The Activator of Cerebroside Sulphatase. Lysosomal Localization

Publisher Website
Google Scholar
Abstract
An activator protein necessary for the enzymic hydrolysis of cerebroside sulfate could be partially purified from unfractionated rat liver. This activator, which is similar to that of human origin, proved to be a heat-stable, non-dialyzable, low molecular weight protein with an isoelectric point of 4.1. Its activity was destroyed by pronase. Rat liver was fractionated by differential centrifugation. The intracellular distribution of the cerebroside sulfatase [EC 3.1.6.8] activator coincided with the lysosomal arylsulfatase [EC 3.1.6.1], indicating a lysosomal localization. This was confirmed using highly purified secondary, i.e., Fe-loaded, lysosomes. After disruption by osmotic shock, these organelles hydrolyzed cerebroside sulfate when incubations were performed under physiological conditions with endogenous and exogenous sulfatase A as enzyme. After subfractionation of the disrupted secondary lysosomes into membrane and lysosol fractions by high speed centrifugation, the activator protein was exclusively associated with the lysosol, but the acid hydrolases were distributed differently between the 2 fractions. The lysosol was further fractionated by semipreparative electrophoresis on polyacrylamide gels. Two protein fractions were obtained: a high molecular weight fraction, containing the activator-free acid hydrolases and a low molecular weight fraction, containing the enzyme-free activator of cerebroside sulfatase. The significance of these findings for the hydrolysis of sphingolipids in the lysosomes is discussed.