Protein Digestion Products Research Articles

Influence of bile salts on the gastrointestinal digestion of agar-casein hybrid systems and the nanoassembly of their digestion products

This manuscript reports on the effect of different bile salts concentration on the gastrointestinal digestion of casein and casein-agar hybrid systems and evaluates the effect on the nanostructural assembly of the digestion products through the application of advanced small angle X-ray scattering analysis (SAXS).The results showed that bile salts promote the hydrolysis of micellar casein upon in vitro gastrointestinal digestions. It is noteworthy that in the presence of agar, bile salts interact with the polysaccharide, leading to a reduction in their proteolytic activity. While structured agar-casein hydrogels were shown to be able to limit the proteolysis during the gastric phase, increasing concentrations of bile salts promoted the diffusion of casein from the gel network during the intestinal phase, hence leading to higher degree of hydrolysis. The released digestion products were seen to interact with bile salts, forming ordered lamellar/micellar nanostructures. In particular, the presence of solubilized agar in the digesta was seen to promote the formation of these nanostructures. These nanostructures were formed at 10 mM bile salts for the hydrogels, and across all concentrations for agar-casein blends. Thus, this work demonstrates the great relevance of bile salts in the nanostructural assembly of protein digestion products, which is expected to have a great relevance in bioavailability and metabolic responses induced by protein-rich foods.

Effects of air frying on protein digestive properties in scallop (Patinopecten yessoensis) adductor muscles and the mechanisms involved

The effects of air frying at different temperatures (140 °C, 160 °C, 180 °C, and 200 °C) on the simulated digestive properties of proteins in scallop adductor muscle (SAM) and the mechanisms involved were investigated. The results of the simulated gastrointestinal digestion showed that the 160 °C-treated SAM group had significantly higher protein digestibility than the fresh SAM group, which was reflected in higher degree of hydrolysis, more low molecular weight (<0.5 kDa) protein digestive products, and less insoluble residues, while the 140 °C-, 180 °C- and 200 °C-treated SAM groups all had opposite results. Moreover, air frying altered the solubility, secondary structure percentage and surface hydrophobicity of SAM proteins, as well as the microstructure of insoluble residues in simulated gastric juice. Correlation analysis showed that the protein digestive level of the air fried SAMs was positively correlated with the ratio of soluble proteins before digestion and the surface hydrophobicity of insoluble residues, but negatively correlated with the content of α-helix of soluble proteins dissolved in simulated gastric juice. Therefore, the highest digestibility of the 160 °C-treated SAM group can be attributed to the lowest α-helix content of soluble proteins, as well as the highest surface hydrophobicity and the largest pores size of insoluble residues. The present study provides a good basis for understanding the effects of air frying temperature on the digestive properties of aquatic muscle foods and the mechanisms involved, which contributes to establish precise air frying method for aquatic muscle foods.

Insights into sensitizing and eliciting capacity of gastric and gastrointestinal digestion products of shrimp (Penaeus vannamei) proteins in BALB/c mice

Shrimp (Penaeus vannamei) proteins have been shown an allergenic potential; however, little information is available on the sensitizing and eliciting capacity of shrimp protein digestion products. In this study, a BALB/c mice model was used to explore the allergenicity of shrimp protein sample (SPS) and their gastric and gastrointestinal digestion products (GDS/GIDS). As compared with the SPS groups, the GDS/GIDS groups caused lower specific immunoglobulins (IgE/IgG1) levels (P < 0.05), but higher than the control groups, indicating that the digestion products sensitized the mice. Meanwhile, spleen index, mouse mast cell protease-1 (mMCP-1) concentration and proportion of degranulated mast cells were significantly reduced in the GDS/GIDS groups (P < 0.05); simultaneously, allergic symptoms, vascular permeability and histopathological changes of tissues were alleviated. Nevertheless, the allergenicity of digestion products cannot be eliminated and still cause systemic allergic reactions in mice. The study showed that the digestion products of shrimp still had high sensitizing and eliciting capacity.

Effect of conventional and microwave heating treatment on antioxidant activity of quinoa protein after simulated gastrointestinal digestion.

