Protein-bound Amino Acids Research Articles

Racemization in Post-Translational Modifications Relevance to Protein Aging, Aggregation and Neurodegeneration: Tip of the Iceberg.

Homochirality of DNA and prevalent chirality of free and protein-bound amino acids in a living organism represents the challenge for modern biochemistry and neuroscience. The idea of an association between age-related disease, neurodegeneration, and racemization originated from the studies of fossils and cataract disease. Under the pressure of new results, this concept has a broader significance linking protein folding, aggregation, and disfunction to an organism’s cognitive and behavioral functions. The integrity of cognitive function is provided by a delicate balance between the evolutionarily imposed molecular homo-chirality and the epigenetic/developmental impact of spontaneous and enzymatic racemization. The chirality of amino acids is the crucial player in the modulation the structure and function of proteins, lipids, and DNA. The collapse of homochirality by racemization is the result of the conformational phase transition. The racemization of protein-bound amino acids (spontaneous and enzymatic) occurs through thermal activation over the energy barrier or by the tunnel transfer effect under the energy barrier. The phase transition is achieved through the intermediate state, where the chirality of alpha carbon vanished. From a thermodynamic consideration, the system in the homo-chiral (single enantiomeric) state is characterized by a decreased level of entropy. The oscillating protein chirality is suggesting its distinct significance in the neurotransmission and flow of perceptual information, adaptive associative learning, and cognitive laterality. The common pathological hallmarks of neurodegenerative disorders include protein misfolding, aging, and the deposition of protease-resistant protein aggregates. Each of the landmarks is influenced by racemization. The brain region, cell type, and age-dependent racemization critically influence the functions of many intracellular, membrane-bound, and extracellular proteins including amyloid precursor protein (APP), TAU, PrP, Huntingtin, α-synuclein, myelin basic protein (MBP), and collagen. The amyloid cascade hypothesis in Alzheimer’s disease (AD) coexists with the failure of amyloid beta (Aβ) targeting drug therapy. According to our view, racemization should be considered as a critical factor of protein conformation with the potential for inducing order, disorder, misfolding, aggregation, toxicity, and malfunctions.

Total and free amino acid contents of popular rice varieties (Oryza sativa L.) consumed in the capital city of Sri Lanka

This study investigated the amino acid composition of eight local and three imported rice varieties frequently consumed by consumers in the capital of Sri Lanka. Investigation included twenty free amino acids (FAAs) analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and sixteen protein bound amino acids analysed using high performance liquid chromatography with diode array detection (HPLC-DAD). The mean total amino acid (TAA) in rice ranged between 64.5 ± 1.9 g/kg and 96.0 ± 5.1 g/kg on dry basis (db). Ponni, Basmathi, Rathu Nadu and Fragrant rice which were parboiled varieties, reported the overall highest mean TAA contents while imported Ponni and Basmathi exhibited the highest mean essential amino acid (EAA) contents depicting high quality protein. Red rice varieties of Nadu, Kekulu and Kekulu Samba reported significantly (p

Use of encapsulated L-lysine-HCl and DL-methionine improves postprandial amino acid balance in laying hens.

The supplementation of dietary limiting amino acids (AA) with crystalline AA makes the use of low-protein diets an option in poultry production. The differing absorption rates of crystalline and protein-bound AA may lead to temporally imbalanced AA in the postabsorptive period. In this study, two experiments were conducted to evaluate the effect of encapsulated L-lysine-HCl (L-Lys-HCl) and DL-methionine (DL-Met) on the laying performance of hens. In exp. 1, a total of 135 forty-seven-wk-old Hy-Line Brown hens were subjected to three dietary treatments for 8 wk: basal diet supplemented with 0.14% L-Lys-HCl and 0.17% DL-Met to satisfy the NRC (1994) total Lys and Met recommendation (control) and basal diet supplemented with encapsulated L-Lys-HCl and DL-Met at the levels of 60% (60CLM, 0.084% L-Lys-HCl and 0.102% DL-Met) or 80% of control (80CLM, 0.112% L-Lys-HCl and 0.136% DL-Met), respectively. In exp. 2, 24 fifty-five-wk-old Hy-Line Brown hens were individually reared in cages and subjected to the same treatments as in exp. 1. The plasma concentrations of free AA and nitrogen metabolites were measured 2, 4, and 6 h after fed. The results showed that dietary AA treatment had no significant influence on body weight (BW), feed intake, laying rate, egg weight, egg mass, or feed efficiency. The expression levels of AA transporters CAT-1, y+LAT1, b0,+AT, B0AT, rBAT, EAAT3, and PepT1 in the duodenum, jejunum, and ileum were not influenced (P > 0.05) by dietary treatment. There was an interaction of dietary AA treatment and time (P < 0.05) and the 80CLM hens exhibited higher concentrations of Lys (P < 0.05) than the controls at 2-h time point. In contrast, plasma Met concentration was not influenced (P > 0.05), while Cys was reduced in the 60CLM hens at every time point. The 80CLM hens had higher taurine concentrations than those receiving the control diet at every postprandial time point. In conclusion, these findings demonstrate that by using encapsulated form, the supplemental levels of synthetic L-Lys-HCl and DL-Met can be effectively reduced by approximately 20% with no negative effect on laying performance. The result suggests that encapsulated Lys and Met may ameliorate the postabsorptive AA balance and contribute to the reduced dietary AA supplemental levels.

