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PubMed1963

SERUM LACTIC DEHYDROGENASE, LEUCINE AMINOPEPTIDASE AND 5-NUCLEOTIDASE ACTIVITIES: OBSERVATION IN PATIENTS WITH CARCINOMA OF THE PANCREAS AND HEPATOBILIARY DISEASE.

Serum lactic dehydrogenase, leucine aminopeptidase, 5-nucleotidase and alkaline phosphatase activities were investigated in a number of diseases involving the hepatobiliary system.Leucine aminopeptidase was found to be a sensitive indicator of biliary obstruction, serum 5-nucleotidase slightly less sensitive, and alkaline phosphatase appreciably less sensitive. Leucine aminopeptidase and 5-nucleotidase activities were often increased by malignant infiltration of the liver and primary hepatic disease even in the absence of jaundice.Serum lactic dehydrogenase was frequently increased in primary hepatic disease and malignant disorders but was not apparently affected by bile duct obstruction per se. Thirty-five of 45 patients with proved malignancy had increased lactic dehydrogenase levels.The highest leucine aminopeptidase levels were encountered in carcinoma of the head of the pancreas. The frequent increase in both serum lactic dehydrogenase and leucine aminopeptidase activities in patients with carcinoma of the head of the pancreas suggests that these combined estimations are useful laboratory procedures in the diagnosis of malignant extrahepatic obstruction.

Aminopeptidases↗
PubMed1991

Leucine aminopeptidase: bestatin inhibition and a model for enzyme-catalyzed peptide hydrolysis.

The three-dimensional structures of native bovine lens leucine aminopeptidase (EC 3.4.11.1) and its complex with bestatin, a slow-binding inhibitor, have been solved and exhaustively refined. The mode of binding of bestatin to leucine aminopeptidase may be similar to that of a tetrahedral intermediate that is thought to form during peptide bond hydrolysis. Bestatin binds in the active site with its alpha-amino group and hydroxyl group coordinated to the zinc ion located in the readily exchangeable divalent cation binding site. Its phenylalanyl side chain is stabilized by van der Waals interactions with Met-270, Thr-359, Gly-362, Ala-451, and Met-454, which appear to form a terminal hydrophobic pocket. The leucyl side chain binds in another hydrophobic cleft lined by Asn-330, Ala-333, and Ile-421. Hydrogen bonds involving active site residues Lys-262, Asp-273, Gly-360, and Leu-362 are responsible for stabilizing the backbone nitrogen and oxygen atoms of bestatin. The mode of bestatin inhibition of leucine aminopeptidase is discussed and correlated with biochemical studies of bestatin analogues. In addition, features of a mechanism of catalysis of peptide hydrolysis by leucine aminopeptidase are discussed.

Amino Acid Sequence↗
PubMed1986

"Leucine aminopeptidase" (neutral arylamidase) in sheep sera: improved resolution with gradient gel electrophoresis.

Electrophoretic resolution of the heterogeneity of sheep serum "leucine aminopeptidase" is greatly improved by the use of gradients of acrylamide polymer, together with enzyme localisation involving L-alanyl beta-naphthylamide and cobaltous ion. The improved resolution contradicts an earlier claim of the existence of only two patterns of individual variation in the heterogeneity of sheep serum "leucine aminopeptidase", with one pattern completely dominant to the other. While the sheep enzyme is unusual among mammalian serum "leucine aminopeptidases" in its complex heterogeneity, it does conform to the typical mammalian pattern of codominant individual variation. The complexity of sheep serum "leucine aminopeptidase" is useful in the study of sheep evolution.

Animals↗
PubMed1983

Human liver L-leucine aminopeptidase: evidence for two forms compared to pig liver enzyme.

Two forms of L-leucine aminopeptidase (E.C. 3.4.11.1) having a specific activity toward L-leucine amide and L-leucylpeptides substrates but not toward chromogenic substrates: L-leucyl paranitroanilide or L-leucyl beta naphthylamide have been evidenced from human liver. Human liver enzymes have been distinguished from pig liver enzyme by DEAE Sephacel chromatography and analytical electrophoresis on cellulose acetate strips. We compared enzymic properties of L-leucine aminopeptidases from human liver with pig liver enzyme: they were activated by Mg2+ and Mn2+ and inhibited by Zn2+ and Co2+, EDTA and citric acid. The optimum pH's were 10. Both human liver L-leucine aminopeptidases were less sensitive to heat elevation than pig liver enzyme.

Animals↗
PubMed1995

Two-metal ion mechanism of bovine lens leucine aminopeptidase: active site solvent structure and binding mode of L-leucinal, a gem-diolate transition state analogue, by X-ray crystallography.

