Investigation of peptide stability upon hydrolysis by of fragments of the organs of the gastrointestinal tract of rats

The hydrolytic stability of a range of cyclic tripeptides, including the therapeutically important dalargin and stemokin, as well as peptides modified by ibuprofen and aspirin, has been studied. The first two experimental systems used utilized purified enzymes (pepsin, trypsin, and chymotrypsin), while the second one utilized fragments of the stomach and small intestine of rats. The linear peptides containing only L-amino acid residues were shown to be hydrolyzed by stomach and intestine fragments, although some of these peptides were resistant to hydrolysis by individual enzymes. The peptides containing D-amino acid residues and cyclic peptides were stable under all of the conditions used, but the peptides modified by aspirin lost the acetyl group of the aspirin moiety in acidic media, this process being accelerated in the presence of pepsin.     

Generation of xenopsin-related peptides during acid extraction of gastric tissues

Biologically active xenopsin-related peptide(s) were shown by radioimmunoassay to be liberated in nearly micromolar concentrations from substrate(s) with Mr greater than 70,000 during acid extraction of mammalian and avian stomach. The reaction displayed an enzyme-like temperature sensitivity, had a pH optimum of 2, and was fully inhibited in the presence of pepstatin A. Treatment of a stabilized gastric substrate with hog pepsin was found to mimic the reaction, producing immunoreactive xenopsin(s) which behaved similarly during high pressure liquid chromatography. Although some of the generated immunoreactivity co-chromatographed with xenopsin, it differed immunochemically when examined with a battery of region-specific antisera. Highly purified rat gastric immunoreactive xenopsin displayed xenopsin-like biologic effects, producing hypotension in the anesthetized rat and contracting the isolated guinea pig ileum. These findings suggest that there may be a renin/angiotensin-like system for the generation of xenopsin-related peptides in the stomach.     

Conformation of disulfide bridges in peptides with pepsin partial sequences.

On the basis of Raman spectra investigation of two model heterodetic cyclic peptides, containing partial sequences of pepsin fragments 45--50 and 206--210 of the chain, it was concluded that the disulfide bridge conformation in pepsin is determined not only by the size and conformation of the peptide loops created by disulfide bridges, but also by the peptide fragments located outside these loops.     

Amino acid sequence of the catalytic subunit of bovine type II adenosine cyclic 3',5'-phosphate dependent protein kinase

The 350-residue amino acid sequence of the catalytic subunit of bovine cardiac muscle adenosine cyclic 3',5'-phosphate dependent protein kinase is described. The protein has a molecular weight of 40 862, which includes an N-tetradecanoyl (myristyl) group blocking the NH2 terminus and phosphate groups at threonine-197 and serine-338. Seven methionyl bonds in the S-carboxymethylated protein were cleaved with cyanogen bromide to yield eight primary peptides. These fragments, and subpeptides generated by cleavage with trypsin, pepsin, chymotrypsin, thermolysin, and Myxobacter AL-1 protease II, were purified and analyzed to yield the majority of the sequence. The primary peptides were aligned by analyses of overlapping peptides, particularly of methione-containing tryptic peptides generated after in vitro [14C]methyl exchange labeling of methionyl residues in the intact protein.     

Accessing the reproducibility and specificity of pepsin and other aspartic proteases

The aspartic protease pepsin is less specific than other endoproteinases. Because aspartic proteases like pepsin are active at low pH, they are utilized in hydrogen deuterium exchange mass spectrometry (HDX MS) experiments for digestion under hydrogen exchange quench conditions. We investigated the reproducibility, both qualitatively and quantitatively, of online and offline pepsin digestion to understand the compliment of reproducible pepsin fragments that can be expected during a typical pepsin digestion. The collection of reproducible peptides was identified from > 30 replicate digestions of the same protein and it was found that the number of reproducible peptides produced during pepsin digestion becomes constant above 5–6 replicate digestions. We also investigated a new aspartic protease from the stomach of the rice field eel (Monopterus albus Zuiew) and compared digestion efficiency and specificity to porcine pepsin and aspergillopepsin. Unique cleavage specificity was found for rice field eel pepsin at arginine, asparagine, and glycine. Different peptides produced by the various proteases can enhance protein sequence coverage and improve the spatial resolution of HDX MS data. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

Does motilin control interdigestive pepsin secretion in the dog?

