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.
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 (15)NH3, [(15)N]APH or [(13)C]APH. Total (15)N incorporation was calculated from separate incubations with (15)NH3 and [(15)N]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.
Atasoglu
C
C
Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, Scotland, UK.
Guliye
A Y
AY
Wallace
R J
RJ
D000596:Amino Acids / Q000096:biosynthesis*; D000641:Ammonia / Q000378:metabolism; D000824:Animal Nutritional Physiological Phenomena; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis; D002244:Carbon / Q000378:metabolism*; D002247:Carbon Isotopes; D004032:Diet; D066298:In Vitro Techniques; D008954:Models, Biological; D009584:Nitrogen / Q000378:metabolism*; D009587:Nitrogen Isotopes; D012417:Rumen / Q000378:metabolism* / Q000382:microbiology; D012756:Sheep / Q000378:metabolism* / Q000382:microbiology; D012995:Solubility
D000596:Amino Acids; D001426:Bacterial Proteins; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D002244:Carbon; D000641:Ammonia; D009584:Nitrogen