IntroductionAlthough the guinea-pig has been an important model for studying histamine function in vitro and in vivo, the enzymes responsible for the inactivation of histamine, diamine oxidase (DAO) and histamine N-methyltransferase (HNMT) [1], have been only poorly characterized at the molecular level for this organism. Therefore, we have cloned and analysed cDNAs encoding guinea-pig DAO and HNMT to learn more about the structure and function of these proteins.
Materials and methodsBioptic samples were obtained from kidney, liver, and intestine of four male Hartley guinea-pigs (mean weight 450 g, mean age 20 weeks, bred in house) immediately post mortem and frozen in liquid nitrogen. Total RNA was prepared from tissue samples using TRI Reagent ® (MRC, Cincinnati, OH) according to manufacturer's instructions and reverse transcribed into cDNA for 90min at 42°C using M-MLV Reverse Transcriptase (Promega, Mannheim, Germany) with (dT) 18 as primer. Partial guinea-pig DAO and HNMT cDNA sequences were amplifi ed by PCR using 35 cycles of 15 s 94°C/15 s 55-65°C/60 s 72°C in reactions containing 5 ng cDNA, 200 µM dNTPs and 200 nM primers derived from the sequences of other mammalian DAO and HNMT proteins, respectively. Full-length or almost full-length DAO and HNMT cDNAs were then obtained by rapid amplifi cation of cDNA ends (RACE) with primers derived from the partial guinea-pig DAO and HNMT cDNA sequences using the SMART ™ RACE cDNA Amplifi cation Kit (Clontech Laboratories, Palo Alto, CA) according to manufacturer's instructions. DNA sequences were determined by cycle sequencing on an ABI Prism ™ 310 Genetic Analyzer using the ABI Prism ® BigDye ™ Terminator Kit (Applied Biosystems, Foster City, CA). The study was carried out according to Austrian regulations on animal experimentation.
Results and discussionUsing primers derived from other mammalian DAO and HNMT protein sequences partial guinea-pig DAO and HNMT cDNAs were amplifi ed by PCR. These cDNA sequences were used to design primers for RACE-PCR that yielded guinea-pig cDNA sequences of almost the complete DAO and the complete HNMT coding regions. The polypeptide sequences deduced from these cDNAs were identical in all guinea-pig tissues analysed and turned out to be highly homologous to other mammalian DAO and HNMT protein sequences determined earlier [2,3].As shown in Figure 1, the guinea-pig DAO sequence determined here includes 692 amino acids and lacks ca. 40 residues at the N-terminus and 20 residues at the C-terminus that could not be determined due to the very low expression levels of DAO in guinea-pig tissues. The guinea-pig DAO sequence is ca. 80 % identical with other mammalian DAO protein sequences and contains all residues that have been shown to be important for catalytic function of copper-containing amine oxidases [2,4].Guinea-pig HNMT has 292 amino acid residues (Fig. 2) and is ca. 80 % identical with other mammalian HNMT proteins [3]. Most but not all residues that have been shown to interact with histamine and S-adenosyl-L-homocysteine in hu...
