Binding characteristics of the Zn-binding membrane protein from common carp

Abstract

This study incorporated the 43 kDa Zn-binding membrane protein isolated from common carp into liposome. The specificity and strength of the binding of 65 Zn to the 43 kDa proteinliposomes, and the binding of the 65 Zn-labeled 43 kDa protein-liposomes to laminin were studied. It was found that 65 Zn was bound to the external side of the 43 kDa protein-liposomes. Specific binding of 65 Zn to the protein-liposomes was detected. The binding parameter of Zn to the protein was found to be: maximum binding site (N max ), 76.7 pmole/µg protein (approx. 3 mole of Zn 2+ /mole); and equilibrium dissociation constant (Kd), 0.19 µM. Of the cations introduced (Ca 2+ , Cd 2+ ,Co 2+ , Cr 2+ , Cu 2+ , Fe 2+ , Hg 2+ , Mg 2+ , Mn 2+ , Ni 2+ , Pb 2+ ), only Co 2+ competed significantly with Zn. The protein-liposomes were also found to bind specifically to laminin with a N max of 1.1 pmole/µg laminin, and Kd of 4.79 µM. No significant protein-liposome binding occurred to other extracellular matrix proteins (fibronectin, fibrinogen or vitronectin). Furthermore, the binding was specifically inhibited by the Arg-Gly-Asp (RGD) peptide or GRGDSPG, while two other analogs (GRGESPG and GRADSPG) were without effect.KEY WORDS: common carp, laminin, Zn-binding protein, Zn-binding specificity and affinity, Zn.

Association of zinc with connective tissue in the digestive tract of common carp

“…It is very possible that when there is more Zn‐binding protein in the fibroblast, the more the collagen and glycosaminoglycans are secreted by the fibroblast. Recently, the authors incorporated the 43 kDa Zn‐binding protein isolated from common carp into liposome, studied its binding characteristics, and found that 65 Zn was bound to the external side of the 43 kDa protein‐liposomes, through SH groups with a Kd of 0.19 µM 17 In the common carp Zn might be bound to the external side of the fibroblast. Binding between the cell and extracellular matrix might be specifically mediated by laminin.…”

“…In the digestive tract of common carp, it seems that the high concentration of Zn comes from the first explanation. Because the 43 kDa Zn‐binding protein is a strong Zn binding agent (Kd = 0.19 µM) and the maximum binding site is approximately 3 mol Zn 2+ per mole, 17 when the Zn leaves intestinal cells, enters the extracellular matrix, it might be bound by the 43 kDa Zn‐binding protein present in the fibroblasts. Before the Zn can enter the blood, most is likely to be adsorbed by the protein.…”

Affinity isolation and mass spectral analysis of 1,10-phenanthroline (OP)-stimulated UV-damaged-DNA binding proteins expressed in zebrafish (Danio rerio) embryos

“…Owing to its Zn-chelating activity, OP has been employed as a probe to determine the importance of Zn as a functional component of metalloproteins (Woods and Roth 1984;Wang and Jeng 2006). Identification of the 30-and 35-kDa polypeptides as homologs of the Lv1 domain of zfVg1 correlated well with the metal-dependent, UV-damaged-DNA binding activities of the two molecules (Lai et al 2006).…”

High zinc in the erythrocyte plasma membranes of common carp Cyprinus carpio

“…The main body of high Zn in animal erythrocyte plasma membranes is not clear, the precise location of Zn in plasma membranes is also unknown 16 . Recently, Jeng and Wang incorporated the 43 kDa Zn‐binding protein isolated from common carp into liposome, and studied its binding characteristics 17 . By treating the protein‐liposome with 1, 10‐phenanthroline, it was found that 65 Zn was bound to the external side of the 43 kDa protein‐liposomes.…”