Brush border peptidases

The intestinal mucosal peptidases are distributed in the brush border and cytosol of the absorptive cell. There are, however, distinct differences between the brush border and cytosolic peptidases [75], The tetrapeptidase activity is associated exclusively with the brush border enzyme. Furthermore, brush border peptidases exhibit more activity against tripeptides than dipeptides, whereas the cytosolic enzymes show greater activity against dipeptides. Studies have demonstrated that more than 50% of dipeptidase activity was detected in the cytosol [76] and just 10% in the brush border membrane [77]. The brush border enzymes include... 

N Triadou, J Bataille, J Schmitz. Longitudinal study of the human intestinal brush border membrane proteins. Distribution of the main disaccharidases and peptidases. Gastroenterology 85 1326-1332, 1983. 

N Tobey, W Heizer, R Yeh, T Huang, C Hoffner. Human intestinal brush border peptidases. Gastroentrology 88 913-926, 1985. 

S Auricchio, A Stellato, B De Vizia. Development of brush border peptidases in human and rat small intestine. Pediatr Res 15 991-995, 1981. 

J. P. F. Bai, Influences of Regional Differences in Activities of Brush-Border Membrane Peptidases within the Rat Intestine on Site-Dependent Stability of Peptide Drugs , Life Sci. 1993,53, 1193-1201. 

Longer, M.A., Woodley, J.F., and Duncan, R., Comparison of the activities of rat small intestine and colon brush border membrane peptidases, Proc. Int. Symp. Contr. Rel Bioact. Mater., 16 225-226 (1989). 

These zinc-dependent endopeptidases (meprin A [EC 3.4.24.18] and meprin B [EC 3.4.24.63] ) are members of the peptidase family M12A. They catalyze the hydrolysis of peptide bonds in proteins and peptide substrates. Meprin A, a membrane-bound enzyme that has been isolated from mouse and rat kidney and intestinal brush borders as well as salivary ducts, acts preferentially on carboxyl side of hydrophobic amino acyl residues. Meprin A and B are insensitive to inhibition by phosphora-midon and thiorphan. 

Kramer, W., et al. 1990. Intestinal uptake of dipeptides and beta-lactam antibiotics. I. The intestinal uptake system for dipeptides and beta-lactam antibiotics is not part of a brush border membrane peptidase. Biochim Biophys Acta 1030 41. 

There are at least three peptidases in the brush border of the small intestine Aminopeptidase A, which has an affinity for peptide-bound acid amino acids (11), aminopeptidase N, which has a broad specificity (12), and dipeptidylpeptidase IV, which releases dipeptides from the N-terminal end of peptides with a preference for X-PRO terminals (13). In Table III are shown the effect of a low concentration ofThe LMW fraction on the activity of these enzymes in extracts of hog intestine. Aminopeptidase N was found to be strongly inhibited by 0.25 mg/ml of the fraction. Aminopeptidase A and dipeptidylpeptidase IV were not inhibited. 

Abstract The major enzymatic barrier to the absorption of macromolecules, particularly therapeutic peptides, is the pancreatic enzymes the peptidases, nucleases, lipases and esterases that are secreted in considerable quantities into the intestinal lumen and rapidly hydrolyse macromolecules and lipids. In the case of the peptidases, they work in a co-ordinated fashion, whereby the action of the pancreatic enzymes is augmented by those in the brush borders of the intestinal cells. The sloughing-off of mucosal cells into the lumen also furnishes a mixture of enzymes that are a threat to macromolecules. As the specificity and activity of the enzymes are not always predictable, during pharmaceutical development it is important to test the stability of therapeutic macromolecules, and novel macromolecular-containing or lipid-containing formulations, in the presence of mixtures of pancreatic enzymes and bile salts, or in animal intestinal washouts or ideally, aspirates of human intestinal contents. 

Figure 1.1 shows the major sites of enzyme activity in the GI tract, and we will consider each of these in turn. While most of the enzymes that hydrolyse macromolecules enter the gut in the pancreatic fluid and hence are found in the lumen of the gut, there is significant peptidase activity located on the membranes of the intestinal cells, the so-called brush border. Consideration should also be given to the enzymes that are located inside the cells of the intestinal mucosa, namely, the epithelial cells or enterocytes. This is for two reasons first, the intestinal mucosa has a turnover of 3-6 days in humans and this means that the enterocytes are constantly being sloughed-off into the lumen of the gut. Thus intracellular enzymes and brush border enzymes will be found in the lumen of the gut, though the precise quantity is difficult to assess (see later in Section 1.6). 

