GENETIC AND BIOCHEMICAL STUDIES ON KRABBE'S DISEASE
The aim of this research is to investigate the enzyme and activator protein which control the lysosomal degradation of galactosylceramide (gal-cer), psychosine monogalactosyldiglyceride and lactosylceramide (lac-cer). This enzyme, gal-cer Beta-galactosidase, is found in very low levels in all tissues from patients with Krabbe disease, an autosomal recessive disease of children, and in leukocyte and fibroblast cultures from some healthy people. We plan to purify this enzyme from control human brain using techniques routinely in use in this laboratory including hydrophobic affinity chromatography, chromatofocusing and specific affinity chromatography using psychosine (galactosylsphingosine) and the activator protein coupled to Sepharose. The purity of our enzyme will be checked by measuring other possible contaminating enzymes, by running polyacrylamide gel electrophoresis on different pH and concentration gels and by reverse phase liquid chromatography. The pure enzyme will be injected into rabbits for the production of antibodies which will then be used for measuring the level of crossreacting material (CRM) in tissues and cells from controls and patients with gal-cer Beta-galactosidase deficiency. These antibodies will then used for studies of processing of the protein chains after cells are given (3H)leucine in the medium. The nature of the mutation will also be examined in tissues and cells after extraction, SDS-PAGE, electroblotting to nitrocellulose filter paper, reaction with specific antibody and then with 125I-Protein A or horse-radish peroxidase conjugated goat antirabbit lgG. A study of this enzyme would not be complete without careful examination of the heat stable protein required for reaction with the lipid substrates. This activator protein will be purified from human brain, liver and spleen using methods currently in use in this laboratory. It's amino acid composition will be determined and its specificity to activate other enzymes will be evaluated. Antibodies to this activator protein will be made in rabbits and the levels of this activator will be determined in tissues and cells using rocket immunoelectrophoresis. The possibility that a defect in the activator protein is responsible for certain genetic diseases or the cause of some clinical heterogeneity will be investigated. Processing and determination of the molecular forms will be studied using the specific antibodies. This research will provide information related to the defect in Krabbe disease and other genetic diseases.