Control of δ-Aminolevulinate Synthetase Activity in Rhodopseudomonas spheroides
III. Partial Purification of the Fraction I Activating Enzyme and the Occurrence of Two Forms of Fraction II
1974 年 76 巻 1 号 p. 157-168
詳細
1974 年 76 巻 1 号 p. 157-168
The activating enzyme of the inactive form of Fraction I of Rhodopseudomonas (R.) spheroides δ-aminolevulinate (ALA) synthetase [EC 2. 3. 1. 37] was purified about 150-fold from extracts of R. spheroides cells grown anaerobically in the light. L-Cystine was also required for the enzyme-catalyzed conversion of the inactive form to the active form. The enzyme activity was lost completely when the enzyme was heated at 100°C for 2min or was treated with trypsin. The initial rate of conversion of the inactive form to the active form was dependent on the concentration of the inactive form, exhibiting a hyperbolic saturation curve. The activity of the activating enzyme in cells grown aerobically in the dark was far lower than in cells grown anaerobically in the light. The activating enzyme may be an inducible enzyme, like ALA synthetase in R. spheroides.
When purified Fraction II was preincubated with L-cystine and the acetone fraction (from the third purification step for Fraction I activating enzyme), the specific activity of Fraction II increased remarkably. DEAE-Sephadex column chromatography of purified Fraction II which was preincubated with L-cystine and the acetone fraction revealed that the Fraction II had been converted to a more active form, The activation of Fraction II appeared to be catalyzed by some enzyme in the acetone fraction, but not by the Fraction I activating enzyme. The apparent molecular weights of both forms of Fraction II were estimated to be about 67, 000 by gel-filtration on Sephadex G-200.
The inhibitory effect of heroin and Mg-protoporphyrin on the four forms of ALA synthetase were studied. The most sensitive form to hemin was the active form The of Fraction I (50% inhibition at 3×10-8M and 90% inhibition at 3×10-7M). Moreover, the inhibition by heroin of the other three forms of ALA synthetase was only partial, i.e., the extents of inhibition were less than 50% even at levels of heroin as high as 1×10-6M. On the other hand, the most sensitive form to Mg-protoporphyrin was the active form of Fraction II. The concentrations of Mg-protoporphyrin required for 50% inhibition were 3.5×10-6M and 1×10-5M for the active form of Fraction II and the other three forms of ALA synthetase, respectively.
