IDENTIFICATION AND PARTIAL CHARACTERIZATION OF A PROTEINASE SPECIFIC FOR INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-3 IN AQUEOUS AND VITREOUS HUMORS

The ICFs (-I and -II) are normally found in serum and other extracellu lar fluids complexed to specific binding proteins (IGFBPs), While seve ral IGFBPs have been identified in vitreous and aqueous humors, the ma jor serum carrier of IGE IGFBP-3, is notably absent from these fluids. To determine if this paucity could be due to an IGFBP-3 proteinase (I GFBP-3ase), samples of bovine vitreous or aqueous humor were mixed wit h serum and incubated at 37 degrees C for 4 h followed by western liga nd blotting. In these experiments, a distinct loss of the 46 kDa band representing IGFBP-3 was observed while other bands present at 35, 28 and 25 kDa were unaltered. The IGFBP-3ase activity is temperature sens itive, has a pH optimum of about 8.0 and is inhibited by EDTA. Acid tr eatment of serum to remove endogenously bound IGF does not affect the specificity or activity of the IGFBP-3 proteinase. Size exclusion chro matography of bovine aqueous indicates an approximate molecular weight of 260 kDa. Incubation of recombinant IGFBP-3 or serum with partially -purified IGFBP-3ase results in the appearance of low molecular weight fragments of approximately 30 kDa. These fragments are undetectable b y western ligand blotting but are readily visualized using an IGFBP-3 specific antibody. Comparison of normal and diabetic vitreous humor re veals the presence of an increased amount of IGFBP-3 proteolytic fragm ents in the diabetic as compared to control. These findings indicate t he presence of a IGFBP-3 proteinase in aqueous and vitreous humors tha t may be important in regulating ocular homeostasis.