With the conventional somatic cell hybridization technique and with the murine galactosylceramidase-deficiency disease, the twitcher, the human gene coded for galactosylceramidase will be mapped to a specific chromosome. Detailed characterization of human galactosylceramidase will be attempted by acrylamide gel electrophoresis and electrofocusing.
The aim i s (1) to distinguish the human enzyme from the mouse enzyme so that the procedure can be used as a supplementary means for the above chromosomal localization study, and (2) to obtain a small amount of relatively pure enzyme to be used as the antigen for monoclonal antibody production and also possibly as the source for sequencing of at least several N-terminal amino acids. More conventional purification will be attempted from the placenta and/or urine with similar eventual aims. The use of the new """"""""suicide"""""""" inhibitor of beta-galactosidase -- galactosylmethyl-p-nitrophenyltriazene (gal-MNT) -- will be explored to differentially inhibit GM1-ganglioside beta-galactosidase. If successful, the conventional fluorogenic substrate, 4-methylumbelliferyl beta-galactoside could be used for assays of galactosylceramidase and also for its localization on the electrophoretic gel. This will facilitate all aspects of the galactosylceramidase studies, including diagnosis of affected patients. Attempts will be made to induce an experimental mouse model of GM1-gangliosidosis with the gal-MNT inhibitor which specifically inhibits GM1-ganglioside beta-galactosidase but not galactosylceramidase. The model will be characterized in detail pathologically and biochemically. Then, the model will be manipulated to answer questions concerning the requirement for the enzyme by developing brain and those concerning the timing of therapeutic intervention in this group of genetic neurological disorders vis-a-vis plasticity of developing brain. Parallel experiments are planned with the use of the organotypic CNS cultures. A possible new factor that confers to glucosylceramidase the capacity to hydrolyze glucosylceramide will be characterized. This is NOT the same factor long known as the """"""""Gaucher activator protein"""""""". By itself, this would be an important development in lipid enzymology. Furthermore, it is conceivable that genetic status resembling Gaucher disease might exist due to abnormality of this factor. Such a possibility will be tested when the factor is adequately characterized.
