During pregnancy, insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) undergoes proteolysis, rendering it undetectable by radioligand binding techniques. This study examines the physical and functional defect in pregnancy IGFBP-3. Ternary complex formation has been measured by the binding of the acid-labile subunit of the circulating IGFBP-3 complex, which also requires IGF-I or IGF-II binding. IGF-depleted pregnancy IGFBP-3, prepared by size-exclusion chromatography at low pH, could not form a ternary complex in the presence of [Tyr60]IGF-I or of an IGF-I analogue extensively altered in the A-domain, whereas analogues altered in the C- or D-domains complexed as well as native IGF-I. After purification by immunoaffinity chromatography, non-pregnancy and pregnancy IGFBP-3 formed ternary complexes with IGF-I equally well, although the pregnancy-proteolysed protein appeared degraded to approximately 30 kDa. On analysis by affinity labelling, cross-linked ternary complexes containing non-pregnancy or pregnancy IGFBP-3 were predominantly 135-140 kDa, with an additional complex of 110-115 kDa in the pregnancy preparation. After reverse-phase h.p.l.c., affinity-isolated pregnancy IGFBP-3 was inactive, whereas the protein from non-pregnancy serum retained activity. Thus pregnancy-proteolysed IGFBP-3 is altered in its specificity for IGF analogues, and is more labile than non-pregnancy IGFBP-3, but shows little impairment in normal IGF binding or ternary complex formation.

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