Effects of microwave and traditional water bath treatment at different temperatures (70, 80, 90 ℃) on in vitro digestion rate and antioxidant activity of digestion products of quinoa protein were investigated. The results indicated microwave treatment at 70 ℃ produced the highest quinoa protein digestion rate and the strongest antioxidant activities of its digestion products (P<0.05), which was further verified by the results of free amino, sulfhydryl group, gel electrophoresis, amino acid profiles and the molecular weight distribution of the digestion products. However, limited exposure of active groups induced by water bath treatment might decrease the susceptibility of digestive enzymes and subsequently lower the digestibility and antioxidant activities of quinoa protein. The results suggested that a moderate microwave treatment could be used as a potential way to enhance the in vitro digestion rate of quinoa protein, as well as increase the antioxidant activities of its digestion products.

Characterization of neonatal and infant enterostomy fluids

The composition of gastrointestinal (GI) fluids is crucial for the dissolution, solubilization, and absorption of orally administered drugs. Disease- or age-related changes in GI fluid composition could significantly affect the pharmacokinetics of oral drugs. However, limited studies have been conducted on the characteristics of GI fluids in neonates and infants due to practical and ethical challenges. The current study collected enterostomy fluids from 21 neonate and infant patients over an extended period of time and from different regions of the small intestine and colon. The fluids were characterized for pH, buffer capacity, osmolality, total protein, bile salts, phospholipids, cholesterol, and lipid digestion products. The study found a large variability in the fluid characteristics among the different patients, in line with the highly heterogeneous study population. Compared to adult intestinal fluids, the enterostomy fluids from neonates and infants had low bile salt concentrations, with an increasing trend as a function of age; no secondary bile salts were detected. In contrast, total protein and lipid concentrations were relatively high, even in the distal small intestine. These findings suggest marked differences in intestinal fluid composition between neonates and infants versus adults, which may affect the absorption of certain drugs.

How dietary amino acids and high protein diets influence insulin secretion.

Glucose homeostasis is the maintenance and regulation of blood glucose concentration within a tight physiological range, essential for the functioning of most tissues and organs. This is primarily achieved by pancreatic secretion of insulin and glucagon. Deficient pancreatic endocrine function, coupled with or without peripheral insulin resistance leads to prolonged hyperglycemia with chronic impairment of glucose homeostasis, most commonly seen in diabetes mellitus. High protein diets (HPDs) are thought to modulate glucose homeostasis through various metabolic pathways. Insulin secretion can be directly modulated by the amino acid products of protein digestion, which activate nutrient receptors and nutrient transporters expressed by the endocrine pancreas. Insulin secretion can also be modulated indirectly, through incretin release from enteroendocrine cells, and via vagal neuronal pathways. Additionally, glucose homeostasis can be promoted by the satiating effects of anorectic hormones released following HPD consumption. This review summarizes the insulinotropic mechanisms by which amino acids and HPDs may influence glucose homeostasis, with a particular focus on their applicability in the management of Type 2 diabetes mellitus.

Food peptides as inducers of CCK and GLP-1 secretion and GPCRs involved in enteroendocrine cell signalling

The strong effect of protein digestion products on gastrointestinal-released hormones is recognised. However, little is known about the specific peptide sequences able to induce gastrointestinal hormone secretion and the receptors involved. Our objective was to identify peptides able to induce the secretion of cholecystokinin (CCK) and glucagon like peptide-1 (GLP-1) in the enteroendocrine cell line STC-1, and to evaluate the involvement of the calcium-sensing receptor and G-protein coupled receptor-93 in this cell signalling. The key role of the amino acidic sequence on CCK and GLP-1 secretion is demonstrated. Removing Ser from the N-terminus of κ-casein 33SRYPS37, or the N-terminal Trp-Ile in lysozyme 123WIRGCRL129 decreased the secretion of both hormones. However, substituting Tyr by Ala in peptide αs1-CN 90RYLG93 enhanced the CCK secretion levels but not the GLP-1 ones. In addition, the involvement of CaSR and GPR93 was evidenced, but our results pointed to the contribution of additional receptors or transporters.

Gastrointestinal digestion products of shrimp (Penaeus vannamei) proteins retain an allergenic potential

The allergenic potential of shrimp (Penaeus vannamei) proteins during gastrointestinal (GI) digestion is currently unclear. In this study, the role of gastric, intestinal, and GI digestion on the sensitizing and eliciting capacity of shrimp proteins was investigated using a BALB/c mice model. Gastric digestion products of shrimp proteins produced lower specific IgE/IgG1, histamine and mMCP-1 levels and significantly reduced the proportion of degranulated mast cells as compared to the shrimp protein extract; furthermore, no histopathological changes were observed in the lung, spleen and jejunum. Conversely, their GI digestion products caused high specific IgE/IgG1 levels, mast cells degranulation and severe allergic symptoms in the mice, similar to those in the shrimp protein extract group. These results may be due to the masking or exposure of shrimp epitopes during digestion. Consequently, the GI digestion products of shrimp proteins retained an allergenic potential, although gastric digestion products showed a lowering effect.

