The overall objective of the proposed research is to gain insight into the biochemical mechanisms responsible for the turnover of palmitate covalently linked to p21(cH-ras) (H-Ras). H-Ras is the protein product of the cellular homologue of the Harvey ras oncogene, which contributes to the development of a wide variety of human cancers. The H-Ras protein undergoes a complex series of post-translational modifications that include carboxy-terminal isoprenylation, proteolysis, methylation and palmitoylation. Palmitoylation has been shown to enhance the transformation efficiency of H-Ras about 10-fold in vivo. A recent study (Magee, A. I., et a1., EMBO J. 6:3353, 1987) has provided strong evidence that the palmitate undergoes a dynamic acylation-deacylation cycle, but details concerning the enzymology of this process and its regulation are lacking. To begin to dissect this process, we have developed an assay for the enzymatic removal of free palmitate from [3H]palmitate-labeled H-Ras. This substrate was produced in a baculovirus expression system and was used to purify to homogeneity a novel 37 kDa enzyme from bovine brain that removes the radiolabeled palmitate. The enzyme recognizes H-Ras as a substrate only when H-Ras is in its native conformation (bound to Mg2+ and guanine nucleotide). The major focus of this proposal is to characterize further this novel enzyme with respect to the requirement for bound Mg2+ and nucleotide, to determine the target protein and fatty acid specificity of the enzyme, and to produce sufficient quantities of the enzyme for protein sequence analysis and cDNA cloning. We will then proceed to characterize the effects of overexpression of the enzyme on palmitate turnover and H- Ras-mediated cellular transformation by transfection of cultured mammalian cells, and use the enzyme to investigate the association of H-Ras with phospholipid bilayers and reconstituted membranes in vitro. The results could be of significant value in understanding normal cellular physiology and malignant transformation by mutated ras oncogenes.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA061823-01
Application #
2102625
Study Section
Pathology B Study Section (PTHB)
Project Start
1994-01-14
Project End
1996-12-31
Budget Start
1994-01-14
Budget End
1994-12-31
Support Year
1
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390
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Schriner, J E; Yi, W; Hofmann, S L (1996) cDNA and genomic cloning of human palmitoyl-protein thioesterase (PPT), the enzyme defective in infantile neuronal ceroid lipofuscinosis. Genomics 34:317-22
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