Localization of Zn-binding protein in the digestive tract tissue of common carp

Abstract

The concentrations of Zn and sulfhydryl (SH) groups in the digestive tract tissue of common carp and some aquatic animals were studied. It was found that Zn and bound SH groups could be used as indicators for detecting the Zn‐binding protein in the digestive tract tissue of common carp. The digestive tract tissue of the fish underwent subcellular fractionation, and it was found that the nuclei/cell debris fraction contained most of the DNA (85%), Na+/K+‐ATPase (82%), organic phosphate (90%) and the Zn‐binding protein (79%), but only part of the 5′‐nucletidase and alkaline phosphatase (<23%). The nuclei/cell debris fraction of the digestive tract tissue of common carp was treated with either collagenase type I or type IV, and subfractionated by sucrose density centrifugation. It was found that treatment with collagenase type IV could release more than 50% of the Zn‐binding protein, Na+/K+‐ATPase and organic phosphate from collagen. Sections of digestive tract tissue of common carp were stained for Zn. It was observed that Zn can be found mainly on the edge of the epithelial layer, and everywhere in the ‘membrane‐like’ portion of the submucosal and muscular layers. It is proposed that most of the Zn‐binding protein in the digestive tract tissue of common carp is located on the basolateral plasma membranes of the epithelial cells and on the surrounding muscle cells that are attached to the collagen type IV of basal laminae.

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

“…The digestive tract of common carp could be divided into five layers in histology (i.e. epithelial layer, lamina propria, submucosal layer, muscular layer, serosal layer) 13 . In fishes, a clear distinction between the connective tissue of the lamina propria and that of the submucosa layer is not possible 14 .…”

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

“…reported that over 90% of the Zn in the nuclei/cell debris fraction of common carp digestive tract tissue could be easily extracted by 1 mM EDTA solution 4 . This EDTA‐extractable Zn was closely related to the bound SH groups of the tissue and it is suggested that the common carp tissue has a significant number of ‘active’ SH groups in its protein that bind to Zn 12 . The present study confirms that Zn is mainly bound to SH groups of the 43 kDa protein, since approximately 90% of the specific binding of Zn was abolished after treatment the protein with PCMB (Fig.…”

Separation and characterization of connective tissue cells expressing 43-kDa zinc-binding protein from common carp

“…The zinc concentration was determined by atomic absorption spectrophotometry (Z‐8100, Hitachi, Tokyo, Japan) as described previously 12 . The zinc concentration in the connective tissue cells of the fish was determined as follows: the connective tissue cell suspension was centrifuged at 2000 × g for 10 min, the precipitated cells were then mixed with 2 mL of 6 mM EDTA dissolved in 50 mM Tris‐HCl buffer, pH 8.0 and centrifuged at 10 000 × g for 10 min.…”