September 1996
Volume 37, Issue 10
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Articles  |   September 1996
Proteolytic activity directed toward pigment epithelium-derived factor in vitreous of bovine eyes. Implications of proteolytic processing.
Author Affiliations
  • Y Q Wu
    Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-2740, USA.
  • S P Becerra
    Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-2740, USA.
Investigative Ophthalmology & Visual Science September 1996, Vol.37, 1984-1993. doi:
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      Y Q Wu, S P Becerra; Proteolytic activity directed toward pigment epithelium-derived factor in vitreous of bovine eyes. Implications of proteolytic processing.. Invest. Ophthalmol. Vis. Sci. 1996;37(10):1984-1993.

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Abstract

PURPOSE: Experiments were designed to identify proteolytic activities that cleave pigment epithelium-derived factor (PEDF), a member of the serpin (serine protease inhibitor) family. METHODS: Proteins in vitreous humor from bovine eyes were analyzed by Western blot with antiserum to human recombinant PEDF protein. Protein fractionation was by ammonium sulfate saturation and by S-Sepharose column chromatography. Proteolytic activities were determined by gelatin zymography and by solution assays against PEDF or chromogenic peptide substrates. RESULTS: PEDF protein was identified and purified to near homogeneity from vitreous humor of bovine eyes. Limited proteolysis showed that the vitreal protein has a protease-sensitive region at its serpin-exposed peptide loop. Proteolytic activities that cleave the PEDF 49.5 kDa-polypeptide were identified only when proteins from these extracts were separated by 45% to 70% ammonium sulfate fractionation (P70). The degradation product had an apparent molecular weight of 46 kDa. This result is consistent with cleavage at the serpin-exposed loop. The PEDF-cleavage activity in P70 was inhibited specifically by the serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), but not by aprotinin, EDTA, or pepstatin. The vitreal P70 extracts contained 49- and 53-kDa gelatinolytic activities that also were inhibited by AEBSF and not by EDTA, aprotinin, or pepstatin. The PEDF-cleavage activity did not hydrolyze substrates for thrombin, factor Xa, alpha-chymotrypsin, trypsin, or plasmin, nor did it immunoreact with antibody to urokinase plasminogen activator. CONCLUSIONS: These data indicated that vitreous has a serine-proteolytic activity associated with a novel 49/53-kDa enzyme that cleaves the PEDF protein in a serpinase fashion. In addition to cleavage in vitro, these proteases might play a role in modulating PEDF in vivo.

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