Premium
Purification of GVBD‐Inducing Protein from the Ciliate Tetrahymena thermophila
Author(s) -
SUGII MANABU,
FUJISHIMA MASAHIRO
Publication year - 2001
Publication title -
journal of eukaryotic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 1066-5234
DOI - 10.1111/j.1550-7408.2001.tb00174.x
Subject(s) - biology , germinal vesicle , electroelution , tetrahymena , oocyte , immunoprecipitation , microbiology and biotechnology , cell cycle , polyacrylamide gel electrophoresis , biochemistry , cell , gene , enzyme , embryo
. Germinal‐vesicle‐breakdown (GVBD) was induced if a 132,000‐g supernatant of Tetrahymena thermophila homogenates was injected into Xenopus oocytes. Using this induction of GVBD as a bioassay system, a GVBD‐inducing substance was purified from the Tetrahymena by ultra‐filtration, liquid chromatography, and electroelution from a band on native‐PAGE gel. Proteins eluted from the single band on the native‐PAGE gel induced GVBD in the absence of oocyte protein synthesis. This band resolved into two bands on SDS‐PAGE: 60 and 1 12 kDa. The 60 kDa protein was the active fraction inducing GVBD. Immunoprecipitation of the 60 kDa protein prevented the GVBD‐inducing activity, supporting the conclusion that the 60 kDa protein is the GVBD‐inducing substance. An immunoblot with anti‐60 kDa monoclonal antibody and PSTAIR antibody showed that p 13 sue1 ‐beads could remove cdc2 homologues from T. thermophila supernatant but could not remove the GVBD‐inducing activity. The 60‐kDa protein appeared at the same time as micronuclear division and disappeared at the beginning of the macronuclear division during synchronous cell division. The cyclic appearance of the 60‐kDa protein in the T. thermophila cell cycle suggests that this protein has a cell cycle function. Key Words. cdc2 homologue, cell cycle, M‐phase promoting factor, PSTAIR antibody, p13 sue1 ‐beads, Xenopus oocytes.