The Fate of IgE Epitopes and Coeliac Toxic Motifs during Simulated Gastrointestinal Digestion of Pizza Base.

Understanding how food processing may modify allergen bioaccessibility and the evolution of immunologically active peptides in the gastrointestinal tract is essential if knowledge-based approaches to reducing the allergenicity of food are to be realised. A soy-enriched wheat-based pizza base was subjected to in vitro oral–gastro–duodenal digestion and resulting digests analysed using a combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS). The digestion profile of pizza base resembled that of bread crust where higher temperatures during baking reduced protein solubility but still resulted in the generation of a complex mixture of peptides. MS profiling showed numerous peptides carrying IgE epitopes, and coeliac toxic motifs were in excess of 20–30 residues long and were only released after either 120 min of gastric digestion or a combination of gastric and duodenal digestion. In silico prediction tools showed an overestimated number of cleavage sites identified experimentally, with low levels of atypical peptic and chymotryptic cleavage sites identified particularly at glutamine residues. These data suggest that such alternative pepsin cleavage sites may play a role in digestion of glutamine-rich cereal foods. They also contribute to efforts to provide benchmarks for mapping in vitro digestion products of novel proteins which form part of the allergenicity risk assessment.

Basement membrane proteins as a substrate for efficient Trypanosoma brucei differentiation in vitro.

The ability to reproduce the developmental events of trypanosomes that occur in their mammalian host in vitro offers significant potential to assist in understanding of the underlying biology of the process. For example, the transition from bloodstream slender to bloodstream stumpy forms is a quorum-sensing response to the parasite-derived peptidase digestion products of environmental proteins. As an abundant physiological substrate in vivo, we studied the ability of a basement membrane matrix enriched gel (BME) in the culture medium to support differentiation of pleomorphic Trypanosoma brucei to stumpy forms. BME comprises extracellular matrix proteins, which are among the most abundant proteins found in connective tissues in mammals and known substrates of parasite-released peptidases. We previously showed that two of these released peptidases are involved in generating a signal that promotes slender-to-stumpy differentiation. Here, we tested the ability of basement membrane extract to enhance parasite differentiation through its provision of suitable substrates to generate the quorum sensing signal, namely oligopeptides. Our results show that when grown in the presence of BME, T. brucei pleomorphic cells arrest at the G0/1 phase of the cell cycle and express the differentiation marker PAD1, the response being restricted to differentiation-competent parasites. Further, the stumpy forms generated in BME medium are able to efficiently proceed onto the next life cycle stage in vitro, procyclic forms, when incubated with cis-aconitate, further validating the in vitro BME differentiation system. Hence, BME provides a suitable in vitro substrate able to accurately recapitulate physiological parasite differentiation without the use of experimental animals.

Induction of CCK and GLP-1 release in enteroendocrine cells by egg white peptides generated during gastrointestinal digestion.

The effect of dietary protein on the induction of intestinal hormones is recognised. However, little is known about the nature of the digestion products involved in this intestinal signalling. Our aim was to characterise egg white protein digestion products and study their ability to induce CCK and GLP-1 release in enteroendocrine STC-1 cells. Intestinal digests triggered GLP-1 release at a higher rate than gastric digests. Peptides, but not free amino acids, showed a potent GLP-1 secretagogue effect, while proteins only had a modest effect. CCK was released in response to peptides and free amino acids but not proteins. Two hydrophobic negatively charged peptides triggered CCK release, while the highest GLP-1 response was found with a hydrophobic positively charged peptide, pointing to the involvement of different receptors or active sites. Identifying peptide sequences and receptors involved in hormonal secretion could open up new ways to control food intake and glucose metabolism.

Biochemical Analysis of Coronavirus Spike Glycoprotein Conformational Intermediates during Membrane Fusion.

