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KMID : 0377519800050010001
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Chung-Ang Journal of Medicine
1980 Volume.5 No. 1 p.1 ~ p.14
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Purification and Properties of δ Aminolevulinate Dehydratase from Livers of Wistar Strain of Rats
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Kim Taek-Jun
Chung Kyou-Chull
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Abstract
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δ-Aminolevulinate dehydratase (δ-ALAD; EC 4.2.1.24) was purified from liver homogenate of Wistar strain of rats more than 300 folds with a 22.3% yield by a modification of the procedure of Doyle and Schimke and its physicochemical and kinetic properties were partially characterized as follows. 1. The purified enzyme preparation had a specific activity of 54.4 units/㎎ of protein, which was 5∼6 folds as activity as those purified from ICR mouse liver homogenate. 2. the enzyme had optimum catalytic at pH 7.0 in phosphate buffer and its Km value was 3.0×10^-4 M. It also found that the enzyme was relatively heat stable. 3. The enzyme was relatively stable by storage. The purified enzyme could be stored at 4℃ for 7 days without significant loss of activity, showing halflife of 40 days. The enzyme stored frozen at -10˚ or -30℃ for 50 days maintained their original activity without any evidence of decrease of enzyme activity. 4. Activity of the purified enzyme decreased linearly relative to the frequency of the freezethawing when it was kept frozen at -30℃ in a deep freezer and thawed at a room temperature up to 20 times. 5. After the DEAEcellulose column chromatography in the purification procedure no enzyme activity was shown unless the preparation was previously activated with a thiol reagent, βmercaptoethanol. Maximal activity was obtained with addition of the reducing reagent in concentration of 20 and 100mM. However, the activity was decreased drastically above 100mM. On the contrary, the enzyme activity was inhibited in inverse proportion by increasing concentration of iodoacetamide in the reaction mixture, which suggests that sulfhydryl group was required for the enzyme activity and cystein in the active site is implicated in the catalytic function of the enzyme molecule. 6. Molecular weight of the δ-ALAD was estimated to be 270,000 and 269,000 respectivelly, by both the Sephadex gel filtration and polyacrylamide disc gel electrophoresis.
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