title: Stoichiometry of the human lysosomal carboxypeptidase-beta-galactosidase complex.
abstract: The understanding of the lysosomal beta-galactosidase-carboxypeptidase-neuraminidase multienzymatic complex structure and function requires an efficient system for dissociation and association of its isolated protein components under controlled conditions. In this paper such a system was used to determine the stoichiometry of the two main components of this complex--beta-galactosidase and carboxypeptidase. The complex, after affinity purification from human placenta, was dissociated at pH 7.5 and beta-galactosidase and carboxypeptidase were separated and purified to homogeneity by FPLC anion-exchange chromatography. The 680 kD complex of beta-galactosidase and carboxypeptidase was reconstituted in vitro by mixing the isolated enzymes in a 1:2 molar ratio at pH 7.5 and then progressively acidifying the medium towards the intralysosomal pH value of 4.75. Under the same conditions, beta-galactosidase and carboxypeptidase independently existed as 306 kDa tetramer and 98 kDa dimer, respectively. Reconstitution experiments with various ratios of purified beta-galactosidase and carboxypeptidase allowed us to conclude that the whole complex is made of 4 beta-galactosidase and 8 carboxypeptidase monomers. Cross-linking of the native and reconstituted complexes with dimethylsuberimidate or glutaric dialdehyde suggested that the native and the reconstituted complexes have the same supramolecular structure.
authors.affiliation: Service de Génétique Médicale, Hôpital Sainte-Justine Université de Montréal, Québec, Canada.
authors.forename: A V
authors.initials: AV
authors.lastname: Pshezhetsky
authors.forename: M
authors.initials: M
authors.lastname: Potier
mesh_terms: D002268:Carboxypeptidases / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D001616:beta-Galactosidase / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D001616:beta-Galactosidase; D002268:Carboxypeptidases