Expression of functional membrane-bound and soluble catechol-O-methyltransferase in Escherichia coli and a mammalian cell line.
Human catechol-O-methyltransferase (hCOMT) cDNA was used to express the recombinant hCOMT enzyme in sufficient quantities in prokaryotic as well as in eukaryotic cells to allow kinetic studies. When human membrane-bound catechol-O-methyltransferase (MB-COMT; amino acids 1-271) and the soluble catechol-O-methyltransferase COMT (S-COMT; delta membrane anchor hCOMT; amino acids 27-271), with the latter lacking the first 26 hydrophobic amino acids, were expressed in Escherichia coli, a relatively high-level synthesis of catalytically active enzymes was obtained. Insertion of the human MB-COMT-coding sequence into an eukaryotic expression vector under transcriptional control of the cytomegalovirus (CMV) promoter and enhancer yielded large quantities of hCOMT in human kidney 293 cells. Subcellular fractionation of 293 cells transfected with pBC12/CMV-hCOMT showed hCOMT to be located predominantly in the membrane fraction. The catechol-O-methyltransferase (COMT) activity was measured in cytosolic and membrane fractions at 37 degrees C, giving values of 33 and 114 units/mg of protein, respectively (1 unit produces 1 nmol of guaiacol/h). Km values were 10 microM for MB-COMT and 108 microM for S-COMT, indicating that recombinant MB-COMT exhibits a higher affinity for catechol as the substrate than the soluble form. RNA blot analysis of human hepatome cells (Hep G2), kidney, liver, and fetal brain revealed only one species of hCOMT mRNA of approximately 1.4 kb. Its level in these various tissues was similar to those of COMT protein in each tissue.(ABSTRACT TRUNCATED AT 250 WORDS)
Malherbe
P
P
Pharma Division, Preclinical Research, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Bertocci
B
B
Caspers
P
P
Zürcher
G
G
Da Prada
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002394:Catechol O-Methyltransferase / Q000235:genetics / Q000378:metabolism*; D002396:Catechols / Q000378:metabolism; D002460:Cell Line; D004926:Escherichia coli / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D008745:Methylation; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins; D012995:Solubility; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution
D002396:Catechols; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D002394:Catechol O-Methyltransferase; C034221:catechol