Fonseca et. al. Enzymatic, physicochemical and biological properties of MMP-sensitive alginate hydro

Detailed analysis of ggygpvgyliggk sensitivity to

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Unformatted text preview: nsitivity to different MMPs was carried out here. Moreover, to better elucidate the role of different MMPs in the degradation of MMP-sensitive alginate hydrogels under culture conditions, the prole of MMP expression by human mesenchymal stem cells (hMSC) was screened. MMP-sensitive and MMP-insensitive hydrogels were characterized in terms of viscoelastic and microstructural properties and used to study the effect of 3D culture conditions in terms of hMSC behaviour. The effect of 3D culture on MMP-secretion by hMSC was specically addressed. 2 microplate reader (SynergyÔ Mx, BioTek) at lEx ¼ 320 nm and lEm ¼ 420 nm. A commercial MMPs–substrate with the same cleavage site (Mca-PLGYL-Dpa-AR-NH2, FRET-PLGL, R&D Systems, Minneapolis, MN, USA) was also assayed as a control. Fluorescence readings (RFU, arbitrary units) were normalized to the molar concentration of each MMP. For the determination of kinetic parameters kcat and Km, FRET-PVGLIG digestion was assayed at 37  C in a Varian Cary Elipse spectrouorometer equipped with a Varian Peltier single cell holder. MMPs were pre-incubated in TCNB buffer for 3 min before adding the substrate and peptide-cleavage was followed by measuring the Abz uorescence changes at lEx ¼ 320 nm and lEm ¼ 420 nm under continuous stirring.21 Initial rate determinations were chosen at a level intended to hydrolyze less than 10% of the added substrate over the time course of data collection. The slope of the generated uorescence signal was converted into micromoles of the substrate hydrolyzed per second based on a calibration curve obtained from the complete hydrolysis of FRET-PVGLIG. The kinetic parameters were determined by tting the Michaelis–Menten equation using GratÒ sowareversion 6.0.7 (Erithacus Soware). Materials and methods All reagents are from Sigma (Steinheim, Germany) unless otherwise stated. 2.1 Paper Enzymatic studies 2.1.1 Fluorescence Resonance Energy Transfer (FRET) peptides. The GGYGPVGYLIGGK sequence was synthesized as a highly sensitive FRET substrate, bearing an ortho-aminobenzoyl (Abz) uorescent group and an N-(2,4-dinitrophenyl)-ethylenediamine (EDDnp) quenching group, as the donor/acceptor pair, located at each side21 (Abz-GGYGPVGYLIGGK-Q-EDDnp, hereaer designated FRET-PVGLIG). The scrambled sequence FRET-GIVGPL was synthesized as a control. Both peptides were obtained by solid-phase synthesis using the Fmoc procedure in an automated solid-phase peptide synthesizer (PSSM8, Shimadzu), as described elsewhere.21 Peptides were puried by semi-preparative HPLC, and their molecular weight and purity (95% or higher) were checked by amino acid analysis and MALDI-TOF using a MicroexÀLT mass spectrometer (BrukerÀDaltonics, Billerica). The concentration of the FRET substrates was obtained by colorimetric determination of the EDDnp group (3 ¼ 17 300 MÀ1 cmÀ1 at 365 nm). 2.1.2 PVGLIG cleavage in the presence of different MMPs. Recombinant human pro-enzymes MMP-2, MMP-9 and MMP14 (R&D Systems, Mi...
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This document was uploaded on 09/21/2013.

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