Use of stable isotopes to measure de novo synthesis and turnover of amino acid-C and -N in mixed micro-organisms from the sheep rumen in vitro
2004
Atasoglu, C. | Guliye, A.Y. | Wallace, R.J.
Protein synthesis and turnover in ruminal micro-organisms were assessed by stable-isotope methods in order to follow independently the fate of amino acid (AA)-C and -N in different AA. Rumen fluid taken from sheep receiving a grass hay-concentrate diet were strained and incubated in vitro with starch-cellobiose-xylose in the presence of NH3 and 5 g algal protein hydrolysate (APH)/l, in incubations where the labels were 15NH3, [15N]APH or [13C]APH. Total 15N incorporation was calculated from separate incubations with 15NH3 and [15N]APH, and net N synthesis from the increase in AA in protein-bound material. The large difference between total and net AA synthesis indicated that substantial turnover of microbial protein occurred, averaging 3.5 %/h. Soluble AA-N was incorporated on average more extensively than soluble AA-C (70 v. 50 % respectively, P=0.001); however, incorporation of individual AA varied. Ninety percent of phenylalanine-C was derived from the C-skeleton of soluble AA, whereas the incorporation of phenylalanine-N was 72 %. In contrast, only 15 % aspartate-C + asparagine-C was incorporated, while 45 % aspartate-N + asparagine-N was incorporated. Deconvolution analysis of mass spectra indicated substantial exchange of carboxyl groups in several AA before incorporation and a condensation of unidentified C2 and C4 intermediates during isoleucine metabolism. The present results demonstrate that differential labelling with stable isotopes is a way in which fluxes of AA synthesis and degradation, their biosynthetic routes, and separate fates of AA-C and -N can be determined in a mixed microbial population.
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