Search

Expression of two copper-transporting P1-type ATPases (ATP7A and ATP7B), the CTR1 protein, a high-affinity copper transporter, and ceruloplasmin (CP), a copper-containing ferroxidase. The level of mRNA of these proteins was determined by RT-PCR analysis, the distribution of polypeptides encoded by these genes was determined by immunoblotting, and the type of cells expressing these genes was identified immunohistochemically. It was found that the major product of CP gene in the brain is cell membrane-bound CP. Secretory CP, whose molecule contains the greatest number of weakly associated copper atoms, is synthesized in the vascular plexus. CTR1 mRNA is evenly distributed in the brain; however, its content is twice higher in the vascular plexus. The Atp7a gene is active in all brain sections, whereas the Atp7b gene is active only in the hypothalamus. The membrane-bound CP is expressed in glial cells of all types and in ependyma cells. ATP7b and ATP7a are expressed predominantly in ependyomyocytes and neutrons, respectively. The organization of copper transport in mammalian brain is discussed.

Using immunoblotting method it was found out that ceruloplasmin (Cp) polypeptides are revealed in mitochondria of rats, isolated from brain, liver, testicles and mammary gland. Cp is localized in matrix and inner membranes of mitochondria. Its molecular weight corresponds to the non-glycosilated form of the protein. Computer analysis did not reveal any sequences homologous to the signal peptide for mitochondria protein import (SPMPI) in the rat chromosomal Cp gene. However the analysis of homologous to Cp sequences, presented in databases, detected SPMPI in the human processed Cp pseudogene. Cp pseudogene region of 984 nucleotides long is translated in the only reading frame to the polypeptide of 328 a.a. long including 66 a.a of SPMPI at N-terminus. The predicted protein with the exception of SPMPI is identical to the corresponding Cp fragment at 92%, it has 20 amino acid substitutions, 8 of which are significant. His-X-His motif, typical for copper containing ferroxidases, is located in the centre of a molecule. Potential copper-binding site appears as a result of proline to histidine substitution at 923 position along Cp sequence. The product of Cp pseudogene transcription was detected in the human cultured cells of HepG2 and HuTu 80 cell lines using RT-PCR strategy. 30 kDa polypeptide that interacts with human Cp antibodies was found in mitochondria of HuTu 80 cells. The possible biological role of mitochondrial Cp is under discussion.