Table 1.2 Bond specificity of brush border peptidases Enzyme Bond hydrolysed...

The activities of some of the peptidases in the brush border membranes of rats have been measured in attempts to identify which of the many enzymes present are the most active and hence represent the greatest threat to... 

FIG. 1 Digestion and absorption of proteins in the small intestine. (1) Brush-border peptidases, (2) brush-border amino acid transport systems, (3) brush-border peptide transport systems, (4) cytoplasmic peptidases, (5) basolateral amino acid transport systems, (6) basolateral peptide transport systems. 

Protein and starch digestion, on the other hand, have potent nonpancreatic compensatory mechanisms. Due to the compensatory action of salivary amylase and brush border oligosaccharidases, a substantial proportion of starch digestion can be achieved without pancreatic amylase. Similarly, protein denaturation and hydrolysis is initiated by gastric proteolytic activity (acid and pepsin) and continued by intestinal brush border peptidases, and is thus partly maintained even in the absence of pancreatic proteolytic activity. 

Schntirch-Bernkop, A., Zarti, H., and Walker, G. F. (2001), Thiolation of polycarbophil enhances its inhibition of soluble and intestinal brush border membrane bound amino-peptidase N, J. Pharm. Sci., 90,1907-1914. 

The brush border of the villi hydrolyses oligosaccharides to glucose, fructose and galactose. Microvilli also attach peptidases, which hydrolyse di-and tripeptides to amino acids. The monosaccharides and amino acids are transported into the blood by active transport processes. 

Final digestion of dietary carbohydrates and proteins occurs on the lumen face of small intestinal enterocytes by so-caUed brush border enzymes , including maltase, sucrose, lactase and peptidases. 

A small fraction of the polypeptides contacting the brush border of the small intestine is hydrolyzed by membrane-bound enzymes attached to the outside of the enteroey te. Although a variety of peptidases are bound to the brush border, the most abundant is amlnopeptidase N, which catalyzes the hydrolysis of amino... 

First-pass intestinal metabolism. This includes brush border metabolism and intracellular metabolism. The former occurs at the surface of the enterocytes by the enzymes present within the brush border membrane. Furthermore, the brush border activity is generally greater in the proximal small intestine (duodenum jejunum > ileum colon) and involves enzymes such as alkaline phosphatase, sucrase, isomal-tase, and a considerable number of peptidases. ... 

Luminal and Membrane Metabolism of Peptides and Proteins. In meaningful studies on peptide and protein drug absorption in the small intestine, it is prerequisite to distinguish among cavital, membrane contact, and intracellular drug metabolism.Cavital metabolism takes place in the lumen of the small intestine by enzymes such as trypsin, chymotrypsin, carboxypepti-dase, and elastase, which are secreted by the pancreas. Membrane contact metabolism is carried out by aminopeptidases lo-calized on the brush border membrane. Intracellular metabolism occurs inside of the cells. The known intra-celluar enzymes are cytoplasmic peptidases, prolidase, dipeptidase, and tripeptidase.A more detailed dis-cussion of this topic is presented in section Intestinal Absorption Barriers, later. 

After the break down of proteins by proteolytic enzymes, the pancreas, and brush border peptidases, the di- and trip-eptides are absorbed through the epithelial cell membrane. Many studies have shown that intact di- and tripeptides are absorbed across the epithelial cell membrane by active transport via specific carrier systems. The absorption process is mediated by the hydrogen-coupled peptide transporter (PEPTl) located in the intestinal apical cell membrane. Because there are 20 amino acids, there may be 400... 

To minimize degradation and improve absorption of peptide and protein drugs administered orally, several factors are significant. First, peptide and protein drugs have to be protected from the acidic environment in the stomach and from enzyme degradation in the lumen and on the brush border of the small intestine. This may be accomplished by coadministration of peptidase inhibitors or by chemical modifications, such as analogs or prodrug approaches, or... 

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