Encapsulated crystalline lysine and DL-methionine have higher efficiency than the crystalline form in broilers

Crystalline amino acids (AAs) exhibit high nutritional values when supplemented AA-deficient diets. However, the AAs in crystalline form in the diet are absorbed quickly than protein-bound AAs, which may take an effect on AA utilization efficiency. In this study, 2 experiments were conducted to investigate the effect of encapsulated lysine-HCl (Lys) and DL-methionine (DL-Met) on the growth performance of broiler chickens. In experiment 1, a total of 432 one-day-old male Arbor Acres broilers were subjected to 3 dietary treatments (27 pens; 16 birds per pen) for 42 d. The control group was basal diets supplemented with crystalline Lys and DL-Met, and treatment groups had basal diets supplemented with encapsulated Lys and DL-Met at the levels of 80% and 60% of control diets (80CLM, 60CLM), respectively. The growth performance, intestinal development, and transcription of AA transporters were determined. In experiment 2, 24 broiler chickens were subjected to the same treatments as in experiment 1. The plasma concentrations of free AAs were measured 0, 2, 4, and 6 h after feeding. The results showed that 80CLM treatment had no significant influence on production performance, carcass characteristics, and plasma free AAs content during the experiment compared with the control group (P > 0.05). In addition, the 80CLM group moderately enhanced gut morphology development and increased AAs' absorption capacity. However, broilers fed the 60CLM diet had lower production performance and breast muscle weight than the control group (P < 0.05), but increased villi height and B0AT mRNA expression level (P < 0.05). At h 4 after feeding, the 60CLM broilers exhibited higher concentration of Ala, Cys, and total dispensable AAs than the control group (P < 0.05). In conclusion, the result suggests that the supplemental levels of crystalline Lys and DL-Met can be effectively saved approximately for 20% by using the encapsulated form in broilers, with improvements to AAs utilization efficiency, while posing no detrimental effects on production performance. Encapsulated Lys and DL-Met would have greater potential for application when replacing crystalline AAs in broiler chickens.

Protein Precipitation to Remove Carbohydrates that Interfere in Protein-Bound Tryptophan Quantification in Soybean Seeds

Tryptophan is one of the nine essential amino acids in humans that can only be obtained through diets and supplements. It is a precursor to many biological processes, such as serotonin, melatonin, kynurenin, and niacin (nicotinamide) vitamin synthesis. The content of tryptophan in foods, such as soybean is an important indicator of nutritional value. Therefore, accurate quantification of tryptophan in soybean is crucial to soybean nutritional improvement. Quantification of soybean protein-bound amino acids first involves acid hydrolysis of total protein to liberate amino acids. However, tryptophan quantification following acid hydrolysis is difficult or impossible due to its reactions with soybean carbohydrates. Therefore, removal of carbohydrates from soy proteins prior to acid hydrolysis is necessary. In this study, we compared four common protein precipitation methods (i.e., methanol, acetonitrile, acetone, and trichloroacetic acid (TCA) protein precipitation methods) to determine the best method to separate soy proteins from carbohydrates, and concluded that acetone provided the highest recovery of soy proteins. Tryptophan content in the precipitated proteins was determined after acid hydrolysis of the proteins using liquid chromatography–tandem mass spectrometry multiple reaction monitoring (LC–MS/MS–MRM). No significant difference in the tryptophan content was found among proteins precipitated with methanol, acetonitrile, and TCA, suggesting that these precipitated proteins have similar compositions. A slightly lower, but statistically significant tryptophan content was found in the acetonitrile-precipitated proteins, suggesting that these proteins contain slightly higher glycosylated proteins.

