RAT-LIVER CATECHOL-O-METHYLTRANSFERASE KINETICS AND ASSAY METHODOLOGY

In mammals, catechol-O-methyltransferase (COMT) is distributed through out various organs, the highest activities being found in the liver an d kidney. However, comparisons of the kinetic parameters are difficult to perform, since the experimental procedures in the enzyme assay var y quite considerably. The present work was aimed at studying the optim al liver COMT assay conditions for determining the kinetics of the enz yme. The COMT assay was performed with liver homogenates from 60 days old male Wistar rats with adrenaline (AD) as the substrate. Time cours e 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 10 0 mu M SAMe, V-max (nmol mg protein(-1) h(-1)) and K-m (mu M) values p rogressively 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 d ue to end-product inhibition, Using 2500 mu M AD, K-m values (mu M) fo r 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 chan ge. 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 fo r the liver homogenate COMT assay.