163 Molecular and Cellular Biochemistry 178: 163–168, 1998. © 1998 Kluwer Academic Publishers. Printed in the Netherlands. Purification and characterization of the rat liver gamma-butyrobetaine hydroxylase Stéphane Galland, Françoise Le Borgne, Denis Guyonnet, Pierre Clouet and Jean Demarquoy Université de Bourgogne, Unité de Recherche en Nutrition Cellulaire et Métabolique, BP400, F-21011 Dijon Cedex, France Received 17 February 1997; accepted 11 June 1997 Abstract The biosynthesis of carnitine from lysine and methionine involves five enzymatic reactions. γ-butyrobetaine hydroxylase (BBH; EC 1.14.11.1) is the last enzyme of this pathway. It catalyzes the reaction of hydroxylation of γ-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. (Mol Cell Biochem 178: 163–168, 1998) Key words: carnitine biosynthesis, γ-butyrobetaine hydroxylase, rat liver, fatty acid oxidation Address for offprints: J. Demarquoy, Université de Bourgogne, Unité de Recherche en Nutrition Cellulaire et Métabolique, BP400, F-21011 Dijon Cedex, France Introduction Carnitine plays a major role in the oxidation of fatty acids allowing the transfer of fatty acids through the inner mito- chondrial membrane to the mitochondrial matrix where β- oxidation occurs. Carnitine biosynthesis involves five enzymatic reactions. The ultimate precursors of this synthesis are two amino acids: lysine and methionine [1]. Five enzymes are required for carnitine synthesis. The first reaction is catalyzed by nuclear enzymes (protein-lysine methyltrans- ferase; EC 2.1.1.X, X = 43, 59, 60). During this reaction, S- adenosylmethionine provides three methyl groups for the methylation of peptide-linked lysyl residues to form peptide- linked 6-N-trimethyllysine (TML) [2]. TML is released from protein by protein turnover and then reaches the mitochondria to be hydroxylated to 3-hydroxy 6-N-trimethyllysine, this reaction, catalyzed by 6-N-trimethyllysine dioxygenase (EC 1.14.11.8) [3], takes place in the mitochondrial matrix. The next step occurs in the cytoplasm and during this reaction, catalyzed by the serine transhydroxy methylase, the 3- hydroxy 6-N-trimethyllysine is clived to glycine and γ- butyrobetaine aldehyde [4], that is further oxidized to γ-butyrobetaine. This NAD-dependent reaction is catalyzed by the cytosolic γ-butyrobetaine aldehyde dehydrogenase (EC 1.2.1.47) [5]. Finally, γ-butyrobetaine is hydroxylated to carnitine by γ-butyrobetaine hydroxylase (BBH; EC 1.14.11.1), a cytosolic enzyme [6]. The first four enzymes of the carnitine biosynthesis pathway are expressed in virtually any tissues. On the opposite, in rat, BBH activity was only detected in the liver and to some extent in the testis [7–9]. Our goal was to characterize the rat liver BBH. We first purify the enzyme and determine kinetic parameters of the reaction on purified enzyme. Antibodies directed against the liver enzyme were raised and used to precise BBH tissular location and to relate BBH activity to the presence of immunoreactive material.