Rat liver catechol-O-methyltransferase kinetics and assay methodology.
In mammals, catechol-O-methyltransferase (COMT) is distributed throughout various organs, the highest activities being found in the liver and kidney. However, comparisons of the kinetic parameters are difficult to perform, since the experimental procedures in the enzyme assay vary quite considerably. The present work was aimed at studying the optimal liver COMT assay conditions for determining the kinetics of the enzyme. The COMT assay was performed with liver homogenates from 60 days old male Wistar rats with adrenaline (AD) as the substrate. Time course experiments using 100 microM S-adenosyl-L-methionine (SAMe) and 300 microM AD showed linearity of O-methylation reaction upto 10 min. Using 100 microM SAMe, Vmax (nmol mg protein-1 h-1) and Km (microM) values progressively decreased respectively from 22.1 and 104.8 at 5 min down to 5.8 and 24.62 at 60 min incubation periods. This decrease was not due to end-product inhibition. Using 2500 microM AD, Km values (microM) for the methyl donor SAMe increased progressively from 174 at 5 min upto 1192.5 at 60 min; upto 30 min of incubation Vmax values did not change. When a 5 min incubation period and 500 microM SAMe were used, Vmax and Km values for liver COMT were 63.4 nmol mg protein-1 h-1 and 261.1 microM, respectively. It is concluded that an incubation period of 5 min and a SAMe concentration of 500 microM provide optimal conditions for the liver homogenate COMT assay.
Borges
N
N
Department of Research and Development, BIAL, S. Mamede do Coronado, Portugal. DID.BIAL@mail.telepac.pt
Vieira-Coelho
M A
MA
Parada
A
A
Soares-da-Silva
P
P
D000818:Animals; D002394:Catechol O-Methyltransferase / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008745:Methylation; D009638:Norepinephrine / Q000378:metabolism; D009647:Normetanephrine / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
D009647:Normetanephrine; D002394:Catechol O-Methyltransferase; D009638:Norepinephrine