PROTECTIVE PROTEIN IN THE BOVINE LYSOSOMAL BETA-GALACTOSIDASE COMPLEX

Cathepsin A [EC 3.4.16.1], so called protective protein, occurs as an enzyme complex with lysosomal beta-galactosidase [3.2.1.23] and is inv olved in the stable enzymic expression of lysosomal sialidase [3.2.1.1 8]. In this study we investigated the enzymatic properties of cathepsi n A in the bovine beta-galactosidase complex and how it is involved in the molecular multiplicities of the beta-galactosidase and sialidase complexes. Bovine protective protein homologous to the human protein h ad a molecular weight of 48 kDa on SDS-PAGE and cathepsin A activity o ptimum around pH 6.0. It hydrolyzed dipeptide substrates composed of h ydrophobic amino acids much faster than any other type of substrate te sted. This specificity was found to be conserved from human to a non-m ammal, chicken. Immunoprecipitation using an anti beta-galactosidase a ntibody demonstrated that cathepsin A is a component of both the siali dase and beta-galactosidase complexes. The over 700 kDa sialidase comp lex depolymerized by a brief incubation at pH 7.5 and the sialidase wa s inactivated irreversibly via formation of an enzyme active smaller s pecies of sialidase. The 669 kDa beta-galactosidase complex dissociate d reversibly into a 120 kDa beta-galactosidase and a 170 kDa cathepsin A, but the 120 kDa beta-galactosidase, free from the cathepsin A, for med a 260 kDa aggregate under the same conditions, Inactivation of cat hepsin A by heat treatment did not affect its complex forming activity . The 170 kDa protective protein dissociated into a 50 kDa one at pH 7 .5, which no longer formed the complex. These findings indicate that t he 170 kDa protective protein could be the minimum unit required for i n vitro reconstitution of the complex, and that its complex forming ac tivity is carried in a heat-stable domain. Both beta-galactosidase and cathepsin A activities were labile under the dissociated condition, i ndicating that it physiologically stabilizes not only beta-galactosida se but also itself by forming the complex. (C) 1997 Elsevier Science B .V.