Tay-Sachs disease is a recessive genetic disorder caused by mutations in the gene encoding the alpha-chain polypeptide of the lysosomal enzyme B-hexosaminidase A. One in thirty Ashkenazi Jews are carriers for a severe form of the disease. Dogma assumed that members of this ethnic group harbored the same mutation, but a splice junction defect and insertion defect in two different Ashkenazi patients which could be detected in approximately 20% and 70% of the carriers, respectively was identified. Moreover, the patient used to characterize the insertion defect was heterozygous for it and null for the splice junction defect indicating the presence of a third mutation in the Ashkenazi population. A G to A base change in exon 11 which introduces a premature termination codon was identified. The defect was not found among one hundred Ashkenazi carriers screened. This suggests that the lesion is a very low frequency allele or private family mutation. I-cell disease is a recessive genetic disorder caused by a deficiency of N- acetylglucosaminylphosphotransferase, a membrane bound enzyme involved in equipping lysosomal enzymes with phosphomannosyl residues that serve to route these enzymes to lysosomes. Since the phosphotransferase has been shown to recognize lysosomal enzymes as specific substrates via a protein determinant shared by these enzymes, attempts will be made to affinity purify the phosphotransferase via its binding to the lysosomal enzyme beta hexosaminidase B, which will be linked to a gel matrix. To obtain sufficient quantities of betahexosaminidase-B for this purpose, active enzyme was produced in cultured insect (Sf9) cells by isolation of a recombinant insect virus (Baculovirus) containing the beta-chain cDNA, and infection of Sf9 cells with this construct. The beta-hexosaminidase B was purified and characterized with regards to size, N-terminal sequence, in vivo processing and oligosaccharide structure. It was shown that the recombinant enzyme can serve as a substrate for the phosphotransferase thus it will be used to purify that enzyme.
