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Type IV Collagen Chains a1(IV) and a2(IV) Predominated in Rat Amnion
Extracts of amnions collected on Day 19 of pregnancy were treated with bacterial collagenase and then subjected to SDS PAGE to resolve the NC1 domains. The proteins were transferred to membranes, which were subsequently probed with polyclonal antibodies recognizing specific NC1 sequences in the a1(IV)/a2(IV), a3(IV), a4(IV), a5(IV), and a6(IV) chains. These Western blot analyses demonstrated that a1(IV)/a2(IV) NC1 domain dimers and monomers were most abundant in rat amnion extracts (Fig. 1). Some a3(IV) was detected, whereas a4(IV), a5(IV), and a6(IV) chains were present at negligible levels. The same antibodies detected a1(IV)/a2(IV), a3(IV), a4(IV), a5(IV), and a6(IV) chains in rat kidney extracts (Fig. 1). buy cheap antibiotics
Rat Amnion Basement Membrane Extracts Were More Susceptible to Collagenase Digestion than Rat Kidney Basement Membrane Extracts
In vitro degradation assays indicated that collagens in the rat amnion basement membrane extract were degraded at a higher rate (Fig. 2) than the collagens in rat kidney basement membranes, which are enriched in a3(IV). a4(IV), and a6(IV) chains. Similar observations were also made when purified activated human MMP-9 was used for basement membrane degradation (Fig. 2), with the exception that bacterial collagenase degraded the basement membrane extracts at a faster rate than did MMP-9.
FIG. 1. Repertoire of type IV collagen a chains expressed in rat amnion (A) and rat kidney (B). Amnions and kidneys were detergent-extracted and treated with bacterial collagenase to generate NC1 domain fragments, which were resolved by SDS-PAGE and then subjected to Western blotting using NC1 domain-specific antibodies. The treatment generated NC1 domain monomers (M) and dimers (D). The lanes indicate the specific antisera employed in the Western blot analyses.
FIG. 2. Differential susceptibility of amnion and kidney basement membranes to collagenase. Detergent extracts of amnions collected on Day 1 9 of pregnancy and of adult kidney were incubated with bacterial collagenase or exogenous active human MMP-9, and hydroxyproline release was monitored over time. Left) Amnion basement membrane extract was degraded by bacterial collagenase at a faster rate than adult kidney basement membrane extract. Right) Amnion basement membrane extracts were more rapidly degraded by exogenous MMP-9 than were kidney basement membranes. Values are means ± SE from three separate assays.
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