HUMAN-PREGNANCY SERUM CONTAINS AT LEAST 2 DISTINCT PROTEOLYTIC ACTIVITIES WITH THE ABILITY TO DEGRADE INSULIN-LIKE-GROWTH-FACTOR FINDING PROTEIN-3

The presence of a proteolytic activity in sera from pregnant humans an d rodents capable of degrading insulin-like growth factor binding prot ein-3 (IGFBP-3) has been known for some time. However, the identity of this activity has remained elusive. We have attempted to purify the I GFBP-3 protease activity from pregnant human serum (PHS) using the deg radation of I-125-IGFBP-3 as a marker. Following ammonium sulfate prec ipitation of PHS and further enrichment of active fractions by ion-exc hange, protein-A Sepharose, and size-exclusion chromatography, a prote ase of approximately 70-90 kDa was isolated and subjected to N-termina l analysis. The N-terminal sequence was consistent with plasminogen, a known fibrinolytic enzyme. To further characterize the IGFBP-3 protea se activities in both PHS and nonpregnant human serum (NHS), aliquots of serum were first enriched by polyethylene glycol-precipitation and subjected to size-exclusion chromatography. The size-separated fractio ns were then incubated with I-125-IGFBP3, and proteolytic activity was measured. PHS contained two separate proteases (>150 kDa and 70-90 kD a), whereas NHS contained only one (70-90 kDa) that had a inhibitor pr ofile similar to plasmin. However, inhibitors of plasmin had no effect on the activity of the >150-kDa protease. Plasminogen activators (PAs ) greatly increased the activity of the 70- to 90-kDa protease, but ha d little effect on the >150-kDa protease activity. Addition of PAs gre atly increased the ability of NHS to proteolyze IGFBP-3. In contrast, the ability of plasminogen-depleted plasma to degrade I-125-IGFBP-3 wa s not affected by the addition of PAs. Both urokinase and tissue-type PA had the ability to proteolyze IGFBP-3 and were, in contrast to the >150-kDa protease activity, inhibited by the specific PA inhibitor D-P HE-PRO-ARG chloromethyl ketone. The present data suggest that sera has the ability to proteolyze IGFBP-3, and that this ability, as demonstr ated by NHS, can be regulated by protease inhibitors and PAs. In addit ion, PHS does indeed contain an unique IGFBP-3 protease activity that is not present in NHS, and its identity is unknown at this time.