RESUMO
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-3,4-dihydroxyphenylalanine) from tyrosine and required the pterin compounds (6-methyl-5,6,7,8-tetrahydropterin; 5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterin) as coenzyme. The K(m) values for tyrosine and 6-methyl-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.
Assuntos
Magnoliopsida/enzimologia , Tirosina 3-Mono-Oxigenase/isolamento & purificação , Catálise , Catecol Oxidase/química , Catecol Oxidase/metabolismo , Quelantes/farmacologia , Coenzimas/farmacologia , Técnicas de Cultura , Di-Hidroxifenilalanina/biossíntese , Ferro/farmacologia , Cinética , Magnoliopsida/química , Manganês/farmacologia , Pterinas/farmacologia , Tirosina/metabolismo , Tirosina 3-Mono-Oxigenase/química , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Four cDNA clones were isolated from Vigna mungo seedlings by the screening with cDNA encoding UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT) of Antirrhinum majus as a probe; the product of the gene corresponding to one cDNA was more highly expressed in the first simple leaves than in stems. Nucleotide sequence analysis revealed 1,691 bp (including 326 bp non-reading) containing an open reading frame of 455 amino acids. The deduced amino acid sequence showed 42% and 23% identity with those of A. majus UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT) and Petunia hybrida UDP-rhamnose:anthocyanidin 3-O-glucoside rhamnosyltransferase (RT), respectively. One region of the cDNA (amino acids 325 to 387) showed similarity to ceramide UDP-galactosyltransferases of mice, rats and humans. A crude extract from Escherichia coli, in which the protein was expressed from the cDNA, showed high UF3GaT activity but low UF3GT activity, and was similar in K(m), optimal pH and substrate specificity to UF3GaT from V. mungo. We conclude that we have obtained UDP-galactose:flavonoid 3-O-galactosyltransferase (UF3GaT) cDNA from V. mungo.
Assuntos
Galactosiltransferases/genética , Plantas/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Sequência Consenso , DNA Complementar , Galactosiltransferases/química , Galactosiltransferases/metabolismo , Humanos , Cinética , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta , Plantas/genética , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Zea mays/enzimologia , Zea mays/genéticaRESUMO
Kaempferol 3,4'-diglucoside causing the blueing effects on anthocyanin was isolated as pale yellow needles from the blue seed coats of Ophiopogon jaburan. Petunidin 3-O-ß-(2(G)-glucosylrutinoside)-5'-glucoside (ophionin) and kaempferol 3,4'-diglucoside were present at about 3.3 × 10(-2) M and 3.7 × 10(-2) M, respectively, in the fresh seed coats. At the same concentration, the mixture of both pigments in citrate phosphate buffer solution (pH 5.6) showed stable blue color, and absorption spectrum of the mixture almost coincided with that of the fresh blue seed-coat.
