Purification and partial characterization of an early pregnancy factor-induced suppressor factor (EPF-S1).
OBJECTIVE
The immunomodulatory properties of early pregnancy factor (EPF) are mediated through induction of at least two lymphokines, designated EPF-S1 and EPF-S2 (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-S1 and EPF-S2 might be distinguished from each other and compared with other suppressor factors. In addition, techniques have been developed to purify EPF-S1 to homogeneity.


METHODS
Congenic mouse strains were used to map the genetic restriction of EPF-S2 in the rosette inhibition test and high performance gel permeation chromatography was used to demonstrate that EPF-S1 induces EPF-S2 but not vice versa. Further studies then focused on isolation of this first component of the cascade, EPF-S1, from immune ascites (from growth in athymic mice of the anti-EPF-S1 producing rat-mouse hybridoma R2T gamma, in which EPF-S1 is complexed to antibody). Techniques used were acidification followed by application to Sep-pak C18 cartridges, high performance cation-exchange chromatography and two reversed-phased HPLC steps on a C3 column. Purified material was analyzed by SDS-PAGE and Edman degradation.


RESULTS
Approximately 10 micrograms EPF-S1 were isolated fom 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.


CONCLUSIONS
Definitive primary characterization of EPF-S1 must await the preparation and isolation of proteolytic fragments of the molecule, but the present studies establish conditions which make such structural analysis possible.
Rolfe
B A
BA
University of Queensland, Department of Surgery, Royal Brisbane Hospital, Herston, Australia.
Athanasas-Platsis
S
S
Hoskin
M J
MJ
Morton
H
H
Cavanagh
A C
AC
D000818:Animals; D001201:Ascites / Q000276:immunology; D018835:Chaperonin 10; D017077:Culture Media, Conditioned / Q000032:analysis; D005260:Female; D006825:Hybridomas / Q000737:chemistry / Q000276:immunology; D007166:Immunosuppressive Agents / Q000302:isolation & purification*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008819:Mice, Nude; D010455:Peptides / Q000302:isolation & purification*; D011257:Pregnancy Proteins; D012397:Rosette Formation; D013491:Suppressor Factors, Immunologic
D018835:Chaperonin 10; D017077:Culture Media, Conditioned; D007166:Immunosuppressive Agents; D010455:Peptides; D011257:Pregnancy Proteins; D013491:Suppressor Factors, Immunologic; C025124:early pregnancy factor