| Abstract
| - The in vitro inhibitory activity of the rice Bowman−Birk inhibitor (rBBI) or soybean Bowman−Birkinhibitor (sBBI) against trypsin-catalyzed activation of pro-matrix metalloproteinase 1 or 9 (pro-MMP-1or pro-MMP-9), respectively, was investigated using electrophoresis with silver staining, heparin-enhanced zymography, biotinylated gelatin, Biotrak assay, and fluorescence quenched substratehydrolysis. rBBI at concentrations of 0.08−0.352 mg/mL dose-dependently inhibited the in vitroactivation of 45 μg/mL pro-MMP-1 by trypsin. Heparin-enhanced zymography analysis of pro-MMP-1, trypsin-activated MMP-1, and a mixture of pro-MMP-1−trypsin−rBBI showed clear zones associatedwith trypsin-activated MMP-1 and the absence of clear zones in lanes containing pro-MMP-1 or amixture of pro-MMP-1, trypsin, and rBBI. The results of the Biotrak assay also indicated that rBBIdose-dependently suppressed the activation of pro-MMP-1 by trypsin. sBBI dose-dependently inhibitedthe activation of 100 μg/mL of pro-MMP-9 by trypsin. Biotinylated gelatin assays demonstrated thatpro-MMP-9 or pro-MMP-9 in the presence of trypsin and BBI did not hydrolyze gelatin, whereasp-aminophenylmercury acetate (APMA)-activated MMP-9 and trypsin-activated MMP-9 causedsignificant hydrolysis of gelatin. Quenched fluorescence substrate hydrolysis for total MMP activityshowed that pro-MMP-1 or pro-MMP-9 did not hydrolyze the substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2; active MMP-1 or MMP-9 hydrolyzed the substrate, but lower substrate hydrolysis wasobtained when pro-MMP-1 or pro-MMP-9 was incubated with trypsin in the presence of increasingconcentrations of rBBI. The results are discussed in light of the role of MMP-1 and MMP-9 in theprocess of angiogenesis and the potential of rBBI or sBBI as a functional food ingredient. Keywords: Metalloproteinase 1; metalloproteinase 9; Bowman−Birk inhibitor; angiogenesis; functionalfood ingredient
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