Identification and characterization of proteases involved in specific proteolysis of vitellogenin and yolk proteins in salmonids.
A pepstatin A-sensitive enzyme involved in yolk formation was purified from the masu salmon (Oncorhynchus masou) ovary using in vitro generation of yolk proteins from purified vitellogenin to assay enzymatic activity. Purification of the enzyme involved precipitation of ovarian extracts by water and ammonium sulfate followed by five steps of column chromatography. After SDS-PAGE and Western blotting, the purified enzyme appeared as a single approximately 42 kDa band that was immunoreactive to anti-human cathepsin D. The course of proteolytic cleavage of the three major yolk proteins (lipovitellin, beta'-component, and phosvitin) in fertilized masu salmon and Sakhalin taimen (Hucho perryi) eggs and embryos was visualized by SDS-PAGE and Western blotting using specific antisera. Major yolk protein bands appeared in positions corresponding to 92 kDa, 68 kDa, and 22 kDa (lipovitellin-derived peptides), as well as 17 kDa (beta'-component). During embryo development, the 92 kDa and 22 kDa bands gradually decreased in intensity, becoming undetectable in alevins. The 68 kDa band and a minor 24 kDa band became more intense after the eyed stage. Two additional peptides, corresponding to 40 and 28 kDa, newly appeared in alevins. During embryonic growth, the beta'-component band (17 kDa) persisted and phosvitin appeared to be progressively dephosphorylated. In vitro analysis of lipovitellin proteolysis indicated that the enzyme involved is a Pefabloc SC-sensitive serine protease. These results demonstrate, for the first time, that a cathepsin D-like protease and serine proteases play key roles in yolk formation and degradation, respectively, in salmonid fishes.
Hiramatsu
Naoshi
N
Division of Marine Bioscience, Graduate School of Fisheries Science, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan.
Ichikawa
Norimasa
N
Fukada
Haruhisa
H
Fujita
Toshiaki
T
Sullivan
Craig V
CV
Hara
Akihiko
A
D000818:Animals; D015153:Blotting, Western; D004527:Egg Proteins / Q000096:biosynthesis*; D004530:Egg Yolk / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000201:enzymology; D047108:Embryonic Development; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D005260:Female; D017683:Oncorhynchus / Q000502:physiology*; D010053:Ovary / Q000201:enzymology* / Q000502:physiology; D014819:Vitellogenins / Q000096:biosynthesis*
D004527:Egg Proteins; D014819:Vitellogenins; D010450:Endopeptidases