May 2010
Volume 51, Issue 5
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Biochemistry and Molecular Biology  |   May 2010
High Molecular-Weight Gelatinase Species of Human Bruch's Membrane: Compositional Analyses and Age-Related Changes
Author Affiliations & Notes
  • Ali A. Hussain
    From the Department of Ophthalmology, King's College London, London, United Kingdom.
  • Yunhee Lee
    From the Department of Ophthalmology, King's College London, London, United Kingdom.
  • John Marshall
    From the Department of Ophthalmology, King's College London, London, United Kingdom.
  • Corresponding author: Ali A. Hussain, Department of Ophthalmology, King's College London, St. Thomas' Hospital, Lambeth Palace Road, London SE1 7EH, UK; alyhussain@aol.com
Investigative Ophthalmology & Visual Science May 2010, Vol.51, 2363-2371. doi:10.1167/iovs.09-4259
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      Ali A. Hussain, Yunhee Lee, John Marshall; High Molecular-Weight Gelatinase Species of Human Bruch's Membrane: Compositional Analyses and Age-Related Changes. Invest. Ophthalmol. Vis. Sci. 2010;51(5):2363-2371. doi: 10.1167/iovs.09-4259.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: The structural and functional demise of aging Bruch's membrane is associated with a reduction in the activity of the matrix metalloproteinase (MMP) degradation system. The gelatinase component of the MMP system consists of MMP2 and MMP9 and two high molecular-weight (HMW1, HMW2) species that are yet to be characterized and whose roles in the aging process are yet to be elucidated. The purpose of this study was to determine the age-related changes in levels of expression and subunit characterization of the HMW gelatinase species of Bruch's membrane.

Methods.: Gelatin zymography followed by densitometric scanning was used to quantify the level of the HMW species present. Gel-filtration chromatography allowed the fractionation of the gelatinases according to their molecular weight, and subsequent degradation of the HMW species with a mino-phenyl acetate activation, reduction, and alkylation produced subunit fragments for analysis.

Results.: Most of the HMW1 and HMW2 pool (80% and 87%, respectively) were tightly bound to the matrix. Aging was associated with significant increases in the levels of HMW1 and HMW2 (P < 0.005 and P < 0.05 respectively). On gel filtration, a single large macromolecular complex (LMMC) was observed containing HMW1, HMW2, MMP9, and some MMP2. Activation-mediated fragmentation of HMW1 and HMW2 showed them to be composed of heteropolymers of MMP2 and MMP9.

Conclusions.: The age-related increase of HMW1 and HMW2, together with the formation of LMMC, resulted in the sequestration of MMP2 and MMP9, thereby reducing the free pool for activation. This is likely to contribute to reduced matrix degradation and turnover of Bruch's membrane in both normal aging and age-related macular degeneration.

