scholarly journals A sensitive method for measuring protein turnover based on the measurement of 2-3H-labelled amino acids in protein

1976 ◽  
Vol 156 (3) ◽  
pp. 561-568 ◽  
Author(s):  
T J Humphrey ◽  
D D Davies
Keyword(s):  
Amino Acids ◽  
Acetic Anhydride ◽  
Protein Degradation ◽  
Protein Turnover ◽  
Half Life ◽  
Lemna Minor ◽  
Protein Hydrolysate ◽  
New Method ◽  
Specific Measurement ◽  
Sensitive Method

A method for measuring the rate of protein degradation is described. The method measures the change in 2-3H content of protein with time by racemization of the protein hydrolysate with acetic anhydride. The 3H on C-2 of amino acids is stable in proteins but becomes labile, owing to the action of transaminases, once the amino acids are released by proteolysis. The specific measurement of 2-3H in amino acids largely overcomes problems due to compartmentation and isotope recycling and evidence to support this claim is presented. Values for the half-life of Lemna minor (duckweed) protein determined by the new method are compared with values obtained by other methods.

scholarly journals The measurement of protein turnover by density labelling

1975 ◽  
Vol 152 (2) ◽  
pp. 409-416 ◽  
Author(s):  
Alain Boudet ◽  
Thomas J. Humphrey ◽  
David D. Davies
Keyword(s):  
Amino Acids ◽  
Protein Degradation ◽  
Plant Tissue ◽  
Protein Turnover ◽  
Half Life ◽  
Lemna Minor ◽  
Enzyme Activation ◽  
Considerable Improvement ◽  
Minor Protein ◽  
Isopycnic Centrifugation

A method for measuring the rate of protein degradation in plant tissue is described. The method uses density labelling to avoid difficulties associated with compartmentation and recycling of amino acids. Although the technique cannot be readily adapted to measure the rate of degradation of single proteins, it avoids difficulties of interpretation due to enzyme activation or inactivation. Values for the half-life of Lemna minor protein obtained by this method are compared with values obtained by a number of other methods. To obtain satisfactory results it was necessary to improve the method of isopycnic centrifugation in CsCl gradients. A considerable improvement was achieved by using KBr gradients, and the advantages of using KBr rather than CsCl for the separation of density-labelled protein are discussed.

scholarly journals A new method for the measurement of protein turnover

1975 ◽  
Vol 148 (1) ◽  
pp. 119-127 ◽  
Author(s):  
T J Humphrey ◽  
D D Davies
Keyword(s):  
Amino Acids ◽  
Carbon Atom ◽  
Protein Degradation ◽  
Culture Medium ◽  
Lemna Minor ◽  
Total Soluble Protein ◽  
Short Exposure ◽  
Protein Amino Acids ◽  
A New Technique

A new technique for the determination of rate constants of protein degradation is described. By using the method, half-lives of total soluble protein of Lemna minor during growth on full culture medium and distilled water were measured. The method involves incubating Lemna on a growth medium containing 3H2O. After a short exposure (20 min) to 3H-labelled culture medium, 3H was found in soluble amino acids, especially aspartate, glutamate, glutamine and alanine. After transfer to a 3H-free medium for 30 min, 80% of the 3H originally present in soluble amino acids was lost. These results suggest that 3H enters and leaves amino acids at the α-carbon atom, a conclusion supported by the observed labelling of glutamates. The exchange of H and 3H on the α-carbon atom is catalysed by transaminases and the speed of this exchange ensures that when the 3H2O is removed, the 3H in free amino acids is rapidly lost, thereby eliminating problems connected with metabolic pools and recycling. After an exposure of 20 min to 3H-labelled medium all protein amino acids, except for arginine, were found to be radioactive. The loss of radioactivity from protein amino acids was used to measure protein degradation.

Effects of insulin and amino acids on leg protein turnover in IDDM patients

Diabetes ◽  
1991 ◽  
Vol 40 (4) ◽  
pp. 499-508 ◽  
Author(s):  
W. M. Bennet ◽  
A. A. Connacher ◽  
R. T. Jung ◽  
P. Stehle ◽  
M. J. Rennie
Keyword(s):  
Amino Acids ◽  
Protein Turnover

scholarly journals RAPID COMMUNICATION: Residual feed intake in beef cattle is associated with differences in protein turnover and nutrient transporters in ruminal epithelium