The three-dimensional structures of bovine lens leucine aminopeptidase (blLAP) complexed with L-leucinal and of the unliganded enzyme have been determined at crystallographic resolutions of 1.9 and 1.6 A, respectively. Leucinal binds as a hydrated gem-diol to the active site of b1LAP), resembling the presumed gem-diolated intermediate in the catalytic pathway. One hydroxyl group bridges the two active site metal ions, and the other OH group is coordinated to Zn1. The high-resolution structure of the unliganded enzyme reveals one metal-bound water ligand, which is bridging both zinc ions. Together, these structures support a mechanism in which the bridging water ligand is the attacking hydroxide ion nucleophile. The gem-diolate intermediate is probably stabilized by four coordinating bonds to the dizinc center and by interaction with Lys-262 and Arg-336. In the mechanism, Lys-262 polarizes the peptide carbonyl group, which is also coordinated to Zn1. The Arg-336 side chain interacts with the substrate and the gem-diolate intermediate via water molecules. Near Arg-336 in the b1LAP-leucinal structure, an unusually short hydrogen bond is found between two active site water molecules.

Animals↗
PubMed2000

Computer-aided design and activity prediction of leucine aminopeptidase inhibitors.

The Ligand Design (LUDI) approach has been used in order to design leucine aminopeptidase inhibitors, predict their activity and analyze their interactions with the enzyme. The investigation was based on the crystal structure of bovine lens leucine aminopeptidase (LAP) complexed with its inhibitor--the phosphonic acid analogue of leucine (LeuP). More than 50 potential leucine aminopeptidase inhibitors have been obtained, including the most potent aminophosphonic LAP inhibitors with experimentally known activity, which have been the subject of more detailed studies. A reasonable agreement between theoretical and experimental activities has been obtained for most of the studied inhibitors. Our results confirm that LUDI is a powerful tool for the design of enzyme inhibitors as well as in the prediction of their activity. In addition, for inhibitor-active site interactions dominated by the electrostatic effects it is possible to improve binding energy estimates by using a more accurate description of inhibitor charge distribution.

Animals↗
PubMed1982

The primary structure of leucine aminopeptidase from bovine eye lens.

The amino acid sequence of bovine eye lens leucine aminopeptidase has been determined. Cyanogen bromide fragments, the COOH-terminal hydroxylamine fragment, and a large fragment obtained by digestion with Staphylococcus aureus protease were isolated from reduced and S-alkylated leucine aminopeptidase. The amino acid sequences of these fragments were determined by automated sequence analysis, by manual direct Edman degradation, and by the dansyl-Edman technique. Overlapping peptides were obtained by tryptic digestion of the S-alkylated protein or the citraconylated S-alkylated protein. The polypeptide chain of leucine aminopeptidase comprises 478 residues, corresponding to a molecular weight of 51,691. No significant sequence homology with any other published protein primary structure could be detected. This is the first report of a complete amino acid sequence of an enzyme belonging to the class of two metal peptidases.

Amino Acid Sequence↗
PubMed1993

Leucine aminopeptidase: an inducible component of the defense response in Lycopersicon esculentum (tomato).

A leucine aminopeptidase (EC 3.4.11.1) cDNA clone (DR57) that was induced in response to Pseudomonas syringae pv. tomato (P.s. tomato) infection was isolated using a subtractive hybridization-enriched cDNA probe. Genomic DNA blot analysis showed that the tomato genome had two leucine aminopeptidase genes. The levels of DR57 mRNAs after P.s. tomato infection and mechanical wounding were determined in two inbred tomato lines that exhibit susceptibility and resistance to P.s. tomato. DR57 mRNAs were detected 12 hours after infection and 4 hours after wounding. Furthermore, DR57 mRNAs were systemically induced in response to wounding. DR57 mRNAs were induced in leaves after Spodoptera littoralis feeding but were not detected in detached leaf controls. Possible roles for the DR57 leucine aminopeptidase in the defense reactions are discussed.

Amino Acid Sequence↗
PubMed2002

Purification, characterization, and genetic analysis of a leucine aminopeptidase from Aspergillus sojae.

