The purification of early-pregnancy factor to homogeneity from human platelets and identification as chaperonin 10.
Early-pregnancy factor (EPF), first discovered in the early stages of gestation, is associated with and necessary for cell proliferation in a wide variety of biological situations. Like many other growth factors, EPF is present in platelets, and, by titration studies with a neutralising anti-EPF monoclonal antibody, platelets were identified as an extremely rich source of this growth factor. EPF has been purified from clinically outdated human platelets by heat extraction, ion-exchange and affinity chromatographies on SP-Sephadex and heparin-Sepharose respectively, high-performance hydrophobic interaction chromatography and three reverse-phase HPLC steps, with an average yield of 15 micrograms/100 platelet units (equivalent to approximately 50 1 blood). Using SDS/PAGE, EPF migrated as a single band with approximate M(r) 8500, coincident with biological activity. Mass spectrometry provided an accurate and precise determination of the molecular mass as M(r) 10843.5 +/- 2, along with definitive evidence of the homogeneity of the preparation. Attempts at Edman degradation indicated that the molecule was blocked at the N-terminus and sequencing of proteolytic fragments was undertaken. The amino acid sequence of approximately 70% of the molecule was determined which, with a single exception, is identical with rat chaperonin 10. This structural relationship was shown to extend to functional identity by studies using chaperonin 10 and its functional associate chaperonin 60. Investigations with the latter confirmed that chaperonin 10 is the moiety in pregnancy serum which initiates response in the EPF bioassay. Our studies identify EPF as a member of the highly conserved heat-shock family of molecules and demonstrate a molecular chaperone performing an extracellular role.
Cavanagh
A C
AC
Department of Surgery, University of Queensland, Royal Brisbane Hospital, Australia.
Morton
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002461:Cell Line, Transformed; D018835:Chaperonin 10; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D017077:Culture Media, Conditioned; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006498:Hepatectomy; D006801:Humans; D007668:Kidney; D008099:Liver / Q000378:metabolism; D008115:Liver Regeneration; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010053:Ovary / Q000502:physiology; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011247:Pregnancy / Q000097:blood* / Q000652:urine; D011257:Pregnancy Proteins; D011261:Pregnancy Trimester, First; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013491:Suppressor Factors, Immunologic
D000911:Antibodies, Monoclonal; D018835:Chaperonin 10; D017077:Culture Media, Conditioned; D006360:Heat-Shock Proteins; D010455:Peptides; D011257:Pregnancy Proteins; D013491:Suppressor Factors, Immunologic; C025124:early pregnancy factor