Natural 15N abundance in specific amino acids indicates associations between transamination rates and residual feed intake in beef cattle.

Improving the ability of animals to convert feed resources into food for humans is needed for more sustainable livestock systems. Genetic selection for animals eating less while maintaining their performance (i.e., low residual feed intake [RFI]) appears a smart strategy but its effectiveness relies on high-throughput animal phenotyping. Here, we explored plasma nitrogen (N) isotope ratios in an attempt to identify easily superior young bulls in terms of RFI. For this, 48 Charolais young bulls fed two contrasting diets (corn vs. grass silage diets) were selected from a larger population as extreme RFI animals (24 low-RFI vs. 24 high-RFI) and their plasma analyzed for natural 15N abundance (δ15N) in the whole protein (bulk protein) and in the individual protein-bound amino acids (PbAA). For the first time, we showed that the δ 15N in plasma bulk protein differed (P = 0.007) between efficient (low-RFI) and inefficient (high-RFI) cattle regardless of diet. Furthermore, most analyzed PbAA followed the same trend as the bulk protein, with lower (P < 0.05) δ 15N values in more efficient (low-RFI) compared with less efficient (high-RFI) cattle, again regardless of diet. The only three exceptions were Phe, Met, and Lys (P > 0.05) for which the first metabolic reaction before being catabolized does not involve transamination, a pathway known naturally to enrich AAs in 15N. The contrasted isotopic signatures across RFI groups only in those PbAA undergoing transamination are interpreted as differences in transamination rates and N-use efficiency between low- and high-RFI phenotypes. Natural isotopic N signatures in bulk proteins and specific PbAA can be proposed as biomarkers of RFI in growing beef cattle fed different diets. However, the current study cannot delineate whether this effect only occurs post-absorption or to some extent also in the rumen. Our data support the conclusion that most efficient cattle in terms of RFI upregulate N conservation mechanisms compared with less efficient cattle and justify future research on this topic.

Serum concentration of free amino acids in pigs of similar performance fed diets containing protein-bound or protein-bound combined with free amino acids

The serum concentration (SC) of amino acids (AA), as indicative of their availability, varies depending on the dietary AA source (free or protein-bound). However, the AA availability may not differ when pigs fed low CP-free AA-supplemented diets or high protein-bound AA diets perform similarly. A study was conducted to analyze the SC of free AA at three different postprandial times in pigs with similar performance fed a low protein-AA supplemented or a high protein diet. Twenty-four pigs were randomly assigned to one of two wheat-soybean meal diets: low protein (140 g/kg) plus free l-lysine (Lys), l-threonine (Thr), DL-methionine (Met), l-leucine (Leu), l-isoleucine (Ile), l-valine (Val), l-histidine (His), l-tryptophan (Trp) and l-phenylalanine (Phe) (LP-FAA); high protein (220 g/kg) without added free AA (HP-PBAA). There were two phases; a 21-day growth one followed by a 4-day blood collection phase from 6 pigs fed twice a day (0700 and 1900 h). Blood was collected at 2-h before the morning meal (preprandial) and at 1, 2, and 3-h after the morning meal (postprandial). There was no effect (P ≥ 0.208) of dietary treatment on body weight gain, feed intake, and gain:feed during the 21-day growth phase. Regarding postprandial time, there was a quadratic effect for serum Leu and Lys (P < 0.05), and a tendency (P < 0.10) for arginine (Arg), Met, Phe, Thr, and Val in pigs fed the LP-FAA diet; in pigs fed the HP-PBAA diet, there was a quadratic response for Arg, His, Phe, Thr, and Val (P < 0.05) and a tendency for Ile and Leu (P < 0.10), but Lys and Met were not affected either in a linear or a quadratic way (P> 0.10). In all pigs, the highest values were observed at 2-h postprandial. Serum Lys and Thr, at 2-h postprandial, was higher in pigs fed the LP-FAA diet than in HP-PBAA pigs (P < 0.05). At 1-h postprandial, SC of all AA did not differ from preprandial SC in LP-FAA pigs, but it was higher (P < 0.05) in HP-PBAA pigs. These results confirmed that the SC of AA varies depending on the form (free or protein-bound) dietary AA were ingested. In conclusion growing pigs fed a 140 g protein/kg diet formulated on the basis of nine SID indispensable AA and net energy, performed similarly to those fed a 220 g protein/kg diet, although the overall efficiency of utilization of dietary AA appears to increase when pigs are fed low protein diets supplemented with free AA.

