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 mu M S-adenosyl-L-methionine (SAMe) and 300 mu M AD showed linearity of O-melhylation reaction upto 10 min. Using 100 mu M SAMe, V-max (nmol mg protein(-1) h(-1)) and K-m (mu M) 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 mu M AD, K-m values (mu M) for the methyl donor SAMe increased progressively from 174 at 5 min upto 1192.5 at 60 min; upto 30 min of incubation V-max values did not change. When a 5 min incubation period and 500 mu M SAMe were used, V-max K-m values for liver COIMT were 63.4 nmol mg protein(-1) h(-1) and 261.1 mu M, respectively. It is concluded that an incubation period of 5 min and a SAMe concentration of 500 mu M provide optimal conditions for the liver homogenate COMT assay.