Expression of recombinant soluble and membrane-bound catechol O-methyltransferase in eukaryotic cells and identification of the respective enzymes in rat brain
(1992) In European Journal of Biochemistry 207(2). p.813-821- Abstract
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 antiserum. 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... (More)
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 antiserum. 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 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.
(Less)
- author
- Tilgmann, C. LU ; Melen, K. ; Lundstrom, K. ; Jalanko, A. ; Julkunen, I. ; Kalkkinen, N. and Ulmanen, I.
- publishing date
- 1992
- type
- Contribution to journal
- publication status
- published
- in
- European Journal of Biochemistry
- volume
- 207
- issue
- 2
- pages
- 9 pages
- publisher
- Wiley-Blackwell
- external identifiers
-
- scopus:0026752312
- pmid:1633830
- ISSN
- 0014-2956
- DOI
- 10.1111/j.1432-1033.1992.tb17112.x
- language
- English
- LU publication?
- no
- id
- 03f07272-0ea6-4ee9-b66b-edb292912488
- date added to LUP
- 2016-04-11 14:00:30
- date last changed
- 2024-05-03 22:18:07
@article{03f07272-0ea6-4ee9-b66b-edb292912488, abstract = {{<p>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 antiserum. 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 [<sup>3</sup><sup>5</sup>S]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 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.</p>}}, author = {{Tilgmann, C. and Melen, K. and Lundstrom, K. and Jalanko, A. and Julkunen, I. and Kalkkinen, N. and Ulmanen, I.}}, issn = {{0014-2956}}, language = {{eng}}, number = {{2}}, pages = {{813--821}}, publisher = {{Wiley-Blackwell}}, series = {{European Journal of Biochemistry}}, title = {{Expression of recombinant soluble and membrane-bound catechol O-methyltransferase in eukaryotic cells and identification of the respective enzymes in rat brain}}, url = {{http://dx.doi.org/10.1111/j.1432-1033.1992.tb17112.x}}, doi = {{10.1111/j.1432-1033.1992.tb17112.x}}, volume = {{207}}, year = {{1992}}, }