1994). https://doi.org/10.1007/BF00731305

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Keywords

  • carbohydrate binding proteins
  • lectins
  • stress
  • rat liver
  • CBP33