Hydrolysis of synthetic substrate, L-pyroglutamyl p-nitroanilide is catalyzed solely by pyroglutamyl aminopeptidase I in rat liver cytosol.
Pyroglutamyl aminopeptidase I (PAP-I) is a cytosolic cysteine peptidase, which hydrolytically removes the L-pyroglutamate residue from the amino terminus of endogenous proteins and peptides. L-Pyroglutamyl p-nitroanilide serves as the synthetic substrate of this enzyme, while there is a possibility of other hydrolases being involved in the hydrolysis of this xenobiotic substrate. We cloned a full-length cDNA encoding rat PAP-I from a rat liver cDNA library and expressed this cDNA in Escherichia coli to obtain a recombinant PAP-I as a single protein. The cDNA encoded a sequence of 209 amino acids with a calculated molecular weight of 22913 Da. The homology of the deduced amino acid sequence of rat PAP-I was 98.6 and 94.3% to mouse and human PAP-Is, respectively. The biochemical properties of the recombinant rat PAP-I were almost identical to those of the recombinant mouse and human PAP-Is and the purified rat liver cytosolic PAP-I in terms of the molecular weight, subunit structure, affinity to the substrate, inhibitor profile and pH optimum. Immunoblot analysis using an antibody raised against recombinant rat PAP-I showed that rat PAP-I is present almost exclusively in the cytosolic fraction of the rat liver. Moreover, the hydrolyzing activity for L-pyroglutamyl p-nitroanilide in rat liver cytosolic fraction was completely inhibited by the antibody, strongly suggesting that this xenobiotic substrate is hydrolyzed solely by PAP-I.
Abe
Koji
K
Pharmacokinetics and Drug Delivery Research Laboratories, Sankyo Co., Ltd., Hiromachi, Tokyo 140-8710, Japan. koujia@shina.sankyo.co.jp
Watanabe
Nobuaki
N
Kosaka
Toshiyuki
T
Yamada
Makiko
M
Tokui
Taro
T
Ikeda
Toshihiko
T
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002384:Catalysis; D003600:Cytosol / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008099:Liver / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009574:Nitro Compounds / Q000378:metabolism; D011751:Pyroglutamyl-Peptidase I / Q000235:genetics / Q000378:metabolism*; D011761:Pyrrolidonecarboxylic Acid / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344
D009574:Nitro Compounds; D011751:Pyroglutamyl-Peptidase I; D011761:Pyrrolidonecarboxylic Acid