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Isolation and biochemical characterization of grape malic enzyme
D Possner1, H P Ruffner, D M Rast
1Institut für Pflanzenbiologie, Physiologie, Universität Zürich, Zollikerstraße 107, CH-8008, Zürich, Switzerland.
Planta
|December 5, 2013
Summary
Grape malic enzyme (ME) was purified and characterized. Its regulatory properties, including substrate cooperativity and cofactor effects, are crucial for understanding grape malate metabolism and fruit ripening.
Area of Science:
- Biochemistry
- Plant Physiology
Background:
- Grape berry malate metabolism is vital for fruit ripening and wine quality.
- Malic enzyme (ME) plays a key role in this metabolic pathway.
- Understanding ME's enzymatic properties is essential for elucidating its regulatory functions.
Purpose of the Study:
- To extract and purify malic enzyme (ME) from grape berries.
- To characterize the kinetic and regulatory properties of purified grape ME.
- To discuss the implications of ME's properties for grape metabolism and ripening.
Main Methods:
- Enzyme extraction using Triton X-100.
- Purification via ammonium sulfate fractionation, preparative isoelectric focusing (IEF), and Sephadex G-100 chromatography.
- Identification using thin-layer chromatography (TLC) with [(14)C]malate and determination of cofactor dependence, pH optimum, and substrate affinities.
Main Results:
- Purified grape ME exhibited an isoelectric point (pI) of 5.8.
- ME showed positive cooperativity towards malate at pH 7.4, approaching Michaelis-Menten kinetics at pH 7.0.
- Substitution of Mn(2+) for Mg(2+) enhanced turnover rates and malate affinity.
Conclusions:
- The characterized kinetic and regulatory properties of grape ME suggest a significant role in controlling malate levels during fruit development.
- These findings provide insights into the biochemical mechanisms underlying grape ripening.
- ME's regulatory features are important for managing grape quality and metabolic processes.
