Kidney aminopeptidase

Preliminary information useful in prodrug design has been obtained with amino acids attached to model aromatic amines. Thus, N-(naphthalen-2-yl) amides of amino acids (6.1, R=side chain of amino acid, R =H) proved to be of interest as test compounds to monitor peptidase activity such as ami-nopeptidase M (membrane alanyl aminopeptidase, microsomal aminopepti-dase, EC 3.4.11.2) [16][17], In the presence of purified rabbit kidney aminopeptidase M or human cerebrospinal fluid (CSF) aminopeptidase activity, the rate of hydrolysis decreased in the order Ala-> Leu->Arg->Glu-2-naphthyl-amide. Ala-2-naphthylamide, in particular, proved to be a good test compound, as its rate of hydrolysis was influenced by experimental conditions (preparation, inhibitors, etc.), as was the hydrolysis of a number of low-molecular-weight opioid peptides and circulating vasoactive peptides. 

Porcine kidney leucine aminopeptidase also binds one Zn2+ per subunit, which is essential for catalysis. The activity of the enzyme is regulated by the binding of divalent metal ions at a second site. 

Digestion studies using homogenates of kidneys, intestine, and liver indicated a close relationship between the levels of aminopeptidase activity as measured with a specific synthetic substrate and that based on the enzymatic activity responsible for hydrolyzing the isopeptide bond (see Table V). Purified aminopeptidase N from the intestinal brush border of rabbit (68) and pig (64) hydrolyzed a dipeptide and the related isodipeptide with about the same efficiency (see Table VI). These results may explain the findings that methionine covalently bound... 

A Taylor, T Surgenor, DK Thomson, RJ Graham, H Oettgen (1984) Comparison of leucine aminopeptidase from human lens, beef lens and kidney, and hog lens and kidney, Exp Eye Res 38 217-229... 

Serum creatinine and BUN, the most common indicators of renal function used in both clinical and preclinical safety laboratory panels, are relatively insensitive markers of injury, particularly for the renal tubules. Urinary measurements of alanine aminopeptidase and A-acetyl-beta-D-glucosaminidase and kidney injury molecule-1 (KIM-1) can provide much more sensitivity when nephrotoxicity is a potential safety concern [28,29], These are also suitable for safety monitoring in early-phase human trials if preclinical studies validate such use to monitor product nephrotoxicity. 

The reaction mixture contained in a final volume of 500 fiL 0.03 mmol Tris-HCl (pH 8.0), 0.3 /imol of peptide substrate, and the appropriate amount of microvillous fraction, which was prepared from rabbit kidney. In the aminopeptidase-P assay, 1.0 /imol of manganese chloride was also included. Reactions were run for 30 minutes at 37°C and were stopped by adding 400 fiL of 10% perchloric acid. After centrifugation, 20 / L aliquots were injected. The amount of product formed was linear with amount of enzyme added out to 250 jug and 1000 ng of microvillus protein for dipeptidyl peptidase IV and amino peptidase-P, respectively. 

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... 

The first evidence indicating that leucine aminopeptidase is a metallo-enzyme was obtained only recently (20, 21) although metals have long been known to aflfect its activity (22). Both the lens and the kidney... 

In spite of the large eflFects of or on the activity of lens aminopeptidase (and probably on the kidney enzyme as well), it is doubtful if they play any physiological role. The relative concentrations of Zn ", Mg and Mn " and the pH of the particular tissues in question are such as to preclude formation of any hybrid metalloenzymes in vivo. However, the fact that the activation does occur indicates its potential for occurring in vivo although it may not be brought about by Mg " or Mn2 ... 

Hooper NM, et al. Inhibition by converting enzyme inhibitors of pig kidney aminopeptidase P. Hypertension 1992 19 281-285. 

The specificity of this enzyme from swine kidney has been established from detailed studies with synthetic substrates (reviewed by Smith and Hill, 1960). All peptide bonds formed by L-amino acids which are adjacent to a free a-amino group are susceptible to hydrolysis, although the rates of hydrolysis vary over a several thousandfold range. The best substrates are those which contain amino-terminal leucine and the poorest are those which contain the amino nitrogen of proline in peptide linkage, e.g., glycyl-proline (Hill and Schmidt, 1962). The action of leucine aminopeptidase on protein and polypeptide substrates (Hill and Smith, 1958, 1959) agrees with the specificity established with synthetic substrates. 