Extracellular leucine aminopeptidase (LAP) from Aspergillus sojae was purified to protein homogeneity by sequential fast protein liquid chromatography steps. LAP had an apparent molecular mass of 37 kDa, of which approximately 3% was contributed by N-glycosylated carbohydrate. The purified enzyme was most active at pH 9 and 70 degrees C for 30 min. The enzyme preferentially hydrolyzed leucine p-nitroanilide followed by Phe, Lys, and Arg derivatives. The LAP activity was strongly inhibited by metal-chelating agents, and was largely restored by divalent cations like Zn(2+) and Co(2+). The lap gene and its corresponding cDNA fragment of the A. sojae were cloned using degenerated primers derived from internal amino acid sequences of the purified enzyme. lap is interrupted by three introns and is transcribed in a 1.3-kb mRNA that encodes a 377-amino-acid protein with a calculated molecular mass of 41.061 kDa. The mature LAP is preceded by a leader peptide of 77 amino acids, predicted to include an 18-amino-acid signal peptide and an extra sequence of 59 amino acids. Two putative N-glycosylation sites are identified in Asn-87 and Asn-288. Southern blot analysis suggested that lap is a single-copy gene in the A. sojae genome. The deduced amino acid sequence of A. sojae LAP shares only 11-33.1% identity with those of LAPs from 18 organisms.

Amino Acid Sequence↗
PubMed1985

Distinct behavior of beta-endorphin and corticotropin toward leucine aminopeptidase action.

Reactions of human beta-endorphin, corticotropin and their synthetic analogs with leucine aminopeptidase have been investigated. The results confirmed previous findings that beta-endorphin is resistant to the aminopeptidase action whereas corticotropin is not. Beta-endorphin-(1-5) is completely digested by the enzyme while beta-endorphin-(1-17) is resistant. In contrast, the NH2-terminal 7 residues in corticotropin are removed readily by leucine aminopeptidase. This is confirmed by the observation that human corticotropin-(7-38) is not hydrolyzed by the enzyme. This contrasting behavior of the two hormones toward leucine aminopeptidase may be related to differences in their conformational structures.

Adrenocorticotropic Hormone↗
PubMed1964

LOCALIZATION OF LEUCINE AMINOPEPTIDASE ISOENZYMES.

A simple, sensitive staining procedure for the localization of leucine aminopeptidase isoenzymes after electrophoretic separation on cellulose acetate membranes is described. Separation of leucine aminopeptidase isoenzymes, using cellulose acetate membranes, does not appear to be a rewarding diagnostic procedure in the investigation of hepatobiliary or pancreatic disorders.

Aminopeptidases↗
PubMed1981

Differential characterization of two leucine aminopeptidases in Drosophila melanogaster.

Two leucine aminopeptidases from Drosophila melanogaster larvae have been partially purified. The LAP A and D enzymes have similar biochemical characteristics including molecular weights of approximately equal to 280,000 daltons. Michaelis-Menten constants of approximately equal to 0.05 mM, associations with metal cofactors, and specificities toward natural and chromogenic substrates. They differ in their pH optima and spatial distributions. If the closely linked genes that code for these enzymes have resulted from a tandem gene duplication event, it is suggested that there has been subsequent evolutionary divergence. This would provide Drosophila larvae with two related, but functionally distinct enzymes.

Animals↗
PubMed1992

Identification of human placental leucine aminopeptidase as oxytocinase.

Human placental leucine aminopeptidase (P-LAP) was purified from retroplacental serum for the first time by serial chromatography on columns of Matrex Blue A, DEAE-Sepharose CL-6B, phenyl-Sepharose 4B, chelating-Sepharose, and Sepharose CL-6B. The purified P-LAP was apparently homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the apparent molecular weight (Mr) was estimated to be 210,000. By comparing P-LAP activity with cystine aminopeptidase activity, we concluded that both activities were shared by the same molecule. We also examined the hydrolytic activity of P-LAP using naturally occurring peptide hormones and found that the enzyme hydrolyzed oxytocin, vasopressin, and angiotensin III. These results suggest that P-LAP shows oxytocinase activity and plays an important role in the regulation of the plasma level of these hormones during pregnancy.

Chromatography, Gel↗
PubMed2004

Function of the signal peptide and N- and C-terminal propeptides in the leucine aminopeptidase from Aeromonas proteolytica.