Organic secretion in ductal cells of the sublingual salivary gland of ferret: Histochemical observations

The presence of organic secretion in ductal cells of the sublingual salivary gland of ferret has been questioned, which prompted the present investigation. Paraffin or cryostat sections from aldehyde fixed or quenched sublingual glands of this species were tested for some amino acid residues, mucosubstances, oxidative and hydrolytic enzymes, and lectins. The glands showed inconspicuous ducts of simple appearances on routine histology. The histochemical procedures, however, revealed a granulated substance in the apical (periluminal) region of ductal cells, which contained tryptophan, disulphides, neutral mucosubstances, αFuc and GalNAc, and showed chloroacetate esterase activity. Occurrence of the substance varied between different ducts of the same gland and/or cells of the same duct. The ductal cells also showed diffuse peroxidase and acid phosphatase, and Golgi-like thiamine pyrophosphatase activities. Acetylcholinesterase-positive nerve fibres embraced the ducts. The results support a particular localisation of protein-bound amino acid residues and enzymatic catalytic activities indicative of organic secretion, possibly tissue kallikrein, in sublingual ductal cells of ferret.

Dietary protein-bound or free amino acids differently affect intestinal morphology, gene expression of amino acid transporters, and serum amino acids of pigs exposed to heat stress.

Pigs exposed to heat stress (HS) increase body temperature in which can damage the intestinal epithelia and affect the absorption and availability of amino acids (AA). Protein digestion and metabolism further increase body temperature. An experiment was conducted with six pairs of pigs (of 47.3 ± 1.3 kg initial body weight) exposed to natural HS to assess the effect of substituting dietary protein-bound AA by free AA on morphology and gene expression of intestinal epithelial and serum concentration (SC) of free AA. Treatments were: high protein, 21.9% crude protein (CP) diet (HShp) and low protein, 13.5% CP diet supplemented with crystalline Lys, Thr, Met, Trp, His, Ile, Leu, Phe, and Val (HSaa). The HShp diet met or exceeded all AA requirements. The HSaa diet was formulated on the basis of ideal protein. Pigs were fed the same amount at 0700 and 1900 hours during the 21-d study. Blood samples were collected at 1700 hours (2.0 h before the evening meal), 2030 hours, and 2130 hours (1.5 and 2.5 h after the evening meal). At the end, all pigs were sacrificed to collect intestinal mucosa and a 5-cm section from each segment of the small intestine from each pig. Villi measures, expression of AA transporters (y+L and B0) in mucosa, and SC of AA were analyzed. Ambient temperature fluctuated daily from 24.5 to 42.6 °C. Weight gain and G.F were not affected by dietary treatment. Villi height tended to be larger (P ≤ 0.10) and the villi height:crypt depth ratio was higher in duodenum and jejunum of pigs fed the HSaa diet (P < 0.05). Gene expression of transporter y+L in jejunum tended to be lower (P < 0.10) and transporter B0 in the ileum was lower (P < 0.05) in HSaa pigs. Preprandial (1700 hours) SC of Arg, His, Ile, Leu, Thr, Trp, and Val was higher (P < 0.05), and Phe tended to be higher (P < 0.10) in HShp pigs. At 2030 hours (1.5 h postprandial), serum Lys, Met, and Thr were higher in the HSaa pigs (P < 0.05). At 2130 hours (2.5 h), Arg, His, Ile, Phe, and Trp were lower (P < 0.05); Met was higher (P < 0.05); and Lys tended to be higher (P < 0.10) in HSaa pigs. In conclusion, feeding HS pigs with low protein diets supplemented with free AA reduces the damage of the intestinal epithelia and seems to improve its absorption capacity, in comparison with HS pigs fed diets containing solely protein-bound AA. This information is useful to formulate diets that correct the reduced AA consumption associated with the decreased voluntary feed intake of pigs under HS.