2019 ◽  
Vol 97 (5) ◽  
pp. 2181-2187
Author(s):  
Ahmed A Elolimy ◽  
Emad Abdel-Hamied ◽  
Liangyu Hu ◽  
Joshua C McCann ◽  
Daniel W Shike ◽  
...  
Keyword(s):  
Amino Acid ◽  
Beef Cattle ◽  
Protein Degradation ◽  
Insulin Signaling ◽  
Feed Intake ◽  
Feed Efficiency ◽  
Protein Turnover ◽  
Residual Feed Intake ◽  
Ubiquitin Proteasome ◽  
Ruminal Epithelium

Abstract Residual feed intake (RFI) is a widely used measure of feed efficiency in cattle. Although the precise biologic mechanisms associated with improved feed efficiency are not well-known, most-efficient steers (i.e., with low RFI coefficient) downregulate abundance of proteins controlling protein degradation in skeletal muscle. Whether cellular mechanisms controlling protein turnover in ruminal tissue differ by RFI classification is unknown. The aim was to investigate associations between RFI and signaling through the mechanistic target of rapamycin (MTOR) and ubiquitin-proteasome pathways in ruminal epithelium. One hundred and forty-nine Red Angus cattle were allocated to 3 contemporary groups according to sex and herd origin. Animals were offered a finishing diet for 70 d to calculate the RFI coefficient for each. Within each group, the 2 most-efficient (n = 6) and least-efficient animals (n = 6) were selected. Compared with least-efficient animals, the most-efficient animals consumed less feed (P < 0.05; 18.36 vs. 23.39 kg/d DMI). At day 70, plasma samples were collected for insulin concentration analysis. Ruminal epithelium was collected immediately after slaughter to determine abundance and phosphorylation status of 29 proteins associated with MTOR, ubiquitin-proteasome, insulin signaling, and glucose and amino acid transport. Among the proteins involved in cellular protein synthesis, most-efficient animals had lower (P ≤ 0.05) abundance of MTOR, p-MTOR, RPS6KB1, EIF2A, EEF2K, AKT1, and RPS6KB1, whereas MAPK3 tended (P = 0.07) to be lower. In contrast, abundance of p-EEF2K, p-EEF2K:EEF2K, and p-EIF2A:EIF2A in most-efficient animals was greater (P ≤ 0.05). Among proteins catalyzing steps required for protein degradation, the abundance of UBA1, NEDD4, and STUB1 was lower (P ≤ 0.05) and MDM2 tended (P = 0.06) to be lower in most-efficient cattle. Plasma insulin and ruminal epithelium insulin signaling proteins did not differ (P > 0.05) between RFI groups. However, abundance of the insulin-responsive glucose transporter SLC2A4 and the amino acid transporters SLC1A3 and SLC1A5 also was lower (P ≤ 0.05) in most-efficient cattle. Overall, the data indicate that differences in signaling mechanisms controlling protein turnover and nutrient transport in ruminal epithelium are components of feed efficiency in beef cattle.

scholarly journals Using protein turnover to expand the applications of transcriptomics

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marissa A. Smail ◽  
James K. Reigle ◽  
Robert E. McCullumsmith
Keyword(s):  
Protein Turnover ◽  
Half Life ◽  
Rna Expression ◽  
Protein Abundance ◽  
Life Data ◽  
Research Fields ◽  
Extended Application ◽  
Functional Implications ◽  
Transcriptomics Data ◽  
Insight Into

AbstractRNA expression and protein abundance are often at odds when measured in parallel, raising questions about the functional implications of transcriptomics data. Here, we present the concept of persistence, which attempts to address this challenge by combining protein half-life data with RNA expression into a single metric that approximates protein abundance. The longer a protein’s half-life, the more influence it can have on its surroundings. This data offers a valuable opportunity to gain deeper insight into the functional meaning of transcriptome changes. We demonstrate the application of persistence using schizophrenia (SCZ) datasets, where it greatly improved our ability to predict protein abundance from RNA expression. Furthermore, this approach successfully identified persistent genes and pathways known to have impactful changes in SCZ. These results suggest that persistence is a valuable metric for improving the functional insight offered by transcriptomics data, and extended application of this concept could advance numerous research fields.

Physiologische Bedeutung der „Stringent Control“ bei Escherichia coli unter extremen Hungerbedingungen / Stringent Control and Starvation Survival in Escherichia coli

1989 ◽  
Vol 44 (9-10) ◽  
pp. 838-844 ◽  
Author(s):  
H. Mach ◽  
M. Hecker ◽  
I. Hill ◽  
A. Schroeter ◽  
F. Mach
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Protein Turnover ◽  
Stringent Response ◽  
Phosphate Starvation ◽  
Pleiotropic Effects ◽  
Prolonged Starvation ◽  
Stringent Control ◽  
Starvation Survival

The viability of three isogenic relA+/relA strain pairs of Escherichia coli (CP78/CP79; NF 161/ NF162; CP 107/CP 143) was studied during prolonged starvation for amino acids, glucose or phosphate. After amino acid limitation we found a prolonged viability of all relA+ strains which synthesized ppGpp. We suggest that some ppGpp-mediated pleiotropic effects of the stringent response (e.g. glykogen accumulation, enhanced protein turnover) might be involved in this prolongation of survival. After glucose or phosphate starvation there was no difference in the relA+/relA strains either in the ppGpp content or in the survival.

