Expression of recombinant soluble and membrane-bound catechol O-methyltransferase in eukaryotic cells and identification of the respective enzymes in rat brain.
The rat and human recombinant soluble and membrane-bound catechol O-methyltransferase (S- and MB-COMT, respectively) were expressed using mammalian and baculovirus vectors. Low levels of rat and human S-COMT polypeptides were detected by immunoprecipitation in K-562 cell lines transfected with the S-COMT vectors. From K-562 cells transfected with the rat MB-COMT construct, both S- and MB-COMT recombinant proteins were detected by a rat COMT-specific anti-serum. Infection of lepidopteran Spodoptera frugiperda cells with recombinant S- or MB-COMT baculovirus constructs yielded high amounts of enzymically active and immunoreactive S- or MB-COMT proteins, respectively. Pulse/chase experiments with [35S]methionine-labelled insect cells infected with the MB-COMT baculovirus showed that the 30-kDa recombinant human MB-COMT polypeptide was not processed into the 25-kDa S-COMT form. Subcellular fractionations of insect cells, followed by immunoblotting with COMT antiserum, showed that recombinant S-COMT was found only in the soluble, cytoplasmic fraction, whereas MB-COMT resided both in soluble and membrane fractions. The recombinant MB-COMT sedimented in Percoll gradients at the density of 1.042 g/ml cosedimenting with the plasma-membrane marker. Fractionation and immunoblotting experiments on homogenized total rat brains indicated that the rat S-COMT (24 kDa) and some of the rat MB-COMT (28 kDa) was recovered in soluble fractions, whereas the microsomal material having COMT activity contained the MB-COMT polypeptide. The rat brain microsomal MB-COMT had a density of 1.042 g/ml in Percoll gradients, cosedimenting with the plasma-membrane and rough-endoplasmic-reticulum marker enzymes. The meta/para methylation ratio of dihydroxybenzoic-acid substrate by different recombinant and rat brain COMT-containing subcellular fractions was analysed.
Tilgmann
C
C
Orion-Farmos Pharmaceuticals, Orion Research Center, Helsinki, Finland.
Melen
K
K
Lundström
K
K
Jalanko
A
A
Julkunen
I
I
Kalkkinen
N
N
Ulmanen
I
I
D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D002394:Catechol O-Methyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D006801:Humans; D066298:In Vitro Techniques; D008745:Methylation; D008969:Molecular Sequence Data; D009036:Moths; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D014407:Tumor Cells, Cultured
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D002394:Catechol O-Methyltransferase