The complex response of free and bound amino acids to water stress during the seed setting stage in Arabidopsis

Free amino acids (FAAs) and protein-bound amino acids (PBAAs) in seeds play an important role in seed desiccation, longevity, and germination. However, the effect that water stress has on these two functional pools, especially when imposed during the crucial seed setting stage is unclear. To better understand these effects, we exposed Arabidopsis plants at the seed setting stage to a range of water limitation and water deprivation conditions and then evaluated physiological, metabolic, and proteomic parameters, with special focus on FAAs and PBAAs. We found that in response to severe water limitation, seed yield decreased, while seed weight, FAA, and PBAA content per seed increased. Nevertheless, the composition of FAAs and PBAAs remained unaltered. In response to severe water deprivation, however, both seed yield and weight were reduced. In addition, major alterations were observed in both FAA and proteome compositions, which indicated that both osmotic adjustment and proteomic reprogramming occurred in these naturally desiccation-tolerant organs. However, despite the major proteomic alteration, the PBAA composition did not change, suggesting that the proteomic reprogramming was followed by a proteomic rebalancing. Proteomic rebalancing has not been observed previously in response to stress, but its occurrence under stress strongly suggests its natural function. Together, our data show that the dry seed PBAA composition plays a key role in seed fitness and therefore is rigorously maintained even under severe water stress, while the FAA composition is more plastic and adaptable to changing environments, and that both functional pools are distinctly regulated.

Changes in spatiotemporal protein and amino acid gradients in wheat caryopsis after N-topdressing

Wheat grain nitrogen content displays large variations within different pearling fractions of grains because of radial gradients in the protein content. We identified how spatiotemporal mechanisms regulate this. The protein gradients emerged clearly at 19 days after anthesis, with the highest N content in aleurone and seed coat, followed by outer endosperm, whereas the lowest was in middle and inner endosperm. Laser microdissection, qRT-PCR and LC–MS were used to dissect tissue from aleurone, outer endosperm, middle endosperm, inner endosperm and transfer cells, measure gene expression and levels of free and protein-bound amino acids, respectively. The results showed that different FAA transportation pathways worked in parallel during grain filling stage while the grain protein gradient did not follow spatial expression of storage proteins. Additionally, two nitrogen (N) topdressing timings were conducted, either at the emergence of top third leaf (standard timing) or top first leaf (delayed timing), finding that delayed N topdressing enhanced both amino acids supply and protein synthesis capacity. The results provide insight into protein synthesis and amino acid transport pathways in endosperm and suggest targets for the enhancement of specialty pearled wheat with higher quality.

Floral resources provided by the new energy crop, Silphium perfoliatum L. (Asteraceae)

Flower-visiting insects are important crop pollinators, but their populations in agricultural landscapes are declining. One reason is the decreasing quantity and quality of floral resources. Deterioration of the situation caused by an increasing production of energy crops like maize, a pollen-only resource, may be mitigated by alternative crops such as Silphium perfoliatum L., which produces pollen and nectar over a long-lasting flowering period (July–September). The aim of this study was to assess the floral resources of S. perfoliatum. We calculated pollen grains and nectar sugar mass per inflorescence as well as inflorescences per plant using experimental plots and commercial fields in Germany. In addition, we analyzed free and protein-bound amino acids in pollen as well as sugars and free amino acids in nectar. The amount of pollen and nectar sugar per inflorescence decreased with increasing stem branching. The production of pollen and nectar sugar, however, was greatest in the second half of August due to the high number of inflorescences per plant at that stage. About half of the farmers harvested S. perfoliatum before the end of flowering to gain higher methane yields. With an early harvest around the beginning of September about one sixth of total pollen and one fifth of total nectar sugar production were lost to pollinators. Pollen and nectar showed high amounts of certain essential amino acids, particularly histidine, but total amino acids were low in concentration. Hence, S. perfoliatum should be complemented with different bee fodder plants to ensure a well-balanced diet.

Extending daily feed access intervals does not influence lysine HCl utilization but enhances amino acid digestibilities in broiler chickens

