Solubilization and partial purification of particulate catechol-<i>O</i>-methyltransferase from rat liver

Tong, Jessica; D'Iorio, A.

doi:10.1139/o77-164

Cited by 43 publications

(14 citation statements)

References 7 publications

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“…Though the results obtained in the present work do not allow us to suggest that younger rats are endowed with higher amounts of MB-COMT than older animals, this could be used as an explanation for the observation that O-methylation developing rats is more sensitive to inhibition by tolcapone. Another indication that would fit this suggestion is that Km values differ markedly between MB-COMT and S-COMT; according to Apprile & Malamud (1975) and Tong & d'Iorio (1977) 8.9 and in the liver by a factor of 3.9. Of course the preparations used in the experiments reported here probably constitute a mixture of MB-COMT and S-COMT, the ratio of which has to be determined in future studies.…”

Section: Discussionmentioning

confidence: 88%

“…Though the results obtained in the present work do not allow us to suggest that younger rats are endowed with higher amounts of MB-COMT than older animals, this could be used as an explanation for the observation that O-methylation developing rats is more sensitive to inhibition by tolcapone. Another indication that would fit this suggestion is that Km values differ markedly between MB-COMT and S-COMT; according to Apprile & Malamud (1975) and Tong & d'Iorio (1977) 12.4 (7.9, 16.9) 27.0 (4.9, 49.1) 24.8 (16.0, 33.5) 24.0 (11.7, 36.3) Values are arithmetic means +s.e.mean and geometric means with 95% confidence intervals of 5 experiments per group. Significantly different from Vmax values in 3 day old rats (*P<0.05) and significantly different from values in renal tissues ( §P<0.05) using the Newman-Keuls test.…”

Section: Discussionmentioning

confidence: 92%

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Ontogenic aspects of liver and kidney catechol‐O‐ methyltransferase sensitivity to tolcapone

Vieira-Coelho

Soares‐da‐Silva

1996

British J Pharmacology

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1 The present work describes the catechol-O-methyltransferase (COMT) activities in the liver and kidney of developing and adult rats (aged 3, 6, 9, 18, 30 and 60 days; n = 5 per group) and evaluates the enzyme sensitivity to inhibition by tolcapone, a reversible COMT inhibitor. 2 COMT activity, evaluated by the ability to methylate adrenaline to metanephrine, was determined in liver and kidney homogenates prepared in 0.5 mm phosphate buffer (pH = 7.8) containing pargyline (0.1 mM), MgCl2 (0.1 mM), EGTA (1 mM) and S-adenosyl-L-methionine (0.1 mM). Vmax (in nmol mg-' protein h-1) of liver COMT was found to decrease gradually with age, from 5.3 + 0.5 at the age of 3 days up to 2.9 + 0.2 at the age of 60 days; for the same age range, Km values (in yM; geometric means with 95% confidence limits) increased from 3.3 (1.0, 7.5) up to 13.1 (2.1, 24.1). At the age of 3 days, Vmax values for kidney COMT (2.6 + 0.1) were lower than those for the liver COMT. However, Vmax values for kidney COMT were found to increase up to 6.2 + 0.6 at the age of 18 days and then declined by 44% at the age of 30 and 60 days. In kidney, aging was also accompanied by an increase in Km 720 (640, 800) at the age of 60 days. As was found in the liver, IC50 values (in nM) for inhibition of kidney COMT by tolcapone also increased with age, from 8 (6, 10) at the age of 3 days up to 177 (131, 240) at the age of 60 days. In all experimental groups, the IC50 values for inhibition of liver COMT by tolcapone was higher than those for kidney COMT. 4 In conclusion, these results suggest that aging is accompanied by a decrease in liver and kidney COMT affinity for the substrate (evidenced by the increase in Km values) and a decrease in sensivity towards inhibition by tolcapone (evidenced by the increase in IC50 values). Furthermore, kidney COMT is shown to be more sensitive to inhibition by tolcapone than liver COMT, irrespective of the age of the animal.

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Section: Discussionmentioning

confidence: 88%

“…Though the results obtained in the present work do not allow us to suggest that younger rats are endowed with higher amounts of MB-COMT than older animals, this could be used as an explanation for the observation that O-methylation developing rats is more sensitive to inhibition by tolcapone. Another indication that would fit this suggestion is that Km values differ markedly between MB-COMT and S-COMT; according to Apprile & Malamud (1975) and Tong & d'Iorio (1977) 12.4 (7.9, 16.9) 27.0 (4.9, 49.1) 24.8 (16.0, 33.5) 24.0 (11.7, 36.3) Values are arithmetic means +s.e.mean and geometric means with 95% confidence intervals of 5 experiments per group. Significantly different from Vmax values in 3 day old rats (*P<0.05) and significantly different from values in renal tissues ( §P<0.05) using the Newman-Keuls test.…”

Section: Discussionmentioning

confidence: 92%

Ontogenic aspects of liver and kidney catechol‐O‐ methyltransferase sensitivity to tolcapone

