Purification of the glyoxylate cycle enzyme malate synthase from maize (Zea mays L.) and characterization of a proteolytic fragment.
A purification scheme is described for the glyoxylate cycle enzyme malate synthase from maize scutella. With our procedure, large amounts of extremely pure enzyme can easily be prepared. Purification involves a heat denaturation step, followed by ammonium sulfate precipitation, and chromatography on DEAE-cellulose and Blue Dextran-Sepharose. Catalase and malate dehydrogenase, which are the most persistent contaminants, are completely removed by this procedure. Maize malate synthase is an octameric protein with a subunit molecular weight of 64 kDa. Purity of the enzyme preparation was demonstrated by SDS-polyacrylamide gel electrophoresis and by isoelectric focusing (pI = 5.0). Pure malate synthase can be stored without appreciable loss of activity at -70 degrees C in 200 mM Hepes buffer containing 6 mM MgCl2 and 2 mM 2-mercaptoethanol, pH 7.6. Maize malate synthase contains no covalently linked carbohydrate residues. The enzyme requires Mg2+ ions for activity. From circular dichroism measurements we estimate that the secondary structure of the enzyme consists of 30% alpha-helical and almost no (5%) beta-pleated sheet segments. A 45-kDa polypeptide, which contaminates malate synthase preparations if the purification starts from seedlings older than 2.5 days, is shown to be a degradation product of malate synthase. Together with full-length chains, these 45-kDa polypeptides are able to take part in octameric oligomer formation.
Khan
A S
AS
Laboratorium voor Chemie der Proteïnen, Vrije Universiteit Brussel, Sint-Genesius-Rode, Belgium.
Van Driessche
E
E
Kanarek
L
L
Beeckmans
S
S
D015153:Blotting, Western; D002942:Circular Dichroism; D004795:Enzyme Stability; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006038:Glyoxylates / Q000378:metabolism; D008292:Malate Synthase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D003313:Zea mays / Q000201:enzymology*
D006023:Glycoproteins; D006038:Glyoxylates; D010446:Peptide Fragments; D008292:Malate Synthase; C031150:glyoxylic acid