Off-sex, male Ross 308 chickens were offered maize-soy diets without and with 3.5g/kg lysine monohydrochloride (HCl), which contained 10.0 or 12.8g/kg digestible lysine, from 7 to 28 D post-hatch. Birds were permitted access to diets at intervals of 12, 16, and 20h/day. Lysine HCl improved weight gain (1,465 vs. 1,417g/bird; P < 0.025) and feed conversion ratios (1.351 vs. 1.382; P < 0.005). Extending feed access intervals increased weight gain (1,542 vs. 1,303g/bird; P < 0.001) and feed intake (2,142 vs. 1,748g/bird; P < 0.001) but compromised feed conversion ratios (1.390 vs. 1.342; P < 0.001). Extending feed access intervals increased (P < 0.001) both relative crop and gizzard weights and amounts of digesta retained in these organs. Effective lysine HCl utilization in poultry irrespective of feeding frequency, as opposed to pigs, may stem from anticipatory feeding behavior, crop and gizzard functionality, and increased episodes of reverse peristalsis. Collectively, these properties appear to modulate the relative intestinal uptakes of unbound lysine and protein-bound amino acids including lysine. Instructively, extending daily feed access intervals from 12 to 20h increased average ileal digestibility coefficients of 16 amino acids by 12.8% (0.830 vs. 0.736; P < 0.001), which was linearly related (r = -0.834; P < 0.001) to hourly feed intake rates. Birds given 12h feed access consumed relatively more feed on an hourly basis and this may have contributed to lesser amino acid digestibilities. As treatment interactions (P > 0.35) between lysine HCl and feed access intervals for parameters of growth performance were not observed, it was concluded that feed access intervals do not influence lysine utilization. The implications of these findings are discussed.

The Relevance of Starch and Protein Digestive Dynamics in Poultry

SUMMARY The fundamental premise of starch and protein digestive dynamics is that an ideal balance of glucose and amino acids is made available at sites of skeletal muscle protein synthesis to promote efficient growth. Digestive dynamics involve the digestion of protein and starch in the gut lumen, absorption of glucose and amino acids along the small intestine and their transition across the gut mucosa into the portal circulation. However, the post-enteral, bilateral bioavailability of glucose and amino acids is ultimately dependent on their metabolic fates in enterocytes as both may be catabolized in avian enterocytes for energy to drive digestive processes. Importantly, digestive dynamics consider rates and sites of glucose and amino acid absorption along the small intestine in addition to their extents of digestion as determined by static digestibility coefficients. There is considerable interest in the development of low-protein/high-supplemental amino acid diets but the digestive dynamics of supplemental and protein-bound amino acids are inherently different. Therefore, the relevance of starch and protein digestive dynamics in poultry will become increasingly evident if low-protein diets are to be developed successfully in the future.

High-resolution 13C metabolic flux analysis.

Precise quantification of metabolic pathway fluxes in biological systems is of major importance in guiding efforts in metabolic engineering, biotechnology, microbiology, human health, and cell culture. 13C metabolic flux analysis (13C-MFA) is the predominant technique used for determining intracellular fluxes. Here, we present a protocol for 13C-MFA that incorporates recent advances in parallel labeling experiments, isotopic labeling measurements, and statistical analysis, as well as best practices developed through decades of experience. Experimental design to ensure that fluxes are estimated with the highest precision is an integral part of the protocol. The protocol is based on growing microbes in two (or more) parallel cultures with 13C-labeled glucose tracers, followed by gas chromatography-mass spectrometry (GC-MS) measurements of isotopic labeling of protein-bound amino acids, glycogen-bound glucose, and RNA-bound ribose. Fluxes are then estimated using software for 13C-MFA, such as Metran, followed by comprehensive statistical analysis to determine the goodness of fit and calculate confidence intervals of fluxes. The presented protocol can be completed in 4 d and quantifies metabolic fluxes with a standard deviation of ≤2%, a substantial improvement over previous implementations. The presented protocol is exemplified using an Escherichia coli ΔtpiA case study with full supporting data, providing a hands-on opportunity to step through a complex troubleshooting scenario. Although applications to prokaryotic microbial systems are emphasized, this protocol can be easily adjusted for application to eukaryotic organisms.

Determination of Amino Acid Composition in the Harpagophytum procumbens Root

The amino acid composition of the roots of Harpagophytum procumbens was investigated by the method of high performance liquid chromatography (HPLC) with preliminary derivatization. Sixteen free and thirteen bound amino acids were quantitatively determined. The content of protein-bound amino acids was calculated.&#x0D; Dhaka Univ. J. Pharm. Sci. 18(1): 85-91, 2019 (June)

Quantifying the contribution of dietary protein to whole body protein kinetics: examination of the intrinsically labeled proteins method.