Turnover of total proteins and ornithine aminotransferase during liver regeneration in rats

1980 ◽  
Vol 238 (1) ◽  
pp. E46-E52
Author(s):  
S. L. Augustine ◽  
R. W. Swick
Keyword(s):  
Amino Acids ◽  
Liver Regeneration ◽  
Protein Degradation ◽  
Partial Hepatectomy ◽  
Free Amino ◽  
Liver Protein ◽  
Ornithine Aminotransferase ◽  
Fractional Rate

The recovery of approximately 40% of the total liver protein during the first day after partial hepatectomy was shown to be due to the near cessation of protein breakdown rather than to an increase in protein synthesis. The decrease in degradation of total protein was less if rats were adrenalectomized or protein-depleted prior to partial hepatectomy. The effect of these treatments originally suggested that changes in free amino acid levels in liver might be related to the rate of protein degradation. However, no correlation was found between levels of total free amino acids and rates of breakdown. Measurements of individual amino acids during liver regeneration suggested that levels of free methionine and phenylalanine, amino acids that have been found to lower rates of protein degradation in vitro, are not correlated with rates of breakdown in vivo. The difference between the fractional rate of ornithine aminotransferase degradation (0.68/day and 0.28/day in sham-hepatectomized and partially hepatectomized rats, respectively) was sufficient to account for the higher level of this protein 3 days after surgery in the latter group.

scholarly journals Effect of long-term refeeding on protein metabolism in patients with cirrhosis of the liver

1997 ◽  
Vol 77 (2) ◽  
pp. 197-212 ◽  
Author(s):  
Jens Kondrup ◽  
Klaus Nielsen ◽  
Anders Juul
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Protein Degradation ◽  
Protein Metabolism ◽  
Cirrhosis Of The Liver ◽  
N Balance ◽  
Whole Body ◽  
Protein Requirement ◽  
Protein Utilization ◽  
Igf I

Patients with cirrhosis of the liver require an increased amount of protein to achieve N balance. However, the utilization of protein with increased protein intake, i.e. the slope from regression analysis of N balance v. intake, is highly efficient (Nielsen et al. 1995). In the present study, protein requirement and protein utilization were investigated further by measuring protein synthesis and degradation. In two separate studies, five or six patients with cirrhosis of the liver were refed on a balanced diet for an average of 2 or 4 weeks. Protein and energy intakes were doubled in both studies. Initial and final whole-body protein metabolism was measured in the fed state by primed continous [15N]glycine infusion. Refeeding caused a statistically significant increase of about 30% in protein synthesis in both studies while protein degradation was only slightly affected. The increase in protein synthesis was associated with significant increases in plasma concentrations of total amino acids (25%), leucine (58%), isoleucine (82%), valine (72%), proline (48%) and triiodothyronine (27%) while insulin, growth hormone, insulin-like growth factor (IGF)-I and IGF-binding protein-3 were not changed significantly. The results indicate that the efficient protein utilization is due to increased protein synthesis, rather than decreased protein degradation, and suggest that increases in plasma amino acids may be responsible for the increased protein synthesis. A comparison of the patients who had a normal protein requirement with the patients who had an increased protein requirement suggests that the increased protein requirement is due to a primary increase in protein degradation. It is speculated that this is due to low levels of IGF-I secondary to impaired liver function, since initial plasma concentration of IGF-I was about 25% of control values and remained low during refeeding.

