ascorbic-acid and gamma-butyrobetaine

The biosynthesis of carnitine from lysine and methionine involves five enzymatic reactions. Gamma-butyrobetaine hydroxylase (BBH; EC 1.14.11.1) is the last enzyme of this pathway. It catalyzes the reaction of hydroxylation of gamma-butyrobetaine to carnitine. This enzyme had never been purified to homogeneity from rat tissue. This paper describes the purification and characterization of the rat liver BBH. This protein has been purified some 413 fold by ion exchange, affinity and gel-filtration chromatographies and appears as a dimere of 43,000 Daltons subunits by PAGE. The affinity chromatography column used in the purification process utilizes 3-(2,2,2-trimethylhydrazinium)propionate (THP), a BBH inhibitor, as the ligand. Polyclonal antibodies were raised against the liver enzyme. They were able to precipitate BBH activity in either a crude liver extract or a purified fraction of the enzyme. Furthermore, it crossreacts with a 43 kDa protein in the liver. No evidence for extra hepatic enzyme was found.

Topics: Animals; Ascorbic Acid; Betaine; Carnitine; Catalase; Catalysis; Chromatography, Affinity; Enzyme Inhibitors; Ferrous Compounds; gamma-Butyrobetaine Dioxygenase; Hydroxylation; Ketoglutaric Acids; Kinetics; Ligands; Liver; Male; Methylhydrazines; Mixed Function Oxygenases; Molecular Weight; Rats; Rats, Wistar

The effect of ascorbate (AsA) on the synthesis of carnitine from gamma-butyrobetaine (BB) in primary cultured guinea pig hepatocytes was investigated. The hepatocyte monolayers preloaded with AsA were incubated for 4 h in medium with various concentrations of BB as the precursor of carnitine. The accumulation of carnitine reached a maximum when the cells were incubated with 0.05-1.0 mM BB and significantly decreased with excess BB (5 mM). In contrast, increasing concentrations of AsA supplemented to medium led to an increase in carnitine content, but AsA and total AsA contents in cells decreased by BB supplementation. Regarding the enhancement of hydroxylation of BB in the hepatocytes, AsA was the most effective among such other reducing agents as glutathione and dithiothreitol. Although erythorbate (ErA) also stimulated the hydroxylation of BB, carnitine content in cells preloaded with ErA was only 60% of that with AsA. These results suggest that AsA is specifically required for the hydroxylation of BB. Furthermore, AsA can regulate carnitine synthesis in the primary cultured guinea pig hepatocytes.

Topics: Animals; Ascorbic Acid; Betaine; Carnitine; Cells, Cultured; Guinea Pigs; Hydroxylation; Liver; Male