Calcium-binding characteristics and conformational changes in metastasin, a member of S-100 protein family.
Investigation of Ca(2+)-binding characteristics of metastasin (Mts-1) by competition with Fluo-3 revealed two types of Ca(2+)-binding sites in Mts-1 with the geometric mean of their dissociation constant (Kd) value of 2.6 microM for the two EF-sites. The Hill coefficient (nH) is 0.98. A substantial increase in the affinity of Mts-1 for Ca2+ and strong cooperative character of binding (kd = 0.2 microM, nH = 1.91) is observed in the presence of the target protein p37 isolated from mouse adenocarcinoma cell lines CSML-100 and CSML-0. Two different hydrophobic sites of binding with the fluorescent probe 2-(p-toludino)naphthalene-6-sulfonate (TNS) per Mts-1 molecule have been determined. The exposure of the hydrophobic binding sites of the first type are shown to be Ca(2+)-dependent and the hydrophobic binding sites of the second type are exposed independently of Ca2+ concentration. A decrease in the number of Ca(2+)-dependent hydrophobic centers in the presence of p37 protein was detected by measurements of TNS fluorescence.
Dukhanina
E A
EA
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia. buh@genom-ii.eimb.rssi.ru
Dukhanin
A S
AS
Lomonosov MYu
Lukanidin
E M
EM
Georgiev
G P
GP
D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology; D000814:Aniline Compounds; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes; D051379:Mice; D009282:Naphthalenesulfonates; D011485:Protein Binding; D011487:Protein Conformation; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; D014407:Tumor Cells, Cultured; D014966:Xanthenes
D000814:Aniline Compounds; D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; C470944:S100a4 protein, mouse; D014966:Xanthenes; C059715:Fluo-3; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid