Stoichiometry of the Human Lysosomal Carboxypeptidase-β-Galactosidase Complex

Abstract The understanding of the lysosomal β-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 - β-galactosidase and carboxypeptidase. The complex, after affinity purification from human placenta, was dissociated at pH 7.5 and β-galactosidase and carboxypeptidase were separated and purified to homogeneity by FPLC anion-exchange chromatography. The 680 kD complex of β-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 intra-lysosomal pH value of 4.75. Under the same conditions, β-galactosidase and carboxypeptidase independently existed as 306 kDa tetramer and 98 k.Da dimer, respectively. Reconstitution experiments with various ratios of purified β-galactosidase and carboxypeptidase allowed us to conclude that the whole complex is made of 4 β-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.