Pepsin output in the Heidenhain pouch, plasma motilin concentration, and contractile activity in the pouch and the main stomach were investigated in five dogs. During the interdigestive state, the pepsin output was significantly increased with a cyclic increase in contractile activity in both the pouch and main stomach at approximately 100-min intervals. The plasma immunoreactive motilin (IRM) concentration fluctuated during the interdigestive state, and, peaks of IRM concentration coincided with the maximum pepsin secretory activity. Exogenous administration of motilin (0.5 micrograms/kg-hr) increased contractile activity in the main stomach and pouch quite similar to the natural interdigestive migrating contractions (IMC), and increased pepsin output significantly. Atropine pre-treatment suppressed the naturally-occurring and motilin-induced pepsin output and contractions in the pouch. It is concluded that pepsin output and contractions in the Heidenhain pouch increase in close association with the IMC in the main stomach during the interdigestive state and these cyclic motor and secretory events in the vagally denervated fundic pouch are most likely regulated by motilin through the intramural cholinergic pathway.     

Affinity purification and characterization of an anti-PEG IgM

Anti-PEG IgM was purified by affinity chromatography using variable length PEG chains (5, 10, 20 and 30 kDa) as affinity ligands. Maximal binding of anti-PEG IgM was observed using the 30 kDa PEG-derivatized NuGel (single passage). Purified anti-PEG IgM was characterized for binding to PEG functionalized proteins/peptides by surface plasmon resonance, western blotting and ELISA. Anti-PEG IgM, in solution and adsorbed on 20 kDa PEG-derivatized NuGel, was subjected to pepsin digestion followed by affinity chromatography. SDS-PAGE analysis of eluates in both preparations yielded one fragment that was similar in size. However, an additional lower molecular weight band was observed in solution-digested affinity purified material that was not present in the eluate from the material subjected to pepsin digestion on the affinity matrix. The lower MW fragment could be eluted under milder conditions, suggesting loss of binding multiplicity. Analysis by mass spectrometry yielded molecular weights of 132 kDa (both) and 82 kDa (solution) for the respective fragments. N-terminal sequencing of both fragments resulted in primary sequences (heavy and light chains) that were not only identical to each other but also to those of native IgM. The anti-PEG IgM fragments were characterized for binding to pegylated interferon alfa-2a by ELISA. The results from these studies suggest that affinity purified anti-PEG IgM and fragments can be used as probes in detection assays for PEG functionalized biotherapeutics in pre-clinical and clinical studies.     

Iron absorption from concentrated hemoglobin hydrolysate by rat

Although heme iron is highly bioavailable, the low iron content of hemoglobin prevents its use for dietary fortification; on the other hand, purified heme has low solubility and absorption rate. The present study was designed to assess the interactions between concentrated heme iron and peptides released during globin hydrolysis and cysteine and their relation with iron absorption. Hemoglobin was hydrolyzed by pepsin or subtilisin, and then, heme iron was concentrated by ultrafiltration. Iron absorption was studied in a Ussing chamber; gluconate was used as control. Iron uptake from nonconcentrated pepsin hydrolysate and gluconate was lower than from other groups. Cysteine significantly enhanced iron uptake except from the concentrated subtilisin hydrolysate. There was no significant difference between cysteine-supplemented groups. According to the different hydrolysis pathways of enzymes, it is assumed that the presence of hydrophobic peptides and the strength of heme-peptide interactions are both determining factors of heme iron absorption. These interactions occur mainly before iron uptake, as emphasized by the effect of cysteine.     

