Phenoloxidase activity in Apis mellifera honey bee pupae, and ecdysteroid-dependent expression of the prophenoloxidase mRNA.
Phenoloxidase (monophenol, l-dopa: oxygen oxidoreductase, EC 1.14.18.1) is a multicopper oxidase, which plays an important role in melanin synthesis, necessary for defense against intruding microorganisms and parasites, wound healing and cuticle pigmentation. A phenoloxidase from the hemolymph of honey bee pupae exhibited an apparent molecular mass of 70 kDa, as estimated by gel filtration and SDS-PAGE. Optimal pH and temperature were 6.5 and 20 degrees C, respectively. Activity was fully stable for 30 min at 50 degrees C. Like phenoloxidases from the hemolymph of other insects, the honey bee enzyme was activated by trypsin and inhibited by protease inhibitors and phenylthiourea. Only high concentrations of sodium azide effectively inhibited the detected activity. A low concentration (5 microM) of Ca2+, Mg2+, and Mn2+ had a stimulatory effect on the activity. Single Michaelis-Menten curves were observed for l-dopa and dopamine oxidation, but the affinity of the enzyme for dopamine was greater than for L-dopa. Semiquantitative RT-PCR and Southern blot analysis using a 359 bp labeled probe, and quantification of the prophenoloxidase mRNA levels by real-time PCR showed increased amounts of transcripts in hemocytes and integument from young pupae injected with 20-hydroxyecdysone.
Zufelato
Maria Salete
MS
Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, 14040-901 Ribeirão Preto, SP, Brazil.
Lourenço
Anete P
AP
Simões
Zilá L P
ZL
Jorge
João A
JA
Bitondi
Márcia M G
MM
D000818:Animals; D001516:Bees / Q000201:enzymology* / Q000254:growth & development; D004156:Catechol Oxidase / Q000096:biosynthesis*; D004441:Ecdysterone / Q000494:pharmacology / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000096:biosynthesis*; D004795:Enzyme Stability; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D006458:Hemolymph / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D011679:Pupa / Q000201:enzymology; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019810:Sodium Azide / Q000494:pharmacology; D013696:Temperature
D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D008670:Metals; D012333:RNA, Messenger; D004441:Ecdysterone; D019810:Sodium Azide; C038451:pro-phenoloxidase; D004156:Catechol Oxidase; D014442:Monophenol Monooxygenase