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A latent proteinase in mouse kidney membranes. Characterization and relationship to meprin
1Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond 23298-0614.
Abstract:
Inbred mice can be phenotypically divided into two groups: those that contain high levels of a kidney metallo-endopeptidase activity (meprin-a) and those with low meprin-a activity. In studies to investigate the molecular basis for the heterogeneity in the expression of this proteinase activity, we found a latent metallo-proteinase activity associated with kidney membranes of C3H/HeJ mice, a low activity strain. The latent proteinase was activated by treatment of kidney brush border membranes with trypsin and was purified from solubilized C3H kidney membranes. Purified preparations of the C3H latent proteinase (referred to as meprin-b) contained three major proteins of subunit molecular weights 90,000, 140,000, and 160,000. In the absence of reducing agents, four 90,000-Da subunits are covalently linked by S-S bridges. The two higher molecular mass proteins are not covalently linked to each other or to the 90,000-Da subunits. However, cross-linking and affinity chromatography studies indicated that the proteins in the meprin-b preparation were tightly associated. By contrast, purified meprin-a contains only 85,000-Da subunit proteins linked by S-S bridges to form a tetramer. Endoglycosidase F treatment decreased the mass of the 90,000-Da meprin-b subunit and the 85,000-Da meprin-a subunit to polypeptides of 65,000-70,000 Da. The 90,000- and 85,000-Da subunits are immunologically similar, in that polyclonal antibodies prepared against one of the subunits cross-react with the other. The substrate specificities and inhibitor profiles of purified preparations of meprin-a and meprin-b are also similar. These data are consistent with the proposition that meprin-b is a polymorphic form of meprin-a that is incompletely processed in vivo.
Insights
Mice exhibit varying kidney metallo-endopeptidase activity (meprin-a). Researchers identified a latent form, meprin-b, in low-activity mice, suggesting it
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Inbred mice display phenotypic differences in kidney metallo-endopeptidase activity (meprin-a), categorized as high or low activity strains.
- Understanding the molecular basis of this heterogeneity is crucial for elucidating enzyme expression and function.
- Previous studies noted low meprin-a activity in C3H/HeJ mice, prompting investigation into their kidney membrane proteinase activities.
Purpose of the Study:
- To investigate the molecular basis for differential expression of kidney metallo-endopeptidase activity (meprin-a) in inbred mice.
- To identify and characterize a latent metallo-proteinase activity found in low-activity mouse strains.
- To compare the biochemical and immunological properties of the latent proteinase (meprin-b) with the known active form (meprin-a).
Main Methods:
- Kidney brush border membranes from C3H/HeJ mice (low meprin-a activity strain) were treated with trypsin to activate latent proteinase activity.
- The activated latent proteinase, designated meprin-b, was purified from solubilized kidney membranes.
- Purified meprin-a and meprin-b were analyzed for subunit composition, molecular weight, inter-subunit linkages (S-S bridges, cross-linking), and immunological similarity using polyclonal antibodies. Endoglycosidase F treatment was used to assess glycosylation.
- Substrate specificities and inhibitor profiles of both meprin forms were compared.
Main Results:
- A latent metallo-proteinase activity (meprin-b) was identified and purified from kidney membranes of C3H/HeJ mice.
- Purified meprin-b consisted of 90,000, 140,000, and 160,000 Da subunits, with the 90,000 Da subunits forming a tetramer linked by S-S bridges. Purified meprin-a comprised only 85,000 Da subunits in a tetrameric structure.
- Endoglycosidase F treatment reduced the mass of both meprin-b (90,000 Da) and meprin-a (85,000 Da) subunits, indicating glycosylation. The subunits were immunologically similar.
- Meprin-a and meprin-b exhibited similar substrate specificities and inhibitor profiles.
Conclusions:
- Meprin-b represents a distinct polymorphic form of meprin-a.
- The data suggest that meprin-b is an incompletely processed precursor of meprin-a, explaining the observed low activity in certain mouse strains.
- The structural and functional similarities support the hypothesis of meprin-b being a precursor form of meprin-a.