Molecular cloning of insect pro-phenol oxidase: a copper-containing protein homologous to arthropod hemocyanin.
Pro-phenol oxidase [pro-PO; zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)] is present in the hemolymph plasma of the silkworm Bombyx mori. Pro-PO is a heterodimeric protein synthesized by hemocytes. A specific serine proteinase activates both subunits through a limited proteolysis. The amino acid sequences of both subunits were deduced from their respective cDNAs; amino acid sequence homology between the subunits was 51%. The deduced amino acid sequences revealed domains highly homologous to the copper-binding site sequences (copper-binding sites A and B) of arthropod hemocyanins. The overall sequence homology between silkworm pro-PO and arthropod hemocyanins ranged from 29 to 39%. Phenol oxidases from prokaryotes, fungi, and vertebrates have sequences homologous to only the copper-binding site B of arthropod hemocyanins. Thus, silkworm pro-PO DNA described here appears distinctive and more closely related to arthropod hemocyanins. The pro-PO-activating serine proteinase was shown to hydrolyze peptide bonds at the carboxyl side of arginine in the sequence-Asn-49-Arg-50-Phe-51-Gly-52- of both subunits. Amino groups of N termini of both subunits were indicated to be N-acetylated. The cDNAs of both pro-PO subunits lacked signal peptide sequences. This result supports our contention that mature pro-PO accumulates in the cytoplasm of hemocytes and is released by cell rupture, as for arthropod hemocyanins.
Kawabata
T
T
Osaka Research Laboratories, Wako Pure Chemical Industries, Ltd., Hyogo, Japan.
Yasuhara
Y
Y
Ochiai
M
M
Matsuura
S
S
Ashida
M
M
D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D003300:Copper / Q000032:analysis*; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000737:chemistry*; D006433:Hemocyanins / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000096:biosynthesis* / Q000737:chemistry*; D016366:Open Reading Frames; D010802:Phylogeny; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D003300:Copper; D006433:Hemocyanins; D014442:Monophenol Monooxygenase