Identification and purification of a stress associated nuclear carbohydrate binding protein (Mr 33000) from rat liver by application of a new photoreactive carbohydrate probe |
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Authors: | Gordan Lauc Mirna Flögel Bärbel Diehl-seifert Heinz C Schröder Werner E G Müller |
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Institution: | (1) Department of Medical Biochemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb, Domagojeva 2, Zagreb, Croatia;(2) Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität, Duesbergweg 6, 55099 Mainz, Germany |
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Abstract: | A photoreactive -D-glucose probe has been designed for the specific detection of carbohydrate binding proteins (CBPs). The probe consists of four parts: (i) an -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 -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 -D-glucose affinity column).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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Keywords: | carbohydrate binding proteins lectins stress rat liver CBP33 |
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