Wilson's disease, an autosomal recessive disorder, is characterized by the excessive accumulation of copper in the liver. WND (ATP7B) gene, which encodes a putative copper transporting P-type ATPase, is defective in the patients. To investigate the in vivo function of WND protein as well as its intracellular localization, WND cDNA was introduced to the Long-Evans Cinnamon rat, known as a rodent model for Wilson's disease, by recombinant adenovirus-mediated gene delivery. An immunofluorescent study and a subcellular fractionation study revealed the transgene expression in liver and its localization to the Golgi apparatus. Moreover, since the synthesis of holoceruloplasmin is disturbed in the LongEvans Cinnamon rat, the plasma level of holoceruloplasmin, oxidase-active and copper-bound form, was examined to evaluate the function of WND protein with respect to the copper transport. Consequently, the appearance of holoceruloplasmin in plasma was confirmed by Western blot analysis and plasma measurements for the oxidase activity and the copper content. These findings indicate that introduced WND protein may function in the copper transport coupled with the synthesis of ceruloplasmin and that the Golgi apparatus is the likely site for WND protein to manifest its function.Wilson's disease, an autosomal recessive disorder, is characterized by the excessive accumulation of copper in the liver (1). This phenomenon is thought to be due to reduced biliary excretion of copper and disturbed incorporation of copper into ceruloplasmin (CPN).1 Hepatic copper at toxic levels causes liver cirrhosis, and extrahepatic copper toxicity occurs especially in the brain due to the released copper from the damaged liver. WND (officially designated ATP7B), identified as the gene responsible for this disease, encodes a putative copper transporting P-type ATPase (2-5). The observations of single base changes or small deletions within WND of Wilson's disease patients have already been reported (3, 6).The Long-Evans Cinnamon (LEC) rat, known as an animal model for Wilson's disease, shows some of the clinical features similar to Wilson's disease, including hepatic copper accumulation, reduced biliary copper excretion, reduced copper in plasma, and a remarkable decrease of serum CPN activity (7,8). Atp7b, the rat gene homologous to WND, has been cloned, and a partial deletion at the 3Ј end in this gene is reported in the LEC rat (9). It is also known that the expression of Atp7b mRNA is absent in the LEC rat (10).CPN, a blue copper oxidase in plasma, contains 90 -95% of plasma copper. This protein is synthesized mainly in hepatocytes and secreted into plasma with 6 atoms of copper per molecule as the oxidase active holoprotein (11-13). The reduced levels of oxidase activity of CPN in the circulation of Wilson's disease patients and LEC rats is due to the secretion of apoceruloplasmin, copper-free and oxidase-inactive form, resulting from the disturbed incorporation of copper atoms into the protein (1, 14), while the intracellular synthesis of CPN...

A novel method useful for selective isolation of the C-terminal peptide from a tryptic digestion mixture of a protein has been developed by taking advantage of a unique property of anhydrotrypsin, which has a strong specific affinity for the peptides containing arginine or lysine at their C-termini. Briefly, peptides produced by tryptic digestion of a protein are fractionated by affinity chromatography on a column of immobilized anhydrotrypsin. The C-terminal peptide is recovered in a breakthrough fraction, while the remainders are adsorbed on the column (unless the protein ends in arginine or lysine). The breakthrough fraction is then subjected to reversed-phase high-performance liquid chromatography in order to purify the C-terminal peptide. Using this method, we have successfully isolated the C-terminal peptides from tryptic digests of the sheath protein (gp 18) and the tube protein (gp 19) of bacteriophage T4. The analytical results on these peptides, together with the information on the N-terminal structures of the original proteins and on the nucleotide sequences of genes 18 and 19, allowed us to establish the complete primary structures of the two proteins.

The nucleotide sequence of gene 18 of bacteriophage T4 was determined by the Maxam-Gilbert method, partially aided by the dideoxy method. To confirm the deduced amino acid sequence of the tail sheath protein (gpl8) that is encoded by gene 18, gpl8 was extensively digested by trypsin or lysyl endopeptidase and subjected to reverse-phase high-performance liquid chromatography. Approximately 40 peptides, which cover 88% of the primary structure, were fractionated, the amino acid compositions were determined, and the corresponding sequences in DNA were identified. Furthermore, the amino acid sequences of 10 of the 40 peptides were determined by a gas phase protein sequencer, including N-and C-terminal sequences. Thus, the complete amino acid sequence of gp18, which consists of 658 amino acids with a molecular weight of 71,160, was determined. * Corresponding author. "Achromobacter lyticus" M467-1, was purchased from Wako Junyaku Co. Polynucleotide kinase and all of the restriction enzymes used were purchased from Takara Shuzo Co. Methanesulfonic acid (4 M) was purchased from Pierce Chemical Co., and all other reagents were analytical grade, from either Nakarai Chemicals or Wako Junyaku Co. Nutrient broth, tryptone, yeast extract, Bacto-Agar, and Casamino Acids were purchased from Difco Laboratories. Medium and buffer. M9A medium was used to grow E. coli. It contained, per liter of water, 6 g of Na2HPO4, 3 g of KH2PO4, 0.2 g of MgSO4. 7H20, 0.5 g of NaCl, 1 g of NH4C1, 4 g of glucose, and 10 g of Casamino Acids. Phages were stored in B+ buffer, which contained, per liter of water, 7 g of Na2HPO4, 4 g of NaCl, and 3 g of KH2PO4, supplemented with 1 mM MgSO4 for use. Phage, plasmids, and bacterial strains. T4D.23amH11 is from our collection. T4D.18am mutants (Fig. 1) were kindly supplied by W. B. Wood and J. King. E. coli BE was used as a nonpermissive host for amber mutant phages, and E. coli CR63 was the permissive host. E. coli JM103 was used to propagate M13 phages and to prepare replicative-form DNA of M13 phage. Three E. coli BE strains, which carry plasmids p662, p655, and p664, respectively, were kind gifts from Tom Mattson. Each of the three plasmids contains an EcoRI fragment which forms approximately one-third of gene 18 (Fig. 1). Marker rescue test. Marker rescue of T4D.18am mutants by plasmids was performed as described by Mattson et al. (21). The results are also shown in Fig. 1. DNA preparations for sequencing. DNA preparations for the Maxam-Gilbert method have been described by Christensen and Young (8). DNA was 5' end labeled with polynucleotide kinase, and the strands were then separated by polyacrylamide gel electrophoresis, or alternatively, the double-stranded DNA was cut with a restriction enzyme and the desired fragment was purified by polyacrylamide gel electrophoresis. DNA sequencing procedures. The Maxam-Gilbert method was done essentially as described by Maxam and Gilbert (22). The dideoxy method was carried out as described by Messing (23). Preparation of gpl8. Gpl8 was prepared as described ...

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