Bruch's membrane constitutes a thin extracellular matrix (ECM) that lies between the retinal pigment epithelium (RPE) and the fenestrated choriocapillaries of the choroidal circulation. It therefore represents the first passive barrier in the transport chain for delivery of essential blood-borne nutrients to the photoreceptor cells of the neural retina. 1 Conversely, it is the final barrier for the efficient clearance of waste metabolites (generated in the retina and RPE) into the circulation. 
Aging is associated with marked thickening of Bruch's membrane from approximately 2 μm in the young to approximately 5 μm in the elderly. 2,3 A simple expansion of the ECM would be expected to correspondingly reduce the driving diffusional gradients for metabolites across the membrane. However, in aging Bruch's membranes, the situation is further complicated by the deposition of debris rich in lipids, an increase in collagen cross-links, and the accumulation of advanced glycation end products and advanced lipid end products. 48 These changes are thought to underlie the age-related demise in the transport potential of Bruch's and, if advanced, may provide the initial metabolic insult in the pathogenesis of age-related maculopathy. 912  
Matrix remodeling of Bruch's membrane is an important physiological phenomenon for maintaining its structural and functional integrity. Coupled processes of synthesis and degradation tightly regulate the turnover of the membrane. The degradation pathway is mediated by a family of Zn2+-containing, Ca2+-dependent enzymes referred to as the matrix metalloproteinases (MMPs). 13,14 Their activity is checked by the presence of tissue inhibitors (TIMPs). Current opinion holds that it is the TIMP/MMP ratio that determines the relative rate of degradation. 15 Disturbance in the regulation of these pathways can lead to devastating outcomes. For example, in Sorsby's fundus dystrophy, a mutation in the TIMP-3 gene leads to the deposition of a thick lipid-rich deposit on the inner aspects of a thickened Bruch's membrane. 16,17 Pathologic features are similar to those of age-related macular degeneration but occur at an earlier age, leading eventually to the loss of central vision. 
Several studies have previously demonstrated the occurrence of various members of the MMP family within Bruch's membrane and have shown the release of these enzymes from adjacent tissue compartments of the pigment epithelium and choroid. 1821 These enzymes are generally released as inactive pro-zymogens and require the removal of a small propeptide for activation. Under zymographic conditions, inactive proenzymes are partially activated on binding sodium dodecyl sulfate (SDS) and are therefore easily identified on substrate SDS-PAGE gels (zymograms). 
The gelatinase component of the MMP family, namely MMP2 and MMP9, has been studied extensively. In aging Bruch's, the level of pro-enzymes was increased. but active forms essential for matrix turnover were rarely encountered in macular regions of the aging fundus. 18 This may relate partially to increased levels of TIMP-3 in aging eyes. 15 Thus, despite the presence of much altered collagenous substrate (which in the elderly may account for nearly 50% of total collagen), the gelatinase machinery would appear to be ineffective in combating these aging changes leading to the thickening of Bruch's membrane. 4  
In addition to MMP2 and MMP9, our previous work has identified two high molecular-weight gelatinase species (HMW1 and HMW2) in Bruch's membrane, but their physiological significance in relation to the remodeling process remains undetermined. 18 In the present communication, we present results indicating an age-related increase in level of these species, and we show that they exist predominantly bound to the matrix, consist of heteropolymers of MMP2 and MMP9, and occur physiologically as large macromolecular complexes (LMMCs). In effect, these high molecular-weight species appear to sequester MMP2 and MMP9, thereby diminishing the free pool for activation and, hence, impacting on the remodeling processes in Bruch's membrane. 
Materials and Methods
Tissue Preparation
All eyes used in the study were obtained from the Bristol Eye Bank (Bristol, UK). Eyes, with corneas removed for transplantation surgery were transported in saline-filled containers in an icebox and used if they were free handling artifacts and arrived in the laboratory within 72 hours postmortem. The 72-hour postmortem limit was applied because previous work had shown structural and functional survival of Bruch's for the 50 hours postmortem time examined and the survival of human RPE for at least 72 hours postmortem. 9,2224 Of the 29 donor eyes used in the aging study, the postmortem times were 36 hours for two donors and 57 hours for one donor; the remainder were within the 37 to 50 hour time-window. 
After a preliminary fundal examination with a dissecting microscope to ensure suitability for further study, the fovea was located, and an 8-mm full-thickness trephine was obtained from the nasoperipheral region lying on the meridian joining the fovea, optic disc, and the cut edges of the globe. The isolated tissue button was transferred to a Petri dish containing PBS supplemented with an antibiotic/antimycotic mixture (Sigma-Aldrich, Poole, UK). Retinal tissue was removed with a fine pair of forceps, and the RPE monolayer was brushed with a camel's hair brush. The Bruch's-choroid compartment was then gently teased away from the underlying sclera by blunt dissection. The remaining globe was cut into quadrants and similarly processed to obtain preparations of peripheral Bruch's choroid. 
MMP Extraction and Zymographic Analyses
Twenty-nine donor eyes (age range, 21–99 years) were used for aging studies. For quantification of free and bound pools of gelatinase species, isolated 8-mm trephines were placed in an Eppendorf tube containing 100 μL PBS and were vortexed five times for periods of 1 minute each. Samples were then centrifuged for 5 minutes at 10,000g, and the supernatant, representing the free pool of MMPs, was removed. Twenty microliters of the supernatant was mixed with 40 μL non-reducing SDS sample buffer, and 20 μL of this mixture was applied to zymographic gels. The remaining tissue disc was washed several times with 1-mL aliquots of PBS and was reconstituted with 20 μL water and 40 μL non-reducing SDS sample buffer. Samples were vortexed as described and spun at 10,000g for 5 minutes, and 20 μL supernatant representing the bound or SDS-extracted fraction was applied to zymographic gels. 
For zymography, 10% SDS-PAGE gels (1 mm thick) containing a 4% stacking layer and 0.1% gelatin in the separating layer were prepared. Samples for analysis were loaded into lanes. together with prestained protein molecular weight markers (Invitrogen, Paisley, UK) and 20% fetal calf serum (FCS; Sigma-Aldrich) as an internal standard to correct for gel-to-gel variation in background staining. Electrophoresis was performed (X Cell SureLock mini-Cell system; Invitrogen). 
After electrophoresis (150 V, 1 hour), the gels were removed from their cassettes, rinsed in distilled water, and incubated for two half-hour periods in 2.5% Triton X-100 to remove SDS and to renature the proteins. They were then transferred to reaction buffer (50 mM Tris-HCl, 10 mM CaCl2, 75 mM NaCl, 0.02% NaN3, pH 7.4) and incubated at 37°C for 20 hours to allow proteolytic digestion of the gelatin substrate. Gels were rinsed again in distilled water and stained (SimplyBlue SafeStain containing Coomassie G-250; Invitrogen) for 3 hours. Destaining was carried out with distilled water for 1.5 hours. 
MMP activity was observed as clear bands on a blue background. These gels were scanned at a resolution of 2400 dpi (3490 scanner; Epsom, Surrey, UK) and stored in jpg format. Color images were uploaded into the software package (Quantiscan, version 3.0; Biosoft, Cambridge, UK) in grayscale format. Colors were inverted so that MMPs were now visualized as dark bands against a whitish background, and the area under individual gelatinase bands was quantified. For normalization purposes, the MMP-9 band of the fetal serum sample was chosen as a reference because the alternative MMP-2 band often showed distortion and skewing effects. Areas under the various gelatinase bands were thus normalized and corrected for dilution, allowing the expression of MMP activity per 8-mm disc of tissue. 
Gel Filtration Chromatography of Gelatinases
In electrophoretic separations, proteins are solubilized in buffers containing SDS, an anionic detergent. Thus macromolecular complexes containing monomeric species that are loosely bound either by electrostatic (including hydrogen bonding) or hydrophobic interactions are disrupted, leading to complex degradation on gels. However, such macromolecular complexes can be separated in an intact configuration by size-exclusion gel filtration chromatography. 
For gel filtration chromatography, pooled tissue samples were macerated using a pestle and mortar in Tris-HCl buffer (100 mM Tris, 0.15 M NaCl, 10 mM CaCl2, 0.02% sodium azide, pH 7.5). The extract was centrifuged at 10,000g for 5 minutes, and the supernatant was removed for analysis. Altogether four preparations (preparations 1–4) were obtained (preparation 1, from two eyes, donor ages 72 and 79 years; preparation 2, from three eyes, donor ages 71, 73, and 84 years; preparation 3, from two eyes, donor ages 67 and 81 years; and preparation 4, from eight eyes, donor ages 69–84 years). Extracted supernatant volumes ranged from 1.5 mL for preparation 1 to 3 mL for preparation 4. 
Supernatant aliquots were applied to a filtration column (30 cm × 1.5 cm inner diameter; Sepharose CL-6B; Sigma-Aldrich) pre-equilibrated with Tris-HCl buffer. Flow rate was adjusted to 0.6 mL/ min, and fractions were collected every 2 minutes for 1.5 to 2 hours. The protein profile of the chromatography run was obtained by measuring the absorbance of the fractions at a wavelength of 280 nm. Fraction aliquots were then processed for zymographic analysis as described earlier. The intensity of the bands of individual gelatinases was plotted as a function of fraction number to deter mine their mobilities on the column. 
Activation of Gelatinases
MMPs are released as proenzymes bound to their respective TIMPs. Physiological activation results in the loss of a small propeptide that normally blocks the active site on these enzymes. In vitro, these inhibitory constraints can be removed by 4-amino phenylmercuric acetate (APMA) activation or by reduction and alkylation of TIMPs. 13,25 Our previous work had demonstrated that reduction/alkylation enhanced the activation by APMA; therefore, the combined procedures were applied to tissue extracts and fractions from gel filtration containing enriched levels of the high molecular-weight species. Samples were activated by incubation with 1 mM APMA for 1 hour at 37°C followed by reduction with 2 mM dithiothreitol and alkylation with 5 mM iodoacetate; the latter incubations lasted 30 minutes each. After activation, samples were mixed with an equal volume of nonreducing SDS buffer and were processed for zymography. 
Double-Electrophoretic Separations
Previous work had demonstrated that activation of tissue extracts with APMA resulted in loss of gelatinase activity associated with the high molecular-weight species. One possibility was that these high molecular-weight species were homopolymers or heteropolymers of MMP2 and MMP9 and that, on activation, the complexes were reduced to their monomers. The likelihood of this phenomenon was assessed by initially separating the gelatinases by electrophoresis using a single-lane gel. After electrophoresis, the gel was removed from the cassette and incubated with 2.5% Triton X-100 to remove SDS and renature the proteins. The gel was then divided in two, and half was activated/reduced/alkylated as described. A thin slither of the gel (40 mm × 5 mm) containing all the gelatinase species was cut out and mixed with nonreducing SDS buffer for 10 minutes (Fig. 1). It was then loaded onto a gelatin zymography gel containing only the separating layer. Electrophoresis was performed a second time, and the gel was processed to identify the resultant gelatinase species. 
Figure 1.
 
