PURIFICATION AND CHARACTERIZATION OF THE RAT-LIVER GAMMA-BUTYROBETAINE HYDROXYLASE

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 h ydroxylation of gamma-butyrobetaine to carnitine. This enzyme had neve r been purified to homogeneity from rat tissue. This paper describes t he purification and characterization of the rat liver BBH. This protei n has been purified some 413 fold by ion exchange, affinity and gel-fi ltration chromatographies and appears as a dimere of 43,000 Daltons su bunits by PAGE. The affinity chromatography column used in the purific ation process utilizes 3-(2,2,2-trimethylhydrazinium)propionate (THP), a BBH inhibitor, as the ligand. Polyclonal antibodies were raised aga inst the liver enzyme. They were able to precipitate BBH activity in e ither a crude liver extract or a purified fraction of the enzyme. Furt hermore, it crossreacts with a 43 kDa protein in the liver. No evidenc e for extra hepatic enzyme was found.