Intrinsically labeled dietary proteins have been used to trace various aspects of digestion and absorption, including quantifying the contribution of dietary protein to observed postprandial amino acid and protein kinetics in human subjects. Quantification of the rate of appearance in peripheral blood of an unlabeled (tracee) amino acid originating from an intrinsically labeled protein (exogenous Ra) requires the assumption that there is no dilution of the isotope enrichment of the protein-bound amino acid in the gastrointestinal tract or across the splanchnic bed. It must also be assumed that the effective volume of distribution into which the tracer and tracee appear can be reasonably estimated by a single value and that any recycling of the tracer is minimal and thus does not affect calculated rates. We have assessed these assumptions quantitatively using values from published studies. We conclude that the use of intrinsically labeled proteins as currently described to quantify exogenous Ra systematically underestimates the true value. When used with the tracer-determined rates of amino acid kinetics, underestimation of exogenous Ra from the intrinsically labeled protein method likely translates to incorrect conclusions regarding protein breakdown, including the effect of a protein meal and the anabolic impact of the speed of digestion and absorption of amino acids. Estimation of exogenous Ra from the bioavailability of ingested protein has some advantages as compared with the intrinsically labeled protein method. We therefore conclude that the bioavailability method for estimating exogenous Ra is preferable to the intrinsically labeled protein method.

Adaptive responses of amino acid metabolism to the combination of desiccation and low nitrogen availability in Sporobolus stapfianus.

Depending on nitrogen availability, S. stapfianus uses different amino acid metabolism strategies to cope with desiccation stress. The different metabolic strategies support essential processes for the desiccation tolerance phenotype. To provide a comprehensive assessment of the role played by amino acids in the adaptation of Sporobolus stapfianus to a combination of desiccation and nitrogen limitation, we used an absolute quantification of free and protein-bound amino acids (FAAs and PBAAs) as well as their gamma-glutamyl (gg-AA) derivatives in four different tissues grown under high- and low-nitrogen regimes. We demonstrate that although specific FAAs and gg-AAs increased in desiccating immature leaves under both nitrogen regimes, the absolute change in the total amount of either is small or negligible, negating their proposed role in nitrogen storage. FAAs and PBAAs decrease in underground tissues during desiccation, when nitrogen is abundant. In contrast, PBAAs are drastically reduced from the mature leaves, when nitrogen is limiting. Nevertheless, the substantial reduction in PBAA and FAA fractions in both treatments is not manifested in the immature leaves, which strongly suggests that these amino acids are further metabolized to fuel central metabolism or other metabolic adjustments that are essential for the acquisition of desiccation tolerance (DT).

Drosophila melanogaster nonribosomal peptide synthetase Ebony encodes an atypical condensation domain

The protein Ebony from Drosophila melanogaster plays a central role in the regulation of histamine and dopamine in various tissues through condensation of these amines with β-alanine. Ebony is a rare example of a nonribosomal peptide synthetase (NRPS) from a higher eukaryote and contains a C-terminal sequence that does not correspond to any previously characterized NRPS domain. We have structurally characterized this C-terminal domain and have discovered that it adopts the aryl-alkylamine-N-acetyl transferase (AANAT) fold, which is unprecedented in NRPS biology. Through analysis of ligand-bound structures, activity assays, and binding measurements, we have determined how this atypical condensation domain is able to provide selectivity for both the carrier protein-bound amino acid and the amine substrates, a situation that remains unclear for standard condensation domains identified to date from NRPS assembly lines. These results demonstrate that the C terminus of Ebony encodes a eukaryotic example of an alternative type of NRPS condensation domain; they also illustrate how the catalytic components of such assembly lines are significantly more diverse than a minimal set of conserved functional domains.

Studying trans-acting enzymes that target carrier protein-bound amino acids during nonribosomal peptide synthesis.

Nonribosomal peptide biosynthesis is a complex enzymatic assembly responsible for producing a great diversity of bioactive peptide natural products. Due to the recurring arrangement of catalytic domains within these machineries, great interest has been shown in reengineering these pathways to produce novel, designer peptide products. However, in order to realize such ambitions, it is first necessary to develop a comprehensive understanding of the selectivity, mechanisms, and structure of these complex enzymes, which in turn requires significant in vitro experiments. Within nonribosomal biosynthesis, some modifications are performed by enzymatic domains that are not linked to the main nonribosomal peptide synthetase but rather act in trans: these systems offer great potential for redesign, but in turn require detailed study. In this chapter, we present an overview of in vitro experiments that can be used to characterize examples of such trans-interacting enzymes from nonribosomal peptide biosynthesis: Cytochrome P450 monooxygenases and flavin-dependent halogenases.