Protein Pepsin

Answer Pepsin proteins have a relatively low pi (near the pH of gastric juice) in order to remain soluble and thus functional in the stomach. (Pepsin—the mixture of enzymes—has a pi of 1.) As pH increases, pepsins acquire a net charge and undergo ionic interactions with oppositely charged molecules (such as dissolved salts), causing the pepsin proteins to precipitate. Pepsin is active only in the stomach. In the relatively high pH of the intestine, pepsin proteins precipitate and become inactive. 

Multiple sequence alignment of BACE family members and human pepsin (Protein Data Bank entry Ipso, used as the basis of homology modeling). Identical residues are shaded gray, highly conserved residues in yellow, and active-site residues in the model in contact with the peptide substrate are indicated by an asterisk ( ). BACE residue Arg 296, which is most often serine or threonine at this position in... 

A protocol to improve pepsin immobilization was proposed by Busby et al. [17], Rather than directly using pepsin, they immobilized pepsinogen to POROS AL-20 support under optimal coupling conditions of pH 6.7 where pepsinogen is stable (in contrast with pepsin). Subsequently, they converted the coupled pepsinogen into active pepsin. Protein assays demonstrated that more enzyme was bound to the POROS AL-20 resin coupled with pepsinogen at pH 6.7 than pepsin at pH 5. 

Absorption of iron is never very efficient and is influenced by many factors. Hydrochloric acid, pepsin, proteins and their digestion products, and ascorbic acid appear to be concerned in the reduction of iron to the ferrous state in which form it is absorbed. Absorption takes place largely in the duodenum (Chapter 20). Iron absorption may be increased by administration of ascorbic acid and decreased by phosphates and perhaps by phytates. 

Urease is one of the enzymes which have been obtained in the crystalline state. This has been done by stirring jack bean meal with 30°o aqueous acetone, filtering and allowing the filtrate to remain at o for several hours. The urease which crystallises out is separated by centrifuging and is then recrystallised. Like crystalline pepsin and trypsin, it is a protein. 

Pish protein concentrate and soy protein concentrate have been used to prepare a low phenylalanine, high tyrosine peptide for use with phenylketonuria patients (150). The process includes pepsin hydrolysis at pH 1.5 ptonase hydrolysis at pH 6.5 to Hberate aromatic amino acids gel filtration on Sephadex G-15 to remove aromatic amino acids incubation with papain and ethyl esters of L-tyrosine and L-tryptophan, ie, plastein synthesis and ultrafiltration (qv). The plastein has a bland taste and odor and does not contain free amino acids. Yields of 69.3 and 60.9% from PPG and soy protein concentrate, respectively, have been attained. 

A pepsin hydrolysate of flounder fish protein isolate has been used as the substrate (40% w/v) for plastein synthesis, using either pepsin at pH 5 or alpha chymotrypsin at pH 7, with an enzyme—substrate ratio of 1 100 w/v at 37°C for 24 h (151). The plastein yields for pepsin and alpha chymotrypsin after precipitation with ethanol were 46 and 40.5%, respectively. 

Pish silage prepared by autolysis of rainbow trout viscera waste was investigated as a substrate for the plastein reaction using pepsin (pH 5.0), papain (pH 6—7), and chymotrypsin (pH 8.0) at 37°C for 24 h (152). Precipitation with ethanol was the preferred recovery method. Concentration of the protein hydrolysate by open-pan evaporation at 60°C gave equivalent yields and color of the final plastein to those of the freeze-dried hydrolysate. 

The enzymatic hydrolysates of milk casein and soy protein sometimes have a strong bitter taste. The bitter taste is frequently developed by pepsin [9001 -75-6] chymotrypsin [9004-07-3] and some neutral proteases and accounted for by the existence of peptides that have a hydrophobic amino acid in the carboxyhc terminal (226). The relation between bitter taste and amino acid constitution has been discussed (227). 

Enzymes Degrading Macromolecules. Enzymes that degrade macromolecules such as membrane polysaccharides, stmctural and functional proteins, or nucleic acids, have all shown oncolytic activity. Treatment strategies include the treatment of inoperable tumors with pepsin (1) antitumor activity of carboxypeptidase (44) cytotoxicity of ribonudease (45—47) oncolytic activity of neuraminidase (48—52) therapy with neuraminidase of patients with acute myeloid leukemia (53) antitumor activity of proteases (54) and hyaluronidase treatment in the management of human soHd tumors (55). 

For precipitated protein, buffered solutions containing chaotropic reagents such as 0.1% SDS, 8 M urea, or 6 M guanidine or proteolytic enzymes such as pepsin may be used. However, an extended washing with buffer is required to remove SDS and guanidine. Unexpected elution behavior can occur if these reagents are not removed completely. 