The leucine aminopeptidase from Aeromonas proteolytica (also known as Vibrio proteolyticus) (AAP) is a metalloenzyme with broad substrate specificity. The open reading frame (ORF) for AAP encodes a 54 kDa enzyme, however, the extracellular enzyme has a molecular weight of 43 kDa. This form of AAP is further processed to a mature, thermostable 32 kDa form but the exact nature of this process is unknown. Over-expression of different forms of AAP in Escherichia coli (with AAP's native leader sequence, with and without the N- and/or C-terminal propeptides, and as fusion protein) has allowed a model for the processing of wild-type AAP to be proposed. The role of the A. proteolytica signal peptide in protein secretion as well as comparison to other known signal peptides reveals a close resemblance of the A. proteolytica signal peptide to the outer membrane protein (OmpA) signal peptide. Over-expression of the full 54 kDa AAP enzyme provides an enzyme that is significantly less active, due to a cooperative inhibitory interaction between both propeptides. Over-expression of AAP lacking its C-terminal propeptide provided an enzyme with an identical kcat value to wild-type AAP but exhibited a larger Km value, suggesting competitive inhibition of AAP by the N-terminal propeptide (Ki approximately 0.13 nM). The recombinant 32 kDa form of AAP was characterized by kinetic and spectroscopic methods and was shown to be identical to mature, wild-type AAP. Therefore, the ease of purification and processing of rAAP along with the fact that large quantities can be obtained now allow new detailed mechanistic studies to be performed on AAP through site-directed mutagenesis.

Aeromonas↗
PubMed1986

Serum leucine aminopeptidase for monitoring viral infections with plasmacytoid reaction.

Analysis of data on 9 cases with active cytomegalovirus infection in patients with kidney grafts showed a positive association of serum leucine aminopeptidase activity concentration with the appearance of plasmacytoid lymphocytes in blood. Additional studies indicate that like the liver, the lymphocytes contain leucine aminopeptidase in relatively large quantities and that this enzyme is increased about 3-fold in plasmacytoid lymphocytes when compared with the activity in normal lymphocytes. In contrast, the 'hepatic' enzyme alanine aminotransferase is practically absent in both lymphocytes and plasmacytoid cells. Therefore, the difference in serum between the relative increases of leucine aminopeptidase and alanine aminotransferase may be attributed to proliferating plasmacytoid lymphocytes. Earlier observations on a large number of cases of acute viral hepatitis A or B lend credence to this assumption. However, in this disease, the serum enzyme changes reflect the much greater involvement of the liver and the relatively slight, but significant, proliferation of plasmacytoid lymphocytes. Our hypothesis is confirmed by the recent observation of 3 cases of acute EBV infection (infectious mononucleosis) in otherwise healthy individuals showing greatly elevated leucine aminopeptidase in contrast to normal or slightly raised alanine aminotransferase in serum.

Adult↗
PubMed1996

In vitro UVB irradiation of bovine crystalline lens causes cell damage and reduction in leucine aminopeptidase activity in lens epithelium.

Past studies in our laboratory have shown that low levels of UVB can cause changes in the optical properties of organ cultured ocular lenses, while other research has shown that in vitro UV radiation causes decreases in leucine aminopeptidase activity in homogenates of crystalline lens material. Therefore we have investigated whether there is a relationship between such decreases in enzyme activity and changes in lens optics and structure. Organ cultured bovine lenses were irradiated with low doses of UVB, and lens optics, histology and leucine aminopeptidase activity (leucine beta-naphthylamide hydrolysis at pH 7.5) were assessed daily. Lenses irradiated with 0.1 J cm-2 UVB showed a decrease of about 30% in leucine aminopeptidase activity 1 h after irradiation, while changes in lens optics were not observed until at least 24 h after irradiation. Histological examination of the lens anterior epithelium revealed changes in epithelial cells ranging from pyknotic nuclei to large areas of cell fragmentation. The results of this study suggest that a decrease in soluble aminopeptidase activity in lens epithelial cells may be a direct result of the epithelial cell damage rather than an effect of UVB on the enzyme per se.

Animals↗
PubMed1999

A bicarbonate ion as a general base in the mechanism of peptide hydrolysis by dizinc leucine aminopeptidase.

The active sites of aminopeptidase A (PepA) from Escherichia coli and leucine aminopeptidase from bovine lens are isostructural, as shown by x-ray structures at 2.5 A and 1.6 A resolution, respectively. In both structures, a bicarbonate anion is bound to an arginine side chain (Arg-356 in PepA and Arg-336 in leucine aminopeptidase) very near two catalytic zinc ions. It is shown that PepA is activated about 10-fold by bicarbonate when L-leucine p-nitroanilide is used as a substrate. No activation by bicarbonate ions is found for mutants R356A, R356K, R356M, and R356E of PepA. In the suggested mechanism, the bicarbonate anion is proposed to facilitate proton transfer from a zinc-bridging water nucleophile to the peptide leaving group. Thus, the function of the bicarbonate ion as a general base is similar to the catalytic role of carboxylate side chains in the presumed mechanisms of other dizinc or monozinc peptidases. A mutational analysis shows that Arg-356 influences activity by binding the bicarbonate ion but is not essential for activity. Mutation of the catalytic Lys-282 reduces k(cat)/K(m) about 10,000-fold.

Aminopeptidases↗