Research Report Differential tissue distribution of tryptophan hydroxylase isoforms 1 and 2 as revealed with monospecific antibodies Stacey A. Sakowski b , Timothy J. Geddes a,d , David M. Thomas a,d , Edi Levi c , James S. Hatfield c , Donald M. Kuhn a,b,d, ⁎ a Department of Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine, USA b Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, USA c Pathology and Laboratory Service, John D. Dingell VA Medical Center, Detroit, MI, USA d Research and Development Service, John D. Dingell VA Medical Center, Detroit, MI, USA ARTICLE INFO ABSTRACT Article history: Accepted 13 February 2006 Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of the neurotransmitter serotonin. Once thought to be a single-gene product, TPH is now known to exist in two isoforms—TPH1 is found in the pineal and gut, and TPH2 is selectively expressed in brain [Walther, D.J., Peter, J.U., Bashammakh, S., Hortnagl, H., Voits, M., Fink, H., Bader, M., 2003. Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science 299, 76.]. Heretofore, probes used for localization of TPH protein or mRNA could not distinguish between the TPH isoforms because of extensive homology shared by them at the nucleotide and amino acid level. We have produced monospecific polyclonal antibodies against TPH1 and TPH2 using peptide antigens from nonoverlapping sequences in the respective proteins. These antibodies allow the differentiation of TPH1 and TPH2 upon immunoblotting, immunoprecipitation, and immunocytochemical staining of tissue sections from brain and gut. TPH1 and TPH2 antibodies do not cross-react with either tyrosine hydroxylase or phenylalanine hydroxylase. Analysis of mouse tissues confirms that TPH1 is the predominant form expressed in pineal gland and in P815 mastocytoma cells with a molecular weight of 51 kDa. TPH2 is the predominant enzyme form expressed in brain extracts from mesencephalic tegmentum, striatum, and hippocampus with a molecular weight of 56 kDa. Antibody specificity against TPH1 and TPH2 is retained across mouse, rat, rabbit, primate, and human tissues. Antibodies that distinguish between the isoforms of TPH will allow studies of the differential regulation of their expression in brain and periphery. © 2006 Elsevier B.V. All rights reserved. Keywords: Tryptophan hydroxylase TPH1 TPH2 Monospecific antibodies Serotonin Abbreviations: 5HT, serotonin αTPH1, antibody against TPH isoform 1 αTPH2, antibody against TPH isoform 2 BH 4 , tetrahydrobiopterin DTT, dithiothreitol MW, molecular weight PBS, phosphate-buffered saline TBST, Tris-buffered saline containing Triton X-100 TPH, tryptophan hydroxylase TPH1, TPH isoform 1 TPH2, TPH isoform 2 BRAIN RESEARCH XX (2006) XXX – XXX ⁎ Corresponding author. John D. Dingell VA Medical Center, Research and Development Service (11R), 4646 John R, Detroit, MI 48201, USA. E-mail address: donald.kuhn@wayne.edu (D.M. Kuhn). BRES-35248; No. of pages: 8; 4C: 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.02.047 available at www.sciencedirect.com www.elsevier.com/locate/brainres ARTICLE IN PRESS