A fusion protein expressed on the surface of enveloped viruses mediates fusion of the viral and cellular membranes to facilitate virus infection. Pre- and postfusion structures of viral fusion proteins have been characterized, but conformational changes between them remain poorly understood. Here, we examined the intermediate conformation of the murine coronavirus fusion protein, called the spike protein, which must be cleaved by a cellular protease following receptor binding. Western blot analysis of protease digestion products revealed that two subunits (67 and 69 kDa) are produced from a single spike protein (180 kDa). These two subunits were considered to be by-products derived from conformational changes and were useful for probing the intermediate conformation of the spike protein. Interaction with a heptad repeat (HR) peptide revealed that these subunits adopt packed and unpacked conformations, respectively, and two-dimensional electrophoresis revealed a trimeric assembly. Based on biochemical observations, we propose an asymmetric trimer model for the intermediate structure of the spike protein. Receptor binding induces the membrane-binding potential of the trimer, in which at least one HR motif forms a packed-hairpin structure, while membrane fusion subunits are covered by the receptor-binding subunit, thereby preventing the spike protein from forming the typical homotrimeric prehairpin structure predicted by the current model of class I viral fusion protein. Subsequent proteolysis induces simultaneous packing of the remaining unpacked HRs upon assembly of three HRs at the central axis to generate a six-helix bundle. Our model proposes a key mechanism for membrane fusion of enveloped viruses.IMPORTANCE Recent studies using single-particle cryo-electron microscopy (cryoEM) revealed the mechanism underlying activation of viral fusion protein at the priming stage. However, characterizing the subsequent triggering stage underpinning transition from pre- to postfusion structures is difficult because single-particle cryoEM excludes unstable structures that appear as heterogeneous shapes. Therefore, population-based biochemical analysis is needed to capture features of unstable proteins. Here, we analyzed protease digestion products of a coronavirus fusion protein during activation; their sizes appear to be affected directly by the conformational state. We propose a model for the viral fusion protein in the intermediate state, which involves a compact structure and conformational changes that overcome steric hindrance within the three fusion protein subunits.

Dezhou donkey (Equus asinus) milk a potential treatment strategy for type 2 diabetes.

Donkey (Equus asinus) milk has become a medical and nutrient product since ancient times. In addition, donkey milk was regarded as a medicinal food and substitute product for infant formula in some ancient western countries. Chinese ancient medical books documented the medicinal value of donkey milk, using donkey milk to treat diabetes, cough and jaundice. To investigate the donkey milk's components and anti-diabetic effect of donkey milk in vitro and in vivo and to study the molecular mechanism of donkey milk was an anti-diabetic medication. In this study, the gastrointestinal digested donkey milk was simulated in vitro and its products of protein digestion were analyzed by SDS-PAGE. We then performed cell viability assay, insulin secretion assay, animal experiments and ELISA assays to study the anti-diabetic effect of donkey milk in vitro and in vivo. Donkey milk's anti-diabetic molecular mechanism and specific targets were detected by using quantitative real time PCR. Lysozyme (LZ) and α-lactalbumin (α-La) exhibited significantly lower digestibility and higher retention than the other components of donkey milk. In vitro, 500 μg/mL of donkey milk could improve damaged β-cells viability significantly (P < 0.0001). In vivo, the blood glucose and HOMA-IR of diabetic rats treated with donkey milk were 14.23 ± 5.18 mM and 74.94 ± 23.62, respectively, whereas the diabetic group were 22.18 ± 2.23 mM and 112.16 ± 18.44, respectively (P < 0.01). The SOD value of donkey milk group was 265.87 ± 21.29 U/L, while the SOD value of diabetic group was 193.20 ± 52.07 U/L (P < 0.05). These results indicated that the blood glucose was reduced, the ability of the body to eliminate free radicals was enhanced, antioxidant levels in the body was increased, insulin resistance was improved in type 2 diabetic rats after donkey milk powder fed for 4 weeks. Furthermore, donkey milk could treat diabetes through down-regulating phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose-6-phosphatase (G6PC). Donkey milk has played an important role in the treatment of type 2 diabetes, and contributed to the development of the donkey milk products.

