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Protein Metabolism I

Protein Metabolism I. ANS 520. Topics. Amino acid metabolism Microbial protein contributions Ruminal N digestion. Protein Pathways. Ruminant Protein Metabolism. Nitrogenous feed component, non-protein nitrogen components, endogenous Ammonia for bacterial growth

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Protein Metabolism I

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  1. Protein Metabolism I ANS 520

  2. Topics • Amino acid metabolism • Microbial protein contributions • Ruminal N digestion

  3. Protein Pathways

  4. Ruminant Protein Metabolism • Nitrogenous feed component, non-protein nitrogen components, endogenous • Ammonia for bacterial growth • Amino acids (AA) for animal needs (absorbed in small intestine)

  5. Feed Protein AcronymsNRC Publications Crude protein Total N x 6.25 DIP (RDP) Degraded intake protein UIP (RUP) Undegraded intake protein SolP, % CP Soluble protein NPN, % CP Nonprotein nitrogen NDFIP, % CP Neutral detergent fiber insoluble protein ADFIP, % CP Acid detergent fiber insoluble protein B1, B2, B3, % hr Rate constants for degradable fractions

  6. Protein • Analysis: Determine total N by Kjeldahl • All N NH4+ • Determine as NH3 • Total N x 6.25 = crude protein • Peptide bond: NH2 R1-C-C-NH O C-C=O R2 N-C-COOH H R3

  7. Nitrogenous Compounds in Feeds • True proteins • Polymers of AA (18 to 20 AA) linked by peptide bonds • Essential AA • Have to be present in the diet (absorbed) • Arg Lys TrpLeu Ile Val Met ThrPhy His • PVT TIM HALL • Nonessential amino acids (dispensable) • Synthesized in body tissues • GluGly Asp Pro Ala Ser Cys Tyr • Proteins Peptides Amino acids

  8. Nitrogenous Compounds in Feeds • Nonprotein nitrogen • Nitrogen not associated with protein • Free amino acids, nucleic acids, amines, ammonia, nitrates, nitrites, urea • Crude protein • Total nitrogen x 6.25 • Proteins on average contain 16% nitrogen

  9. Protein Degradation in the Rumen • Feed proteins Peptides Amino acids • Undegraded feed proteins • Escaped feed proteins • “Bypass proteins” • Enzymes from protozoa and bacteria • Many species of bacteria involved • Bacterial enzymes are extracellular • Enzymes not in cell free rumen fluid • Both exopeptidase and endopeptidase activity • Assumption in CNCPS: Enzymes (microorganisms) in excess – substrate limited

  10. Factors Affecting Ruminal Protein Degradation • Chemical nature of the proteins • Solubility – More soluble proteins degraded faster • Exceptions might include • egg ovalbumin, serum proteins • 3-dimensional structure – Affects solubility & availability • Chemical bonding • Disulfide bonds – Reduces degradation

  11. Factors Affecting Ruminal Protein Degradation • Physical barriers • Cell walls of plants • Cross linking of peptide chains – Reduces degradation • Aldehydes, Tannins • Feed intake • Rate of passage – Time proteins remain in the rumen • Feed processing • Rate of passage • Heat damage – Complexes with carbohydrates

  12. Estimating Ruminal Protein Degradation • 1. In situ digestion • Feed placed in Dacron bags suspended • in the rumen • Measure protein lost over time • 2. Cannulated animals (rumen & duodenum) • Measure protein flowing through duodenum • Need to differentiate feed from microbes • 3. In vitro incubation with rumen microbes • Relative differences among proteins • 4. In vitro digestion with fungal enzymes

  13. Protein Degradation In situ Log, % N remaining A - All degraded B - Partly degraded Slope = degradation rate C - Not degraded Digestion time, hr

  14. Protein Degradation • DIP (RDP) = A + B[Kd/(Kd+Kp)] • DIP = Degraded intake protein • Kd = degradation rate, %/h • Kp = passage rate, %/h • UIP (RUP) = B[Kp/(Kd+Kp)] + C • UIP = Undegraded intake protein

  15. Feed Protein Fractions (CNCPS & NRC) NPN - A Sol Proteins - B1 Insoluble - B2 Insoluble - B3 Indigestible - C Soluble Insoluble Feed

  16. Protein Fractions In FeedsLaboratory Analysis • A - Soluble in buffer (borate-phosphate) and not • precipitated by tungstic acid • B1 - Soluble in buffer and precipitated by tungstic acid • B2 - Insoluble in buffer • = (Insol protein) - (protein insol in neutral detergent) • B3 - Insoluble in buffer • = (Insol in neutral detergent) - (Insol in acid detergent) • C - Insoluble in buffer and acid detergent

  17. Kd Values for Feed Proteins FractionKd, %/h A Infinity B1 120 to 400 B2 3 to 16 B3 0.06 to 0.55 C Not degraded

  18. Kp Values Wet forages Kp = 3.054 + 0.614X1 Dry forages Kp = 3.362 + 0.479X1 – 0.007X2 – 0.017X3 Concentrates Kp = 2.904 + 1.375X1 – 0.020X2 X1 = DMI, % Body Wt X2 = Concentrate, % of ration DM X3 = NDF of feedstuff, % DM

  19. “Bypass proteins” • Proteins that are not extensively degraded in the rumen • Natural • Corn proteins, blood proteins, feather meal • Modification of feed proteins to make them less • degradable • Heat - Browning or Maillard reaction • Expeller SBM, Dried DGS, Blood meal • Chemical • Formaldehyde • Polyphenols • Tannins • Alcohol + heat • Usually some loss in availability of amino acids - lysine

