Reexamination of properties of prophenoloxidase isolated from larval hemolymph of the silkworm Bombyx mori.
Prophenoloxidase in hemolymph of the silkworm (Bombyx mori) was purified by the method of Ashida (Ashida, M. (1971) Arch. Biochem. Biophys. 144, 749-762) with slight modifications to further increase the purity, and its properties were reinvestigated. The purified prophenoloxidase gave two discrete bands in isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) (pI 4.95 and 4.98) and in native-polyacrylamide gel electrophoresis with 4.5% separating gel. Each band in IEF-PAGE was separated into two bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with mobilities corresponding to 71.5- and 71-kDa polypeptides. In HPLC on octadecyl column the prophenoloxidase preparation gave two well-separated symmetrical peaks (proPO polypeptide I and proPO polypeptide II). The molecular masses of the proPO polypeptides I and II were determined to be 71.5 and 71 kDa in SDS-PAGE and 78,880 and 81,105 Da by matrix-assisted laser desorption ionization mass spectrometry, respectively. Native prophenoloxidase was eluted at a position corresponding to 126-kDa protein in gel permeation chromatography. Amino acid compositions and peptide mappings of proPO polypeptides indicated that both polypeptides differ in their primary structures. These results are discussed in relation to the subunit structure, the presence of bicopper cluster, and the polymorphism of prophenoloxidase in silkworm hemolymph.
Yasuhara
Y
Y
Biochemical Laboratory, Hokkaido University, Sapporo, Japan.
Koizumi
Y
Y
Katagiri
C
C
Ashida
M
M
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D012831:Bombyx / Q000201:enzymology*; D002241:Carbohydrates / Q000032:analysis; D004156:Catechol Oxidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006458:Hemolymph / Q000201:enzymology*; D007525:Isoelectric Focusing; D007814:Larva / Q000201:enzymology; D015394:Molecular Structure; D008970:Molecular Weight; D010449:Peptide Mapping; D011110:Polymorphism, Genetic; D011487:Protein Conformation
D000596:Amino Acids; D002241:Carbohydrates; D004792:Enzyme Precursors; C038451:pro-phenoloxidase; D004156:Catechol Oxidase