Schematic representation of the double-electrophoretic run to examine the degradation products after in-gel activation of the gelatinase species of Bruch's membrane by APMA. A thin slither (5 mm × 40 mm) of gel was removed from both control and activated half-gels (A) and, after a 10-minute incubation in nonreducing SDS sample buffer, was carefully placed on top of a 0.1% gelatin gel for standard zymography (B).
Figure 1.
 
Schematic representation of the double-electrophoretic run to examine the degradation products after in-gel activation of the gelatinase species of Bruch's membrane by APMA. A thin slither (5 mm × 40 mm) of gel was removed from both control and activated half-gels (A) and, after a 10-minute incubation in nonreducing SDS sample buffer, was carefully placed on top of a 0.1% gelatin gel for standard zymography (B).
Statistical Analysis
Standard linear and nonlinear regression analyses were performed with a commercial statistical package (Fig-Sys; Biosoft, Cambridge, UK) that used the Marquardt-Levenberg algorithm. Significance levels of the Pearson's correlation coefficient were obtained using the t-statistic with N-2 degrees of freedom. 
Results
Results of the analyses undertaken are presented in the following order. Identities of the gelatinase species normally found in Bruch's membrane are given in Figure 2, followed by aging changes in the level of total and bound forms of the high molecular-weight species (Figs. 3, 4). Native gelatinase species (i.e., without SDS-mediated dissociation) were separated according to molecular weight by gel filtration chromatography, and their constituent gelatinase activities were determined by zymography (Figs. 5 67). Dissociation of the high molecular-weight species on APMA activation and analysis of the breakdown products are presented in Figures 8 to 10
Figure 2.
 
The releasable pool of gelatinase species of Bruch's membrane. Bruch's-choroid proteins were isolated from two pairs of eyes (donor ages, 75 and 83 years). After mixing with an equal volume of SDS sample buffer, 10, 15, and 20 μL were loaded into lanes 1, 2, and 3 respectively. Altogether five gelatinase species were clearly identifiable. Log plots were used to calculate the molecular weights of HMW2 and HMW1 as 348 and 150 kDa, respectively.
Figure 2.
 
The releasable pool of gelatinase species of Bruch's membrane. Bruch's-choroid proteins were isolated from two pairs of eyes (donor ages, 75 and 83 years). After mixing with an equal volume of SDS sample buffer, 10, 15, and 20 μL were loaded into lanes 1, 2, and 3 respectively. Altogether five gelatinase species were clearly identifiable. Log plots were used to calculate the molecular weights of HMW2 and HMW1 as 348 and 150 kDa, respectively.
Figure 3.
 
Zymograms for quantification of free and bound gelatinase activity of Bruch's membrane. The free (A) and bound (B) pools of gelatinases were extracted from 8-mm tissue discs. MW, molecular weight standards; FCS, fetal calf serum.
Figure 3.
 
Zymograms for quantification of free and bound gelatinase activity of Bruch's membrane. The free (A) and bound (B) pools of gelatinases were extracted from 8-mm tissue discs. MW, molecular weight standards; FCS, fetal calf serum.
Figure 4.
 
Age-related variation in the total and bound levels of high molecular-weight gelatinase species of Bruch's membrane. Gelatinase activity is expressed on the vertical axis of each plot as the area under the corresponding zymographic bands after correction for volumes used to obtain the extracts. (A) Total and bound levels of HMW2 species were observed to increase linearly with age (P < 0.005). (B) Despite the large scatter in the data, total and bound levels of HMW1 also showed age-related increases (P < 0.05 and P < 0.01, respectively).
Figure 4.
 
Age-related variation in the total and bound levels of high molecular-weight gelatinase species of Bruch's membrane. Gelatinase activity is expressed on the vertical axis of each plot as the area under the corresponding zymographic bands after correction for volumes used to obtain the extracts. (A) Total and bound levels of HMW2 species were observed to increase linearly with age (P < 0.005). (B) Despite the large scatter in the data, total and bound levels of HMW1 also showed age-related increases (P < 0.05 and P < 0.01, respectively).
Figure 5.
 
Gel-filtration chromatography of released proteins from Bruch's membrane. (A, B) The absorbance of each fraction was read at 280 nm and was plotted as a function of fraction number.
Figure 5.
 
Gel-filtration chromatography of released proteins from Bruch's membrane. (A, B) The absorbance of each fraction was read at 280 nm and was plotted as a function of fraction number.
Figure 6.
 
Zymography of fractions after gel filtration chromatography of the free gelatinase pool of Bruch's membrane. Highest levels of HMW2 were present in the early fractions, together with the presence of HMW1 and MMP9. This was followed by fractions containing very little gelatinase activity except for traces of HMW2. MMP9, together with some HMW1/HMW2, was then released and was accompanied in later fractions by MMP2. (A) Extract from three eyes (donor ages, 71, 73, and 84 years). (B) Extract from eight eyes (donor ages, 69–84 years).
Figure 6.
 
Zymography of fractions after gel filtration chromatography of the free gelatinase pool of Bruch's membrane. Highest levels of HMW2 were present in the early fractions, together with the presence of HMW1 and MMP9. This was followed by fractions containing very little gelatinase activity except for traces of HMW2. MMP9, together with some HMW1/HMW2, was then released and was accompanied in later fractions by MMP2. (A) Extract from three eyes (donor ages, 71, 73, and 84 years). (B) Extract from eight eyes (donor ages, 69–84 years).
Figure 7.
 