Protein‐oligosaccharide conjugates as novel prebiotics

Maintaining a good proportion of gut probiotic bacteria is imperative to health. This may be achieved by consuming “prebiotics,” e.g., galacto‐oligosaccharides (GOS) that selectively promote probiotic bacteria, as they often uniquely express transporters for such oligosaccharides. Proteins are an important source for amino acids essential to probiotic bacteria. As most protein digestion products are absorbed in the small intestine, and there is great competition on the residuals by colonic bacteria, amino acids are scarce (&lt;0.01 mM) in the colonic intercellular fluid, thus limiting probiotics' proliferation. However, no existing prebiotic product contains protein. Herein, we propose a new type of prebiotics: protein‐oligosaccharide conjugates. These conjugates were designed to be selectively targeted to probiotic bacteria in the colon, for enhancing their competitive advantage over undesired microorganisms. The approach was inspired by active targeting of chemotherapy, achieved by conjugating drugs to ligands, which selectively bind to proteins uniquely expressed on cancer cells; except here, we aimed to promote, not eliminate, the targeted cells. We formed these conjugates by mild Maillard‐reaction‐based covalent conjugation of GOS to lactoferrin hydrolysate (LFH), formed by peptic digestion, hence it resists gastric digestion. LFH‐GOS conjugates comprised 76% ± 1% LFH and 25% ± 4% GOS, and self‐assembled into 0.2 to 1.5‐μm microparticles. Most of the conjugates' protein content endured simulated gastrointestinal digestion, hence is expected to reach the colon. Remarkably, we found that the growth rate of a model probiotic bacterium (Lactobacillus casei) on the conjugates was double that on the unconjugated components (0.082 and 0.041 h−1, respectively). This study proposes the next generation of prebiotics.

Intestinal Signaling of Proteins and Digestion-Derived Products Relevant to Satiety.

Luminal nutrients stimulate enteroendocrine cells through the activation of specific receptors to release hormones that inhibit appetite and promote glucose homeostasis. While food protein is the macronutrient with the highest effect on satiety, the signaling on the protein digestion products at the gut is poorly understood. This perspective aims to highlight the existing gaps in the study of protein digestion products as signaling molecules in gastrointestinal enteroendocrine cells. Because dietary protein digestion can be modulated by the technological processes applied to food, it is possible to target gut receptors to control food intake by formulating specific food ingredients or protein preloads.

The effect of meat processing methods on changes in disulfide bonding and alteration of protein structures: impact on protein digestion products.

We investigated the effects of different pork preparation methods (cooked pork, emulsion-type sausage, dry cured pork, and stewed pork) on protein structures and in vitro digestion. Compared with raw meat, processed meats contained lower levels of free sulfhydryl groups (P < 0.05). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed different protein profiles for pork products treated without or with 5% βME, which indicated different extents of disulfide bond formation. Emulsion-type sausage showed significantly higher α-helix content and lower β-sheet, β-turn, and random coil contents than cooked pork (P < 0.05). Correspondingly, emulsion-type sausage and dry-cured pork had the highest values of surface hydrophobicity (P < 0.05). Proteome data showed that the long salting and drying times used for dry-cured pork as well as long-term high-temperature cooking of stewed pork might alter the accessibility of digestive proteolytic enzymes to the protein cleavage sites.

Dynamic gastric digestion of a commercial whey protein concentrate†

A dynamic gastrointestinal simulator, simgi® , has been applied to assess the gastric digestion of a whey protein concentrate. Samples collected from the outlet of the stomach have been compared to those resulting from the static digestion protocol INFOGEST developed on the basis of physiologically inferred conditions. Progress of digestion was followed by SDS-PAGE and LC-MS/MS. By SDS-PAGE, serum albumin and α-lactalbumin were no longer detectable at 30 and 60 min, respectively. On the contrary, β-lactoglobulin was visible up to 120 min, although in decreasing concentrations in the dynamic model due to the gastric emptying and the addition of gastric fluids. Moreover, β-lactoglobulin was partly hydrolysed by pepsin probably due to the presence of heat-denatured forms and the peptides released using both digestion models were similar. Under dynamic conditions, a stepwise increase in number of peptides over time was observed, while the static protocol generated a high number of peptides from the beginning of digestion. Whey protein digestion products using a dynamic stomach are consistent with those generated with the static protocol but the kinetic behaviour of the peptide profile emphasises the effect of the sequential pepsin addition, peristaltic shaking, and gastric emptying on protein digestibility. © 2017 Society of Chemical Industry.

