The overall objective is to clarify the pathway of Beta- hydroxyisobutyrate catabolism and provide insight into the regulation of metabolism of this compound. The working hypothesis is that beta-hydroxyisobutyrate is made from valine in peripheral tissues and used by the liver for gluconeogenesis. It is proposd that 3-hydroxyisobutyrate dehydrogenase (E.C. 1.1.1.31) is important in controlling interorgan catabolism of beta-hy- droxyisobutyrate. It is further proposed that an enzyme catalyzing the same reaction as methylmalonate semialdehyde dehydrogenase (E.C. 1.2.1.27) of pseudomonads is present in mammalian tissues. Studies will be conductied with purified enzymes, isolated hepatocytes, and intact animals in different nutritional, hormonal, and developmental states. The detailed specific aims are: (a) to clone and sequence cDNA for 3-hy- droxyisobutyrate dehydrogenase; (b) to determine the effects of nutritional, hormonal, and developmental states on tissue amounts of 3-hydroxyisobutyrate dehydrogenase and its mRNA; (c) to develop an enzymatic assay for beta-hydroxyisobutyrate based on the reaction catalyzed by 3-hydroxyisobutyrate dehydrogenase; (d) to determine effects of nutritional and hormonal states on blood levels of beta-hydroxyisobutyrate; (e) to determine effects of other substrates (ethanol, oleate, beta-hydroxybutyrate) and hormones on the metabolism of beta-hydroxyisobutyrate by isolated hepatocytes; (f) to determine whether tissue specific isozymes of 3-hydroxyisobutyrate dehydrogenase exist; (g) to determine whether some enzyme other than 3-hydroxyisobutyrate dehydrogenase is responsible for the oxidation of R-3- hydroxyisobutyrate (intermediate in thymine catabolism); (h) to identify cysteine residue(s) involved in substrate binding by 3- hydroxyisobutyrate dehydrogenase; (i) to isolate and characterize rat liver methylmalonate semialdehyde dehydrogenase; (j) to clone and sequence cDNA for methylmalonate semialdehyde dehydrogenase; and (k) to determine the effects of nutritional, hormonal, and developmental states on tissue amounts of methylmalonate semialdehyde dehydrogenase and its mRNA. These studies should further our understanding of the possible role of beta-hydroxyisobutyrate as a source of carbon for hepatic gluconeogenesis, establish whether methylmalonate semialdehyde dehydrogenase is present in tissues of higher organisms, and explain the accumulation of beta-hydroxyisobutyrate and related compounds under conditions of ketoacidosis and in various inherited organic acidemias.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK040441-03
Application #
3240715
Study Section
Biochemistry Study Section (BIO)
Project Start
1988-09-01
Project End
1993-08-31
Budget Start
1990-09-01
Budget End
1991-08-31
Support Year
3
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Type
Schools of Medicine
DUNS #
005436803
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Popov, K M; Shimomura, Y; Hawes, J W et al. (2000) Branched-chain alpha-keto acid dehydrogenase kinase. Methods Enzymol 324:162-78
Hawes, J W; Crabb, D W; Chan, R J et al. (2000) Mammalian 3-hydroxyisobutyrate dehydrogenase. Methods Enzymol 324:218-28
Kedishvili, N Y; Goodwin, G W; Popov, K M et al. (2000) Mammalian methylmalonate-semialdehyde dehydrogenase. Methods Enzymol 324:207-18
Shimomura, Y; Murakami, T; Nakai, N et al. (2000) 3-hydroxyisobutyryl-CoA hydrolase. Methods Enzymol 324:229-40
Nakai, N; Kobayashi, R; Popov, K M et al. (2000) Determination of branched-chain alpha-keto acid dehydrogenase activity state and branched-chain alpha-keto acid dehydrogenase kinase activity and protein in mammalian tissues. Methods Enzymol 324:48-62
Hawes, J W; Harper, E T; Crabb, D W et al. (1997) Structural and mechanistic aspects of a new family of dehydrogenases, the beta-hydroxyacid dehydrogenases. Adv Exp Med Biol 414:395-402
Jaskiewicz, J; Zhao, Y; Hawes, J W et al. (1996) Catabolism of isobutyrate by colonocytes. Arch Biochem Biophys 327:265-70
Hawes, J W; Jaskiewicz, J; Shimomura, Y et al. (1996) Primary structure and tissue-specific expression of human beta-hydroxyisobutyryl-coenzyme A hydrolase. J Biol Chem 271:26430-4
Hawes, J W; Harper, E T; Crabb, D W et al. (1996) Structural and mechanistic similarities of 6-phosphogluconate and 3-hydroxyisobutyrate dehydrogenases reveal a new enzyme family, the 3-hydroxyacid dehydrogenases. FEBS Lett 389:263-7
Taniguchi, K; Nonami, T; Nakao, A et al. (1996) The valine catabolic pathway in human liver: effect of cirrhosis on enzyme activities. Hepatology 24:1395-8

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