TY - JOUR
T1 - Purification and characterization of maturation-promoting factor in fish
AU - Yamashita, M.
AU - Fukada, S.
AU - Yoshikuni, M.
AU - Bulet, P.
AU - Hirai, T.
AU - Yamaguchi, A.
AU - Lou, Y. H.
AU - Zhao, Z.
AU - Nagahama, Y.
N1 - Funding Information:
We are grateful to Professor P. Nurse and Dr. J. Hayles (University of Oxford) for providing the E. coli strain over-producing p13”‘, and to Professor Y. Ohba and Dr. H. Yasuda (Kanazawa University) for their collaboration in the early stages of these experiments, including technical guidance for measuring Hl kinase activity. We also thank Dr. H. Onozato, National Research Institute of Aquaculture, for help in collecting mature carp oocytes and Dr. I. G. Gleadall for critical comments on the manuscript. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (0120210 to Y.N.), the Naito Foundation, and the Japan Health Sciences Foundation.
PY - 1992/1
Y1 - 1992/1
N2 - Maturation-promoting factor (MPF) activity has been demonstrated for the first time in fish oocytes. We purified MPF from a 100,000g supernatant of crushed, naturally spawned carp oocytes using four chromatography columns: Q-Sepharose Fast-Flow, p13sucl-affinity Sepharose, Mono S, and Superose 12. The final preparation was purified over 1000-fold with a recovery of about 1%. On Superose 12, MPF eluted as a single peak with an apparent molecular weight of 100 kDa. SDS-PAGE analysis of the active fractions after Superose 12 revealed the presence of four proteins of 33, 34, 46, and 48 kDa. A monoclonal antibody against the PSTAIR sequence of cdc2 kinase recognized the 33- and 34-kDa proteins for which the 46- and 48-kDa proteins are endogenous substrates. The 46- and 48-kDa proteins were recognized by a monoclonal antibody against Escherichia coli-produced goldfish cyclin B, but not by an anti-cyclin A antibody. When oocytes were matured in the presence of 32P, the labeling was seen with the 34-kDa protein, but not with the 33-kDa protein. The 34-kDa protein corresponded to the MPF activity, but the 33-kDa protein did not. These findings indicate that carp MPF is a complex of cdc2 kinase and cyclin B, and further that active MPF contains the phosphorylated form of cdc2 kinase.
AB - Maturation-promoting factor (MPF) activity has been demonstrated for the first time in fish oocytes. We purified MPF from a 100,000g supernatant of crushed, naturally spawned carp oocytes using four chromatography columns: Q-Sepharose Fast-Flow, p13sucl-affinity Sepharose, Mono S, and Superose 12. The final preparation was purified over 1000-fold with a recovery of about 1%. On Superose 12, MPF eluted as a single peak with an apparent molecular weight of 100 kDa. SDS-PAGE analysis of the active fractions after Superose 12 revealed the presence of four proteins of 33, 34, 46, and 48 kDa. A monoclonal antibody against the PSTAIR sequence of cdc2 kinase recognized the 33- and 34-kDa proteins for which the 46- and 48-kDa proteins are endogenous substrates. The 46- and 48-kDa proteins were recognized by a monoclonal antibody against Escherichia coli-produced goldfish cyclin B, but not by an anti-cyclin A antibody. When oocytes were matured in the presence of 32P, the labeling was seen with the 34-kDa protein, but not with the 33-kDa protein. The 34-kDa protein corresponded to the MPF activity, but the 33-kDa protein did not. These findings indicate that carp MPF is a complex of cdc2 kinase and cyclin B, and further that active MPF contains the phosphorylated form of cdc2 kinase.
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U2 - 10.1016/0012-1606(92)90259-J
DO - 10.1016/0012-1606(92)90259-J
M3 - Article
C2 - 1728595
AN - SCOPUS:0026537556
SN - 0012-1606
VL - 149
SP - 8
EP - 15
JO - Developmental Biology
JF - Developmental Biology
IS - 1
ER -