Purification and characterization of tyrosinase from gill tissue of Portabella mushrooms.
A group of tyrosinase isoforms with isoelectric points between 4.9 and 5.2 was isolated from gill tissue of Portabella mushrooms. Use of protease inhibitors was not able to increase the amount of latent forms significantly in crude extracts or to preserve latent tyrosinase activity during purification. The tyrosinase in gill tissue extracts showed latent activity above pH 5.5 and suppressed or displayed no latent activity below pH 5.5 when assayed in the presence of SDS. The purified isoforms showed monophenolase activity toward 4-hydroxyanisole but practically no activity toward tyrosine or tyramine. The purified isoforms showed greater activity toward catechol than either 4-methylcatechol, dopa, dopamine, chlorogenic acid, t-butylcatechol, or catechin. The Km for catechol was similar for the group of isolated isoforms (4.3 mM) compared to the isoforms in crude extracts (5.3 mM). Crude extracts showed several isoforms ranging from 50 to 230 kDa after partially denaturing SDS PAGE, while the purified isoforms showed molecular weights of 70 kDa.
Fan
Yan
Y
Chemistry Department, Science Building, Indiana State University, Terre Haute, IN 47809, USA.
Flurkey
William H
WH
D000364:Agaricus / Q000201:enzymology*; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007527:Isoenzymes; D007700:Kinetics; D014442:Monophenol Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010636:Phenols / Q000737:chemistry / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013379:Substrate Specificity
D007527:Isoenzymes; D010636:Phenols; D011480:Protease Inhibitors; D012967:Sodium Dodecyl Sulfate; D014442:Monophenol Monooxygenase