A history of pepsin and related enzymes

Studies on gastric digestion during 1820-1840 led to the discovery of pepsin as the agent which, in the presence of stomach acid, causes the dissolution of nutrients such as meat or coagulated egg white. Soon afterward it was shown that these protein nutrients were cleaved by pepsin to diffusible products named peptones. Efforts to isolate and purify pepsin were spurred by its widespread adoption for the treatment of digestive disorders, and highly active preparations were available by the end of the nineteenth century. There was uncertainty, however, as to the chemical nature of pepsin, for some preparations exhibited the properties of proteins while other preparations failed to do so. The question was not settled until after 1930, when Northrop crystallized swine pepsin and provided convincing evidence for its identity as a protein. The availability of this purified pepsin during the 1930s also led to the discovery of the first synthetic peptide substrates for pepsin, thus providing needed evidence for the peptide structure of native proteins, a matter of debate at that time. After 1945, with the introduction of new separation methods, notably chromatography and electrophoresis, and the availability of specific proteinases, the amino acid sequences of many proteins, including pepsin and its precursor pepsinogen, were determined. Moreover, treatment of pepsin with chemical reagents indicated the participation in the catalytic mechanism of two aspartyl units widely separated in the linear sequence. Studies on the kinetics of pepsin action on long chain synthetic peptides suggested that the catalytic site was an extended structure. Similar properties were found for other "aspartyl proteinases," such as chymosin (used in cheese making), some intracellular proteinases (cathepsins), and plant proteinases. After 1975, the three-dimensional structures of pepsin and many of its relatives were determined by means of x-ray diffraction techniques, greatly extending our insight into the mechanism of the catalytic action of these enzymes. That knowledge has led to the design of new inhibitors of aspartyl proteinases, which are participants in the maturation of human immunodeficiency virus and in the generation of Alzheimer's disease.     

Gastric mucosal integrity: gastric mucosal blood flow and microcirculation. An overview

The stomach is in a state of continuous exposure to potentially hazardous agents. Hydrochloric acid together with pepsin constitutes a major and serious threat to the gastric mucosa. Reflux of alkaline duodenal contents containing bile and pancreatic enzymes are additional important injurious factors of endogenous origin. Alcohol, cigarette smoking, drugs and particularly aspirin and aspirin-like drugs, and steroids are among exogenous mucosal irritants that can inflict mucosal injury. The ability of the stomach to defend itself against these noxious agents has been ascribed to a number of factors constituting the gastric mucosal defense. These include mucus and bicarbonate secreted by surface epithelial cells, prostaglandins, sulfhydryl compounds and gastric mucosal blood flow. The latter is considered by several researchers to be of paramount importance in maintaining gastric mucosal integrity. The aim of this paper is to review the experimental and clinical data dealing with the role of mucosal blood flow and in particular the microcirculation in both damage and protection of the gastric mucosa.     

Fragments produced by digestion of human immunoglobulin G subclasses with pepsin in urea.

It was previously shown that digestion of human IgG1/kappa myeloma proteins with pepsin in the presence of 8 M-urea produces fragments which differ from other proteolytic fragments of IgG, including those produced by peptic digestion in aqueous buffers. The two large urea/pepsin fragments each consist of three peptides, and together account for all of the constant region of the light chains and most of the constant region of the heavy chains. Myeloma proteins of subclasses IgG2, IgG3 and IgG4 with kappa light chains were digested with pepsin in 8 M-urea, and the resulting fragments compared with those produced from IgG1/kappa proteins. Gel filtration, starch- and polyacrylamide-gel electrophoresis and sequence analysis have shown that the peptides from each subclass are analogous with those from IgG1. A brief investigation of the products of urea/pepsin digestion of myeloma proteins with lambda light chains has shown that in these proteins light-chain cleavage occurs at residue leucine-182, instead of or as well as at residue 117, where cleavage takes place in kappa chains. Comparison of sequences around sites of urea/pepsin cleavage has shown that pepsin has quite restricted specificity under these conditions.     

Purification and some properties of rabbit stomach myosin.

Myosin from rabbit stomach was highly purified by ammonium sulfate fractionation in the presence of ATP and MgCl2, ultracentrifugation and Sepharose 4B chromatography. The myosin composed of one heavy and two light chains as determined by SDS-gel electrophoresis. The molecular weights of the light chains were the same as those of gizzard myosin, about 20,000 and 17,000, respectively. The pH-activity curve and the KCl concentration dependency of Ca-ATPase of the stomach myosin were similar to those of other smooth muscle myosins. The stomach myosin was more resistant to pepsin digestion than skeletal myosin. Other proteolytic enzymes, trypsin, chymotrypsin, papain, and nagarse, digested the myosin in the same way as skeletal myosin.     

