| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1989: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1988: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
In the present study, it was difficult to isolate the cDNA coding for arylalkylamine N-acetyltransferase (NAT), which is concerned in the melatonin biosynthesis. However, we could clone two new cDNAs coding for arylamine NATs from the chicken pineal gland by screening a pineal cDNA library using the cDNA of liver NAT as a probe. The nucleotide sequences of these cDNAs were determined, from which amino acid sequences were deduced. Both cDNAs coded for 290 amino acids. Similarities in amino acid sequences were about 60% between p- NAT-3, p-NAT-10 and liver NATs. Northern blot analysis indicated that p-NAT-3 cDNA detected intensively a 2.2-kb band with a poly(A)^+ RNA from the brain and gut, while p-NAT-10 cDNA hybridized only with the poly(A)^+ RNA from the kidney. Genomic Southern blot analysis showed that p-NAT-3, p-NAT-10 and liver NATs were encoded in a separate single gene. Properties of the enzymes expressed in the transfected cells were compared with NATs from the pineal gland, br
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ain and kidney. On a DEAE-cellulose column, the kidney and p-NAT enzymes appeared in an effluent fraction, whereas the brain and p-NAT-3 enzymes were eluted from the column with a gradient elution at 0.08 M NaCl. Supernatant of the pineal gland obtained in the daytime showed two peaks appearing in an effluent fraction and an eluate fraction at 0.08 M NaCl. Substrate specificities of these enzymes were examined with some amine substrates. All the enzymes preferred arylamines to arylalkylamines, indicating that both p-NAT-3 and p-NAT-10 cDNAs encoded arylamine NAT.
There were very similar amino acid sequences between p-NAT-3, p-NAT-10 and liver NATs. Among them, especially interesting is the sequence from amino acids 61-70, in which four basic amino acids were located three-amino acids apart from cysteine residue. It is proposed that the catalytic site of NAT should contain a cysteine residue and might be rich in basic amino acids. Therefore, the use of the cDNAs of pineal arylamine NATs isolated here and the oligonucleotides for the putative catalytic site of the enzyme might be promising ways to clone the cDNA of arylalkylamine NAT in the pineal gland. Less
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