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Synthesis of [¹⁵N]Glutamate from [¹⁵N]H₄⁺ and [¹⁵N]Glycine by Mitochondria Isolated from Pea and Corn Shoots ¹

Institute for Agricultural and Biological Sciences, Okayama University, Kurashiki, Okayama 710, Japan, Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1, Department of Horticulture, Purdue University, West Lafayette, IN 47907

Metabolically competent mitochondria were isolated from pea and corn shoots on Percoll discontinuous density gradients. Rates of synthesis of [¹⁵N]glutamate were measured by gas chromatography-mass spectrometry after the incubation of mitochondria with either 2 millimolar [¹⁵N] H₄⁺ or [¹⁵N]glycine in the presence of 1 millimolar citrate as the respiratory substrate. When [¹⁵N]H₄⁺ was provided, mitochondria isolated from light-grown pea shoots synthesized [¹⁵N]glutamate with a rate of 2.64 nanomoles per hour per milligram mitochondrial protein. Corn mitochondria produced [¹⁵N]glutamate at a rate approximately 11 times greater than the pea mitochondria. Dark treatment during growth for the last 24 hours caused a slight reduction in the rate of synthesis in both species. When [¹⁵N]glycine was used, pea mitochondria synthesized [¹⁵N]glutamate with a rate of 6.32 nanomoles per hour per milligram protein. Rapid disappearance of [¹⁵N]glycine and synthesis of [¹⁵N]serine was observed with a molar ratio of 2 glycine to 0.78 serine. The rate of glutamate synthesis was only 0.2% that of serine, due in part to the dilution of [¹⁵N]H₄⁺ by the [¹⁴N]H₄⁺ pool in the mitochondria. The majority of the [¹⁵N]H₄⁺ released from glycine appears to have been released from or remains unmetabolized in the mitochondria. Corn mitochondria showed no apparent disappearance of [¹⁵N]glycine and little synthesis of [¹⁵N]serine, indicating that our preparation originated primarily from mesophyll cells. Under our conditions of glycine/serine conversion, [¹⁵N]glutatmate was synthesized at a rate of 7% of that of [¹⁵N]serine synthesis by corn mitochondria.

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