Effect of Intraduodenal Starch Infusion on Net Portal Absorption of Amino Acids in Growing steers Fed Lucerne

1994 ◽  
Vol 1994 ◽  
pp. 28-28
Author(s):  
C.J. Seal ◽  
D.S. Parker ◽  
J.C. MacRae ◽  
G.E. Lobley
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gastrointestinal Tract ◽  
Protein Turnover ◽  
Portal Blood ◽  
Intestinal Lumen ◽  
Short Term ◽  
Net Uptake ◽  
Amino Acid Requirements ◽  
Glucose Availability

Amino acid requirements for energy metabolism and protein turnover within the gastrointestinal tract are substantial and may be met from luminal and arterial pools of amino acids. Several studies have demonstrated that the quantity of amino acids appearing in the portal blood does not balance apparent disappearance from the intestinal lumen and that changing diet or the availability of energy-yielding substrates to the gut tissues may influence the uptake of amino acids into the portal blood (Seal & Reynolds, 1993). For example, increased net absorption of amino acids was observed in animals receiving exogenous intraruminal propionate (Seal & Parker, 1991) and this was accompanied by changes in glucose utilisation by the gut tissues. In contrast, there was no apparent change in net uptake of [l-13C]-leucine into the portal vein of sheep receiving short term intraduodenal infusions of glucose (Piccioli Cappelli et al, 1993). This experiment was designed to further investigate the effects on amino acid absorption of changing glucose availability to the gut with short term (seven hours) or prolonged (three days) exposure to starch infused directly into the duodenum.

Measurement of protein turnover in the small intestine of lambs. 2. Effects of feed intake

1997 ◽  
Vol 128 (2) ◽  
pp. 233-246 ◽  
Author(s):  
S. A. NEUTZE ◽  
J. M. GOODEN ◽  
V. H. ODDY
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Small Intestine ◽  
Experimental Model ◽  
Feed Intake ◽  
Protein Turnover ◽  
Jugular Vein ◽  
Specific Radioactivity ◽  
Whole Body ◽  
Body Protein

This study used an experimental model, described in a companion paper, to examine the effects of feed intake on protein turnover in the small intestine of lambs. Ten male castrate lambs (∼ 10 months old) were offered, via continuous feeders, either 400 (n = 5) or 1200 (n = 5) g/day lucerne chaff, and mean experimental liveweights were 28 and 33 kg respectively. All lambs were prepared with catheters in the cranial mesenteric vein (CMV), femoral artery (FA), jugular vein and abomasum, and a blood flow probe around the CMV. Cr-EDTA (0·139 mg Cr/ml, ∼ 0·2 ml/min) was infused abomasally for 24 h and L-[2,6-3H]phenylalanine (Phe) (420±9·35 μCi into the abomasum) and L-[U-14C]phenylalanine (49·6±3·59 μCi into the jugular vein) were also infused during the last 8 h. Blood from the CMV and FA was sampled during the isotope infusions. At the end of infusions, lambs were killed and tissue (n = 4) and digesta (n = 2) samples removed from the small intestine (SI) of each animal. Transfers of labelled and unlabelled Phe were measured between SI tissue, its lumen and blood, enabling both fractional and absolute rates of protein synthesis and gain to be estimated.Total SI mass increased significantly with feed intake (P < 0·05), although not on a liveweight basis. Fractional rates of protein gain in the SI tended to increase (P = 0·12) with feed intake; these rates were −16·2 (±13·7) and 23·3 (±15·2) % per day in lambs offered 400 and 1200 g/day respectively. Mean protein synthesis and fractional synthesis rates (FSR), calculated from the mean retention of 14C and 3H in SI tissue, were both positively affected by feed intake (0·01 < P < 0·05). The choice of free Phe pool for estimating precursor specific radioactivity (SRA) for protein synthesis had a major effect on FSR. Assuming that tissue free Phe SRA represented precursor SRA, mean FSR were 81 (±15) and 145 (±24) % per day in lambs offered 400 and 1200 g/day respectively. Corresponding estimates for free Phe SRA in the FA and CMV were 28 (±2·9) and 42 (±3·5) % per day on 400 g/day, and 61 (±2·9) and 94 (±6·0) on 1200 g/day. The correct value for protein synthesis was therefore in doubt, although indirect evidence suggested that blood SRA (either FA or CMV) may be closest to true precursor SRA. This evidence included (i) comparison with flooding dose estimates of FSR, (ii) comparison of 3H[ratio ]14C Phe SRA in free Phe pools with this ratio in SI protein, and (iii) the proportion of SI energy use associated with protein synthesis.Using the experimental model, the proportion of small intestinal protein synthesis exported was estimated as 0·13–0·27 (depending on the choice of precursor) and was unaffected by feed intake. The contribution of the small intestine to whole body protein synthesis tended to be higher in lambs offered 1200 g/day (0·21) than in those offered 400 g/day (0·13). The data obtained in this study suggested a role for the small intestine in modulating amino acid supply with changes in feed intake. At high intake (1200 g/day), the small intestine increases in mass and CMV uptake of amino acids is less than absorption from the lumen, while at low intake (400 g/day), this organ loses mass and CMV uptake of amino acids exceeds that absorbed. The implications of these findings are discussed.

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