The enzyme involved in the biosynthesis and targeting of enzymes to lysosomal organelles is the GlcNAc phosphotransferase. The absence of severe reduction of this enzyme's activity is responsible for the two autosomally inherited disorders, l-cell disease (lCD) and pseudo-Hurler polydystrophy (PHP). Although a single enzyme deficiency has been proposed for these disorders, the existence of several complementation groups suggests that more than one gene can regulate the expression of this enzyme. Our long range objective is to examine the effect of the gene mutation(s) on the structure and function of the GlcNAc phosphotransferase. Lymphoblast cells, which can be grown in large quantities, demonstrate normal properties for the GlcNAc phosphotransferase thus making it a good system from which to purify and study the enzyme. Preliminary purification results reveal that the enzyme is a glycoprotein of greater than 300,000 molecular weight. Our immediate goal is to purify the normal enzyme to apparent homogeneity by steps which include anion exchange chromatography, concanavalin A-affinity chromatography, gel filtration chromatography, affinity chromatography and gel electrophoresis. Characterization of the kinetic, chemical and physical properties of the purified enzyme will yield information concerning it's structure and function. These studies are necessary for understanding the alterations at the protein level responsible for the different complementation groups. An integral part of this approach will require polyclonal antisera to the purified enzyme. The antibody will be used in conjunction with labelled amino acids or mannose to determine whether the mutation(s) responsible for the complementation groups affect the biosynthesis and/or post-translational processing of the GlcNAc phosphotransferase in lCD and PHP cultured fibroblasts. The polyclonal antibody will also make it possible for us to establish collaborations to map and clone the gene for the enzyme. The lymphoblast system will also allow us to study the oligosaccharide structures on normal, lCD and PHP acid hydrolases within the lysosomes as they relate to the existence of an alternate recognition system or marker to phosphorylated mannose in targeting enzymes to lysosomes. These studies will utilize subcellular fractionation by density gradient centrifugation on colloiday silica of lysosomal enzymes that had been labeled previously with 3H-mannose.