Purification, characterization and molecular cloning of prophenoloxidases from Sarcophaga bullata

Prophenoloxidase (PPO) is a key enzyme associated with both melanin biosynt hesis and sclerotization in insects. This enzyme is involved in three physi ologically 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 precipitatio n, Phenyl Sepharose chromatography, DEAE-Sepharose chromatography, and Seph acryl S-200 column chromatography. The purified enzyme exhibited two closel y moving bands on 7.5% SDS-PAGE under denaturing conditions. From the estim ates of molecular weight on Sephacryl S-100, TSK-3000 HPLC column and SDS-P AGE, which ranged from 90,000 to 100,000, it was inferred that the enzyme i s made up of a single polypeptide chain. Activation of PPO (K-a=40 mu M) wa s achieved by the cationic detergent, cetyl pyridinium chloride below its c ritical micellar concentration (0.8 mM) indicating that the detergent molec ules are binding specifically to the PPO and causing the activation. Neithe r anionic, nor nonionic (or zwitterionic) detergents activated the PPO. The active enzyme exhibited wide substrate specificity and marked thermal unst ability. Using primers designed to conserved amino acid sequences from know n PPOs, we PCR amplified and cloned two PPO genes from the sarcophagid larv ae. The clones encoded polypeptides of 685 and 691 amino acids. They contai ned two distinct copper binding regions and lacked the signal peptide seque nce. They showed a high degree of homology to dipteran PPOs. Both contained putative thiol ester site, two proteolytic activation sites and a conserve d C-terminal region common to all known PPOs. (C) 2000 Elsevier Science Ltd . All rights reserved.