An assay for human erythrocyte catechol-o-methyltransferase activity using a catechol estrogen as the substrate

G. William Bates, Clare D. Edman, John C. Porter, John M. Johnston, Paul C. Macdonald

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Abstract

A radiometric assay for catechol-O-methyltransferase (COMT) activity in human erythrocytes is described that employs 2-hydroxy[3H]estrone, and nonradiolabeled S-adenosylmethionine (SAM) as the cosubstrates. The ease of separation of the product of the reaction, 2-methoxy[3H]estrone from 2-hydroxy-[3H] estrone makes it possible to achieve low reaction blanks. The assay is very sensitive, and only 200 μl of whole blood are used per determination. The assay is highly reproducible. The interassay variability (coefficient of variation) was 6.5% for 24 assays of COMT activity in red blood cells in blood obtained daily for 24 days from one person. In incubations conducted at 37°C for 30 min, the catechol-O-methyltransferase activity was a linear function of enzyme concentration (equivalent to 11 to 180 μl of packed red blood cells). Employing this assay, we evaluated the catalytic conversion of 2-hydroxyestrone to 2-methoxyestrone by catechol-O-methyltransferase from human red blood cells and found that the apparent Michaelis constant and the apparent maximal rate of reaction were 3 × 10-7 M and 6.7 × 10-9 mol · ml-1 erythrocytes · h-1, respectively. The catechol-O-methyltransferase activity measured in erythrocytes obtained from 100 healthy subjects (men and nonpregnant women) was 8.2 ± 0.17 (mean ± S.E.) nmol 2-methoxyestrone · ml-1 erythrocytes · h-1.

Original languageEnglish (US)
Pages (from-to)63-71
Number of pages9
JournalClinica Chimica Acta
Volume94
Issue number1
DOIs
StatePublished - May 16 1979

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Catechol Estrogens
Catechol O-Methyltransferase
Methyltransferases
Assays
Blood
Erythrocytes
Estrone
Substrates
Cells
S-Adenosylmethionine
catechol
Human Activities
Healthy Volunteers
Enzymes

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry

Cite this

An assay for human erythrocyte catechol-o-methyltransferase activity using a catechol estrogen as the substrate. / Bates, G. William; Edman, Clare D.; Porter, John C.; Johnston, John M.; Macdonald, Paul C.

In: Clinica Chimica Acta, Vol. 94, No. 1, 16.05.1979, p. 63-71.

Research output: Contribution to journalArticle

Bates, G. William ; Edman, Clare D. ; Porter, John C. ; Johnston, John M. ; Macdonald, Paul C. / An assay for human erythrocyte catechol-o-methyltransferase activity using a catechol estrogen as the substrate. In: Clinica Chimica Acta. 1979 ; Vol. 94, No. 1. pp. 63-71.
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AB - A radiometric assay for catechol-O-methyltransferase (COMT) activity in human erythrocytes is described that employs 2-hydroxy[3H]estrone, and nonradiolabeled S-adenosylmethionine (SAM) as the cosubstrates. The ease of separation of the product of the reaction, 2-methoxy[3H]estrone from 2-hydroxy-[3H] estrone makes it possible to achieve low reaction blanks. The assay is very sensitive, and only 200 μl of whole blood are used per determination. The assay is highly reproducible. The interassay variability (coefficient of variation) was 6.5% for 24 assays of COMT activity in red blood cells in blood obtained daily for 24 days from one person. In incubations conducted at 37°C for 30 min, the catechol-O-methyltransferase activity was a linear function of enzyme concentration (equivalent to 11 to 180 μl of packed red blood cells). Employing this assay, we evaluated the catalytic conversion of 2-hydroxyestrone to 2-methoxyestrone by catechol-O-methyltransferase from human red blood cells and found that the apparent Michaelis constant and the apparent maximal rate of reaction were 3 × 10-7 M and 6.7 × 10-9 mol · ml-1 erythrocytes · h-1, respectively. The catechol-O-methyltransferase activity measured in erythrocytes obtained from 100 healthy subjects (men and nonpregnant women) was 8.2 ± 0.17 (mean ± S.E.) nmol 2-methoxyestrone · ml-1 erythrocytes · h-1.

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