Expression of Putative Umami/L‐Amino Acid Receptors in Mouse Intestine and Colon, and a Murine Enteroendocrine Cell Line

The ability of the gastrointestinal tract to adequately regulate motility and secretion is dependent on the interplay of both sensory and motor system. Chemosensation of nutrients in luminal contents is critical to this process and is largely dependent on activation of receptors present on enteroendocrine cells. G‐protein coupled nutrient receptors for free fatty acids, carbohydrates, protein and proteolytic digestion products as well as receptors for other components of luminal contents such as bile acids have been identified in the gut and postulated to play a role in chemosensation. Several receptors have been postulated to play a role in the sensing of L‐amino acids (L‐AA) in the gut as well as the initiation of umami taste sensation in taste buds. These include the metabotropic glutamate receptors 1 and 4 (mGluR1 and mGluR4), the calcium‐sensing receptor (CaR), the GPRC family C group 6 subtype A receptor (GPRC6A), and the taste receptor heterodimer T1R1/T1R3.AimTo test the hypothesis that the putative L‐AA receptors CaR, mGluR1, mGluR4, and T1R1 are differentially expressed in mouse intestine, colon, and a murine enteroendocrine cell line, the STC‐1 cells.MethodsIntestine and colon were removed from mice, and separated into the mucosal layer containing enteroendocrine cells (EEC) and the smooth muscle layer. Expression of mRNA for each receptor was measured in mRNA isolated from each layer by RT‐PCR using specific primers. STC‐1 cells were grown in DMEM‐10; mRNA for each receptor type was measured by RT‐PCR and protein expression was measured by immunohistochemistry. Co‐localization of receptors with each other and with endocrine peptides Peptide YY (PYY) and Glucagon‐Like Peptide 1 (GLP‐1) was also examined.ResultsAll receptors were expressed in intestine and colon. The pattern of mRNA expression was similar for CaR, mGluR1, and mGluR4 such that (1) there was greater expression in colon than in intestine and (2) there was greater expression in mucosa than in muscle. In contrast, T1R1 mRNA expression was greater in intestine than colon although, similar to other L‐AA receptors, T1R1 expression in mucosa was greater than in muscle. In the EEC cell line (STC‐1), mRNA for all receptors was expressed; however, the rank order of expression was CaR&gt;mGluR1=mGlu4&gt;T1R1. Immunohistochemical studies showed that protein for all receptors was expressed on EEC membranes and demonstrated a high degree of co‐expression of CaR with mGluR1 and mGluR4. In contrast, T1R1 receptors were rarely co‐expressed with CaR or mGluR1 and mGluR4. STC‐1 cells expressing each receptor type also expressed GLP‐1 and PYY confirming the EEC phenotype of these STC‐1 cells.ConclusionMultiple L‐amino acid sensing receptors are expressed in murine intestine and colon, and a murine enteroendocrine cell line. CaR and mGluRs are more likely to be co‐localized in enteroendocrine cells and mediate L‐AA sensing in colon whereas T1R1 may be more important in intestine. The co‐localization of these receptors with important gut hormones in EEC suggests a role of nutrient L‐AA in regulating the release of GLP‐1 and PYY.Support or Funding InformationSupported by DK15564, DK28300, and DK34153

Structural Characteristics of in Vitro Digestion Products of Heat-Treated Soybean Protein

Structural Characteristics of in Vitro Digestion Products of Heat-Treated Soybean Protein

Molecular identification and functional characteristics of peptide transporters in the bonnethead shark (Sphyrna tiburo).

Elasmobranchs are considered to be top marine predators, and in general play important roles in the transfer of energy within marine ecosystems. Despite this, little is known regarding the physiological processes of digestion and nutrient absorption in these fishes. One topic that is particularly understudied is the process of nutrient uptake across the elasmobranch gastrointestinal tract. Given their carnivorous diet, the present study sought to expand knowledge on dietary nutrient uptake in elasmobranchs by focusing on the uptake of products of protein digestion. To accomplish this, a full-length cDNA encoding peptide transporter 1 (PepT1), a protein previously identified within the brush border membrane of vertebrates that is responsible for the translocation of peptides released during digestion by luminal and membrane-bound proteases, was isolated from the bonnethead shark (Sphyrna tiburo). A cDNA encoding the related peptide transporter PepT2 was also isolated from S. tiburo using the same methodology. The presence of PepT1 was then localized in multiple components of the bonnethead digestive tract (esophagus, stomach, duodenum, intestine, rectum, and pancreas) using immunohistochemistry. Vesicle studies were used to identify the apparent affinity of PepT1 and to quantify the rate of dipeptide uptake by its H(+)-dependent cotransporter properties. The results of this study provide insight into the properties of peptide uptake within the bonnethead gut, and can facilitate future work on physiological regulation of protein metabolism and absorption including how these processes may vary in elasmobranchs that exhibit different feeding strategies.