Homo- and heterotetrameric forms of the membrane-bound metalloendopeptidases meprin A and B.
Meprin A and B are disulfide-linked, tetrameric metalloendopeptidases in renal brush border membranes. Meprin A contains 90-kDa subunits (alpha subunits) and is expressed in random-bred and some inbred strains of mice. Meprin B contains subunits of 110 kDa (beta subunits) in situ, and the enzyme from C3H mice, a strain that does not express alpha subunits, has been characterized. Evidence from this and previous studies indicate that beta subunits are expressed in all mouse strains. The tetrameric organization of these meprins was examined in brush border membrane fractions from a random-bred strain (ICR) and two inbred strains of mice (C57BL/6 and C3H/He). Lectin blotting using biotinylated concanavalin A revealed that membranes from the random-bred strain contained three oligomeric complexes of approximately 390, 440, and 490 kDa as determined after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of reducing agents. The subunits in all three oligomers were linked by disulfide bridges. Western blotting using an anti-alpha monoclonal antibody indicated that alpha subunits (90 kDa) were present in the 390- and 440-kDa complexes. Western blotting with a polyclonal antibody specific for beta subunits (110 kDa) indicated the presence of these subunits in the 440- and 490-kDa complexes. Electroelution of the individual oligomers followed by SDS-PAGE under reducing conditions confirmed that the 390- and 490-kDa molecules are homotetramers of alpha and beta subunits, respectively, and that the 440-kDa molecule is a heterotetramer consisting of disulfide-bridged alpha and beta subunits. C57BL/6 mice expressed both alpha and beta subunits and contained tetramers composed of alpha 4 and alpha 2 beta 2. C3H/He mice expressed only the 110-kDa beta subunits and the beta 4 oligomer. This type of multimeric organization of disulfide-linked subunits is unique for the known endopeptidases.
Gorbea
C M
CM
Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg 24061-0308.
Flannery
A V
AV
Bond
J S
JS
D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D007668:Kidney / Q000201:enzymology; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry*; D008666:Metalloendopeptidases / Q000737:chemistry*; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D008819:Mice, Nude; D008871:Microvilli / Q000187:drug effects / Q000201:enzymology; D008970:Molecular Weight; D011487:Protein Conformation
D008565:Membrane Proteins; D008666:Metalloendopeptidases; C053833:meprin A