The role of albumin in the release of platelet-activating factor by mouse preimplantation embryos in vitro.
Platelet-activating factor (PAF) produced by embryos remained associated with mouse four-cell embryos after culture in vitro and was also released into the medium. The release of PAF into medium required albumin as a media supplement and the amount of PAF released increased (P < 0.05) with increasing albumin concentration. There was a trend for the amount of PAF remaining associated with embryos to decrease as the extracellular albumin concentration increased. The association of released PAF with albumin was confirmed by size fractionation with size exclusion membranes and high performance gel filtration, and by affinity chromatography (Cibacron blue and anti-BSA) and native PAGE. PAF released from embryos was not degraded by serum PAF:acetylhydrolase (PAF:AH; E.C. 3.1.1.47) after exposure for 24 h to the serum in vitro, while an equivalent concentration of synthetic PAF added to identical media was readily degraded, suggesting that PAF released from the embryo was protected from PAF:AH action. However, when the medium was subjected to organic extraction by the Bligh-Dyer (methanol/chloroform) method and the resulting extract added to equivalent media, embryo-derived PAF was readily degraded by PAF:AH. Furthermore, PAF in embryo-conditioned medium (30 two-cell embryos for 24 h) could not be detected after direct assay of the culture medium by radioimmunoassay or bioassay (platelet aggregation in vitro), yet after extraction, purification and addition to medium with BSA, the embryo-derived PAF was readily detected in either assay. To determine whether the different behaviour of synthetic PAF and embryo-derived PAF resulted from differences in the nature of their binding to albumin, the location to which PAF bound was assessed by limited proteolytic digestion of albumin. Digestion with pepsin or trypsin showed that embryo-derived PAF was located exclusively between amino acids 240 and 386 (domain II) of albumin. Most synthetic PAF added to equivalent medium not exposed to embryos was not at this location, suggesting that PAF released from embryos bound to a site on albumin not generally accessible to synthetic PAF added to similar media.
Ammit
A J
AJ
Department of Physiology, University of Sydney, Royal North Shore Hospital of Sydney, St Leonards, NSW, Australia.
O'Neill
C
C
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D000418:Albumins / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D000818:Animals; D001665:Binding Sites; D001755:Blastocyst / Q000187:drug effects / Q000378:metabolism*; D002850:Chromatography, Gel; D017077:Culture Media, Conditioned; D005109:Extracellular Matrix / Q000378:metabolism; D005260:Female; D006868:Hydrolysis; D051379:Mice; D008815:Mice, Inbred Strains; D010741:Phospholipases A / Q000378:metabolism; D010972:Platelet Activating Factor / Q000032:analysis / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism
D000418:Albumins; D017077:Culture Media, Conditioned; D010972:Platelet Activating Factor; D012710:Serum Albumin, Bovine; D010741:Phospholipases A; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase