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Purification and characterization of D-glycerate-3-kinase from maize leaves

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Abstract

Glycerate kinase (GK; EC 2.7.1.31) from maize (Zea mays L.) leaves was purified by a sequence of ammonium-sulfate precipitations and chromatography on diethylaminoethyl-cellulose, hydroxyapatite, Sephadex G-75SF and dye ligand (Green A) columns. The purest preparation was almost 1300-fold enriched and had a specific activity of 68 μmol · min-1 · (mg protein) -1. The enzyme was a monomer of a relative molecular mass (Mr) of 44 kDa (kdalton) as determined by gel filtration, electrophoresis in dissociating conditions and by immunoblots. The enzyme was only weakly recognized by polyclonal antibodies against purified spinach GK, indicating substantial differences in molecular structure of the two proteins. Highly reducing conditions stabilized GK activity and were required for activation of crude leaf enzyme. The enzyme had a broad pH optimum of 6.8–8.5, and formed 3-phosphoglycerate and ADP as reaction products. Apparent K ms for D-glycerate and Mg-ATP were 0.11 and 0.25 mM, respectively. The enzyme was strongly affected by a number of phosphoesters, especially by 3-phosphoglycerate (K i= 0.36 mM), fructose bisphosphates and nucleoside bisphosphates. Inhibition by 3-phosphoglycerate was competitive to Mg-ATP and noncompetitive to D-glycerate. Pyruvate was found noncompetitive to D-glycerate (K is=4 mM). The ratio of stromal concentration of Mg-ATP to phosphoesters, particularly to 3-phosphoglycerate, may be of importance in the regulation of GK during C4-photosynthesis.

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Abbreviations

DEAE:

diethylaminoethyl

kDa:

kdalton

GAP-DH:

glyceraldehyde phosphate dehydrogenase

GK:

glycerate kinase

LDH:

lactate dehydrogenase

2-ME:

2-mercaptoethanol

Mr :

relative molecular mass

PEP:

phosphoenolpyruvate

PGA(PK):

phosphoglycerate (phosphokinase)

PK:

pyruvate kinase

SDS-PAGE:

sodium dodecyl sulfatepolyacrylamide gel electrophoresis

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Author notes

  1. Leszek A. Kleczkowski

    Present address: Institute of Plant Biology, Warsaw Agricultural University, Rakowiecka 26/30, 02-528, Warsaw, Poland

Authors and Affiliations

  1. Department of Biochemistry, Univerity of Missouri, 117 Schweitzer Hall, 65211, Columbia, MO, USA

    Leszek A. Kleczkowski & Douglas D. Randall

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  1. Leszek A. Kleczkowski
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  2. Douglas D. Randall
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Kleczkowski, L.A., Randall, D.D. Purification and characterization of D-glycerate-3-kinase from maize leaves. Planta 173, 221–229 (1988). https://doi.org/10.1007/BF00403014

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  • DOI: https://doi.org/10.1007/BF00403014

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