Premium
Purification and Properties of Bovine Brain Dopamine β‐Hydroxylase
Author(s) -
Matsui Hiroaki,
Yamamoto Chosaburo,
Nagatsu Toshiharu
Publication year - 1982
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1982.tb11498.x
Subject(s) - sephadex , chemistry , chromatography , size exclusion chromatography , agarose , sepharose , concanavalin a , enzyme , dopamine , biochemistry , tyramine , ammonium sulfate , biology , endocrinology , in vitro
Dopamine β‐hydroxylase (DBH) was purified from bovine brain by a series of steps including extraction with 0.5% Triton X‐100, ammonium sulfate fractionation, and serial chromatographies with Concanavalin A (Con A)‐Sepharose, Biogel A‐1.5 m, DEAE‐Sephadex, and phenyl‐Sepharose. The overall purification was approximately 4200‐fold and the final specific activity was 147 nmol/min/mg protein. Bovine brain DBH was apparently a glycoprotein and interacted with immobilized Con A. Furthermore, the enLyme bound to phenyl‐substituted agarose by hydrophobic interaction. An approximate molecular weight was estimated to be 400,000 by gel filtration; the protein eluted earlier than bovine adrenal DBH with a molecular weight estimated to be 290,000. The K m values toward tyramine and ascorbate were 1.53 and 1.42 mM, respectively, the optimal pH was 5.0 in the presence of 20 mM tyramine as substrate. Immunological titration studies indicated that bovine brain and adrenal DBH had common antigenic sites. Our data showed a close similarity between the bovine brain and adrenal enzymes.