In related work, Dong and Martin developed an assay that detects the catalytic activity of the enzymes pig liver esterase and porcine kidney leucine aminopeptidase by using substrates which have been labeled with metal-binding ligands [57], The enzymes catalyze changes in the substrates that affect their ability to bind to non-luminescent Ru complexes to form mixed-ligand complexes capable of ECL. 

Leucine arylamidase (= leucine aminopeptidase) is a ubiquitous enzyme, mainly localized in the liver and bile ducts as well as in the pancreas, breast, intestine and kidney. During pregnancy (as from trimester), LAP increases. Arylamidases are predominantly localized in the cytoplasm, while aminopeptidases are found in the microsomes of hepatocytes. LAP splits aminoterminal amino acids off from peptides. High LAP activities are detectable in the epithelia of the bile ducts. 

Saier MH, Jr. Growth and differentiated properties of a kidney epithelial cell line (MDCK). Am J Physiol 240 Cl 06-109,1981. Louvard D. Apical membrane aminopeptidase appears at site of cell-cell contact in cultured kidney epithelial cells. Proc Natl Acad Sci U S A 77 4132-4136,1980. 

Although the Edman method is by far the most common method of sequencing peptides from the /V-terminus, some enzymic methods are used occasionally. Several aminopeptidases are available commercially, which differ in their specificities. One aminopeptidase from porcine kidney preferentially releases amino acids such as leucine with hydrophobic side-chains. This enzyme does not release /V-terminal Arg or Lys or any amino acid that is followed by Pro. Another enzyme, aminopeptidase M, which is obtained from the microsomal fraction of porcine kidney cells, is less specific and perhaps more useful. It is advisable to examine aliquots of the hydrolysate at intervals by chromatography to determine the order in which amino acids are being released. 

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. 

Leucine aminopeptidase Hepatobiliary system, intestine, pancreas, kidney... 

Measurement of serum y-GT activity has clinical significance. The enzyme is present in all tissues, but the highest level is in the kidney however, the serum enzyme originates primarily from the hepatobiliary system. Elevated levels of serum y-GT are found in the following disorders intra- and posthepatic biliary obstruction (elevated serum y-GT indicates cholestasis, as do leucine aminopeptidase, 5 -nucleotidase, and alkaline phosphatase) primary or disseminated neoplasms some pancreatic cancers, especially when associated with hepatobiliary obstruction alcohol-induced liver disease (serum y-GT may be exquisitely sensitive to alcohol-induced liver injury) and some prostatic carcinomas (serum from normal males has 50% higher activity than that of females). Increased activity is also found in patients receiving phenobarbital or phenytoin, possibly due to induction of y-GT in liver cells by these drugs. 

Leucine aminopeptidase Porcine kidney 300,000 6 N-terminal exopeptidase Zn binds at two sites, one for catalysis, the other for regulation. 

Louvard D. Apical membrane aminopeptidase appears at site of cell-cell contact in cultured kidney epithelial cells. Proc Natl Acad Sci 1980 77 4132-4136. 

The plasma half-life of most administered proteins is relatively short, because they are susceptible to a wide variety of metabolic reactions. Rapid hydrolytic degradation of peptide bonds by both nonspecific enzymes and highly structurally selective aminopeptidases, carboxypeptidases, deamidases, and proteinases occurs at the site of administration, while crossing the vascular endothelia, at the site of action, in the liver, in the blood, in the kidney, and in fact, in most tissues and fluids of the body. Overall, the metabolic products of most proteins are not considered to be a safety issue. They generally are broken down into amino acids and reincorporated into new, endogenously biosynthesized proteins. 

Leucine aminopeptidase EC 3.4.11.1 Leu-X- (AA-X-) Bovine and pig lens and kidney Activated by Mg, Mn basic pH optimum chromogenic substrates not hydrolysed inhib. by 1,10 phenanthroline, acti-nonin, bestatin 6 subunits of M, 53000. 

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