This work focuses on electrophoretic variants of arylsulfatase A (ASA; E.C. 3.1.6.1.) which have been proposed as a predisposing factor for the neuropathological effects of alcohol. This enzyme catalyzes the degradation of sulfatides to cerebrosides and sulfate. The studies in this grant application will examine genetic, metabolic, and structural aspects related to the proposed defect in alcoholic patients. To investigate the possibility that variant ASA's may be under genetic control, blood samples will be taken from first and second degree relatives of probands with variant ASA's. The platelet lysates will be analyzed for ASA banding patterns by discontinuous electrophoresis, isoelectric focusing, and two-dimensional gel electrophoresis. The possible metabolic consequences of the variant ASA's will be examined, first, by measuring the urinary levels of sulfatides in subjects with variant and normal ASA's and, second, by determining the kinetic parameters of the variant and normal ASA's in vitro using sulfatides as substrate in the presence of activator proteins. Extensive structural studies will be performed which include glycan, protein, and nucleic acid analyses. The effect of specific hydrolytic enzymes and lectin binding on variant and normal ASA's will be studied to explore the importance of structural elements of the glycan moiety on the electrophoretic pattern. The sugar content of the native enzyme as well as selected polypeptides will be determined after alkylation and fragmentation of the variant and normal ASA's. Complete structure of the glycan moiety of selected glycopeptides will be determined. Similarly, protein analysis, including comparisons of the variant forms by peptide mapping and microsequencing, will be completed for the native and deglycosylated ASA's. It will be determined if the 2.0 and 3.5 kb mRNA's for ASA arise from a single gene or two closely homologous genes. If the results from the peptide and carbohydrate analyses suggest that the variant forms of the enzyme have a different peptide backbone, the cDNA's will be used as probes to the DNA from patients with variant ASA's in order to determine if a mutation has occurred in their genome.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
3R01AA007799-02S1
Application #
2044128
Study Section
Biochemistry, Physiology and Medicine Subcommittee (ALCB)
Project Start
1990-01-01
Project End
1992-12-31
Budget Start
1992-06-01
Budget End
1992-12-31
Support Year
2
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Psychiatry
Type
Schools of Medicine
DUNS #
622146454
City
Piscataway
State
NJ
Country
United States
Zip Code
08854
Stoecker, P W; Manowitz, P; Harvey, R et al. (1998) Determination of galactose and galactocerebroside using a galactose oxidase column and electrochemical detector. Anal Biochem 258:103-8
Ricketts, M H; Amsterdam, J D; Park, D S et al. (1996) A novel arylsulfatase A protein variant and genotype in two patients with major depression. J Affect Disord 40:137-47
Park, D S; Poretz, R D; Stein, S et al. (1996) Association of alcoholism with the N-glycosylation polymorphism of pseudodeficient human arylsulfatase A. Alcohol Clin Exp Res 20:228-33
Park, D S; Manowitz, P; Stein, S et al. (1996) Structural characterization of variant forms of arylsulfatase A that associate with alcoholism. Alcohol Clin Exp Res 20:234-9
Park, D S; Poretz, R D; Ricketts, M H et al. (1996) Arylsulfatase A: relationship of genotype to variant electrophoretic properties. Biochem Genet 34:149-61
Ricketts, M H; Goldman, D; Long, J C et al. (1996) Arylsulfatase A pseudodeficiency-associated mutations: population studies and identification of a novel haplotype. Am J Med Genet 67:387-92
Ricketts, M H; Zhang, X; Manowitz, P (1995) A method for rapid detection of arylsulfatase A pseudodeficiency mutations. Hum Hered 45:235-40
Manowitz, P; Stoecker, P W; Yacynych, A M (1995) Galactose biosensors using composite polymers to prevent interferences. Biosens Bioelectron 10:359-70
Poretz, R D; Yang, R S; Canas, B et al. (1992) The structural basis for the electrophoretic isoforms of normal and variant human platelet arylsulphatase A. Biochem J 287 ( Pt 3):979-83