Enzymes leucine aminopeptidase

The effects of CPH-treatment of rats (1200 mg/ kg/d for 3d) on the polypeptide composition of renal brush border from the proximal tubule cells enzymatic activities and transport systems of the brush border membrane vesicles (BBMV) were investigated [77]. The results of these studies showed that CPH-treatment induces a 20-30% decrease in the specific activities of renal brush border enzymes leucine aminopeptidase and D-glutamyltransferase. SDS-gel electrophoresis showed that CPH-treatment induced a decrease of the intensity of 3 brush border polypeptides of molecular weights of 72,000, 58,000 and 39,000 [77]. 

Non-corrin cobalt has a number of interesting applications in the chemical industry, for example in the hydroformylation (OXO) reaction between CO, H2 and olefins. A number of non-corrin Co-containing enzymes have been described, including methionine aminopep-tidase, prolidase, nitrile hydratase and glucose isomerase. We describe the best characterized of these, namely the E. coli methionine aminopeptidase, a ubiquitous enzyme, which cleaves N-terminal methionine from newly translated polypeptide chains. The active site of the enzyme (Figure 15.13) contains two Co(II) ions that are coordinated by the side-chain atoms of five amino acid residues. The distance between the two Co2+ is similar to that between the two Zn2+ atoms in leucine aminopeptidase, and indeed the catalytic mechanism of methionine aminopeptidase shares many features with other metalloproteases, in particular leucine aminopeptidases. 

A novel concept of using bioadhesive polymers as enzyme inhibitors has been developed [97]. Included are derivatives of poly acrylic acid, polycarbophil, and car-bomer to protect therapeutically important proteins and peptides from proteolytic activity of enzymes, endopeptidases (trypsin and a-chymotrypsin), exopeptidases (carboxypeptidases A and B), and microsomal and cytosolic leucine aminopeptidase. However, cysteine protease (pyroglutamyl aminopeptidase) is not inhibited by polycarbophil and carbomer [97]. 

Enzymes activities are particularly sensitive to the anticoagulant used in collecting the specimen. Heparin inhibits acid phosphatase (W16) and muramidase (Z5). Amylase activity is inhibited by oxalate or citrate (MIO), and lactic dehydrogenase and acid phosphatase lose activity in oxalate (C2). Alkaline phosphatase is stable in oxalate, oxalate-fluoride, or heparin, but 25 mAf citrate inhibits 50% of the activity, and as little as 50 mlf EDTA is completely inhibitory (B19). Leucine aminopeptidase is inhibited by EDTA, as is creatine phosphokinase (F3). Amylase activity has been reported to be only 83% of that in serum when oxalate or citrate-plasma is used (MIO). Heparin plasma appears to have no inhibitory effect. Despite the fact that clotting factor V is not stable in oxalate or EDTA, these are often used as anticoagulants to obtain plasma for prothrombin determinations (Z2, Z4). 

Thiourea probably acts in a manner comparable to that of Naddtc and should also be administered after cis-Pt treatment (78). Like Naddtc, thiourea is able to remove platinum from platinated enzymes, such as leucine aminopeptidase (76, 128), y-glutamyltranspeptidase (76,128), and fumarase (129) (Fig. 9), and from Pt-methionine model adducts (Table III) (131). However, thiourea appears to be less useful as an inhibitor of nephrotoxicity it also reacts quite rapidly with platinum-DNA cross-links (56). 

Many aminopeptidases are metalloenzymes.437 Most studied is the cytosolic leucine aminopeptidase which acts rapidly on N-terminal leucine and removes other amino acids more slowly. Each of the subunits of the hexameric enzyme contains two divalent metal ions, one of which must be Zn2+ or Co2+438/439 A methionine aminopeptidase from E. colt contains two Co2+ ions440/441 and a proline-specific aminopeptidase from the same bacterium two Mn2+.442 In all of these enzymes the metal ions are present as dimetal pairs similar to those observed in phosphatases and discussed in Section D,4 and to the Fe-Fe pairs of hemerythrin and other diiron proteins (Fig. 16-20). A hydroxide ion that bridges the metal ions may serve as the nucleophile in the aminopeptidases.438 A bound bicarbonate ion may assist.4383... 

Leucine aminopeptidase may be isolated from Aspergillus oryzae.1300 The RMM (37 500) of this enzyme is significantly smaller than that from mammalian sources, but, like the latter, it is a metalloenzyme. 