Distribution of gelatinase species in the various fractions of the gel filtration run. Gelatinase band intensities (obtained from zymograms as in Fig. 6) have been plotted as a function of fraction number. In every preparation examined, the earliest fractions corresponding to the highest molecular–weight species always contained a mixture of HMW2, HMW1, and MMP9, indicating that these species moved as one macromolecular entity. Three of the four preparations displayed showed that the free MMP9 fractions were also associated with some degree of HMW1 and HMW2. MMP2, the smallest species, was eluted last but with considerable overlap with the slightly larger MMP9 species. Preparation 1, extract from two eyes (donor ages, 72 and 79 years); preparation 2, extract from three eyes (donor ages, 71, 73, and 84 years; gel A in Fig. 6); preparation 3, extract from two eyes (donor ages, 67 and 81 years); preparation 4, extract from eight donor eyes (donor ages, 69–84 years; gel B in Fig. 6).
Figure 7.
 
Distribution of gelatinase species in the various fractions of the gel filtration run. Gelatinase band intensities (obtained from zymograms as in Fig. 6) have been plotted as a function of fraction number. In every preparation examined, the earliest fractions corresponding to the highest molecular–weight species always contained a mixture of HMW2, HMW1, and MMP9, indicating that these species moved as one macromolecular entity. Three of the four preparations displayed showed that the free MMP9 fractions were also associated with some degree of HMW1 and HMW2. MMP2, the smallest species, was eluted last but with considerable overlap with the slightly larger MMP9 species. Preparation 1, extract from two eyes (donor ages, 72 and 79 years); preparation 2, extract from three eyes (donor ages, 71, 73, and 84 years; gel A in Fig. 6); preparation 3, extract from two eyes (donor ages, 67 and 81 years); preparation 4, extract from eight donor eyes (donor ages, 69–84 years; gel B in Fig. 6).
Figure 8.
 
Activation of gelatinase species. A concentrated extract was obtained from 11 eyes (donor ages, 58–91 years) and on zymography showed the ample presence of all gelatinase species except active MMP9 (Con lanes). Activation resulted in the complete loss of HMW1, with a major reduction in the intensity of the HMW2 band. These changes were accompanied by increases in the levels of activated MMP2 and MMP9. MW, molecular–weight standards; FCS, fetal calf serum; Con, control; Act, activated samples.
Figure 8.
 
Activation of gelatinase species. A concentrated extract was obtained from 11 eyes (donor ages, 58–91 years) and on zymography showed the ample presence of all gelatinase species except active MMP9 (Con lanes). Activation resulted in the complete loss of HMW1, with a major reduction in the intensity of the HMW2 band. These changes were accompanied by increases in the levels of activated MMP2 and MMP9. MW, molecular–weight standards; FCS, fetal calf serum; Con, control; Act, activated samples.
Figure 9.
 
Activation of gelatinases in the HMW2-enriched fraction. Fraction 20 of preparation 3 (Fig. 7) that contained predominantly HMW2 and some MMP9 was activated. (A) Resultant zymogram. The HMW2 band was lost with a concomitant increase in levels of activated MMP2. (B, C) The underlying changes are made clearer. (B) Presence of HMW2 and MMP9 but not of HMW1 or MMP2. (C) Activation shows loss of HMW2, increase in the region of active and inactive MMP9, and a prominent increase in level of active MMP2.
Figure 9.
 
Activation of gelatinases in the HMW2-enriched fraction. Fraction 20 of preparation 3 (Fig. 7) that contained predominantly HMW2 and some MMP9 was activated. (A) Resultant zymogram. The HMW2 band was lost with a concomitant increase in levels of activated MMP2. (B, C) The underlying changes are made clearer. (B) Presence of HMW2 and MMP9 but not of HMW1 or MMP2. (C) Activation shows loss of HMW2, increase in the region of active and inactive MMP9, and a prominent increase in level of active MMP2.
Figure 10.
 
Compositional analysis of the breakdown products of HMW1 and HMW2 after activation using the double-electrophoretic technique. (A) Initial separation (to obtain the slithers) was performed by standard SDS-PAGE. In the control portion, there is evidence of partial breakdown of HMW1 and HMW2 (arrows) and some activation of MMP2 (asterisk). Activation in the absence of gelatin (and under the conditions used) resulted in marked loss of gelatinase activity. Nonetheless, activation resulted in the release of MMP9 species from both HMW1 and HMW2 (arrows). (B) Initial separation was performed on a gelatin zymography gel and resulted in better preservation of gelatinase activities (control section). Activation resulted in breakdown of HMW1 and HMW2, giving rise to MMP9 species (arrows).
Figure 10.
 
