Premium
Isolation and characterization of cytoplasmic NADP + ‐malic enzyme from Lupinus luteus leaves
Author(s) -
Tomaszewska Barbara,
Schramm Ryszard W.,
Kokot Anna,
Mazurowa Hanna
Publication year - 1983
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1983.tb06293.x
Subject(s) - malic enzyme , sephadex , enzyme , chromatography , enzyme assay , affinity chromatography , divalent , oxalate , citrate synthase , fractionation , malate dehydrogenase , size exclusion chromatography , chemistry , biochemistry , biology , dehydrogenase , organic chemistry
NADP + ‐dependent malic enzyme (L‐malate : NADP + oxidoreductase, decarboxylating, EC 1.1.1.40) was extracted from the leaves of yellow lupine. The purification procedure included fractionation with (NH 4 ) 2 SO 4 and Sephadex G‐25 chromatography, followed by purification on DEAE‐cellulose and Sephadex G‐200 columns. The enzyme was purified 122‐fold. The enzyme affinity towards L‐malate was found to be significantly higher with Mn 2+ than with Mg 2+ . The Hill coefficient for Mg 2+ depended on concentration and was 1.6 for the lower and 3.9 for the higher concentrations. The dependence of the enzyme activity on NADP + followed a hyperbolic curve. K m values and Hill coefficients for NADP + were similar with both Mn 2+ and Mg 2+ . The enzyme activity was strictly dependent on divalent cations and followed a sigmoidal curve at least for Mg 2+ . The enzyme had 4‐fold higher affinity towards Mn 2+ than towards Mg 2+ , the K m values being 0.3 and 1.15 m M respectively. Of several tested organic acids, oxalate was the most effective inhibitor followed by oxaloacetate while succinate was the strongest activator.