Purification and characterisation of tyrosine decarboxylase and aromatic-L-amino-acid decarboxylase.
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.
Børresen
T
T
Technical Laboratory, Technical University of Denmark, Lyngby.
Klausen
N K
NK
Larsen
L M
LM
Sørensen
H
H
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D013293:Enterococcus faecalis / Q000201:enzymology*; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D000637:Transaminases / Q000302:isolation & purification* / Q000378:metabolism; D014445:Tyrosine Decarboxylase / Q000302:isolation & purification* / Q000378:metabolism
D000637:Transaminases; C019729:aromatic amino acid aminotransferase; D014445:Tyrosine Decarboxylase