Børresen T, Klausen N K, Larsen L M, Sørensen H
Technical Laboratory, Technical University of Denmark, Lyngby.
Biochim Biophys Acta. 1989 Oct 13;993(1):108-15. doi: 10.1016/0304-4165(89)90149-9.
Microbial tyrosine decarboxylase (EC 4.1.1.25) and mammalian aromatic-L-amino-acid decarboxylase (EC 4.1.1.28) catalyse the formation of tyramine from L-tyrosine. These enzymes were characterised after isolation to purity by methods including fast polymer liquid chromatography (FPLC). Tyrosine decarboxylase was isolated from Streptococcus faecalis by FPLC anion exchange chromatography (11-times purification; 72% recovery; 23.2 U/mg protein). FPLC on Phenyl-Superose resulted in purification to 115 U/mg protein. Aromatic-L-amino-acid decarboxylase was isolated from pig kidney by ammonium sulfate fractionation, DEAE chromatography, and FPLC anion exchange chromatography (21-times purification; 22% recovery; 0.71 U/mg protein). By FPLC chromatofocusing, tyrosine decarboxylase eluted at pH 4.3 and aromatic-L-amino-acid decarboxylase at pH 5.0. Isoelectric focusing of tyrosine decarboxylase gave two bands (pI 4.4 and 4.5). With pyridoxal 5'-phosphate removed by ultrafiltration, only one band (pI 4.4) appeared, and SDS polyacrylamide electrophoresis confirmed the purity. FPLC gel filtration resulted in molecular weights 143,000 and 86,000, respectively, for tyrosine decarboxylase and aromatic-L-amino-acid decarboxylase. In SDS electrophoresis, tyrosine decarboxylase had the monomer molecular weight 75,000, showing a dimer structure for the enzyme.
微生物酪氨酸脱羧酶(EC 4.1.1.25)和哺乳动物芳香族L-氨基酸脱羧酶(EC 4.1.1.28)催化由L-酪氨酸形成酪胺。这些酶通过包括快速聚合物液相色谱法(FPLC)在内的方法分离至纯后进行了表征。通过FPLC阴离子交换色谱法从粪链球菌中分离出酪氨酸脱羧酶(纯化11倍;回收率72%;23.2 U/mg蛋白质)。在苯基-琼脂糖凝胶上进行FPLC可将其纯化至115 U/mg蛋白质。通过硫酸铵分级沉淀、DEAE色谱法和FPLC阴离子交换色谱法从猪肾中分离出芳香族L-氨基酸脱羧酶(纯化21倍;回收率22%;0.71 U/mg蛋白质)。通过FPLC色谱聚焦法,酪氨酸脱羧酶在pH 4.3时洗脱,芳香族L-氨基酸脱羧酶在pH 5.0时洗脱。酪氨酸脱羧酶的等电聚焦产生两条带(pI 4.4和4.5)。通过超滤去除磷酸吡哆醛5'-磷酸后,仅出现一条带(pI 4.4),SDS聚丙烯酰胺电泳证实了其纯度。FPLC凝胶过滤法测得酪氨酸脱羧酶和芳香族L-氨基酸脱羧酶的分子量分别为143,000和86,000。在SDS电泳中,酪氨酸脱羧酶的单体分子量为75,000,表明该酶具有二聚体结构。