Improved purification and some properties of bovine lysosomal carboxypeptidase B.

Lysosomal carboxypeptidase B (peptidyl-L-amino-acid hydrolase, EC 3.4.18.1) from bovine spleen purified to apparent homogeneity was found to have a molecular weight of 52 000 in the absence and of 25 000 in the presence of urea, determined by gel filtration, indicating the existence of two subunits of identical size. The amount of approx. 15% carbohydrate estimated after cleavage by endoglycosidase H was shown to be insignificant for enzymatic activity. The isoelectric focusing separated lysosomal carboxypeptidase B into several protein bands - each enzymatically active - with a range of isoelectric points between 4.6 and 5.2. The titration of the sulphydryl group in the active site of the enzyme with the proteinase inhibitor E-64 yielded one thiol group per molecule. A maximum of activation was achieved by the addition of selenocystamine together with dithioerythritol and EDTA in the incubation solution. Under these conditions the carboxypeptidase hydrolyzed benzoylglycylarginine (80 kat/mol enzyme), benzoylarginine amide (38 kat/mol enzyme) and carbobenzoxyglutaryltyrosine (110 kat/mol enzyme). Slight enzymatic activities towards benzoylarginine 2-naphthylamide and benzoylarginine p-nitroanilide could be measured. With the oxidized insulin B chain, lysosomal carboxypeptidase B exhibited only carboxypeptidase activity.