Optimum pH for enzymes in stomach

Activity and Specificity

Pepsin B hydrolyzes gelatin efficiently but has a very weak proteolytic activity towards hemoglobin in contrast to pepsin A [5,6]. Porcine (at pH 3) and canine pepsin B (at pH 2) showed about 4% of the activity of porcine and human pepsin A (Chapter 3) against acid-denatured hemoglobin [5,6]. After chromatography, porcine pepsin B was originally detected through its activity against Ac-Phe↓Tyr(I2), which shows an optimum at pH 2. Relative to pepsin A, pepsin B shows a restricted specificity in hydrolysis of the B chain of oxidized insulin, but it liquefies gelatin much more readily than does pepsin A [1]. Porcine pepsin B preparations have been detected through its milk-clotting activity [5]. Canine pepsin B has a remarkable alkaline stability, retaining more than 90% activity at pH 8.0 at 15°C and even 50% after 16 hours [7]. It hydrolyzes a variety of peptides with a preference for an aromatic amino acid at the P1 position [6,7]. The substrate specificity has been investigated in detail both for wild-type canine pepsin B and several mutants [6]. The isoelectric point has not been determined, but the theoretical pI of canine pepsinogen B is 4.43 and 3.87 for the enzyme.

Biological Aspects

Pepsin F mRNA has been identified in extraembryonic membranes of horses, cats and mice [2–4] and the neonatal stomach of rabbits, horses, rats and mice [1,2,4,5]. Immunolocalization of pepsin F in these organs has only been examined in horses and mice. The expression pattern of pepsin F in extraembryonic membranes is species dependent. In the horse, pepsin F is predominantly localized to trophoblast cells of the chorion (the outer layer of the placenta) [4]. In the mouse, pepsin F expression is not present in trophoblast; rather it is localized to endodermal cells of the yolk sac [2]. In the horse, pepsin F expression is detectable in both early pregnancy (day 25) and in term placenta [4]. In the mouse placenta, pepsin F expression is predominantly detectable during the latter half of pregnancy, with maximal expression from day 13 to term [2].

Pepsin F expression in the stomach is localized to chief cells of the glandular stomach [2]. In all species in which it has been examined, the expression of pepsin F in the stomach is restricted to fetal (just prior to term) and neonatal developmental stages [1,2,5].

148.2.2.1 Pepsin (EC 3.4.23.1)

Pepsin, the first animal enzyme discovered (Florkin, 1957), is an acidic protease that catalyzes the breakdown of proteins into peptides in the stomach, while it does not digest the body’s own proteins. It is produced and stored in the chief cells of the gastric mucosa in its inactive form, pepsinogen, and then released as needed. Pepsinogen is converted into pepsin by an autocatalytic cleavage of a 44-amino-acid peptide. Pepsin, which degrades proteins preferentially at carboxylic groups of aromatic amino acids, such as phenylalanine and tyrosine, will not act on bonds containing valine, alanine, or glycine.

We measured pepsin activity by a discontinuous direct spectrophotometer assay utilizing denatured hemoglobin as substrate, and subtracting the endogenous pepsinogen activity present in pepsin, according to Kay (1975).

In order to evaluate the effect of oleuropein on this protease, pepsin enzymatic activity was assessed both in the absence and in the presence of oleuropein, and the data obtained were used to calculate the enzyme kinetic constants. Figure 148.1 reports the Lineweaver-Burk plot and evidences that the polyphenol oleuropein exerts a positive effect on pepsin enzymatic activity.

Digestive Action of Gastric Secretion

Pepsin. The main digestive action of gastric juice is exerted by pepsin, which catalyzes the partial hydrolysis of proteins. Pepsin is secreted in the state of pepsinogen by glands in the stomach’s body and fundus.

Pepsinogen (42.5 kDa) is a proenzyme, or zymogen, activated by H+ ions in gastric secretions. Its activity is further potentiated by its active form, pepsin. This mechanism, by which an enzyme activates its own zymogen, is called autocatalysis.

