Kenneth Lundström

Characteristics of catechol O-methyltransferase (COMT) and properties of selective COMT inhibitors

The enzyme-catalyzed O-methylation of catecholamines was first described by Axelrod and coworkers in the late 1950’s [1–3]. They called the responsible enzyme catechol O-methyltransferase (COMT). During the subsequent 15 years the enzyme was partially purified, its distribution was established, several reaction mechanisms were proposed, and a number of inhibitors were described. The results of this study period were extensively reviewed by Guldberg and Marsden in 1975 [4].

Semliki Forest virus vectors for rapid and high-level expression of integral membrane proteins

Abstract Semliki Forest virus (SFV) vectors have been applied for the expression of recombinant integral membrane proteins in a wide range of mammalian host cells. More than 50 G protein-coupled receptors (GPCRs), several ion channels and other types of transmembrane or membrane-associated proteins have been expressed at high levels. The establishment of large-scale SFV technology has facilitated the production of large quantities of recombinant receptors, which have then been subjected to drug screening programs and structure–function studies on purified receptors. The recent Membrane Protein Network (MePNet) structural genomics initiative, where 100 GPCRs are overexpressed from SFV vectors, will further provide new methods and technologies for expression, solubilization, purification and crystallization of GPCRs.

The human catechol-O-methyltransferase (COMT) gene maps to band q11.2 of chromosome 22 and shows a frequent RFLP with BglI

We have been able to assign the human catechol-O-methyltransferase gene (COMT) to chromosome 22q11.2 by using Southern blot analysis of panels of somatic cell hybrids and chromosomal in situ hybridization. Furthermore, Southern blot analysis of DNA from blood and bone marrow samples of a patient with chronic myeloid leukemia (CML), having an extra Philadelphia chromosome (Ph1) in addition to the one produced by the reciprocal translocation between chromosomes 9 and 22, showed increased COMT and BCR gene dosage as compared to DNAs originating from CML patients with only one Ph1 chromosome or from chromosomally normal individuals. Control hybridizations of the same blot with TCRG- and TCRA-specific probes showed corresponding signal intensities in all samples. A relatively frequent two-allele COMT gene RFLP (PIC = 0.37) was recognized in DNAs digested with BglI. Our gene mapping result is in concordance with that previously reported by Brahe et al. (1986), who used an autoradiozymogram assay on different somatic cell hybrids to map this gene to chromosome 22.

Organization of the mouse 5-HT3 receptor gene and functional expression of two splice variants

The structure of the mouse 5-HT3 receptor gene, 5-HT3R-A, is most similar to nicotinic acetylcholine receptor (nAChR) genes, in particular to the gene encoding the neuronal nAChR subunit alpha 7. These genes share among other things the location of three adjacent introns, suggesting that 5-HT3R-A and nAChR genes arose from a common precursor gene. The alternative use of two adjacent splice acceptor sites in intron 8 creates, in addition to the original 5-HT3R-A cDNA (5-HT3R-AL), a shorter isoform (5-HT3R-AS) which lacks six codons in the segment that translates into the major intracellular domain. This splice consensus sequence is not found in human genomic DNA. In mouse, we demonstrate by RNAse protection assay that 5-HT3R-AS mRNA is approximately 5 times more abundant than 5-HT3R-AL mRNA in both neuroblastoma cell lines and neuronal tissues. We used the Semliki Forest virus expression system for electrophysiological characterization of 5-HT3R-AS and 5-HT3R-AL in mammalian cells. No differences in electrophysiological characteristics, such as voltage dependence, desensitization kinetics, or unitary conductance were found between homomeric 5-HT3R-AS and 5-HT3R-AL receptors. Their properties are very similar to those of 5-HT3 receptors in mouse neuroblastoma cell lines.

Expression of enzymatically active rat liver and human placental catechol-O-methyltransferase in Escherichia coli; purification and partial characterization of the enzyme

To produce sufficient amounts of recombinant catechol-O-methyltransferase (COMT) for structural and functional studies the coding regions of the rat liver and human placental COMT genes have been introduced into a bacterial expression vector pKEX14. Recombinant COMT was produced in Escherichia coli up to 10% of total bacterial protein after the induction of the T7 RNA polymerase gene with isopropyl-beta-D-thiogalactopyranoside. Both the rat and human enzymes were enzymatically active, soluble and reacted with anti-COMT antiserum in Western blotting. Both enzymes were purified from E. coli cells and partially characterized by determining their specific activity, apparent molecular weight and pI.