Meprin A and B, metalloproteases consisting of evolutionarily related ␣ and/or  subunits, are membranebound and secreted enzymes expressed by kidney and intestinal epithelial cells, leukocytes, and cancer cells. Previous work established that the multidomain meprin subunits (each ϳ80 kDa) form disulfide-bridged homoand heterodimers, and differ in substrate and peptide bond specificities. The work herein clearly demonstrates that meprin dimers differ markedly in their ability to oligomerize. Electrophoresis, light scattering, size exclusion chromatography, and electron microscopy were used to characterize quaternary structures of recombinant rat meprins. Meprin B, consisting of meprin  subunits only, was dimeric under a wide range of conditions. By contrast, meprin ␣ homodimers formed heterogeneous multimers (ring-, circle-, spiral-, and tube-like structures) containing up to 100 subunits, with molecular masses at protein peaks ranging from ϳ1.0 to 6.0 MDa. The size of the meprin ␣ homo-oligomers was dependent on protein concentration, ionic strength, and activation state. Meprin ␣ heterodimers tended to form tetramers but not higher oligomers. Thus, the presence of meprin , which has a transmembrane domain in vivo, restricts the oligomerization potential of meprin molecules and localizes meprins to the plasma membrane. By contrast, the propensity of secreted meprin ␣ homodimers to self-associate concentrates proteolytic potential into high molecular mass multimers and thus allows for autocompartmentalization. The work indicates that different mechanisms exist to localize and concentrate the proteolytic activity of membrane-bound and secreted meprin metalloproteinases.Proteolytic enzymes are essential components of many cellular and extracellular processes from maturation of proteins to cell death (1). Their activities and localities, however, must be highly regulated because of their destructive potential. Regulation of proteases is accomplished through several mechanisms including zymogen formation, inhibition, localization to specific compartments, and transcriptional regulation. The structures of proteases themselves have revealed mechanisms to regulate proteolytic activity, and this has been amply demonstrated in the high molecular mass oligomeric structures of the proteasome, tripeptidyl peptidase II, and the tricorn protease (2-4). These multimeric serine and threonine proteolytic complexes are homo-or hetero-oligomeric, have molecular masses of 0.7-9 MDa, and are found intracellularly. The structures serve to restrict, localize, and concentrate proteolytic activity. Proteases at the cell surface are known to form transient oligomeric complexes, such as those between membranetype 1 matrix metalloproteinase, tissue inhibitor of metalloproteinase 2, and matrix metalloproteinase 2 that lead to the activation of matrix metalloproteinase 2 (5). However, stable secreted, multimeric proteolytic complexes were not described until recently, when homo-oligomers of meprin A were found to form multimers of ϳ0.9 MD...
A subset of eukaryotic aminoacyl-tRNA synthetases (a-RS) are contained in a multienzyme complex for which little structural detail is known. Three reversible chemical crosslinking reagents have been used to investigate the arrangement of polypeptides within this particle as isolated from rabbit reticulocytes. Identification of the crosslinked protein pairs was accomplished by two-dimensional SDS diagonal gel electrophoresis. Seventeen neighboring protein pairs have been identified. Eight are seen with at least two reagents: K-RS:p38, D-RS:K-RS, R-RS dimer, K-RS dimer, K-RS:Q-RS, E/P-RS:K-RS, E/P-RS:I-RS, and Q-RS with one of the nonsynthetase proteins. Nine more are observed with one reagent: D-RS dimer, R-RS:p43, D-RS:Q-RS, D-RS:M-RS, K-RS:L-RS, I-RS:R-RS, D-RS:E/P-RS, I-RS:Q-RS, I-RS:L-RS. One trimeric association is seen: E/P-RS:I-RS:L-RS.The observed neighboring protein pairs suggest that the polypeptides within the aminoacyl-tRNA synthetase complex are distributed in three structural domains of similar mass. These can be arranged in a U-shaped particle in which each ''arm'' is considered a domain and the third forms the "base" of the structure. The arms have been termed domain I (D-RS, M-RS, Q-RS) and domain I1 (K-RS, R-RS), with domain III (E/P-RS, I-RS, L-RS) assigned to the base. The smaller proteins (p38, p43) may bridge the domains. This proposed spatial relationship of these domains, as well as their compositions, are consistent with earlier studies. Thus, this study provides an initial three-dimensional working model of the arrangement of polypeptides within the multienzyme aminoacyl-tRNA synthetase complex.Keywords: aminoacyl-tRNA synthetase complex; multienzyme complex; protein-protein interaction; protein structure; reversible chemical crosslinking Aminoacyl-tRNA synthetases are a family of enzymes characterized by their role in coupling each amino acid with the correct tRNA for subsequent use in protein biosynthesis. In addition to this fundamental role in translation, these enzymes are widely studied as models for the evolution of modular proteins and for insight into the mechanisms of RNA-protein recognition. These enzymes also have additional cellular functions, such as synthesis of signaling dinucleotides. There are several recent reviews of these topics (Mirande, 1991;Schulman, 1991;Cusack, 1995).One of the most intriguing and least understood properties of aminoacyl-tRNA synthetases from higher eukaryotes is the existence of a high molecular mass multienzyme complex of a subset Reprint requests to: Mona T. Norcum, Biochemistry Department, University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi 39216-4505; e-mail: mnorcum@fiona.umsmed.edu.Abbreviations: a-RS, aminoacyl-tRNA synthetase, a is the one-letter ambo acid abbreviation; cis-DDP, cis-dichlorodiammine platinum; EGS, erhylene glycobis(succinimydy1succinate); SMPT, 4-succinimydyl oxycarbonyl-2-pyridyldithiotoluene. of these proteins (reviewed in Kisselev & Wolfson, 1994;Yang, 1996). As isolated fr...
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