The activator of cerebroside sulphatase. Lysosomal localization.
1) An activator protein necessary for the enzymic hydrolysis of cerebroside sulphate 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 could be destroyed by pronase. 2) For elucidation of the subcellular localization of the activator, rat liver was fractionated by differential centrifugation. The intracellular distribution of the cerebroside sulphatase activator was compared to the distribution patterns of marker enzymes for different cell organelles and found to coincide with the lysosomal arylsulphatase, thus indicating a lysosomal localization. 3) This was confirmed using highly purified secondary, i.e. iron-loaded, lysosomes. After disruption by osmotic shock, these organelles hydrolyzed cerebroside sulphate when incubations were performed under physiological conditions with endogenous as well as exogenous sulphatase A as enzyme. 4) After subfractionation of the disrupted secondary lysosomes into membrane and lysosol fractions by high speed centrifugation, it was found that the activator protein was exclusively associated with the lysosol, whereas the acid hydrolases were distributed differently between the two fractions. 5) The lysosol was further fractionated by semi-preparative 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 sulphatase. 6) The significance of these findings for the hydrolysis of sphingolipids in the lysosomes is discussed.
D000818:Animals; D002553:Cerebroside-Sulfatase / Q000378:metabolism*; D004789:Enzyme Activation; D006867:Hydrolases / Q000302:isolation & purification; D007501:Iron; D008099:Liver; D008247:Lysosomes / Q000032:analysis* / Q000201:enzymology; D008297:Male; D008566:Membranes / Q000201:enzymology; D011092:Polyethylene Glycols; D011506:Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D051381:Rats; D013429:Sulfatases / Q000378:metabolism*
D011506:Proteins; D011092:Polyethylene Glycols; D007501:Iron; D006867:Hydrolases; D013429:Sulfatases; D002553:Cerebroside-Sulfatase