The flavonoids fisetin and genistein exhibit antiangiogenic activity as evaluated in in vivo studies [279]. Flavonoids may also exert a limiting effect on tumor metastasis by way of their inhibitory activity on proteolytic enzymes such as trypsin, leucine aminopeptidase and other... 

Ribonuclease Ti is fairly resistant to proteases. The threonine residue at the carboxyl terminal of the enzyme can be removed by carboxy-peptidase A without loss of activity (67). Leucine aminopeptidase does not release amino acids from the amino terminal (68). Ribonuclease Ti is not inactivated by trypsin or chymotrypsin in the presence of 0.2 M phosphate (69), which probably binds the enzyme and protects it from inactivation (67). Treatment of the enzyme with trypsin in the absence of phosphate inactivates it (67). Ribonuclease Tj is hydrolyzed by pepsin with progressive loss of activity (69). 

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. 

Aminopeptidases that catalyze the hydrolysis of cysteinyl peptides are known. The membrane-bound aminopeptidases are glycoproteins, usually with molecular weights of about 100,000 daltons. They appear to be metalloproteins, one of the better known being a zinc-containing enzyme. Other enzymes, such as the leucine aminopeptidase, are cytosolic but, at least in this case, are also zinc-containing. The substrate specificity of these enzymes varies but most are relatively nonspecific. 

FIGURE 5 Relative scopes (measured as quartile range/median) for an array of extracellular enzymes and for bacterial production and respiration. Dependent variables shown along the X-axis are (left to right) leucine aminopeptidase, phosphatase, fatty acid esterase, (3-glucosidase, a-glucosidase, bacterial production, and bacterial respiration. Data are derived from four DOM experimental amendments. 

Interestingly, compounds which have been investigated for their penetration-enhancing effect at the absorbing membrane have also been shown to decrease the metabolism of certain peptides. By denaturing leucine aminopeptidase and preventing enzyme-substrate complex formation, the bile salt sodium glycocholate has been shown to protect insulin from proteolysis in the rat nasal mucosa. 

Isoenzymes or isozymes are enzymes from a single species that have the same kind of enzymatic activity but differ in chemical structure. In addition, they may differ in quantitative characteristics such as possessing different Km s with the same substrate and may differ in response to temperature and effectors. Isozymes of more than 100 enzymes have been demonstrated in humans. The most important of these for diagnostic purposes are the isozymes of LDH, CK, alkaline phosphatase, leucine aminopeptidase, acid phosphatase, and aldolase. These have been exploited for differential organ diagnosis. 

Leucine aminopeptidase (LAP, E.C.3.4.11.1) is one of the first discovered and the most widely studied aminopeptidase with respect to sequence, structure and mechanism of action.59 -63 LAP is a zinc containing exopeptidase that catalyzes the removal of amino acids from the N-terminus of peptides or proteins. Similar to other aminopeptidases, this enzyme is of significant biological and medical importance because of its key role in protein modification, activation, and degradation as well as in the metabolism of biologically active peptides and activity regulation of hormonal... 

Leucine aminopeptidase is an enzyme for which the most systematic and detailed computational studies regarding enzyme-inhibitor interactions have been performed.54,56-73,74 This results both from the availability of the crystal structure of LAP with bound inhibitors, including phosphonic acid analogue of leucine -LeuP (structure encoded as llcp in PDB, Figure 8-4), and from the existence of binding data for many LAP inhibitors. Such a set of experimental results enabled to evaluate the effectiveness of theoretical methods both empirical and quantum chemical in designing and activity prediction of LAP inhibitors. 

Application and Principle This procedure is used to determine leucine aminopeptidase activity in enzyme preparations derived from Lactococcus lactis. The assay is based on the rate of absorbance change over 5 min at 30° the change in absorbance is due to liberatedp-nitroaniline from the hydrolysis of leucine p-nitroanilide. 

The direct acylation and methylation of the free 01-NH2 groups of proteins have been proposed to be useful in providing resistance toward proteolytic attacks. Although the basis for this explanation is not always readily apparent from the known specificities of proteases, it may be valid in some cases. Thus, the acetylated N-terminus of a-crystallin, the major protein found in eye lens (17), is presumably important for the protein to survive in an environment rich in leucine aminopeptidase. On the other hand, it is difficult to rationalize that the acetylated N-terminus of bovine pancreatic a-amylase is in any way responsible for the fact that the enzyme is exceedingly stable against tryptic and chymotryptic digestion (18). The function of the acetylation is, in this case, as obscure as is the basis on which a-amylase is selected for acetylation among the many non-acetylated companion pancreatic proteins. 

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... 

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