Purification and molecular cloning of rat 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase.
2-Amino-3-carboxymuconate-6-semialdehyde decarboxylase (ACMSD; EC 4.1.1.45) is one of the important enzymes regulating tryptophan-niacin metabolism. In the present study, we purified the enzyme from rat liver and kidney, and cloned the cDNA encoding rat ACMSD. The molecular masses of rat ACMSDs purified from the liver and kidney were both estimated to be 39 kDa by SDS/PAGE. Analysis of N-terminal amino acid sequences showed that these two ACMSDs share the same sequence. An expressed sequence tag (EST) of the mouse cited from the DNA database was found to be identical with this N-terminal sequence. Reverse transcription-PCR (RT-PCR) was performed using synthetic oligonucleotide primers having the partial sequences of the EST, and then cDNAs encoding rat ACMSDs were isolated by using subsequent 3'-rapid amplification of cDNA ends and RT-PCR methods. ACMSD cDNAs isolated from liver and kidney were shown to be identical, consisting of a 1008 bp open reading frame (ORF) encoding 336 amino acid residues with a molecular mass of 38091 Da. The rat ACMSD ORF was inserted into a mammalian expression vector, before transfection into human hepatoma HepG2 cells. The transfected cells expressed ACMSD activity, whereas the enzyme activity was not detected in uninfected parental HepG2 cells. The distribution of ACMSD mRNA expression in various tissues was investigated in the rat by RT-PCR. ACMSD was expressed in the liver and kidney, but not in the other principal organs examined.
Tanabe
Atsushi
A
Graduate School of Science and Technology, Chiba University, 648 Matsudo, Matsudo, Chiba 271-8510, Japan. atanabe@i.bekkoame.ne.jp
Egashira
Yukari
Y
Fukuoka
Shin-Ichi
S
Shibata
Katsumi
K
Sanada
Hiroo
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D002262:Carboxy-Lyases / Q000235:genetics* / Q000302:isolation & purification*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D020224:Expressed Sequence Tags; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013997:Time Factors; D014018:Tissue Distribution; D014162:Transfection
D000596:Amino Acids; D018076:DNA, Complementary; D012333:RNA, Messenger; D002262:Carboxy-Lyases; C024396:aminocarboxymuconate-semialdehyde decarboxylase