Abstract
Purpose: :
The LMMC particle containing high molecular weight species HMW1&2 and pro-MMP9 effectively sequesters free MMPs from the activation process leading to diminished turnover of Bruch’s membrane in patients with age-related macular degeneration (AMD). The stability of the LMMC complex together with susceptibility for activation of its components with mercurial compounds was assessed. The potential for ginseng saponin mediated dispersion of the LMMC complex was also assessed.
Methods: :
LMMC particles were isolated from macerated Bruch’s-choroid preparations from four eyes (of donors aged 72-84 years) by gel-filtration chromatography on Sepharose CL-6B columns. Isolated LMMC particles were activated with APMA (amino-phenyl mercuric acetate) by incubation for 1 hour and the mixture re-chromatographed to assess stability of the complex. Elution fractions were examined by gelatin zymography to determine if the MMP components of the particles could be activated. Similarly, LMMC particles were incubated with 4% ginseng for 24 hours and stability and nature of any MMP conversion determined.
Results: :
Isolated intact LMMC particles contained only HMW2, HMW1 and pro-MMP9 species. Incubation of these particles for 24 hours at 37oC did not lead to particle disintegration (as judged by gel filtration) but resulted in partial auto-activation of pro-MMP9. Incubation with ginseng did not affect the stability of the particles but blocked the conversion of pro-MMP9 to its activated form. APMA incubation resulted in complete disintegration of the LMMC particle with hydrolysis of both HMW components. The released pro-MMP9 fraction was fully activated but the corresponding pro-MMP2 (from HMW1&2 hydrolysis) was resistant to activation.
Conclusions: :
The LMMC particle is labile with partial activation of the pro-MMP9 species. Since ginseng can block this conversion, it may be useful in inhibiting the neovascularisation process. The study shows the potential for drug-based disintegration of the LMMC complex in an attempt to increase the levels of active MMPs to improve the dynamics of Bruch’s membrane.
Keywords: protein purification and characterization • extracellular matrix • enzymes/enzyme inhibitors