Purification and characterization of two insecticyanin-type proteins from the larval hemolymph of the Eri-silkworm, Samia cynthia ricini.
Two different biliverdin-binding proteins, designated BBP-I and BBP-II, were purified from the larval hemolymph of the Eri-silkworm, Samia cynthia ricini. These proteins were readily isolated from the hemolymph of fifth instar larvae using two chromatographic steps, hydrophobic interaction chromatography and ion exchange chromatography. Both BBPs were easily separated by Q-Sepharose HP column chromatography. BBP-I has an apparent molecular weight of 24 kDa, as determined by gel-filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Native BBP-II had a molecular weight of 48 kDa estimated by gel-filtration. SDS-PAGE revealed a single band with a molecular weight of 26 kDa. Moreover, the molecular weights of BBP-I and BBP-II were determined to be 20,468 and 22,708 by MALDI-TOF/MS (matrix-assisted laser desorption ionization-time of flight/mass spectrometry), respectively. On this basis, BBP-I and BBP-II molecules are assumed to be a monomer and a dimer, respectively. The blue color of BBPs collected from the hemolymph is attributed to the presence of biliverdin IX gamma, which is non-covalently and stoichiometrically bound to the apoprotein, based on absorbance maxima at 359 and 695 nm in methanol:HCl (95:5, v/v). One molecule of BBP-I contains one molecule of biliverdin IX gamma, whereas BBP-II contains two molecules of biliverdin IX gamma. The amino acid compositions of BBP-I and BBP-II are different, although the N-terminal sequences of both BBPs have a 48% identity. These BBPs were found in the hemolymph of fourth and fifth instar larvae. The newly molted fifth instar larvae had the highest concentration of BBP-I in the hemolymph. This gradually decreased during larval development. In contrast to BBP-I, the level of BBP-II was low, and increased slightly at the same developmental stage in S. cynthia ricini larvae.
Saito
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Department of Insect Genetics and Breeding, National Institute of Sericultural and Entomological Science, Ibaraki, Japan. saito 13@ss.nises.affrc.go.jp
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D012831:Bombyx / Q000254:growth & development / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006458:Hemolymph / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007447:Invertebrate Hormones / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007814:Larva / Q000254:growth & development / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
D000596:Amino Acids; D002352:Carrier Proteins; D019476:Insect Proteins; D007447:Invertebrate Hormones; C043409:insecticyanin protein, insect