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Purification and Partial Characterization of an Early Pregnancy Factor‐Induced Suppressor Factor (EPF‐S 1 )
Author(s) -
ROLFE BARBARA E.,
ATHANASASPLATSIS STAVROSIA,
HOSKIN MEGAN J.,
MORTON HALLE,
CAVANAGH ALICE C.
Publication year - 1995
Publication title -
american journal of reproductive immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.071
H-Index - 97
eISSN - 1600-0897
pISSN - 1046-7408
DOI - 10.1111/j.1600-0897.1995.tb00911.x
Subject(s) - lymphokine , biology , antigen , sepharose , microbiology and biotechnology , chemistry , chromatography , immunology , biochemistry , enzyme
PROBLEM : The immunomodulatory properties of early pregnancy factor (EPF) are mediated through induction of at least two lymphokines, designated EPF‐S 1 and EPF‐S 2 (previously estimated M r 15,000 and 55,000 respectively). The activity of the former is MHC‐restricted while the latter is restricted to a locus (or loci) outside the MHC. The present study established further criteria by which EPF‐S 1 and EPF‐S 2 might be distinguished from each other and compared with other suppressor factors. In addition, techniques have been developed to purify EPF‐S 1 to homogeneity. METHOD : Congenic mouse strains were used to map the genetic restriction of EPF‐S 2 in the rosette inhibition test and high performance gel permeation chromatography was used to demonstrate that EPF‐S 1 induces EPF‐S 2 but not vice versa. Further studies then focused on isolation of this first component of the cascade, EPF‐S 1 , from immune ascites (from growth in athymic mice of the anti‐EPF‐S 1 ‐producing rat‐mouse hybridoma R2Tγ, in which EPF‐S 1 is complexed to antibody). Techniques used were acidification followed by application to Sep‐pak C 18 cartridges, high performance cation‐exchange chromatography and two reversed‐phase HPLC steps on a C 3 column. Purified material was analyzed by SDS‐PAGE and Edman degradation. RESULTS : Approximately 10 μg EPF‐S 1 were isolated from 60 ml ascitic fluid. Homogeneity of the purified material was demonstrated by SDS‐PAGE, where it ran as a single band of approximate M r 12,000 coincident with biological activity. Attempts at Edman degradation indicate that the molecule is N‐blocked. CONCLUSION : Definitive primary characterization of EPF‐S 1 must await the preparation and isolation of proteolytic fragments of the molecule, but the present studies establish conditions which make such structural analysis possible.