Characterization of aminopeptidase N from the brush border membrane of the larvae midgut of silkworm, Bombyx mori as a zinc enzyme.
Three GPI-anchored proteins, aminopeptidase N, alkaline phosphatase and alkaline phosphodiesterase I were released from the midgut brush border membrane of Bombyx mori by phosphatidylinositol-specific phopholipase C and the aminopeptidase N was purified to a homogeneous state. N-terminus and 6 internal sequences, one of which possessed part of zinc-binding motif, showed homology with those from other species. The zinc content in purified aminopeptidase N was estimated as approximately 0.72 mol/mol of the protein and 1,10-phenanthroline completely inhibited the enzyme activity, suggesting zinc requirement for the activity. The aminopeptidase N activity was inhibited not only by probestin and actinonin, but also strongly depressed by amastatin, while leuhistin and bestatin were less inhibitory. These suggest that the active site of aminopeptidase N might be structurally different from those of mammals. Calcium and magnesium ions stimulated the aminopeptidase N activity, but copper ion was rather inhibitory. Zinc ion showed bi-modal effect on the activity, i.e., stimulatory at low concentration, but inhibitory at higher than 100 microM. This inhibition was completely restored by EDTA. These results suggest that the aminopeptidase N possesses two zinc ion-binding sites with high and low affinity as essential and inhibitory one, as well as some regulatory metal-binding sites.
Hua
G
G
Department of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Nagoya City University, Aichi, Japan.
Tsukamoto
K
K
Taguchi
R
R
Tomita
M
M
Miyajima
S
S
Ikezawa
H
H
D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000201:enzymology*; D018826:CD13 Antigens / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D008666:Metalloendopeptidases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism
D018826:CD13 Antigens; D008666:Metalloendopeptidases; D015032:Zinc