Purification and Properties of Tryptophan 5‐Monooxygenase from Rat Brain‐Stem

Abstract

Tryptophan 5-monooxygenase was purified .apprx. 5500-fold, to apparent homogeneity with a specific activity of 374 nmol min-1 mg-1 at 30.degree. C, from rat brain-stem using Sepharose CL-6B, DEAE-Sepharose CL-6B and pteridine-agarose chromatography. Two distinct active forms were separable by DEAE-Sepharose CL-6B and designated as form I and form II based on their order of elution from the gel column. The apparent MW of form I was determined to 300,000 by gel filtration on Ultrogel AcA 34 and 288,000 by gradient polyacrylamide gel electrophoresis. The enzyme gave a single band on sodium dodecylsulfate/polyacrylamide gel electrophoresis, the MW of which was estimated to be 59,000, indicating that the enzyme might be composed of 4 identical subunits. The tetrameric structure of the enzyme was further suggested by crosslinking studies using dimethyl suberimidate as a bifunctional reagent. The enzyme activity was stimulated .apprx. 3.5-fold by the addition of Fe2+. Kinetics studies revealed that this activation was associated with an increase of V value. The purified enzyme had an activity of phenylalanine hydroxylation but not an activity of tyrosine hydroxylation.