Compositional analysis of the breakdown products of HMW1 and HMW2 after activation using the double-electrophoretic technique. (A) Initial separation (to obtain the slithers) was performed by standard SDS-PAGE. In the control portion, there is evidence of partial breakdown of HMW1 and HMW2 (arrows) and some activation of MMP2 (asterisk). Activation in the absence of gelatin (and under the conditions used) resulted in marked loss of gelatinase activity. Nonetheless, activation resulted in the release of MMP9 species from both HMW1 and HMW2 (arrows). (B) Initial separation was performed on a gelatin zymography gel and resulted in better preservation of gelatinase activities (control section). Activation resulted in breakdown of HMW1 and HMW2, giving rise to MMP9 species (arrows).
Gelatinase Species of Bruch's Membrane
The releasable gelatinase pool of MMP enzymes from human Bruch's membrane is shown in the representative zymogram of Figure 2, obtained from the tissue of two donors aged 75 and 83 years. Five species were clearly discernible: 92-kDa pro-MMP9, 65-kDa MMP2, 58-kDa activated form of MMP2, 150-kDa HMW1, and 348-kDa HMW2. A more comprehensive analysis of molecular weights determined individually and in pooled samples from several donors has provided molecular weights for HMW1 and HMW2 of 122 ± 9 kDa and 344 ± 22 kDa (mean ± SD; n = 24), respectively. 
Age-Related Changes in the HMW Gelatinase Pool of Bruch's Membrane
Investigations to assess the effect of aging on both the total and the bound pool of high molecular-weight species were undertaken in a population sample comprising 29 eyes (donor age range, 21–99 years). Representative zymographic gels of free and bound fractions for a donor subset (age range, 22–87 years) are shown in Figure 3. Visual inspection of the gels showed considerable individual variation between donors regarding the level of gelatinase species present. Generally, the free pool was dominated by the presence of pro-MMP9 but complete absence of activated MMP-9 on all the gels examined; active MMP2 was present in 48% of samples. The bound fraction was dominated by the presence of HMW2 and pro-MMP9, with active MMP-9 appearing in 45% of the samples. Active MMP2 species was present in the bound fraction of all the donors examined. 
Activities of the high molecular-weight species, expressed as area per 8-mm trephine, are presented as a function of donor age in Figure 4. Aging of Bruch's was associated with an increase in both total and bound levels of HMW2 (P < 0.005). Levels of HMW1 showed considerable variation between donors, but, despite the scatter, aging resulted in an increase of both total and bound fractions (P < 0.05 and P < 0.01 respectively). Across the donor age range examined, the bound fractions of HMW1 and HMW2 species constituted 87% ± 10% and 80% ± 20% (mean ± SD), respectively, of total species activity. HMW1 and HMW2 accounted for 23% ± 20% of gelatinase activity in the releasable pool and 43% ± 8% in the bound fraction. 
Gel Filtration Chromatography of Extracts of Bruch's Membrane
Protein elution profiles from two preparations are shown in Figure 5. Higher molecular–weight entities were eluted early in the chromatographic run, followed by fractions of decreasing molecular weight. Eluted fractions were then subjected to gelatin zymography to identify the nature of the gelatinase activities present (Fig. 6). From the zymographic run, the fractions could be divided into roughly four groups. The first group, representing the early fractions (numbers 15–20), contained high levels of HMW2, together with lesser amounts of HMW1 and MMP9. MMP2 was conspicuously absent. The second group generally contained only traces of HMW2. Group 3 contained all the major fractions of gelatinases, and group 4 contained MMP2 and MMP9 only. These gels were subjected to densitometry; the resultant profile of individual gelatinases across the elution spectrum for four preparations is given in Figure 7
In all the preparations examined, the earliest fraction contained HMW2, HMW1, and high levels of MMP9. The fact that the low molecular–weight components were present with HMW2 at the forefront of the elution profile implies that the three species interact to form LMMC. The presence of HMW2 lower in the elution profile (Gp2-fraction) may be indicative of a spectrum of LMMC species with varying degrees of MMP9 incorporation. However, this cannot account for the presence of HMW2 in regions of the elution profile that normally house free MMP9 species (Gp3). MMP2 was present in group 3 and 4 fractions and generally was the last to be eluted out of the column, in keeping with its lower molecular weight relative to the other gelatinase species. 
Subunit Characterization of High Molecular-Weight Species
Activation of gelatinases results in loss of a propeptide. Therefore, on zymography, these forms run faster because of the lowered molecular weight. Activation, reduction, and alkylation of MMPs from Bruch's membrane were associated with the movement of the respective pro-MMP bands toward lower molecular-weight regions (Fig. 8). More important, the high molecular-weight species were either completely eliminated (HMW1) or were considerably reduced in level (HMW2), with concomitant increases in the levels of activated MMP2 and MMP9. 
To determine the source of the activated MMP2 (Fig. 8), a fraction from the elution profiles (fraction 20, preparation 3) enriched in HMW2 with trace amounts of HMW1 was used. Activation resulted in loss of the HMW2 band accompanied by a large increase in activated MMP2 (Fig. 9). 
Attempting to activate these species within the gel and then separating the breakdown products by a second electrophoretic run undertook further analysis of the composition of HMW1 and HMW2. Initially, the gelatinase extract was separated by standard SDS-PAGE and following activation/reduction/alkylation; a slither of the gel containing all the MMP species was loaded for zymographic analysis (Fig. 10A). In the control half of the gel (with a slither that was not activated), the lengthy electrophoretic procedure resulted in partial breakdown of both HMW1 and HMW2 and some activation of MMP2 (Fig. 10A, asterisk). In the activated portion of the gel, gelanolytic activity was considerably subdued. Furthermore, activation under these circumstances, in which the presence of SDS had already altered the conformation of the MMP molecule, did not result in propeptide loss from the pro-MMP9 species. It did, however, result in breakup of the high molecular-weight species. Nonetheless, breakdown products from both HMW1 and HMW2 ran in alignment with endogenous MMP9. 
Subsequently, primary separation of the gelatinases was undertaken in the presence of gelatin substrate (Fig. 10B). Even under these conditions, the HMW2 species showed some breakdown on the control portion of the gel. Activation resulted in the breakdown of HMW1 and HMW2 to yield gelatinase products with electrophoretic mobilities comparable to those of MMP9. There was considerable loss of endogenous MMP2 activity in the activated portion of the gel. Therefore, the likelihood of detecting released MMP2 from HMW1 and HMW2 (if any) was minimal under the conditions used. 
Discussion
Continuous remodeling of Bruch's is essential for maintaining the structural and functional integrity of the membrane. Aging is associated with increasing thickness, accumulation of denatured collagens, and nonspecific cross-links, scarcity of active forms of MMPs, and an increase in the level of TIMP-3. 4,15,18 Compromised degradation has been assumed to be the result of an increased ratio of TIMP3/MMP. 15 In the present investigation, using peripheral tissue samples of Bruch's membrane, analysis of the free or mobile MMP pool showed the presence of active MMP2 in 48% of samples, but active MMP9 was totally absent. In the corresponding bound pool, active MMP2 was present in all samples examined, and active MMP9 was present in 45% of samples. Thus, despite the aging changes noted earlier, activated forms of MMPs were present. Whether they were functionally active or simply trapped or “locked” to their substrates remains to be determined. The possibility also exists that these “endogenous” active forms could be released during endothelial cell invasion, enhancing the neovascular episodes associated with age-related macular degeneration. 
Little is known of the activation mechanism for pro-MMP9 after its release from the RPE, but that for pro-MMP2 has been elucidated. Activation occurs on the basolateral surfaces of the RPE cell and requires—in addition to membrane-bound MT14—free and mobile levels of TIMP2 and pro-MMP2. 26,27 The mechanism requires two molecules of MMP-14; the first MMP-14 molecule binds TIMP-2, and this enables the formation of the ternary complex with pro-MMP-2. The second MMP-14 molecule then cleaves the pro-form to release active MMP-2. Thus, efficient activation requires the presence of MMPs and TIMPs in optimal concentrations near the basolateral surface of the RPE. Age-related alterations that reduce the free level of MMPs are therefore likely to compromise the activation of the MMP degradation system. 