Vieira-Coelho

Soares‐da‐Silva

1996

British J Pharmacology

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“…The soluble, cytoplasmic COMT (S-COMT) is responsible for the majority of enzyme activity in all tissues studied, whereas the membrane-bound or particulate COMT (MB-COMT) represents a minority of the overall COMT activity (Guldberg and Marsden, 1975;Kopin, 1986). The two enzymes have many properties in common (Jeffery and Roth, 1984), but differences also exist in the kinetic characteristics, particularly in the K, and in the position of the methylation of substrates (Aprille and Malamud, 1975;Nissinen, 1984;Tong and D'Iorio, 1977;Rivett et al, 1983). Cross-reactivity of S-and MB-COMT with polyclonal and monoclonal anti-COMT antisera has suggested structural similarity between the two enzyme forms (Grossmann et al, 1985;Bertocci et al, 1990;1991 a).…”

mentioning

confidence: 99%

Expression of recombinant soluble and membrane‐bound catechol O‐methyltransferase in eukaryotic cells and identification of the respective enzymes in rat brain

Tilgmann

Melén

Lundström

et al. 1992

European Journal of Biochemistry

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The rat and human recombinant soluble and membrane-bound catechol 0-methyltransferase (Sand 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 [' 5S]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 roughendoplasmic-reticulum marker enzymes. The metalpara methylation ratio of dihydroxybenzoic-acid substrate by different recombinant and rat brain COMT-containing subcellular fractions was analysed.Catechol 0-methyltransferase (COMT) inactivates catecholamine neurotransmitters (dopamine, epinephrine and norepinephrine) and catechol steroids (e. g.

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“…In this paper we describe further studies on the use of the method in the large-scale preparation of catechol-0-methyl transferase and some properties of the purified enzyme. The previous method [5] has been improved by the use of a more stable ligand attached by a flexible 'spacer' arm to an agarose support.Various workers have suggested that catechol-0-methyl transferase may aggregate [6], possibly in the presence of membrane-derived material [7]. Experiments have been carried out on highly purified catechol-O-methyl transferase to investigate this possibility.…”

mentioning

confidence: 99%

“…Various workers have suggested that catechol-0-methyl transferase may aggregate [6], possibly in the presence of membrane-derived material [7]. Experiments have been carried out on highly purified catechol-O-methyl transferase to investigate this possibility.…”

mentioning

confidence: 99%

The Purification and Properties of Pig‐Liver Catechol‐O‐Methyl Transferase

Gulliver

Tipton

1978

European Journal of Biochemistry

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A procedure utilising affinity chromatography is described for the large-scale purification of pigliver catechol-0-methyl transferase. The enzyme prepared by this method appears to be homogeneous by polyacrylamide gel electrophoretic criteria and gel chromatography. It is stable for prolonged periods when stored at -5 "C in 20% (v/v) glycerol. The enzyme has a molecular weight of about 23000 and does not appear to be a compound of subunits, or to associate to any appreciable degree. The pH optimum of the enzyme's activity is approximately pH 7.1 -7.4, it does not catalyse the methylation of benzimidazole and has a K,,, of 0.64 mM and 0.056 mM towards 3,4-dihydroxyphenylacetic acid and S-adenosyl-L-methionine, respectively. Amino acid analysis showed the presence of five cysteine residues.Catechol-0-methyl transferase catalyses the methylation of a wide range of catechol substrates, mainly on the meta-hydroxyl using S-adenosyl-L-methionine (Ado-Met) as methyl donor and magnesium cations as cofactor. In the liver, the enzyme has been shown to have a central role in terminating the action of circulatory catecholamines 11 1.Purification of the enzyme from a variety of mammalian tissues has been attempted, but yields and overall purification has proved disappointing, even in the presence of protecting agents such as mercaptoethanol, dithiothreitol (21 or serum albumin 131.The application of affinity chromatography techniques to the purification of catechol-0-methyl transferase is made difficult by the instability of the coenzyme and catechol substrates at neutral or alkaline pH. However, two attempts have proved comparatively successful [4,5]. In this paper we describe further studies on the use of the method in the large-scale preparation of catechol-0-methyl transferase and some properties of the purified enzyme. The previous method [5] has been improved by the use of a more stable ligand attached by a flexible 'spacer' arm to an agarose support.Various workers have suggested that catechol-0-methyl transferase may aggregate [6], possibly in the presence of membrane-derived material [7]. Experiments have been carried out on highly purified catechol-O-methyl transferase to investigate this possibility.Enzyme. Catechol-0-methyl transferase (EC 2 1 .I .6) MATERIALS AND METHODSReagents used were of analytical quality (BDH, Poole, England) and dissolved in deionised distilled water. All pH measurements are relative to 20 "C. AssaysCatechol-0-methyl transferase was assayed at 37 "C either radiochemically [8] or by the adenosinedeaminase-coupled spectrophotometric assay [9] as modified in 181, in the presence of 0.2 M triethanolamine hydrochloride buffer pH 8.00. One unit (U) of enzyme activity is defined as that amount of enzyme which catalyses the formation of 1 pmol product in 1 min.Protein concentrations above 1 mg/ml were determined by the method of Gornall et al. [lo] and at lower concentrations by the method of MejbaumKatzenellenbogen and Dobryszycka [1 I]. Standard curves were prepared using bovine serum albumin ...

show abstract

Solubilization and partial purification of particulate catechol-O-methyltransferase from rat liver

Cited by 43 publications

References 7 publications

Ontogenic aspects of liver and kidney catechol‐O‐ methyltransferase sensitivity to tolcapone

Ontogenic aspects of liver and kidney catechol‐O‐ methyltransferase sensitivity to tolcapone

Expression of recombinant soluble and membrane‐bound catechol O‐methyltransferase in eukaryotic cells and identification of the respective enzymes in rat brain

The Purification and Properties of Pig‐Liver Catechol‐O‐Methyl Transferase

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