title: Purification and properties of cathepsin D from rat Yoshida ascites hepatoma AH-130.
abstract: Cathepsin D was affinity-purified on pepstatin-Sepharose from control rat liver, from Yoshida ascites hepatoma (AH-130) cells, and from the liver of AH-130 tumour-bearing rats. Apparent molecular mass and immunological reactivity, as determined by SDS-PAGE and immunoblotting, were identical for the three enzyme preparations. The active enzyme concentrations were determined by active-site titration. Catalytic parameters were measured for the three enzymes using two synthetic chromogenic peptides as substrates, and inhibition constants were determined for the proteinases with a number of naturally-occurring as well as synthetic inhibitors. All three enzymes were clearly distinguished from cathepsin E, since none of them was affected by the protein inhibitor from Ascaris lumbricoides. The cathepsin D isolated from AH-130 cells was indistinguishable in its kinetic properties from rat liver cathepsin D, except in its susceptibility to inhibition by isovaleryl-pepstatin. On isoelectrofocusing, the isoenzyme pattern of the tumour enzyme was shifted somewhat towards more basic pI values by comparison with rat liver cathepsin D. These findings are considered with respect to the possibility of an alteration in the S4 subsite of the enzyme active site cleft.
authors.affiliation: Dipartimento di Medicina e Oncologia Sperimentale, Università di Torino, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Bonelli
authors.forename: J
authors.initials: J
authors.lastname: Kay
authors.forename: L
authors.initials: L
authors.lastname: Tessitore
authors.forename: R A
authors.initials: RA
authors.lastname: Jupp
authors.forename: C
authors.initials: C
authors.lastname: Isidoro
authors.forename: C G
authors.initials: CG
authors.lastname: Norey
authors.forename: R
authors.initials: R
authors.lastname: Autelli
authors.forename: A D
authors.initials: AD
authors.lastname: Richards
authors.forename: F M
authors.initials: FM
authors.lastname: Baccino
mesh_terms: D000818:Animals; D002384:Catalysis; D002402:Cathepsin D / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D014407:Tumor Cells, Cultured / Q000201:enzymology
chemical_list: D002402:Cathepsin D