cDNA cloning and expression of chicken aminopeptidase H, possessing endopeptidase as well as aminopeptidase activity.
Chicken aminopeptidase H is a cysteine protease possessing endopeptidase as well as aminopeptidase activity [Rhyu, M. R., Nishimura, T., Kato, Y., Okitani, A. & Kato, H. (1992) Eur. J. Biochem. 208, 53-59]. This enzyme exhibits molecular masses of 400 kDa on gel filtration and 52 kDa on SDS/PAGE, indicating that it consists of eight subunits with the same molecular mass. In the current study, we cloned the cDNA for the catalytic subunit of chicken aminopeptidase H. The open reading frame of the aminopeptidase H gene consists of 1362 base pairs encoding a 52-kDa protein consistent with the molecular mass determined on SDS/PAGE; the deduced amino acid sequence contains all the partial sequences determined for the purified enzyme. The sequence is similar to that of the bleomycin hydrolase of rabbit lung, which has been partially determined. The recombinant 52-kDa protein expressed in COS7 cells exhibited both aminopeptidase and endopeptidase activities, which were inhibited by monoiodoacetic acid. Furthermore, the expression of aminopeptidase H in COS7 cells was also recognized on immunoblotting. This gene is the first one for aminopeptidase H in an animal tissue whose sequence has been completely determined.
Adachi
H
H
The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Japan.
Tsujimoto
M
M
Fukasawa
M
M
Sato
Y
Y
Arai
H
H
Inoue
K
K
Nishimura
T
T
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002645:Chickens; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology; D011817:Rabbits; D016415:Sequence Alignment; D014162:Transfection
D018076:DNA, Complementary; D010450:Endopeptidases; D000626:Aminopeptidases; C030596:hydrolase H