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

C. Atasoglu, A. Y. Guliye, R. J. Wallace

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

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 (NH3)-N-15, [N-15]APH- or [C-13]APH. Total N-15 incorporation was calculated from separate incubations with (NH3)-N-15 and [N-15]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 C-2 and C-4 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.

Original languageEnglish
Pages (from-to)253-261
Number of pages9
JournalBritish Journal of Nutrition
Volume91
Issue number2
DOIs
Publication statusPublished - Feb 2004

Keywords

  • amino acids
  • carbon skeletons
  • rumen
  • sheep
  • volatile fatty acids
  • microbial-growth
  • phenylacetic acid
  • ruminal bacteria
  • 2,2'-diaminopimelic acid
  • nitrogen-metabolism
  • anaerobic bacteria
  • invitro
  • peptides
  • protein

Cite this

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. / Atasoglu, C.; Guliye, A. Y.; Wallace, R. J.

In: British Journal of Nutrition, Vol. 91, No. 2, 02.2004, p. 253-261.

Research output: Contribution to journalArticle

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abstract = "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 (NH3)-N-15, [N-15]APH- or [C-13]APH. Total N-15 incorporation was calculated from separate incubations with (NH3)-N-15 and [N-15]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 C-2 and C-4 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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T1 - 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

AU - Atasoglu, C.

AU - Guliye, A. Y.

AU - Wallace, R. J.

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N2 - 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 (NH3)-N-15, [N-15]APH- or [C-13]APH. Total N-15 incorporation was calculated from separate incubations with (NH3)-N-15 and [N-15]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 C-2 and C-4 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.

AB - 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 (NH3)-N-15, [N-15]APH- or [C-13]APH. Total N-15 incorporation was calculated from separate incubations with (NH3)-N-15 and [N-15]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 C-2 and C-4 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.

KW - amino acids

KW - carbon skeletons

KW - rumen

KW - sheep

KW - volatile fatty acids

KW - microbial-growth

KW - phenylacetic acid

KW - ruminal bacteria

KW - 2,2'-diaminopimelic acid

KW - nitrogen-metabolism

KW - anaerobic bacteria

KW - invitro

KW - peptides

KW - protein

U2 - 10.1079/BJN20031040

DO - 10.1079/BJN20031040

M3 - Article

VL - 91

SP - 253

EP - 261

JO - British Journal of Nutrition

JF - British Journal of Nutrition

SN - 0007-1145

IS - 2

ER -