The purification and physicochemical characterization of maize (Zea mays L.) isocitrate lyase.
A purification scheme is described for the glyoxylate cycle enzyme isocitrate lyase from maize scutella. Purification involves an acetone precipitation and a heat denaturation step, followed by ammonium sulfate precipitation and chromatography on DEAE-cellulose and on blue-Sepharose. The latter step results in the removal of the remaining malate dehydrogenase activity, and of a high molecular mass (62 kDa) but inactive degradation product of isocitrate lyase. Catalase can be completely removed by performing the DEAE-cellulose chromatography in the presence of Triton X-100. Pure isocitrate lyase can be stored without appreciable loss of activity at -70 degrees C in 5 mM triethanolamine buffer containing 6 mM MgCl2, 7 mM 2-mercaptoethanol, and 50% (v/v) glycerol, pH 7.6. Maize isocitrate lyase is a tetrameric protein with a subunit molecular mass of 64 kDa. Purity of the enzyme preparation was demonstrated by polyacrylamide gel electrophoresis in the presence of dodecylsulfate, in acid (pH 3.2) urea and by isoelectric focusing (pI = 5.1). Maize isocitrate lyase is devoid of covalently linked sugar residues. From circular dichroism measurements we estimate that its structure comprises 30% alpha-helical and 15% beta-pleated sheet segments. The enzyme requires Mg2+ ions for activity, and only Mn2+ apparently is able to replace this cation to a certain extent. The kinetics of the isocitrate lyase-catalyzed cleavage reaction were investigated, and the amino acid composition of the maize enzyme was determined. Finally the occurrence of an association between maize isocitrate lyase and catalase was observed. Such a multienzyme complex may be postulated to play a protective role in vivo.
Khan
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AS
Laboratorium voor Chemie der Proteïnen, Vrije Universiteit Brussel, Belgium.
Van Driessche
E
E
Kanarek
L
L
Beeckmans
S
S
D000596:Amino Acids / Q000032:analysis; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002942:Circular Dichroism; D003902:Detergents; D006863:Hydrogen-Ion Concentration; D007522:Isocitrate Lyase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D017830:Octoxynol; D010944:Plants / Q000201:enzymology; D011092:Polyethylene Glycols; D011487:Protein Conformation; D003313:Zea mays / Q000201:enzymology*
D000596:Amino Acids; D003902:Detergents; D046911:Macromolecular Substances; D011092:Polyethylene Glycols; D008345:Manganese; D017830:Octoxynol; D007522:Isocitrate Lyase; D008274:Magnesium