Purification and Properties of A New Lysosomal Protease in Rat Liver

Abstract

1. A new lysosomal protease which hydrolyzes calf thymus histone was found in rat liver, and it differed from some cathepsins: cathepsin B₁ [EC 3,4,22,1] cathepsin L [EC 3,4,22, -], cathepsin H [EC 3,4,22, -], α-N-benzoyl-d-1-arginine amidohydrolase. This protease was purified about 80-fold from the lysosomal extract, by DEAE-cellulose and CM-cellulose column chromatographies, and Sephadex G-100 and Sephadex G-75 gel filtrations. The purified preparation appeared to be homogeneous on polyacrylamaide gel electrophoresis in the presence of SDS.
2. This protease was activated by 2-mercaptoethanol or dithiothreitol and inhibited by monoiodoacetate or p-hydroxymercuribenzoate. Further, this protease was strongly inhibited by leupeptin and chymostatin, but not by pepstatin.
3. The molecular weight of this pro tease was determined to be 27,000 by gel filtration on sephadex G-75. Optimum pH of this protease for the hydrolysis of histone was pH 6.0, and the isoelectric points were found to be 4.90,6.04 and 6.78.
4. This protease strongly hydrolyzed calf thymus histo ne, milk casein, bovine serum albumin, and acid denatured hemoglobin, but not hydrolyzed α-N-carbobenzoxy-L-glutamyl-Lphenylalanine, α-N-benzoyl-L-arginine amide, glycyl-L-tyrosine amide, and a-N-carbobenzoxy-L-glutamyl-L-tyrosine, substrates of cathepsin A [EC 3,4,2, -], B₂ [EC 3,4,22,1], C [EC 3,4,14,1], IV, respectively, and this protease also inactivated glucokinase [EC 2,7,1,2], and aldolase [EC 4,1,2,13]. The rates of inactivation of glucokinase and aldolase by this protease were lower than those of the new cathepsin and cathepsin B1. Further, this protease did not inactivate glucose-6-phosphate dehydrogenase [EC 1,1,49].