Interaction of calcyclin and its cyanogen bromide fragments with annexin II and glyceraldehyde 3-phosphate dehydrogenase.
The structural properties of calcyclin protein are quite well characterized but its function remains obscure. To help elucidate the biological role of calcyclin we have performed the in vitro studies of the Ca(2+)-dependent interaction of Ehrlich ascites tumor cells calcyclin and its cyanogen bromide fragments with two potential calcyclin targets: annexin II and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The binding of annexin II, evidenced by the reaction with 125I-calcyclin, was found to be very weak and occurred only for intact calcyclin. On the other hand the interaction between calcyclin and GAPDH was of high affinity and could be assigned to the N-terminal region of calcyclin. Intact calcyclin and its N-terminal fragment bound to GAPDH in the gel overlay and affinity chromatography assay. When examined in the presence of a crosslinking agent the interaction resulted in the formation of 46K covalent adduct between calcyclin monomer and GAPDH subunit. Fluorescence of 5-iodoacetamido-fluorescein-labelled calcyclin was efficiently quenched by GAPDH in the presence of Ca2+. Titration experiments revealed the stoichiometry of one calcyclin monomer binding to each of GAPDH subunits with a binding constant of 10(8) M-1. The results of this work suggest that the binding between calcyclin and GAPDH may have bearing on calcyclin function.
Filipek
A
A
Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
Wojda
U
U
Leśniak
W
W
D000595:Amino Acid Sequence; D000818:Animals; D017306:Annexin A2 / Q000378:metabolism*; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002286:Carcinoma, Ehrlich Tumor; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide; D005470:Fluorometry; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D009418:S100 Proteins; D014407:Tumor Cells, Cultured
D017306:Annexin A2; D002135:Calcium-Binding Proteins; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D009418:S100 Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003488:Cyanogen Bromide