Isolation and characterization of plasmin-generated bioactive fragments ofIGFBP-3

Insulin-like growth factor-binding protein-3 (IGFBP-3) was digested with pl asmin, and the proteolytic fragments were isolated by HPLC and tested for b ioactivity as measured by stimulation of glucose uptake in microvessel endo thelial cells. Two Of the pooled fractions of the digest stimulated glucose uptake. The major bioactive pool, at an estimated protein concentration <5 0 ng/ml, stimulated glucose uptake to 150% of control with greater stimulat ion and 220% of control at similar to 250 ng/ml. Two fragments were present in the bioactive fraction, the dominant one migrating at similar to 20,000 and the other at similar to 8,000. Bath fragments bound I-125-labeled insu lin-like growth factor and [H-3]heparin. NH2-terminal amino acid analysis o f the bioactive peak yielded two sequences. One, representing the majority of the material, had an NH2-terminal sequence identical to IGFBP-3; the sec ond fragment began at amino acid 202 of IGFBP-3. In contrast to the bioacti ve fragments, intact IGFBP-3, at concentrations up to 130 mu g/ml, had no b ioactivity. These findings demonstrate that IGFBP-3 can be degraded into fr agments that have potent bioactivities that. are not present in the intact IGFBP-3 molecule.