Regulation of lysosomal hydrolase degradation by cathepsins

Abstract

This study proposed a generalized mechanism for the reversible binding of lysosomal enzymes to the lysosomal membrane. Native lysosomes were partially purified from rabbit liver by standard techniques of homogenization and centrifugation. A fraction (HM) containing lysosomes and mitochondria was further purified by centrifugation on a discontinuous sucrose gradient with a density range of 1.12 to 1.23. Band 5, which was isolated at the interface between sucrose layers of density 1.20 and 1.23, was utilized for the remaining studies. Autolytic degradation of samples was more rapid at pH 4.0 than pH 6.5. At pH 6.5 the samples containing lysosomal membranes were degraded more slowly than soluble enzyme fractions. Samples of HM and band 5 were incubated at 22°C for 30 minutes in 0.02 M acetate buffer between pH 3.5 and 7.0. Maximum binding of the lysosomal enzymes (cathepsins B1 and C and β-glucuronidase) to the membrane occurred between pH 4.0 and 5.0, the region of their maximum hydrolytic activity. Band 5, purified from the HM fraction, showed a greater degree of binding than the HM fraction. Elution of enzyme activities from the membrane with NaCl at pH 4.0 further demonstrated the ionic character of the binding. Band 5 required approximately eight times the concentration of NaCl to remove the enzymes from the membrane when compared with the HM fraction. This fact, in conjunction with the different degrees of binding displayed in the pH curves, indicates that contaminating proteins and other ionic molecules contributes to the elution of lysosomal enzymes from the lysosomal membrane..