Determination of interchain crosslinkages in insulin B-chain dimers by fast atom bombardment mass spectrometry

New methodology for identifying and locating crosslinkages in peptides is described. Pepsin is used to cleave insulin and B-chain dimers of insulin into fragments under conditions which retain the original peptide crosslinkages. After partial separation by HPLC, the peptides are analyzed by fast atom bombardment mass spectrometry (FABMS) to determine their molecular weights. The molecular weights of peptide fragments expected from the pepsin digests of human insulin and related model compounds are calculated from the amino acid sequence of the intact peptide. Digestion products are identified by matching their molecular weights, as determined by FABMS, with calculated molecular weights. Locations of interchain crosslinkages are deduced after the peptide fragments have been assigned to specific segments of the parent peptide. The validity of the method has been established by correctly identifying structurally important products in the pepsin digests of model compounds such as human, bovine, and porcine insulins. Procedures developed with the model compounds were used to identify crosslinkages in peptides of unknown structure isolated from an insulin A-chain/B-chain combination reaction mixture. Evidence is presented for formation of three different types of crosslinkages, disulfide, lanthionine, and sulfoxide.     

The fragments of bovine high molecular weight kininogen promote osteoblast proliferation in vitro

High molecular weight (HMW) kininogen is known to be a large plasma protein and cleaved by plasma proteinase kallikrein, then it generates four fragments in the blood coagulation cascade: heavy chain, bradykinin, fragment 1.2, and light chain. The fragment 1.2 has also been found in the basic protein fraction of bovine milk as a bioactive protein which promotes osteoblast proliferation. The milk basic protein has been shown to be a multi functional edible protein which promotes bone formation and inhibits bone resorption. In the present study, we purified the fragment 1.2 from bovine plasma and assessed it could promote osteoblast proliferation and posses the activity after pepsin digestion. Purified plasma HMW kininogen did not promote the proliferation, however, the kallikrein-cleaved HMW kininogen promoted the proliferation. The fragment 1.2, purified from the proteolysate, also promoted the proliferation. The pepsin digestion was performed according to the method of the assessment of allergenesity of genetically modified crops. After pepsin digestion, the fragment 1.2 generated resistant fragments and showed the promoting activity of osteoblast proliferation. These results suggest that the enzymatically-digested fragments of bovine HMW kininogen are able to be a naturally occurred active protein that promotes the bone formation by oral administration.     

The specificity of some pig and human pepsins towards synthetic peptide substrates.

1. The peptidase activities of pig pepsins A and C and human pepsin and gastricsin were compared. 2. The peptides studied had the general formula A Leu Val-His-B. Hydrolysis at 37 degrees C and pH 2.07 occurred at the amino side of the leucine residue for all the enzymes and all the peptides. 3. When A was Ac-Ala the peptides were hydrolysed under these conditions slowly by pig pepsin C only. 4. Pig pepsin A and human pepsin were unable to hydrolyse the tyrosine-containing peptides under the conditions tested. Gastricsin (human pepsin C) had about one-third of the activity of pig pepsin C with these substrates. 5. The increase in the rate of hydrolysis caused by the extension of the chain by a single alanine residue was most marked for pig pepsin A and human pepsin.     

Isolation and covalent structure of the aspirin-modified, active-site region of prostaglandin synthetase

Aspirin (acetylsalicylic acid) inhibits prostaglandin synthesis by acetylating a single internal serine residue of the initial enzyme in the biosynthetic pathway, prostaglandin synthetase. In this study, the region of the enzyme that is modified by aspirin has been isolated, and its amino acid sequence has been determined. Sheep vesicular gland [acetyl-3H]prostaglandin synthetase was purified following treatment with [acetyl-3H]aspirin and digest with pepsin. An acetyl-3H-labeled peptic peptide of approximately 25 residues was isolated by high-pressure liquid chromatography, and its amino acid sequence was determined to be Ile-Glu-Met-Gly-Ala-Pro-Phe-Ser-Leu-Lys-Gly-Leu-Gly-Asn-Pro-Ile-Glu-Ser-Pro-Glu-Tyr. The acetylated serine residue was located at position 8 in this sequence. The current study marks this polypeptide sequence as a region related to an active site of the enzyme.     

