Purification of the Glyoxylate Cycle Enzyme Malate Synthase from Maize (Zea mays L) and Characterization of a Proteolytic Fragment

• Published in: Protein expression and purification Vol. 4; no. 6; pp. 519 - 528
• Main Authors: Khan, A.S., Vandriessche, E., Kanarek, L., Beeckmans, S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.1993
• Subjects: Blotting, Western; Circular Dichroism; Enzyme Stability; Glycoproteins - chemistry; Glycoproteins - isolation & purification; Glycoproteins - metabolism; Glyoxylates - metabolism; Malate Synthase - chemistry; Malate Synthase - isolation & purification; Malate Synthase - metabolism; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Zea mays - enzymology
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.1993.1068

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°C in 200 mM Hepes buffer containing 6 mM MgCl2 and 2 mM 2-mercaptoethanol, pH7.6. 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% α-helical and almost no (5%) β-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.