Isolation and purification of tyrosine hydroxylase from callus cultures of Portulaca grandiflora

• Published in: Plant and cell physiology 2001-09, Vol.42 (9), p.969-975
• Main Authors: Yamamoto, K. (Kumamoto Univ. (Japan)), Kobayashi, N, Yoshitama, K, Teramoto, S, Komamine, A
• Format: Article
• Language: English
• Subjects: 4-dihydroxyphenylalanine; 6-methyl-5; 6MPH4; 7-dimethyl-5; 8-tetrahydrobiopterin; 8-tetrahydropterin; BH4; CALLUS; Catalysis; Catechol Oxidase - chemistry; Catechol Oxidase - metabolism; Chelating Agents - pharmacology; Coenzymes - pharmacology; Culture Techniques; Dihydroxyphenylalanine - biosynthesis; dithiothreitol; DMPH4; DOPA; DTT; ENZYMES; Iron - pharmacology; Key words: Betacyanin — DOPA — Portulaca grandiflora — Polyphenol oxidase (EC 1.10.3.1) — Pterin — Tyrosine hydroxylase (EC 1.14.16.2); Kinetics; l-3; Magnoliopsida - chemistry; Magnoliopsida - enzymology; Manganese - pharmacology; PORTULACA; Pterins - pharmacology; PURIFICATION; TISSUE CULTURE; TYROSINE; Tyrosine - metabolism; Tyrosine 3-Monooxygenase - chemistry; Tyrosine 3-Monooxygenase - isolation & purification; Tyrosine 3-Monooxygenase - metabolism
• ISSN: 0032-0781; 1471-9053
• DOI: 10.1093/pcp/pce125

Tyrosine hydroxylase was separated from polyphenol oxidase activity and was highly purified from betacyanin producing callus cultures of Portulaca grandiflora. The purified enzyme catalyzed the formation of DOPA (L-3,4dihydroxyphenylalanine) from tyrosine and required the pterin compounds (6-methy1-5,6,7,8-tetrahydropterin; 5,6,7,8-tetrahydrobiopterin; 6,7-dimethy1-5,6,7,8-tetrahydropterin) as coenzyme. The Km values for tyrosine and 6-methy1-5,6,7,8-tetrahydropterin were 0.5 mM and 0.15 mM, respectively. This enzyme was activated by Fe**2+ and Mn**2+, and inhibited by metal chelating agents.