Human pregnancy serum contains at least two distinct proteolytic activities with the ability to degrade insulin-like growth factor binding protein-3.
The presence of a proteolytic activity in sera from pregnant humans and rodents capable of degrading insulin-like growth factor binding protein-3 (IGFBP-3) has been known for some time. However, the identity of this activity has remained elusive. We have attempted to purify the IGFBP-3 protease activity from pregnant human serum (PHS) using the degradation of 125I-IGFBP-3 as a marker. Following ammonium sulfate precipitation of PHS and further enrichment of active fractions by ion-exchange, protein-A Sepharose, and size-exclusion chromatography, a protease of approximately 70-90 kDa was isolated and subjected to N-terminal analysis. The N-terminal sequence was consistent with plasminogen, a known fibrinolytic enzyme. To further characterize the IGFBP-3 protease 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 fractions were then incubated with 125I-IGFBP-3, and proteolytic activity was measured. PHS contained two separate proteases (>150 kDa and 70-90 kDa), whereas NHS contained only one (70-90 kDa) that had a inhibitor profile 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 had little effect on the >150-kDa protease activity. Addition of PAs greatly increased the ability of NHS to proteolyze IGFBP-3. In contrast, the ability of plasminogen-depleted plasma to degrade 125I-IGFBP-3 was 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-PHE-PRO-ARG chloromethyl ketone. The present data suggest that sera has the ability to proteolyze IGFBP-3, and that this ability, as demonstrated by NHS, can be regulated by protease inhibitors and PAs. In addition, PHS does indeed contain an unique IGFBP-3 protease activity that is not present in NHS, and its identity is unknown at this time.
Bang
P
P
Department of Women and Child Health, Karolinska Hospital and Institute, Stockholm, Sweden. peter.bang@netg.se
Fielder
P J
PJ
D000595:Amino Acid Sequence; D004492:Edetic Acid / Q000494:pharmacology; D010450:Endopeptidases / Q000097:blood* / Q000737:chemistry; D005260:Female; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010960:Plasminogen Activators / Q000494:pharmacology; D015849:Plasminogen Inactivators / Q000494:pharmacology; D011247:Pregnancy; D011480:Protease Inhibitors / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000378:metabolism / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology
D018972:Insulin-Like Growth Factor Binding Protein 3; D010446:Peptide Fragments; D015849:Plasminogen Inactivators; D011480:Protease Inhibitors; D004492:Edetic Acid; D010450:Endopeptidases; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator; C080473:insulin-like growth factor binding protein-3 protease; D015032:Zinc