The activation of pepsin is accomplished by hydrolysis of the peptide bond between residues 42 and 43 of the zymogen, releasing a 42 amino acid segment from the N-terminus of the protein. Active pepsin has a mass of 35 kDa.

Pepsinogen secretion is stimulated by the same factors that activate HCl release: acetylcholine (vagal neurotransmitter), gastrin, and histamine. Approximately 99% of pepsinogen produced in the principal glands is secreted into the gastric lumen. The remaining 1% moves to the interstitial fluid and blood, eventually reaching the kidney to be excreted into urine as uropepsin. Determination of uropepsin in urine serves as an index of stomach peptic activity.

Pepsin action. Pepsin acts on virtually all proteins except keratins, mucoproteins, and protamines. It catalyzes the hydrolysis of peptide bonds located in the interior of the protein chain. Due to this action, pepsin belongs to a family of enzymes known as endopeptidases. The product of protein hydrolysis catalyzed by pepsin are polypeptide fragments of high molecular weight, which were originally named proteoses and peptones. Although pepsin can hydrolyze virtually any peptide bond, it has certain preferences, selectively targeting bonds that contain the amine group of an aromatic amino acid (tryptophan, phenylalanine, and tyrosine). The optimum pH for pepsin activity of 1.0–2.0 is maintained in the stomach by HCl. When the pH of the medium increases to values greater than 3.0, pepsin is almost completely inactivated.

In young children, gastric acidity is usually higher than in normal adults. In the first few months of life the gastric pH is approximately 5.0. Proteolytic action does not depend on pepsin but on other proteinases, including cathepsins, which are present in the lysosomes of almost all cells and released from desquamated gastric mucosa cells. In addition, a proteinase capable to act at near neutral pH has also been described in gastric juice of children.

Lab ferment or rennin. This proteolytic enzyme, also known as chymosin, is secreted by the fourth stomach of ruminants at early stages of life. It produces milk coagulation, acting on the most abundant protein of milk, casein. Rennin transforms casein into paracasein in the presence of Ca2+ ions, forming a calcium paracaseinate precipitate. This change in casein conformation facilitates its digestion by other proteases.

In humans, pepsin catalyzes the same reaction at a pH of 4.0. Perhaps pepsin is responsible for milk coagulation in an infant’s stomach.

Lipase. This enzyme is secreted by cells of the gastric fundus. Its optimum pH fluctuates between 3 and 6. It catalyzes the hydrolysis of ester bonds at positions 1 and 3 of triacylglycerides, especially those that have short or medium length chain fatty acids; their products are free fatty acids and diacylglycerols. Together with lingual lipase, which continues its action in the stomach, they contribute to the degradation of fats. These lipases are not essential and their absence does not produce clinical alterations because the pancreas secretes another lipase that is sufficient to meet the needs of digestion. Gastric and lingual lipases become important in neonates and young infants who have not yet fully developed their pancreatic function.

Mucus. This substance is secreted by cells of the principal gastric glands and mucosa. Mucus consists of glycoproteins; it cannot be digested by pepsin and helps protecting the gastric mucosa.

Parietal glands produce a glycoprotein with a molecular mass of 55 kDa, called intrinsic factor, which forms a complex with vitamin B12. Formation of this complex is required for the absorption of vitamin B12 in the ileum. Vitamin B12 is an essential factor for normal erythropoiesis (p. 677). Lack of intrinsic factor abolishes vitamin B12 absorption and causes a type of anemia known as pernicious anemia.