Polymerization to high molecular-weight forms would in effect serve to sequester the free pool of MMPs. In the soluble fraction, HMW1 and HMW2 accounted for 23% ± 20% of total gelatinase activity, with the percentage increasing in the bound fraction to 43% ± 8%. Furthermore, aging was observed to increase the level of these species, and the large scatter in the data of Figure 4 is worth mentioning. Our data were expressed per unit area of the Bruch's-choroid complex, but the level of MMPs should be proportional to the mass of the extracellular matrix (i.e., its thickness). The high scatter in thickness measurements observed in several studies may underlie the high dispersion of data points in the present study. 2,3 Thus, if the high molecular-weight components represent monopolymers or heteropolymers of MMP2 and MMP9, their presence and age-related increase would signify considerable sequestration, removing the monomeric species from the activation process. 
Gel-filtration chromatography was undertaken in the expectation that the high molecular-weight species could be separated, allowing a more detailed investigation of their composition. In keeping with the molecular weights of individual species, HMW2 was eluted in the earlier fraction (group 1) followed by MMP9 (group 3), with MMP2 eluting last (group 4). However, group 1 fractions, containing the highest level of HMW2, were always contaminated by HMW1, MMP9, and in the peak fractions, with a trace presence of MMP2. The fact that they eluted together in the high molecular-weight range of the fractionation implies that they moved as a single entity; this large macromolecular MMP complex has been referred to as LMMC. HMW1 and HMW2 were also present much later in the chromatographic run in regions containing high levels of MMP9 (group 3). If these high molecular-weight species ran as isolated forms, then their higher molecular weights would lead to elution before the peak MMP9 region. Thus, their presence in the group 3 region suggests spontaneous polymerization of MMP9 (with perhaps MMP2) during the period between fractionation on the column and the subsequent zymographic run. 
On SDS-PAGE, LMMC breaks down into its components of HMW1 and HMW2, MMP9, and a trace presence of MMP2. As the plots of Figure 7 show, the LMMC entities contained large amounts of MMP9 that had been sequestered, diminishing the free pool for activation. 
Aging of Bruch's membrane is associated with increased thickening and reduced transport properties. 911 This inevitably leads to increased “dwell” times for traversing molecules and may provide the opportunity for polymerization of MMPs and their sequestration. 
Previous work from our laboratories has shown that chemical activation of HMW1 and HMW2 by APMA leads to reduction in the molecular weights of these species. 18 Furthermore, subsequent reduction and alkylation leads to the loss of the high molecular-weight species and an increase in the levels of activated MMP2 and MMP9. These results have been confirmed in Figures 8 implying that the high molecular-weight species may represent heteropolymers of MMP2 and MMP9. 
Separating HMW1 from HMW2 would have allowed direct analysis of the monomer composition within each species, but this was experimentally difficult. Alternatively, a chromatographic fraction (fraction 20, preparation 3) enriched in HMW2 with trace amounts of HMW1 and MMP9 was activated/reduced and alkylated. Results showed the loss of HMW2 was accompanied by an increase in active MMP-2, indicative of the presence of this species in the HMW2 complex. The double-electrophoretic procedure clearly demonstrated the presence of MMP9 in both HMW1 and HMW2, but the running conditions were not suitable for the detection of MMP2. After exposure to SDS in the first electrophoretic run, APMA activation did not lead to truncation of the proenzymes but did result in the breakdown of the HMW1 and HMW2 complexes. Furthermore, prolonged exposure to SDS appeared to elicit the activation of MMP2 (Fig. 10B, control), and chemical activation under these conditions led to a reduction in or loss of MMP2. Thus, further studies are planned to use Western blot analysis and immunolabeling to detect the presence of MMP2 in the breakdown products of HMW1 and HMW2. 
In summary, the high molecular-weight gelatinase species of Bruch's membrane consist of monopolymers or heteropolymers of MMP2 and MMP9. They aggregate as macromolecular complexes consisting of HMW1, HMW2, MMP9, and some MMP2 (LMMCs) and are easily disrupted by anionic detergents such as SDS. The formation of HMW1 and HMW2 and enlargement to LMMCs results in the sequestration of MMP monomers, thereby reducing the potential for pro-MMP activation. This process may underlie the reduced MMP degradation potential in normal aging and in age-related macular degeneration. 
Footnotes
 Supported by The Guide Dogs for the Blind Association (GDBA) and Fight for Sight (UK).
Footnotes
 Disclosure: A.A. Hussain, None; Y. Lee, None; J. Marshall, None
References
Marshall J Hussain AA Starita C Moore DJ Patmore A . Ageing and Bruch's membrane. In: Marmor MF Wolfensberger TJ eds. Retinal Pigment Epithelium: Function and Disease. New York: Oxford University Press; 1998:669–692.
Ramratten RS van der Schaft TL Mooy CM de Bruijn WC Mulder PGH de Jong PTVM . Morphometric analysis of Bruch's membrane, the choriocapillaris and the choroid in ageing. Invest Ophthalmol Vis Sci. 1994;35:2857–2864. [PubMed]
Okubo A Rosa RH Bunce CV . The relationships of age changes in retinal pigment epithelium and Bruch's membrane. Invest Ophthalmol Vis Sci. 1999;40:443–449. [PubMed]
Karwatowski WSS Jefferies TE Duance VC Albon J Bailey AJ Easty DL . preparationof Bruch's membrane and analysis of the age related changes in the structural collagens. Br J Ophthalmol. 1995;79:944–952. [CrossRef] [PubMed]
Pauleikhoff D Zuels S Sheraidah G Marshall J Wessing A Bird AC . Correlation between biochemical composition and fluorescein binding of deposits in Bruch's membrane. Ophthalmology. 1992;99:1548–1553. [CrossRef] [PubMed]
Holz FG Sheraidah GS Pauleikhoff D Bird AC . Analysis of lipid deposits extracted from human macular and peripheral Bruch's membrane. Arch Ophthalmol. 1994;112:402–406. [CrossRef] [PubMed]
Handa JT Verzijl N Matsunaga H . Increase in advanced glycation end product pentosidine in Bruch's membrane with age. Invest Ophthalmol Vis Sci. 1999;40:775–779. [PubMed]
Glenn JV Mahaffy H Wu K . Advanced glycation endproduct (AGE) accumulation on Bruch's membrane: links to age-related RPE dysfunction. Invest Ophthalmol Vis Sci. 2009;50:441–451. [CrossRef] [PubMed]
Moore DJ Hussain AA Marshall J . Age-related variation in the hydraulic conductivity of Bruch's membrane. Invest Ophthalmol Vis Sci. 1995;36:1290–1297. [PubMed]
Starita C Hussain AA Pagliarini S Marshall J . Hydrodynamics of ageing Bruch's membrane: implications for macular disease. Exp Eye Res. 1996;62:565–572. [CrossRef] [PubMed]
Moore DJ Clover GM . The effect of age on the macromolecular permeability of human Bruch's membrane. Invest Ophthalmol Vis Sci. 2001;42:2970–2975. [PubMed]
Hussain AA Rowe L Marshall J . Age-related alterations in the diffusional transport of a mino acids across the human Bruch's-choroid complex. J Opt Soc Am. 2002;19:166–172. [CrossRef]
Woessner JF . Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J. 1991;5:2145–2154. [PubMed]
Birkedal-Hansen H Moore WGL Bodden MK . Matrix metalloproteinases: a review. Crit Rev Oral Biol Med. 1993;4:197–250. [PubMed]
Kamei M Hollyfield JG . TIMP-3 in Bruch's membrane: changes during aging and in age-related macular degeneration. Invest Ophthalmol Vis Sci. 1999;40:2367–2375. [PubMed]
Fariss RN Apte SS Luthert PJ Bird AC Milam AH . Accumulation of tissue inhibitor of metalloproteinases-3 in human eyes with Sorsby's fundus dystrophy or retinitis pigmentosa. Br J Ophthalmol. 1998;82:1329–1334. [CrossRef] [PubMed]
Weber BH Vogt G Pruett RC Stohr H Felbor U . Mutations in the tissue inhibitor of metalloproteinase-3 (TIMP-3) in patients with Sorsby's fundus dystrophy. Nat Genet. 1994;8:352–356. [CrossRef] [PubMed]
Guo L Hussain AA Limb GA Marshall J . Age-dependent variation in the metalloproteinase activity of isolated Bruch's membrane and choroid. Invest Ophthalmol Vis Sci. 1999;40:2676–2682. [PubMed]
Hunt RC Fox A Pakalnis VAL . Cytokines cause cultured retinal pigment epithelial cells to secrete metalloproteinases and to contract collagen gels. Invest Ophthalmol Vis Sci. 1993;34:3179–3186. [PubMed]
Alexander JP Bradley JMB Gabourel JD Acott TS . Expression of matrix metalloproteinases and inhibitor by human retinal pigment epithelium. Invest Ophthalmol Vis Sci. 1990;31:2520–2528. [PubMed]
Vranka JA Johnson E Zhu X . Discrete expression and distribution pattern of TIMP-3 in the human retina and choroid. Curr Eye Res. 1997;16:102–110. [CrossRef] [PubMed]
Edwards RB . Culture of mammalian retinal pigment epithelium and neural retina. Methods Enzymol. 1982;81:39–43. [PubMed]
Hussain AA Voaden MJ . Postenucleation survival of taurine uptake by pigment epithelium and choroid of the baboon eye. Exp Eye Res. 1985;40:643–646. [CrossRef] [PubMed]
Hussain AA Marshall J . Taurine transport pathways in the outer retina in relation to ageing and disease. In: Tombran-Tink J Barnstable CJ eds. Ocular Transporters in Ophthalmic Diseases and Drug Delivery. Totowa, NJ: Humana Press; 2008:217–234.
Brown D Hamdi H Bahri S Kenney C . Characterization of an endogenous metalloproteinase in human vitreous. Curr Eye Res. 1994;13:639–647. [CrossRef] [PubMed]
Strongin AY Collier I Bannikov G Marmer BL Grant GA Goldberg GI . Mechanism of cell surface activation of 72kDa type IV collagenase: isolation of the activated form of the membrane metalloproteinase. J Biol Chem. 1995;270:5331–5338. [CrossRef] [PubMed]
Butler GS Butler MJ Atkinson SJ . The TIMP-2 membrane type I metalloproteinase ‘receptor’ regulates the concentration and efficient activation of progelatinase A: a kinetic study. J Biol Chem. 1998;273:871–880. [CrossRef] [PubMed]
Figure 1.
 