  20. Average RuminalDegradation of Several ProteinsUsed in Level 1 Soybean meal (Solvent processed) 75% Soybean meal ( Expeller processed) 50% Alfalfa 80% Corn proteins 62% Corn gluten meal 42% Corn gluten feed 80% Dried distillers grains 55% Blood meal 20% Feather meal 30% Urea 100%

  21. Degradation of NPN Compounds • Activity associated with microorganisms • Urea CO2 + 2 NH3 • High concentrations of urease activity • in the rumen • Low concentrations of urea in the rumen • Biuret 2 CO2 + 3 NH3 • Low activity in the rumen • NO3 NH3

  22. Fate of Free Amino Acids in the Rumen • Amino acids not absorbed from the rumen • Concentrations of free AA in the rumen very low • Amino acids and small peptides (up to 5 AA) • transported into bacterial cells • Na pumped out of cells – Uses ATP • Na gradient facilitates transport of AA by a carrier • Utilized for synthesis of microbial proteins • Amino acids metabolized to provide energy

  23. Amino Acid Degradation in the Rumen • NH3 CO2 • Amino acids Keto acids VFA • Enzymes from microorganisms • Intracellular enzymes • Peptides probably hydrolyzed to amino acids • and then degraded • NH3, VFA and CO2 absorbed from rumen

  24. Amino Acid Fermentation Valine Isobutyrate Leucine Isovalerate Isoleucine 2-methybutyrate Alanine, glutamate, histidine, aspartate, glycine, serine, cystein and tryptophan pyruvate Threonine, homoserine, homocyseine and methionine Ketones

  25. Control of Amino Acid Fermentation • When CHOH is ample for growth, incorporation • of amino acids into protein is favored • Majority of transported amino acids and • peptides do not go through ammonia pool • When CHOH supply is limiting growth, amino • acids are fermented for energy • There is an increase in amino acids going • through the ammonia pool

  26. Amino Acid Fermenters in the Rumen High numbers Low numbers Low activity High activity Butrivibrio fibrisolvens Clostridium aminophilum Measphaera elsdenii Clostridium sticklandii Selenomonas ruminantium Peptostreptococuss anaerobius 109 per ml107 per ml 10 to 20 NMol NH3 300 NMol NH3 per min per min per mg protein per mg protein Monensin resistant Monensin sensitive Involved in CHOH Ferment CHOH slowly or fermentation not at all

  27. Microbial Protein Synthesis • End product of protein degradation is mostly NH3 • Protein synthesis • Fixation of N in organic form • Synthesis of amino acids • Synthesis of protein(s)

  28. Rumen microbes • Bacteria (50% CP) • Protozoa (20-60%, avg 40% CP) • Bacteria major player, % of microbial N entering SI from protozoa < 10% • N source for microbes • Diet protein • Non protein N • Recycled N

  29. Microbial N • Microbial N entering SI (% of non-ammonia N) • High protein diets -40% • Low protein diets -60% • Exclusive NPN diet -100% • Limiting factors would include C and/or energy source

  30. Nutritive Value of Microbial N • Increases value of low quality feed N • Decreases value of high quality feed N • Animal can survive on non-protein N • Can survive on low amounts of recycled N

  31. Bacterial ProteinSynthesis in the Rumen NH3 Amino acids & Peptides VFA Amino acids Microbial Fermentation proteins CHOH VFA • Microbial protein synthesis related to: • 1. Available NH3 and amino acids (DIP) • 2. Fermentation of CHOH - Energy

  32. Microbial RequirementsBacteria • Nitrogen • Mixed cultures • NH3 satisfies the N requirement • Cross feeding can supply amino acids • Pure cultures • Fiber digesters require NH3 • Starch digesters require NH3 and amino acids • Peptides can be taken up by cells • Branched-chain fatty acids • Required by major rumen cellulolytic bacteria • Energy from fermentation • Need energy for synthesis of macromolecules

  33. Role of Protozoa • Do not use NH3 directly • Engulf feed particles and bacteria • Digest proteins • Release amino acids and peptides into rumen • Use amino acids for protein synthesis • Protozoa engulf bacteria • Protozoa lyse easily – May contribute little • microbial protein to the animal

  34. Efficiency of Microbial Growth • Grams microbial N/100 g organic matter digested • Ranges from 1.1 to 5.0 • 1. Kind of diet Forages > Grain • 2. Level of feeding High > Low • 3. Rate of passage Fast > Slow • 4. Turnover of microbial cells • Younger cells turnover less than aging cells • Maintenance requirement of cells • Microbes use energy to maintain cellular integrity • Energy spilling • Dissipation of energy different from maintenance • Most apparent when energy is in excess

  35. Efficiency of Microbial Growth Slow Low rumen passage pH Bacteria Low quality use energy to forages slow pump protons passage G BCP/100 g TDN 8 13 TDN, % feed DM

  36. Microbial Growth in The Rumen • Nutrients available to microbes • DIP - NH3, peptides, amino acids • CNCPS adjusts for inadequate available N • 2. Energy from the fermentation • Growth rate related to Kd of CHOH • Quantity of cells related to CHOH digested • CNCPS assumes microbes digesting • non-fiber and fiber CHOH both have • a maximum yield of 50g cells/100g • CHOH fermented • 3. Other - branched-chain acids, minerals

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