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NADP-linked malic enzyme. Purification from maize leaves, Mr and subunit composition
1Department of Biochemistry, University of Oxford, U.K.
The Biochemical Journal
|August 15, 1988
Summary
NADP-linked malic enzyme from maize leaves was purified and characterized. Its molecular weight and subunit composition were studied, revealing different forms in solution depending on buffer conditions and dithiothreitol presence.
Area of Science:
- Biochemistry
- Plant Physiology
- Enzymology
Background:
- NADP-linked malic enzyme (EC 1.1.1.40) plays a role in plant metabolism.
- Understanding enzyme structure and behavior is crucial for elucidating its function.
Purpose of the Study:
- To isolate and purify NADP-linked malic enzyme from maize leaves.
- To investigate the enzyme's molecular weight (Mr) and subunit composition.
- To determine how buffer conditions and reducing agents affect enzyme quaternary structure.
Main Methods:
- Affinity chromatography using N6-aminohexyl-2',5'-bisphosphoadenosine-agarose.
- Gel filtration with Sephadex G-100.
- Ion-exchange chromatography on DEAE-Sephadex A-50.
- Sedimentation-equilibrium and sedimentation-velocity studies.
Main Results:
- The enzyme was purified 140-fold with a 30% yield.
- Different molecular weight forms were observed in solution.
- In triethanolamine buffer with dithiothreitol, a tetramer (subunit Mr 60,000) was detected.
- In phosphate buffer, a dimer (Mr 120,000) was observed.
- Gel filtration indicated a Mr of approximately 340,000 in both buffer types.
Conclusions:
- Maize NADP-linked malic enzyme exhibits quaternary structural heterogeneity.
- The enzyme's oligomeric state is sensitive to buffer composition and the presence of dithiothreitol.
- Further studies are needed to fully understand the functional implications of these structural variations.