Schematic representation of the double-electrophoretic run to examine the degradation products after in-gel activation of the gelatinase species of Bruch's membrane by APMA. A thin slither (5 mm × 40 mm) of gel was removed from both control and activated half-gels (A) and, after a 10-minute incubation in nonreducing SDS sample buffer, was carefully placed on top of a 0.1% gelatin gel for standard zymography (B).
Figure 1.
 
Schematic representation of the double-electrophoretic run to examine the degradation products after in-gel activation of the gelatinase species of Bruch's membrane by APMA. A thin slither (5 mm × 40 mm) of gel was removed from both control and activated half-gels (A) and, after a 10-minute incubation in nonreducing SDS sample buffer, was carefully placed on top of a 0.1% gelatin gel for standard zymography (B).
Figure 2.
 
The releasable pool of gelatinase species of Bruch's membrane. Bruch's-choroid proteins were isolated from two pairs of eyes (donor ages, 75 and 83 years). After mixing with an equal volume of SDS sample buffer, 10, 15, and 20 μL were loaded into lanes 1, 2, and 3 respectively. Altogether five gelatinase species were clearly identifiable. Log plots were used to calculate the molecular weights of HMW2 and HMW1 as 348 and 150 kDa, respectively.
Figure 2.
 
The releasable pool of gelatinase species of Bruch's membrane. Bruch's-choroid proteins were isolated from two pairs of eyes (donor ages, 75 and 83 years). After mixing with an equal volume of SDS sample buffer, 10, 15, and 20 μL were loaded into lanes 1, 2, and 3 respectively. Altogether five gelatinase species were clearly identifiable. Log plots were used to calculate the molecular weights of HMW2 and HMW1 as 348 and 150 kDa, respectively.
Figure 3.
 
Zymograms for quantification of free and bound gelatinase activity of Bruch's membrane. The free (A) and bound (B) pools of gelatinases were extracted from 8-mm tissue discs. MW, molecular weight standards; FCS, fetal calf serum.
Figure 3.
 
Zymograms for quantification of free and bound gelatinase activity of Bruch's membrane. The free (A) and bound (B) pools of gelatinases were extracted from 8-mm tissue discs. MW, molecular weight standards; FCS, fetal calf serum.
Figure 4.
 
Age-related variation in the total and bound levels of high molecular-weight gelatinase species of Bruch's membrane. Gelatinase activity is expressed on the vertical axis of each plot as the area under the corresponding zymographic bands after correction for volumes used to obtain the extracts. (A) Total and bound levels of HMW2 species were observed to increase linearly with age (P < 0.005). (B) Despite the large scatter in the data, total and bound levels of HMW1 also showed age-related increases (P < 0.05 and P < 0.01, respectively).
Figure 4.
 
Age-related variation in the total and bound levels of high molecular-weight gelatinase species of Bruch's membrane. Gelatinase activity is expressed on the vertical axis of each plot as the area under the corresponding zymographic bands after correction for volumes used to obtain the extracts. (A) Total and bound levels of HMW2 species were observed to increase linearly with age (P < 0.005). (B) Despite the large scatter in the data, total and bound levels of HMW1 also showed age-related increases (P < 0.05 and P < 0.01, respectively).
Figure 5.
 
Gel-filtration chromatography of released proteins from Bruch's membrane. (A, B) The absorbance of each fraction was read at 280 nm and was plotted as a function of fraction number.
Figure 5.
 
