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

Abstract

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 [15]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] 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]glutamate was synthesized at a rate of 7% of that of [15N]serine synthesis by corn mitochondria.