| Copper is a trace element for all living organisms because this metal serves as a cofactor for activating numerous enzymes critical for homeostasis. When copper exceeds the cellular needs, it is toxic through the production of highly reactive hydroxyl radicals, that have deleterious effects on cellular components including destabilization of plasma membranes and lysosomal membranes. Thus organisms have e-volved various mechanisms to obtain and distribute copper safely and to excrete the excess. Some of these copper homeostatic mechanisms have identified, the advances in knowledge have been catalyzed by the identification of the genes involved in human disorders of copper transport. Wilson' s disease is the consequence of toxic accumulation of copper initially in the liver and later in extrahepatic sites, this disorder is inherited in autosomal recessive manner and is present in 1 in 30000 individuals in all populations. Hepatic copper at toxic levels induces hepatocellular injury, and the released copper from the affected liver causes extrahepatic toxicity in the brain, kidneys and eyes.Up to now little is known about copper excretion from hepatocyte to bile, and this process mobilizes 3/4 of absorbed copper in hepatocyte. Searching for novel proteins involved in copper transport willhelp us to resolve the various cellular pathways for metal transport, and may contribute to better understanding of the intracellular transport of rare metal homeostasis in the body.Materials and Methods1. Isolation of lysosomal fraction from 10 week-old wister rat liver by canullation,homogenization and serious of centrifugation.2. Purification copper binding protein using G - 25 column and copper chelating column.3. Analysis of copper binding protein by SDS - PAGE, three main protein bands observed. The main bands were digested by lysyl endopeptidase and isolate different peptides by HPLC.4. Amino acid sequencing and homology search.5. Preparation of rabbit anti rat lysosomal copper binding protein polyclonal antibody.Result1. We isolated .a novel copper binding protein ( 58 KD).2. We use the novel copper binding protein as antigen, prepared rabbit anti rat copper binding protein polyclonal antibody.DiscussionRecently, a number of proteins, that play significant roles in intracellular copper homeostasis, were identified in various species, ranging from bacteria to humans. These proteins can be classified intocopper chaperons and copper transporters. Copper chaperons deliver copper to specific cytoplasmic compartments and target proteins, preventing inappropriate interactions of copper with other cellular components. Members of copper chaperons include CopZ in Enterococcus hirae, Atx1, CCS, and Cox17 in yeast, HAH1,hCCS and hCOX in humans. On the other hand, copper transporters which are integral membrane proteins mediate cellular uptake and export of copper from cells. Identification of two human copper transporters distributed to understand and reveal the mechanism of Wilson' s disease and Menkes disease.Copper is essential trace metal for living organisms, serves as a cofactor for activating numerous enzymes, including chromosome C ox-idase (a component of mitochondrial electron transport chain) , super-oxide dismutase ( part of the protection against reactive oxygen species) , and lysyl oxidase, which is needed for the crosslinking of collagen and elastin. But more or less than normal copper concentration will lead to diseases, the most typical diseases are Wilson' s disease and Menkes disease. So it is important to reveal the mechanism of uptake and release of copper in hepatocyte, we aimed to purify a novel copper binding protein from rat liver lysosomal fraction. Researches show that copper binding proteins have conserved amino acid sequences - MXCXXC ( X is any amino acid) , can bind with copper. For example ATP7B has five MXCXXC sequence, and it was determined 1 mol ATP7B can interact 5 mol of copper ions. If the novel protein we purified has MXCXXC sequence, and... |
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