Purification and Characterization of Prophenoloxidases from Pupae of Drosophila melanogaster11

Abstract

Two isoforms of prophenoloxidase were isolated from pupae of Oregon-R strain of Drosophila melanogaster. The purification procedure included ammonium sulfate fractionation, Sephacryl S-200 gel chromatography, DEAE-cellulose, and hydroxylapatite column chromatography. The two isoforms, A1 and A3, could be separated by ammonium sulfate fractionation. The isoelectric points of A1 and A3 were determined to be pH 5.8 and 6.7, respectively. The molecular weights of the monomers of A1 and A3 were estimated by SDS-PAGE to be 78 and 77 kDa, respectively. The native states of A1 and A3 are considered to be homodimeric, as judged by gel-filtration chromatography.

Nucleotide sequence of the cDNA encoding the proenzyme of phenol oxidase A1 of Drosophila melanogaster.

“…2). The calculated Mr of 78,903 agrees well with that of the purified protein, which has been estimated by SDS/PAGE (Mr = 78,000) (8). Amino acid sequences of 14 peptides corresponded to the protein sequence deduced from the cDNA sequence, except for 6 residues: Met-12, Asn-422, Asn-425, Asn-430, Asp-432, and Asn-437.…”

“…pro-PO A1 was purified from pupae, 24-48 hr after pupariation, as described (8). Because the NH2-terminal amino acid of pro-PO A1 was found to be blocked, the purified protein was fragmented to obtain partial amino acid sequences.…”

“…Activation of the pro-PO was first found in the fruit fly Drosophila melanogaster (2,3). The pro-PO has been isolated in a homogeneous state from the silkworm Bombyx mori (4), the tobacco hornworm Manduca sexta (5), the saturniid Hyalophora cecropia (6), the housefly Musca domestica (7), and the fruit fly D. melanogaster (8).…”

“…Two molecular forms, A1 and A3, have been isolated from pupae and well characterized (8). The gene encoding A1 (Mox) is mapped at 79.6 on the right arm of chromosome 2, and the deletion mapping indicates the cytological position at 55A-B (10).…”

Identification of copper ligands in Aspergillus oryzae tyrosinase by site-directed mutagenesis

“…The oxidase activity of the activated recombinant tyrosinase with 10 mM -dopa was also determined at pH 5.5 and 40 mC by measuring A %(& with the same spectrophotometric method [27].…”