Intestinal Aminopeptidase

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. 

Limiting essential amino acids covalently attached to proteins by using activated amino acid derivatives can improve the nutritional quality and change the functional properties of proteins. The best chemical methods for incorporating amino acids into water-soluble proteins involve using car-bodiimides, N-hydroxysuccinimide esters of acylated amino acids, or N-carboxy-a-amino acid anhydrides. The last two methods can give up to 75% incorporation of the amount of amino acid derivative used. With the anhydride method, as many as 50 residues of methionine have been linked to the 12 lysine residues of casein. The newly formed peptide and isopeptide bonds are hydrolyzed readily by intestinal aminopeptidase, making the added amino acids and the lysine from the protein available nutritionally. 

Table VI. Kinetic Parameters of the Hydrolysis of a Dipeptide and Its Related Isodipeptide Catalyzed by Intestinal Aminopeptidase N°...

The N-Carboxy-Amino Acid Anhydride Method. Since the isopeptide bond of e-methionyllysine was hydrolyzed readily by intestinal aminopeptidase and the released amino acid was biologically available, we decided to further increase the amount of covalently attached methionine through a polymerization reaction. The most suitable amino acid derivative for this approach is the N-carboxyanhydride or Leuchs anhydride. 

Aminopeptidases are present in many tissues (Table III). Leucine aminopeptidase from intestinal mucosa is very effective in catalyzing the hydrolysis of leucine from the amino terminus of peptides, polypeptides, and proteins. It also hydrolyzes leucine amide and leucine esters (10). The designation leucine aminopeptidase is somewhat of a misnomer because activity is also observed when other amino acids replace leucine. Only the L-isomers of amino acids are substrates, and the presence of a D-amino acid residue or proline in the penultimate position will retard hydrolysis (10). Enzymes having the same specificity as the intestinal aminopeptidase have been identified and/or isolated from kidney, pancreas, muscle, lens, and various bacterial sources (10). The kidney... 

In their work, Posternak and Pollaczek draw attention to the fact that both the relative position of a phosphate group in the peptide chain and the adjacent amino acids may be the factors determining whether or not enzymatic hydrolysis occurs. These authors isolated two phosphopeptones, each of which contained three phosphate groups. One of these, phospho-peptone I, consisted of ten to eleven amino acid residues, whereas phospho-peptone II had only seven. From phosphopeptotie II all three phosphate groups are liberated with the aid of kidney phosphatase, whereas this enzyme removes only two of the phosphorus atoms of peptone I (78). Moreover, it is of interest that, as in the case of the dipeptide, 0-phosphoryl-serylglutamic acid, an intestinal aminopeptidase did not act on these peptones but that after removal of the phosphate groups with the aid of a phosphatase some of the peptide bonds were hydrolyzed by the action of the proteolytic enzyme. 

Furthermore, enzymatic hydrolysis of model isopeptides N -oligo(L-methionyl)-l-lysine from Bio-beads13031 by pepsin, chymotrypsin, cathepsin C (dipeptidyl peptidase IV) and intestinal aminopeptidase N was investigated using high-performance liquid chromatography to identify and quantify the hydrolysis products 3041. 

Dietary proteins are cleaved to amino acids by proteases (see Fig. 2.2, circle 3). Pepsin acts in the stomach, and the proteolytic enzymes produced by the pancreas (trypsin, chymotrypsin, elastase, and the carboxypeptidases) act in the lumen of the small intestine. Aminopeptidases and di- and tripeptidases associated with the intestinal epithelial cells complete the conversion of dietary proteins to amino acids, which are absorbed into the intestinal epithelial cells and released into the hepatic portal vein. 

Pattus, F., Verger, R., and Desnuelle, P., 1976, Comparative study of the interactions of the trypsin and detergent form of the intestinal aminopeptidase with liposomes,... 

Fig. 9.1. Linear relationship between molecular weight logarithm of globular proteins and log n (number of antigenic determinants) (from Louvard et ai, 1976). Abbreviations are as follows RNase pancreatic ribonuclease OV, ovalbumin BSA, bovine serum albumin yG, human IgG immunoglobulin PH, E. coli alkalin phosphatase Cat, liver catalase AP, porcine intestinal aminopeptidase. The following equations apply s = k s — /c2U, log M =...

The use of monovalent Fab fragments can be very useful since equi-molecular antibody-antigen complexes are expected with monospecific antibodies. The Fab fragment was also used in the determination of the number of antigenic determinants of soluble and of membrane-bound intestinal aminopeptidase (Louvard et al, 1976). 