pH值对中国龙虾消化酶活力的影响

采用酶学分析方法研究了pH对中国龙虾胃蛋白酶、类胰蛋白酶、淀粉酶、纤维素酶和脂肪酶活力的影响。结果表明,在设定的pH范围内,中国龙虾各消化酶的活力均随着pH的升高呈现先升后降的变化趋势。其中,胃、肠、肝胰腺内胃蛋白酶最适pH均为2.2,类胰蛋白酶最适pH分别为8.8-9.2、8.4、8.8,淀粉酶最适pH分别为7.0、7.0、7.4,纤维素酶最适pH分别为4.2、4.2-4.6、5.4,脂肪酶最适pH分别为7.2-7.6、7.2、6.8-7.2。同时测得中国龙虾胃、肠、肝胰腺内的生理pH分别为5.33、6.93、6.60。中国龙虾的消化酶活力存在器官特异性。在最适pH下,胃蛋白酶活力顺序为胃>肠>肝胰腺,类胰蛋白酶、纤维素酶、脂肪酶的活力顺序均为肝胰腺>肠>胃,淀粉酶的活力顺序为肠>肝胰腺>胃。     

Inhibition of aspartic proteinases by propart peptides of human procathepsin D and chicken pepsinogen

Two propart peptides of aspartic proteinases, the propart peptide of chicken pepsin and human cathepsin D, respectively, were investigated from the point of view of their inhibitory activity for a set of aspartic proteinases. These peptides display a very broad inhibitory spectrum. The strongest inhibition was observed for pepsin A-like proteinases where propart peptides can be used as titrants of active enzymes.     

Purification and identification of novel angiotensin-I converting enzyme (ACE) inhibitory peptides from cultured marine microalgae (Nannochloropsis oculata) protein hydrolysate

Isolation of bioactive compounds and commercialization of marine microalgae sources are interesting targets in future marine biotechnology. Cultured biomass of the marine microalga, Nannochloropsis oculata, was used to purify angiotensin-I converting enzyme (ACE) inhibitory peptides using proteases including pepsin, trypsin, α-chymotrypsin, papain, alcalase, and neutrase. The pepsin hydrolysate exhibited the highest ACE inhibitory activity, compared to the other hydrolysates and then was separated into three fractions (F1, F2, and F3) using Sephadex G-25 gel filtration column chromatography. First fraction (F1) showed the highest ACE inhibitory activity and it was further purified into two fractions (F1-1 and F1-2) using reverse-phase high-performance liquid chromatography. The IC₅₀ value of purified ACE inhibitory peptides were 123 and 173 μM and identified as novel peptides, Gly-Met-Asn-Asn-Leu-Thr-Pro (GMNNLTP; MW, 728 Da) and Leu-Glu-Gln (LEQ; MW, 369 Da), respectively. In addition, nitric oxide production level (%) was significantly increased by the purified peptide (Gly-Met-Asn-Asn-Leu-Thr-Pro) compared to the purified peptide (Leu-Glu-Gln) and other treated pepsin hydrolysate fractions on human umbilical vein endothelial cells (HUVECs). Cell viability assay showed no cytotoxicity on HUVECs with the treated purified peptides and fractions. These results suggest that the isolated peptides from cultured marine microalga, N. oculata protein sources may have potentiality to use commercially as ACE inhibitory agents in functional food industry.     

Partial purification of pepsins from adult and juvenile salmon fish Oncorhynchus keta. Effect of NaCl on proteolytic activities

1. Two pepsins, designated Pepsin I and Pepsin II, were isolated and partially characterized from the stomach of the adult stage salmon Oncorhynchus keta. This stage is developed in a marine environment. 2. One pepsin, designated Pepsin II, was isolated from the stomach of the juvenile stage salmon Oncorhynchus keta. This stage is developed in an estuarine environment. 3. The enzymes were partially purified by ammonium sulfate precipitation, ion exchange chromatography and gel filtration. 4. Pepsins I and II from adults and Pepsin II from juvenile showed proteolytic activity on acid-denatured hemoglobin with a pH optimum of 3. 5. The mol. wt determined by gel filtration on Sephadex G-100 of Pepsin I from juvenile species was found to be 32,000 whereas a value of 27,000 was determined for Pepsin II from juvenile and adult fish. 6. In contrast with Pepsin II, Pepsin I was activated by NaCl. It is suggested that the appearance of NaCl-activated pepsin would represent and adaptive response of the organism to the change from a low to a high salinity environment.