title: Activation of human brain galactosylceramidase by phosphatidylserine.
abstract: Assays of sphingolipid hydrolases in vitro generally require bile salts or other detergents. A few 'activator proteins' have been reported that can partially replace the detergents in the assay mixture. We report here that phosphatidylserine from bovine brain is a relatively specific activator of human brain galactosylceramidase in the absence of sodium taurocholate (phosphatidylserine system). Activity similar to that obtained with the conventional assay system containing taurocholate and oleic acid (taurocholate system) could be obtained. Other lipids tested generally gave less than 10% of the taurocholate system activity, but sulfatide could activate human brain galactosylceramidase to 20--30% of the taurocholate system. The properties of the reaction in the phosphatidylserine system were examined with human brain whole homogenate, crude soluble post-concanavalin A preparations, and partially purified preparations as the enzyme source and compared with those obtained with the taurocholate system. The pH optimum shifted from 4.2 in the taurocholate system to 4.7 in the phosphatidylserine system. The phosphatidylserine system was superior in the linearity of the reaction with respect to the enzyme protein. Reasonably linear Lineweaver-Burk plots could be obtained. The Km values for the phosphatidylserine system were greater than those for the taurocholate system. The effect of phosphatidylserine was not additive to that of taurocholate. Additional phosphatidylserine to the taurocholate system was either without effect at lower concentrations or inhibitory at higher concentrations. The assays of galactosylceramidase with phosphatidylserine and without taurocholate do not necessarily provide pragmatic advantages but offer a potentially useful system with which to study the mechanism of in vivo degradation of the membrane-bound glycosphingolipid.
mesh_terms: D001921:Brain / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D005696:Galactosidases / Q000378:metabolism*; D005698:Galactosylceramidase / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008055:Lipids / Q000494:pharmacology; D010718:Phosphatidylserines / Q000494:pharmacology*; D013656:Taurocholic Acid / Q000494:pharmacology
chemical_list: D008055:Lipids; D010718:Phosphatidylserines; D013656:Taurocholic Acid; D005696:Galactosidases; D005698:Galactosylceramidase