Purification and characterization of myrosinase from the cabbage aphid (Brevicoryne brassicae), a brassica herbivore.
Aphids are among the most serious insect pests of agricultural crops in the world. They often have specific hosts, and the cabbage aphid (Brevicoryne brassicae) is a specialist on Cruciferae. It has previously been described that certain insects contain the enzyme myrosinase (EC 3.2.3.1), which is considered an important defence enzyme of crucifers. Myrosinase was purified to homogeneity from cabbage aphid soluble extracts using anion-exchange and phenyl-Sepharose chromatography. The protein has an apparent subunit molecular mass of 57-58 kDa and is a dimer. The isoelectric point is 4.9 and the enzyme has a temperature optimum around 40 degrees C. The enzyme was active towards the glucosinolates tested, sinigrin and glucotropaeolin, but was inhibited by ascorbate at concentrations that normally activate plant myrosinases. Using sinigrin as the substrate Km was determined as 0.41 mM, and the kcat as 36 s(-1). With glucotropaeolin the Km and kcat values were determined as 0.52 mM and 22.8 s(-1), respectively. The enzyme was stable upon storage at 4 degrees C for many months, but lost some activity upon freezing. The insect myrosinase did not cross-react with antibodies raised to plant myrosinase. Peptide sequencing of a tryptic digest of the protein showed homology to beta-glucosidases. The presence of myrosinase in an insect pest specialist may be an example of a coevolution process that facilitates host specialization.
Pontoppidan
B
B
Department of Plant Biology, Uppsala Genetic Centre, Swedish University of Agricultural Sciences, Uppsala, Sweden. Bo.Pontoppidan@vbio.slu.se
Ekbom
B
B
Eriksson
S
S
Meijer
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001042:Aphids / Q000201:enzymology*; D001205:Ascorbic Acid / Q000737:chemistry; D015153:Blotting, Western; D001937:Brassica; D004591:Electrophoresis, Polyacrylamide Gel; D005961:Glucosinolates / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013696:Temperature; D001617:beta-Glucosidase / Q000235:genetics
D005961:Glucosinolates; D019476:Insect Proteins; D006026:Glycoside Hydrolases; C022739:thioglucosidase; D001617:beta-Glucosidase; D001205:Ascorbic Acid