Kinetics of rat brain and liver solubilized membrane-bound catechol-O-methyltransferase.
Catechol-O-methyltransferase (COMT), an enzyme involved in the metabolism of catecholamines, is present in mammals as soluble (S-COMT) and membrane-bound (MB-COMT) forms. The kinetic properties of rat liver and brain solubilized MB-COMT were evaluated and compared with the ones of the respective native enzymes. Treatment with Triton X-100 did not affect the affinity of S-COMT for the substrate (adrenaline) or the activity of the enzyme. Conversely, solubilized MB-COMT presented a lower affinity for the substrate than the native protein, as evidenced by a significant increase in the Km values: 9.3 (6.2, 12) vs 2.5 (0.8, 4.3) microM for the liver enzyme and 12 (11, 13) vs 1.4 (1.0, 1.9) microM for the brain enzyme. A 1.6- and 1.5-fold increase in Vmax was also observed for the liver and brain solubilized enzymes, respectively. The actual enzyme concentrations (molar equivalence, Meq) and their efficiency in the O-methylation reaction (catalytic number, Kcat) were determined from Ackermann-Potter plots. Both liver and brain solubilized MB-COMT were more efficient in methylating adrenaline than the respective native enzymes as revealed by higher Kcat values (P < 0.05): 16.4+/-0.9 vs 10.9+/-0.8 min(-1) (brain) and 5.9+/-0.3 vs 3.3+/-0.2 min(-1) (liver). Subjecting liver solubilized MB-COMT to further purification increased the Km of the enzyme to the levels of liver S-COMT, 252 (127; 377) vs 257 (103; 411) microM. The solubilization process significantly alters MB-COMT kinetic properties but only after partial purification does the enzyme present an affinity for the subtrate identical to S-COMT.
Bonifácio
M J
MJ
Department of Research and Development, BIAL, São Mamede do Coronado, Portugal.
Vieira-Coelho
M A
MA
Borges
N
N
Soares-da-Silva
P
P
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002394:Catechol O-Methyltransferase / Q000302:isolation & purification / Q000378:metabolism*; D004837:Epinephrine / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D008745:Methylation; D017830:Octoxynol / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar
D008565:Membrane Proteins; D017830:Octoxynol; D002394:Catechol O-Methyltransferase; D004837:Epinephrine