CALCIUM-PROTEIN INTERACTIONS IN THE EXTRACELLULAR ENVIRONMENT - CALCIUM-BINDING, ACTIVATION, AND IMMUNOLOCALIZATION OF A COLLAGENASE GELATINASE ACTIVITY EXPRESSED IN THE SEA-URCHIN EMBRYO/

Citation
J. Mayne et Jj. Robinson, CALCIUM-PROTEIN INTERACTIONS IN THE EXTRACELLULAR ENVIRONMENT - CALCIUM-BINDING, ACTIVATION, AND IMMUNOLOCALIZATION OF A COLLAGENASE GELATINASE ACTIVITY EXPRESSED IN THE SEA-URCHIN EMBRYO/, Journal of cellular biochemistry, 71(4), 1998, pp. 546-558
Citations number
58
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology,"Cell Biology
ISSN journal
0730-2312
Volume
71
Issue
4
Year of publication
1998
Pages
546 - 558
Database
ISI
SICI code
0730-2312(1998)71:4<546:CIITEE>2.0.ZU;2-N
Abstract
We have purified and characterized a collagenase/gelatinase activity e xpressed during sea urchin embryonic development. The native molecular mass was determined to be 160 kDa, while gelatin substrate gel zymogr aphy revealed an active species of 41 kDa, suggesting that the native enzyme is a tetramer of active subunits. Incubation in the presence of EGTA resulted in nearly complete loss of activity and this effect cou ld be reversed by calcium. Calcium-induced reactivation appeared to be cooperative and occurred With an apparent kd value of 3.7 mM. Two mod es of calcium binding to the 41-kDa subunit were detected; up to 80 mo les of calcium bound with a lid value of 0.5 mM, while an additional 1 20 moles bound with a lid value of 5 mM. Amino acid analysis revealed a carboxy plus carboxyamide content of 24.3 mol/100 mel, indicating th e availability of substantial numbers of weak Ca2+-binding sites. Calc ium binding did not result in either secondary or quaternary structura l changes in the collagenase/gelatinase, suggesting that Ca2+ may faci litate activation through directly mediating the binding of substrate to the enzyme. The collagenase/gelatinase activity was detected in bla stocoelic fluid and in the hyalin fraction dissociated from l-h-old em bryos. Immunolocalization studies revealed two storage compartments in the egg; cortical granules and small granules/vesicles dispersed thro ughout the cytoplasm. After fertilization, the antigen was detected in both the apical and basal extracellular matrices, the hyaline layer, and basal lamina respectively. (C) 1998 Wiley-Liss, Inc.