Purification and properties of δ-aminolevulinate dehydrase from human erythrocytes

P. M. Anderson, R. J. Desnick

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Abstract

Human δ-aminolevulinate dehydrase (5-aminolaevulinate hydro-lyase, EC 4.2.1.24) was purified from erythrocytes more than 38,000-fold with a 69% yield. The purification procedure included DEAE-cellulose chromatography, ammonium sulfate precipitation, hydrophobic chromatography on octyl- and phenyl-Sepharose, and gel filtration. Sequential chromatography on octyl- and phenyl-Sepharose provided enhanced purification by exploitation of the enzyme's unique hydrophobic properties. After prior removal of most other hydrophobic proteins by octyl-Sepharose, the enzyme was bound to and eluted from phenyl-Sepharose. The purified enzyme had a specific activity of 18.5 units/mg of protein (23.8 units/mg in the presence of 0.1 mM zinc) and appeared homogeneous by gel electrophoresis. The molecular weight of the native enzyme was estimated to be 252,000 by gel filtration. Under denaturing conditions, the dissociated enzyme had a single subunit of approximately M(r) ≃ 31,000, indicating that the enzyme is composed of eight apparently identical subunits. The enzyme had a pH optimum of 6.3 to 6.7, an isoelectric point of 4.9, and a K(m) of 0.27 mM. Zinc activated the enzyme at 0.1 and 0.02 mM concentrations but inhibited the enzyme at a concentration of 1.0 mM. Kinetic analysis of the inhibition of activity by ferrous iron (K(i) = 0.26 mM) was consistent with noncompetitive inhibition, whereas pyridoxal 5'-posphate (K(i) = 0.031 mM) was a competitive inhibitor. Lead (K(i) = 0.0017 mM) altered both the K(m) and V(max) values. Spectral analysis suggested tht pyridoxal 5'-phosphate may be bound to the enzyme.

Original languageEnglish
Pages (from-to)6924-6930
Number of pages7
JournalJournal of Biological Chemistry
Volume254
Issue number15
StatePublished - 1979
Externally publishedYes

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