Abstract
delta-Aminolevulinic acid synthetase has been purified from liver mitochondria of young, uninduced rats. After nonionic detergent solubilization of mitochondrial inner membrane-matrix fractions, the enzyme was purified to a specific activity of approximately 2,000 nmol of delta-aminolevulinic acid formed/h/mg of protein at 30 degrees C, by means of ammonium sulfate precipitation, diethylaminoethyl cellulose chromatography, Sephacryl chromatography, and preparative gel electrophoresis. The purified enzyme preparation thus obtained was apparently homogeneous as judged by its migration as a single band with a molecular weight of 58,000 +/- 6,000 upon electrophoresis in sodium dodecyl sulfate polyacrylamide gels. The native enzyme probably exists as a dimer with a molecular weight of approximately 120,000. A pH optimum of 7.5 and an isoelectric point of 4.5 were also determined. Both monovalent cations and hemin strongly inhibited the activity of the purified enzyme.
Highlights
&Aminolevulinic acid synthetase has been purified from liver mitochondria of young, uninduced rats
The purified enzyme preparation obtained was apparently homogeneous as judged by its migration as a single band with a molecular weight of 58,000 f 6,000 upon electrophoresis in sodium dodecyl sulfate polyacrylamide gels
Acid Synthetase Actiuity-The inner membrane-matrix fraction was prepared from washed mitochondria by treatment with digitonin according to the method of Schnaitman and Greenawalt [39]
Summary
&Aminolevulinic acid synthetase has been purified from liver mitochondria of young, uninduced rats. The purified enzyme preparation obtained was apparently homogeneous as judged by its migration as a single band with a molecular weight of 58,000 f 6,000 upon electrophoresis in sodium dodecyl sulfate polyacrylamide gels. A pH optimum of 7.5 and an isoelectric point of 4.5 were determined Both monovalent cations and hemin strongly inhibited the activity of the purified enzyme. These findings may not be applicable to the mechanism of induction in the adult mammalian liver. Dixon have demonstrated striking differences in the pattern of response to inducers of the enzyme in adult and neonate rabbits [20]. We present a method for obtaining reasonable quantities of highly purified
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