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).
Lauc
G
G
Department of Medical Biochemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb, Croatia.
Flögel
M
M
Diehl-Seifert
B
B
Schröder
H C
HC
Müller
W E
WE
D000595:Amino Acid Sequence; D000818:Animals; D037263:Asialoglycoprotein Receptor; D002467:Cell Nucleus / Q000378:metabolism*; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents / Q000737:chemistry; D004076:Digoxigenin / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005947:Glucose / Q000737:chemistry; D037102:Lectins; D008099:Liver / Q000378:metabolism*; D008239:Lysine / Q000031:analogs & derivatives; D008297:Male; D015335:Molecular Probes; D008969:Molecular Sequence Data; D010777:Photochemistry; D051381:Rats; D017208:Rats, Wistar; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D013312:Stress, Physiological / Q000378:metabolism
D037263:Asialoglycoprotein Receptor; D003432:Cross-Linking Reagents; D037102:Lectins; D015335:Molecular Probes; D011956:Receptors, Cell Surface; D005947:Glucose; D008239:Lysine; D004076:Digoxigenin