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A. A. Hussain, Y. Lee, A. Kumar, J. Marshall; Identification and Characterisation of a Large Macromolecular Matrix Metalloproteinase Complex (LMCC) in Human Bruch’s Membrane. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6317.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously shown the presence of various gelatinase species in Bruch’s membrane using SDS-zymography, a technique that disrupts macromolecular complexes into their subunits. Using gel filtration analyses, we have now attempted to separate individual macromolecular species to identify the physiological configuration of these proteases in human Bruch’ membrane.
Peripheral samples of Bruch’s were isolated, homogenised in phosphate buffered saline, and after centrifugation the supernatant containing the matrix metalloproteinase (MMP) species isolated. These were subjected to gel filtration on a calibrated Sepharose CL-6B column and the resulting eluant fractions examined by gelatin zymography. The highest molecular entities present in the first fraction showing MMP activity were deposited onto donor Bruch’s membrane by centrifugation and the resulting preparation examined by scanning electron microscopy.
On gelatin zymography, the molecular weights of the MMP species MMP2, MMP9, HMW1 & HMW2 were determined as 58kDa, 92kDa, 122+/-9kDa and 344+/-22kDa respectively. By gel filtration, the relative molecular weights of MMP species from Bruch’s membrane were 125kDa, 259kDa, 487kDa and 300kDa respectively. Thus in Bruch’s membrane, MMPs 2&9 exist as dimers, HMW1 as a tetramer, and HMW2 as a monomer. A large macromolecular complex containing MMP activity termed ‘LMMC’ was completely excluded from the column material appearing in the void volume space. On zymography, the LMMC complex was observed to be a polymer of HMW1, HMW2 and MMP9. On scanning electron microscopy, these particles were observed as spherical with diameters of 300 +/-50nm.
In human Bruch’s membrane, the MMP system comprises dimers of MMPs 2&9, tetramers of HMW1 and monomers of HMW2. In addition, the membrane houses a large macromolecular complex comprising HMW1, HMW2 and MMP9. The LMMC complex effectively sequesters MMP activity and is expected to impact on the normal turnover of Bruch’s, a function essential for maintaining structural and functional integrity of the membrane.
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