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Contribution of Malic Enzyme, Pyruvate Kinase, Phosphoenolpyruvate Carboxylase, and the Krebs Cycle to Respiration and Biosynthesis and to Intracellular pH Regulation during Hypoxia in Maize Root Tips Observed by Nuclear Magnetic Resonance Imaging and Gas Chromatography-Mass Spectrometry1

Shaune Edwards, Bich-Ty Nguyen, Binh Do, and Justin K.M. Roberts*

Department of Biochemistry, University of California, Riverside, California 92521

In vivo pyruvate synthesis by malic enzyme (ME) and pyruvate kinase and in vivo malate synthesis by phosphoenolpyruvate carboxylase and the Krebs cycle were measured by 13C incorporation from [1-13C]glucose into glucose-6-phosphate, alanine, glutamate, aspartate, and malate. These metabolites were isolated from maize (Zea mays L.) root tips under aerobic and hypoxic conditions. 13C-Nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry were used to discern the positional isotopic distribution within each metabolite. This information was applied to a simple precursor-product model that enabled calculation of specific metabolic fluxes. In respiring root tips, ME was found to contribute only approximately 3% of the pyruvate synthesized, whereas pyruvate kinase contributed the balance. The activity of ME increased greater than 6-fold early in hypoxia, and then declined coincident with depletion of cytosolic malate and aspartate. We found that in respiring root tips, anaplerotic phosphoenolpyruvate carboxylase activity was high relative to ME, and therefore did not limit synthesis of pyruvate by ME. The significance of in vivo pyruvate synthesis by ME is discussed with respect to malate and pyruvate utilization by isolated mitochondria and intracellular pH regulation under hypoxia.

1   This work was supported by National Science Foundation (NSF) grant no. IBN 9310850. S.E. was supported by a NSF Minority Graduate Research Fellowship in Plant Biochemistry.
*   Corresponding author; e-mail jkmr{at}ucrac1.ucr.edu; fax 1-909-787-3590.

Plant Physiol. (1998) 116: 1073-1081
Copyright Clearance Center:   0032-0889/98/116/1073/09
© 1998 American Society of Plant Physiologists




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