There are two main classes of proteolytic digestive enzymes (proteases), with different specificities for the amino acids forming the peptide bond to be hydrolyzed. Endopeptidases hydrolyze peptide bonds between specific amino acids throughout the molecule. They are the first enzymes to act, yielding a larger number of smaller fragments, eg, pepsin in the gastric juice and trypsin, chymotrypsin, and elastase secreted into the small intestine by the pancreas. Exopeptidases catalyze the hydrolysis of peptide bonds, one at a time, fi"om the ends of polypeptides. Carboxypeptidases, secreted in the pancreatic juice, release amino acids from rhe free carboxyl terminal, and aminopeptidases, secreted by the intestinal mucosal cells, release amino acids from the amino terminal. Dipeptides, which are not substrates for exopeptidases, are hydrolyzed in the brush border of intestinal mucosal cells by dipeptidases. 

The proteases are secreted as inactive zymogens the active site of the enzyme is masked by a small region of its peptide chain, which is removed by hydrolysis of a specific peptide bond. Pepsinogen is activated to pepsin by gastric acid and by activated pepsin (autocatalysis). In the small intestine, trypsinogen, the precursor of trypsin, is activated by enteropeptidase, which is secreted by the duodenal epithelial cells trypsin can then activate chymotrypsinogen to chymotrypsin, proelas-tase to elastase, procarboxypeptidase to carboxypepti-dase, and proaminopeptidase to aminopeptidase. 

Aminopeptidase A is another brush border membrane enzyme which has been the subject of various studies [79,81,83-86], It has been found in the intestinal brush border membrane of humans, rabbits, rats, and pigs and is active against peptides with acidic amino acids at the amino terminus. Its activity against dipeptides is more limited. Shoaf et al., isolated three rat brush border aminopeptidases with distinct but somewhat overlapping substrate specificities. These enzymes had preference for dipeptides containing methionine, arginine, or aspartic acid and glycine. The optimal pH for activity of aminopeptidase was reported to be 7-8. 

RE Stratford Jr, VHL Lee. (1985). Aminopeptidase activity in albino rabbit extraocular tissues relative to the small intestine. J Pharm Sci 74 731-734. 

Di- and tripeptides Aminopeptidases Hydrolyze di- and tripeptides into amino acids Absorptive cells of small intestine Brush border of absorptive cells... 

Smith, T.S., Graham, M., Munn, E.A., Newton, S.E., Knox, D.P., Coadwell, W.J., McMichael-Phillips, D., Smith, H., Smith, W.D. and Oliver, J.J. (1997) Cloning and characterisation of a microsomal aminopeptidase from the intestine of the nematode Haemonchus contortus. Biochimica et Biophysica Acta 1338, 295—306. 

Fig. 6.11. Peptide prodrugs (6.20, 6.21, and 6.22) for the intestine-selective delivery of 5-aminosalicylic acid (6.23). The prodrugs undergo selective activation by intestinal brush border enzymes, namely aminopeptidase A and/or carboxypeptidases [39].

The brush border enzymes in the intestine play a major role in peptide hydrolysis. Thus, both aminopeptidase and endopeptidase activities were detected with [Leu5]enkephalin as the substrate [146],... 

These proteolytic enzymes are all endopeptidases, which hydrolyse links in the middle of polypeptide chains. The products of the action of these proteolytic enzymes are a series of peptides of various sizes. These are degraded further by the action of several peptidases (exopeptidases) that remove terminal amino acids. Carboxypeptidases hydrolyse amino acids sequentially from the carboxyl end of peptides. They are secreted by the pancreas in proenzyme form and are each activated by the hydrolysis of one peptide bond, catalysed by trypsin. Aminopeptidases, which are secreted by the absorptive cells of the small intestine, hydrolyse amino acids sequentially from the amino end of peptides. In addition, dipeptidases, which are structurally associated with the glycocalyx of the entero-cytes, hydrolyse dipeptides into their component amino acids. 

The luminal surface of the intestine contains aminopeptidase—an exopeptidase that repeatedly cleaves the N-terminal residue from oligopeptides to produce free amino acids and smaller peptides. 

In the small intestine, proteases released by the pancreas as zymogens become active. Each has a different specificity for the amino acid R-groups adjacent to the susceptible peptide bond. Examples of these enzymes are trypsin, chymotrypsin, elastase, and car-boxypeptidase A and B. The resulting oligopeptides are cleaved by aminopeptidase found on the luminal surface of the intestine. Free amino acids and dipeptides are then absorbed by the intestinal epithelial cells. 

McClellan, J.B.J., and C.W. Garner. 1980. Purification and properties of human intestine alanine aminopeptidase. Biochim Biophys Acta 613 160. 

Bernkop-Schniirch, A., G. Walker, and H. Zarti. 2001. Thiolation of polycarbophil enhances its inhibition of intestinal brush border membrane bound aminopeptidase N. J Pharm Sci 90 1907. 

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