FIGURE 2.16 pH versus enzymatic activity. The activity of enzymes is very sensitive to pH. The pH optimum of an enzyme is one of its most important characteristics. Pepsin is a protein-digesting enzyme active in the gastric fluid. Trypsin is also a proteolytic enzyme, but it acts in the more alkaline milieu of the small intestine. Lysozyme digests the cell walls of bacteria it is found in tears. 

Mammals, fungi, and higher plants produce a family of proteolytic enzymes known as aspartic proteases. These enzymes are active at acidic (or sometimes neutral) pH, and each possesses two aspartic acid residues at the active site. Aspartic proteases carry out a variety of functions (Table 16.3), including digestion pepsin and ehymosin), lysosomal protein degradation eathepsin D and E), and regulation of blood pressure renin is an aspartic protease involved in the production of an otensin, a hormone that stimulates smooth muscle contraction and reduces excretion of salts and fluid). The aspartic proteases display a variety of substrate specificities, but normally they are most active in the cleavage of peptide bonds between two hydrophobic amino acid residues. The preferred substrates of pepsin, for example, contain aromatic residues on both sides of the peptide bond to be cleaved. 

Pepsin Animal stomach Digestion of dietary protein... 

The second enzyme to be crystallized (byjohn Nordrrnp in 1930). Even more than nrease before it, pepsin. study by Northrnp established tirat enzyme activity comes from proteins. fAiso known as rennin, it is tire major pepsinlike enzyine in gastric Jnice of fetal and newborn animals. 

Just as individual amino acids have isoelectric points, proteins have an overall p/ because of the acidic or basic amino acids they may contain. The enzyme lysozyme, for instance, has a preponderance of basic amino acids and thus has a high isoelectric point (p/= 11.0). Pepsin, however, has a preponderance of acidic amino acids and a low- isoelectric point pi 1.0). Not surprisingly, the solubilities and properties of proteins with different pi s are strongly affected by the pH of the medium. Solubility- is usually lowest at the isoelectric point, where the protein has no net charge, and is higher both above and below the pi, where the protein is charged. 

Fig. 13. Relative sorption capacity of proteins by carboxylic CP Biocarb-T vs pH of solution 1) terrilytin, 2) insulin, 3) chymotrypsinogen, 4) pancreatic ribonuclease, 3) pepsin, 6) thymarine, 7) thermolysine, 8) haemoglobin, P) lysozyme. mma, — quantity of protein bonden on Biocarb-T by pHma (...

There are indications that the crystal protein is subject to proteolytic enzymes when separated from the sporangium. The crystal protein has also been shown to be degradable by fairly nonspecific proteases such as pepsin and trypsin. 

By adding 1-alkanols to AOT-based w/o microemulsions, some proteins (ribonucle-ase, lysozyme, alpha-chymotrypsin, pepsin, bovine serum albumin, and catalase) are readily expelled, while the major part of the surfactant remained in solution [171]. 

Reaction of purified Ca " -ATPase with 0.3 mM NBD-Cl in the presence of 1 mM AMP-PNP and 1 mM CaCl2 caused inhibition of ATPase activity with the incorporation of 2= 15 nmol NBD-Cl per mg protein [335]. The inhibition was attributed to the binding of 7-8 nmol NBD-Cl/mg enzyme protein, corresponding to = 1 mol NBD-Cl per mol ATPase. The NBD-labeled enzyme was digested with pepsin and several NBD-labeled peptides were isolated [335]. All peptides contained the Gly-X (Cys) sequence that occurs only in one place in the Ca -ATPase, i.e., at Gly343-Cys344. Therefore NBD-Cl reacts with the same cysteine 344 residue that is also modified by maleimide derivatives [319]. The NBD modified enzyme had only 5-10% of the ATPase activity of the control ATPase, but the steady state concentration of the phosphoenzyme intermediate was only slightly reduced [335]. The Ca ... 

After the food is swallowed, the digestive process continues in the stomach where the food is attacked by stomach acid. In fact, stomach acid is concentrated hydrochloric acid. The hydrochloric acid, along with an enzyme called pepsin, breaks down proteins in the food. Pepsin can only function in the low pH environment of the stomach. The hydrochloric acid is needed to maintain the low pH that pepsin needs to function. 

Pepsinogen is produced by the chief cells. Within the lumen of the stomach, this precursor molecule is split by HCl to form the active enzyme pepsin. Optimally active at an acidic pH (pH = 2), pepsin begins protein digestion by fragmenting proteins into smaller peptide chains. 

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