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 ⁶⁵Zn to the 43 kDa protein-liposomes, and the binding of the ⁶⁵Zn-labeled 43 kDa protein-liposomes to laminin were studied. It was found that ⁶⁵Zn was bound to the external side of the 43 kDa protein-liposomes. Specific binding of ⁶⁵Zn 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 Zn²⁺/mole); and equilibrium dissociation constant (Kd), 0.19 µM. Of the cations introduced (Ca²⁺, Cd²⁺,Co²⁺, Cr²⁺, Cu²⁺, Fe²⁺, Hg²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pb²⁺), only Co²⁺ 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.     

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

ABSTRACT:   Zinc (Zn) concentration in the digestive tract of common carp is always >10 times higher than most animal tissues. In a previous paper, it was reported that this high Zn came from a 43 kDa Zn-binding membrane protein. In this present study it was further found that in the digestive tract of common carp, Zn content was closely associated with the amounts of extracellular macromolecules. The higher the Zn content, the more there is of collagen and glycosaminoglycans. An indirect immunoperoxidase staining method using antibody against the 43 kDa Zn-binding protein, or the fibroblast marker (Thy 1.1 protein) was applied to the sections of digestive tract of fish. It was found that the Zn-binding protein in the digestive tract of common carp is mainly located in the connective tissue of its lamina propria and submucosal layer. Connective tissue cells, probably fibroblasts, hold the 43 kDa Zn-binding protein. In the common carp Zn might be bound to the external side of the fibroblast. The present finding may have a significant meaning on extending the studies of Zn in biology to the field of Zn with extracellular matrix.     

Seasonal variations of free amino acids and nucleotide-related compounds in the muscle of cultured Taiwanese puffer Takifugu rubripes

SUMMARY: Seasonal variation in the level of extractive nitrogenous components was investigated in the muscle of puffer Takifugu rubripes cultured in two different areas of Taiwan. There were no seasonal and regional variations in the proximate composition of the fish. Of the free amino acids (FAA) in the muscle of puffer, the predominant one was taurine, followed by glycine, lysine, and alanine. Among nucleotide-related compounds, inosine monophosphate (IMP) and guanosine monophosphate (GMP) were the most prominent compounds. The total nucleotide-related compounds in the muscle of puffer were higher in July and November than those in the other months. The level of total taste-active components including glycine, alanine, arginine, GMP, IMP, and adenosine 5'-monophosphate was much higher in the muscle of puffer collected from July to January. Therefore, the puffer is probably more palatable in these periods.     

Role of high zinc levels in the stress defense of common carp

We studied the link between high zinc levels and the extreme stress tolerance of common carp. Fish under stress showed much higher plasma cortisol levels than controls. Stress or cortisol injection induced large changes in zinc levels in the common carp but not in grass carp, silver carp or tilapia. The effect of 5 days of anoxia and 4 subsequent days of recovery on cortisol and zinc contents in the common carp was investigated. Elevated plasma cortisol resulting from anoxia was correlated with decreased zinc in digestive tract tissue and increased zinc in the head kidney. Zinc was mobilized in the common carp while under stress. Changes in cortisol and zinc contents were reversible during the subsequent recovery from anoxia. Under stress, protein-bound zinc levels increased in the head kidney cell nuclei of common carp as cortisol increased. Zinc and cortisol were bound to the same protein, which was bound to DNA. The protein is likely a glucocorticoid receptor. An increase in immature red blood cells in stressed common carp was observed. Zinc was involved in the stress erythropoiesis response. Zinc may play an important role in stress defense in the common carp via the glucocorticoid receptor.     

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

ABSTRACT:   The distribution of zinc (Zn) concentration in the blood and erythrocytes of common carp, grass carp, silver carp and tilapia was studied. It was found that in whole blood, the average Zn concentrations in these four species of fish (approx. 6–14 μg/[ml whole blood]) were relatively similar to those in other species of fish and mammals. However, the mean Zn concentration in the erythrocytes of common carp is approximately two times higher than the other three species of fish (approx. 5 vs approx. 2 μg/[ml whole blood]). It was found that approximately 70% of the Zn in the common carp whole blood came from its erythrocytes. In addition, approximately 43% of the Zn in the erythrocytes of common carp was found to be located on its outer plasma membranes. When an antibody against a 43 kDa Zn-binding protein, isolated from the digestive tract tissue of common carp, was used, significant quantities of the protein were shown to be present on the erythrocyte plasma membranes of common carp by an indirect immunofluorescent staining. High Zn on the outer plasma membrane of the common carp erythrocyte most probably comes from the 43 kDa Zn-binding protein.     

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.     

Paralytic toxins in Taiwanese crab Xanthias lividus

ABSTRACT: Paralytic toxicity was detected by tetrodotoxin (TTX) bioassay in four and five specimens of new toxic xanthid crab Xanthia lividus collected from Lanyu, south-east Taiwan in April 1999 and Hsiaoliuchiu south-west Taiwan in November 1999, respectively. The average toxicity of crab specimens collected from Lanyu and Hsiaoliuchiu was 230 ± 94 (mean ± SD) mouse units (MU) and 241 ± 114 MU, respectively. The toxin was partially purified by YM-1 membrane ultrafiltration and Bio-Gel P-2 column chromatography. Electrophoresis, high-performance liquid chromatography and gas chromatography–mass spectroscopy analyses showed that the crab toxin was composed mainly of tetrodotoxin (83%), as well as a small amount of gonyautoxin 1-4 (17%).