Purification and properties of tryptophan 5-monooxygenase from rat brain-stem.

Abstract

Tryptophan 5-monooxygenase was purified approximately 5,500-fold, to apparent homogeneity with a specific activity of 374 nmol min-1 mg-1 at 30 degrees 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 molecular weight of form I was determined to be 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 molecular weight of which was estimated to be 59,000, indicating that the enzyme might be composed of four identical subunits. The tetrameric structure of the enzyme was further suggested by cross-linking studies using dimethyl suberimidate as a bifunctional reagent. The enzyme activity was stimulated approximately 3.5-fold by the addition of Fe2+. Kinetic 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.