Purification, characterization and molecular cloning of prophenoloxidases from Sarcophaga bullata.
Prophenoloxidase (PPO) is a key enzyme associated with both melanin biosynthesis and sclerotization in insects. This enzyme is involved in three physiologically important processes viz., cuticular hardening, defense reactions and wound healing in insects. It was isolated from the larval hemolymph of Sarcophaga bullata and purified by employing ammonium sulfate precipitation, Phenyl Sepharose chromatography, DEAE-Sepharose chromatography, and Sephacryl S-200 column chromatography. The purified enzyme exhibited two closely moving bands on 7.5% SDS-PAGE under denaturing conditions. From the estimates of molecular weight on Sephacryl S-100, TSK-3000 HPLC column and SDS-PAGE, which ranged from 90,000 to 100,000, it was inferred that the enzyme is made up of a single polypeptide chain. Activation of PPO (K(a)=40 microM) was achieved by the cationic detergent, cetyl pyridinium chloride below its critical micellar concentration (0.8 mM) indicating that the detergent molecules are binding specifically to the PPO and causing the activation. Neither anionic, nor nonionic (or zwitterionic) detergents activated the PPO. The active enzyme exhibited wide substrate specificity and marked thermal unstability. Using primers designed to conserved amino acid sequences from known PPOs, we PCR amplified and cloned two PPO genes from the sarcophagid larvae. The clones encoded polypeptides of 685 and 691 amino acids. They contained two distinct copper binding regions and lacked the signal peptide sequence. They showed a high degree of homology to dipteran PPOs. Both contained putative thiol ester site, two proteolytic activation sites and a conserved C-terminal region common to all known PPOs.
Chase
M R
MR
Department of Biology, University of Massachusetts-Boston, Boston, MA 02125, USA.
Raina
K
K
Bruno
J
J
Sugumaran
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004156:Catechol Oxidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004175:Diptera / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D018076:DNA, Complementary; D004792:Enzyme Precursors; C038451:pro-phenoloxidase; D004156:Catechol Oxidase