The long-term aim of his research is to identify genetic predispositions to complications of alcoholism. The immediate goal for this grant period is to elucidate the basis of an abnormality in the thiamin pyrophosphate (TPP)-dependent enzyme transketolase (EC 2.2.1.1). An impaired affinity to TPP (high Km) has been postulated to predispose a subpopulation of alcoholics to the development of a form of thiamin deficiency, the Wernicke-Korsakoff's syndrome. Structural and molecular genetic studies is proposed to characterize the aberrant transketolase in cultured skin fibroblasts from patients with Wernicke-Korsakoff's syndrome or from patients with documented transketolase abnormality. Structural comparisons will be performed by immunoblots of electrophoretic gels (SDS-gels, isoelectric focusing gels and native gels), by activity staining of electrophoretic gels, by peptide mapping and other immunochemical techniques. This proposition will also examine the possibility of multiple forms of human transketolase. Preliminary work has already purified human liver transketolase, raised antibodies and standardized immunochemical procedures. For molecular genetic studies, cDNA for normal human transketolase will be isolated (with the antibody probe) sequenced, and used to isolated genomic DNAs to construct a structural map for the transketolase gene. The cDNA for aberrant transketolase will also be isolated and sequenced to identify the exact site of genetic mutation. The mutation will be further tested at the genomic level using the abnormal genomic fragment amplified with the polymerase chain reaction (PCR) method. These studies will answer the following 2 questions. Is the aberration of Wernicke-Korsakoff transketolase associated with a structural abnormality? If so, is this structural abnormality determined at the genetic level?

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
1R01AA008246-01
Application #
3112270
Study Section
Biochemistry, Physiology and Medicine Subcommittee (ALCB)
Project Start
1990-01-01
Project End
1991-12-31
Budget Start
1990-01-01
Budget End
1990-12-31
Support Year
1
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Winifred Masterson Burke Med Research Institute
Department
Type
DUNS #
City
White Plains
State
NY
Country
United States
Zip Code
10605
Sheu, K F; Calingasan, N Y; Lindsay, J G et al. (1998) Immunochemical characterization of the deficiency of the alpha-ketoglutarate dehydrogenase complex in thiamine-deficient rat brain. J Neurochem 70:1143-50
Sheu, K F; Calingasan, N Y; Dienel, G A et al. (1996) Regional reductions of transketolase in thiamine-deficient rat brain. J Neurochem 67:684-91
Calingasan, N Y; Sheu, K F; Baker, H et al. (1995) Heterogeneous expression of transketolase in rat brain. J Neurochem 64:1034-44
Jung, E H; Sheu, K F; Blass, J P (1993) An enzymatic and immunochemical analysis of transketolase in fibroblasts from Wernicke-Korsakoff syndrome. J Neurol Sci 114:123-7