Gel-filtration chromatography of released proteins from Bruch's membrane. (A, B) The absorbance of each fraction was read at 280 nm and was plotted as a function of fraction number.
Figure 6.
 
Zymography of fractions after gel filtration chromatography of the free gelatinase pool of Bruch's membrane. Highest levels of HMW2 were present in the early fractions, together with the presence of HMW1 and MMP9. This was followed by fractions containing very little gelatinase activity except for traces of HMW2. MMP9, together with some HMW1/HMW2, was then released and was accompanied in later fractions by MMP2. (A) Extract from three eyes (donor ages, 71, 73, and 84 years). (B) Extract from eight eyes (donor ages, 69–84 years).
Figure 6.
 
Zymography of fractions after gel filtration chromatography of the free gelatinase pool of Bruch's membrane. Highest levels of HMW2 were present in the early fractions, together with the presence of HMW1 and MMP9. This was followed by fractions containing very little gelatinase activity except for traces of HMW2. MMP9, together with some HMW1/HMW2, was then released and was accompanied in later fractions by MMP2. (A) Extract from three eyes (donor ages, 71, 73, and 84 years). (B) Extract from eight eyes (donor ages, 69–84 years).
Figure 7.
 
Distribution of gelatinase species in the various fractions of the gel filtration run. Gelatinase band intensities (obtained from zymograms as in Fig. 6) have been plotted as a function of fraction number. In every preparation examined, the earliest fractions corresponding to the highest molecular–weight species always contained a mixture of HMW2, HMW1, and MMP9, indicating that these species moved as one macromolecular entity. Three of the four preparations displayed showed that the free MMP9 fractions were also associated with some degree of HMW1 and HMW2. MMP2, the smallest species, was eluted last but with considerable overlap with the slightly larger MMP9 species. Preparation 1, extract from two eyes (donor ages, 72 and 79 years); preparation 2, extract from three eyes (donor ages, 71, 73, and 84 years; gel A in Fig. 6); preparation 3, extract from two eyes (donor ages, 67 and 81 years); preparation 4, extract from eight donor eyes (donor ages, 69–84 years; gel B in Fig. 6).
Figure 7.
 
Distribution of gelatinase species in the various fractions of the gel filtration run. Gelatinase band intensities (obtained from zymograms as in Fig. 6) have been plotted as a function of fraction number. In every preparation examined, the earliest fractions corresponding to the highest molecular–weight species always contained a mixture of HMW2, HMW1, and MMP9, indicating that these species moved as one macromolecular entity. Three of the four preparations displayed showed that the free MMP9 fractions were also associated with some degree of HMW1 and HMW2. MMP2, the smallest species, was eluted last but with considerable overlap with the slightly larger MMP9 species. Preparation 1, extract from two eyes (donor ages, 72 and 79 years); preparation 2, extract from three eyes (donor ages, 71, 73, and 84 years; gel A in Fig. 6); preparation 3, extract from two eyes (donor ages, 67 and 81 years); preparation 4, extract from eight donor eyes (donor ages, 69–84 years; gel B in Fig. 6).
Figure 8.
 
Activation of gelatinase species. A concentrated extract was obtained from 11 eyes (donor ages, 58–91 years) and on zymography showed the ample presence of all gelatinase species except active MMP9 (Con lanes). Activation resulted in the complete loss of HMW1, with a major reduction in the intensity of the HMW2 band. These changes were accompanied by increases in the levels of activated MMP2 and MMP9. MW, molecular–weight standards; FCS, fetal calf serum; Con, control; Act, activated samples.
Figure 8.
 
Activation of gelatinase species. A concentrated extract was obtained from 11 eyes (donor ages, 58–91 years) and on zymography showed the ample presence of all gelatinase species except active MMP9 (Con lanes). Activation resulted in the complete loss of HMW1, with a major reduction in the intensity of the HMW2 band. These changes were accompanied by increases in the levels of activated MMP2 and MMP9. MW, molecular–weight standards; FCS, fetal calf serum; Con, control; Act, activated samples.
Figure 9.
 
Activation of gelatinases in the HMW2-enriched fraction. Fraction 20 of preparation 3 (Fig. 7) that contained predominantly HMW2 and some MMP9 was activated. (A) Resultant zymogram. The HMW2 band was lost with a concomitant increase in levels of activated MMP2. (B, C) The underlying changes are made clearer. (B) Presence of HMW2 and MMP9 but not of HMW1 or MMP2. (C) Activation shows loss of HMW2, increase in the region of active and inactive MMP9, and a prominent increase in level of active MMP2.
Figure 9.
 
Activation of gelatinases in the HMW2-enriched fraction. Fraction 20 of preparation 3 (Fig. 7) that contained predominantly HMW2 and some MMP9 was activated. (A) Resultant zymogram. The HMW2 band was lost with a concomitant increase in levels of activated MMP2. (B, C) The underlying changes are made clearer. (B) Presence of HMW2 and MMP9 but not of HMW1 or MMP2. (C) Activation shows loss of HMW2, increase in the region of active and inactive MMP9, and a prominent increase in level of active MMP2.
Figure 10.
 
Compositional analysis of the breakdown products of HMW1 and HMW2 after activation using the double-electrophoretic technique. (A) Initial separation (to obtain the slithers) was performed by standard SDS-PAGE. In the control portion, there is evidence of partial breakdown of HMW1 and HMW2 (arrows) and some activation of MMP2 (asterisk). Activation in the absence of gelatin (and under the conditions used) resulted in marked loss of gelatinase activity. Nonetheless, activation resulted in the release of MMP9 species from both HMW1 and HMW2 (arrows). (B) Initial separation was performed on a gelatin zymography gel and resulted in better preservation of gelatinase activities (control section). Activation resulted in breakdown of HMW1 and HMW2, giving rise to MMP9 species (arrows).
Figure 10.
 
Compositional analysis of the breakdown products of HMW1 and HMW2 after activation using the double-electrophoretic technique. (A) Initial separation (to obtain the slithers) was performed by standard SDS-PAGE. In the control portion, there is evidence of partial breakdown of HMW1 and HMW2 (arrows) and some activation of MMP2 (asterisk). Activation in the absence of gelatin (and under the conditions used) resulted in marked loss of gelatinase activity. Nonetheless, activation resulted in the release of MMP9 species from both HMW1 and HMW2 (arrows). (B) Initial separation was performed on a gelatin zymography gel and resulted in better preservation of gelatinase activities (control section). Activation resulted in breakdown of HMW1 and HMW2, giving rise to MMP9 species (arrows).
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