Purification and characterization of prophenoloxidase from the hemolymph of coleopteran insect, Holotrichia diomphalia larvae.
Prophenoloxidase (pro-PO), a precursor of phenol oxidase (PO), was purified from the hemolymph of coleopteran Holotrichia diomphalia larvae. The enzyme was purified to apparent homogeneity, in six steps of chromatography, using Sephadex G-100, CM-52, Dextran-sulfate Sepharose CL-6B, Phenyl Sepharose CL-4B, Sephacryl S-200, and a Mono-Q column. The preparation exhibited a single band on SDS-PAGE. The proenzyme had a molecular weight of 158 kDa, as estimated by gel filtration. On SDS-PAGE under reducing conditions, it gave a 79 kDa band, indicating that it forms a dimer of the 79 kDa protein. On the other hand, the purified pro-PO gave two well-separated peaks, named pro-PO-1 and pro-PO-2, on reverse-phase HPLC. Amino acid compositions of both proteins were indistinguishable, thereby suggesting the presence of an allelic variant or an isoprotein. On dextran-sulfate Sepharose CL-6B chromatography, a fraction containing prophenoloxidase activating enzyme(s) (PPAE fraction), free from pro-PO, was also separated. In reconstitution experiments, the activation of purified pro-PO by PPAE fraction, was observed in the presence of 5 mM Ca2+, with a specific limited proteolysis. The NH2-terminal sequence of generated PO was determined to be NH2-Phe-Gly-Glu-Asp-Asp-. The activated PO oxidized o-diphenols but did not oxidize mono-phenol and p-diphenol substrates. The purified pro-PO was not activated by trypsin, alpha-chymotrypsin, and SDS.
Kwon
T H
TH
College of Pharmacy, Pusan National University, Korea.
Lee
S Y
SY
Lee
J H
JH
Choi
J S
JS
Kawabata
S
S
Iwanaga
S
S
Lee
B L
BL
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004156:Catechol Oxidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001517:Coleoptera / Q000201:enzymology*; D019281:Dimerization; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D006458:Hemolymph / Q000201:enzymology; D007814:Larva / Q000201:enzymology; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D000596:Amino Acids; D004792:Enzyme Precursors; C038451:pro-phenoloxidase; D004156:Catechol Oxidase