Digestion

Proteins are high molecular weight substances composed of up to 20 different amino acids, joined together in peptide linkages (see Fig. 8.10). In the adult, most protein is degraded in the digestive tract to small peptides and amino acids. This is accomplished by a variety of proteolytic enzymes. These can be divided into two categories, endopeptidases and exopeptidases. Endopeptidases cleave peptide bonds in the centre of the peptide chains, the initial products being mostly large peptides, which are subsequently degraded to oligopeptides. Exopeptidases cleave bonds at the ends of the peptide chain, splitting off amino acids one by one, in a stepwise manner: carboxypeptidases act at the C-terminal, and aminopeptidases at the N-terminal. Enzymes that specifically attack dipeptides and tripeptides are also present. The combined actions of these enzymes digest proteins to small peptides and amino acids.

Determination of Proteinases (Proteases)

Proteinases catalyze the hydrolysis of peptide bonds in proteins. The selectivity of action of some proteinases is shown in Figure 1.

Pepsin, pepsin-like enzymes, chymosin, rennin, and other acid proteinases have an activity optimum at pH 2.0–3.5; papain, trypsin, chymotrypsin, and similar enzymes are most active at neutral pH (pH 6–8). Subtilisin BPN, pancreatic elastase, leucine aminopeptidase (cytosol), and other alkaline proteinases work best at pH >8.

Many proteinases cleave not only peptide bonds but also ester bonds. Determinations of trypsin, chymotrypsin, papain, and some other enzymes are based on this property. The standard substrate for activity determination of trypsin and papain is N-benzoyl-l-arginine ethyl ester (BAEE), and for chymotrypsin, N-benzoyl-l-tyrosine ethyl ester. For example:

[XIII]N−Benzoyl−L−arginineethylester++H2O→N−Benzoyl−L−arginine+C2H5OH+H+

The reaction may be monitored by spectrophotometry or microcalorimetry.

Some proteinases use high relative molecular mass substrates as standards. For example, hemoglobin is a standard substrate for pepsin, angiotensinogen is a substrate for renin, elastin for elastase, and casein for chymosin.

Antigen Retrieval

Pepsin Pretreatment (for Ior egfR-3) is as follows:

Drain excess TBS, dry area around tissue with a Kimwipe, and encircle the section using a hydrophobic pen.

Incubate sections with 0.4% pepsin in 0.1 N hydrochloric acid at 37°C, 30 min.

Rinse gently in tap water, then in TBS for 5 min.

An alternative method for antigen retrieval is as follows:

Place slides inside Coplin jar, fully filled with 1 mM EDTA (pH 8.0). Using a microwave, heat sections in 1 mM EDTA (pH 8.0) for 1 min at full power, then heat for 14 min at medium power.

Cool slides for 20 min at room temperature.

Wash in distilled water 3×, 5 min each.

5.1.3 Some other monomeric enzymes

Pepsin A, like the pancreatic serine proteases, plays a role in the digestion of proteins eaten by mammals. It is called an acid protease because it functions at the low pH values found in the stomach. Peptide fragments are removed from the inactive form, pepsinogen, by the action of acid or other pepsin molecules to produce the active enzyme. This has a preference for cleaving bonds with a nonpolar amino acid residue on either side. Another acid protease found in the stomach is chymosin (rennin). Others are found in micro-organisms.

A group of thiol proteases, similar in structure to each other, are found in plants. These include papain, from the papaya fruit, and fìcain (formerly ficin), from figs. Other thiol proteases, of different structure, are found in bacteria and mammalian lysosomes. The essential cysteine residue in each of these enzymes plays a similar role to that of serine in the serine proteases.

Several exopeptidases, which remove terminal amino acid residues from polypeptide chains, are well known. Bovine pancreatic carboxypeptidase A, a monomeric enzyme containing one zinc ion per molecule, will break the peptide bonds linking C-terminal non-polar amino acids to the rest of the chain. It is produced when trypsin removes peptide fragments from the zymogen, procarboxypeptidase A. A very similar enzyme, carboxypeptidase B, which has a specificity for C-terminal amino acids with basic side chains, is also secreted as a zymogen by bovine pancreas.