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PubMed1988

Increased cytosol aminopeptidase and lactate dehydrogenase in serum originating from lymphocytes in measles and rubella infection.

We determined cytosol aminopeptidase (c-AP; EC 3.4.11.1) and lactate dehydrogenase (LDH) levels in serum; these enzymes are known to originate from lymphocytes in patients with measles and rubella. In patients with measles (n = 19), both enzyme levels increased markedly with the onset of rash: mean (+/- SD) c-AP was 269.7 +/- 103.5 U/L and LDH was 1149.5 +/- 255.2 U/L. In patients with rubella, activities of both enzymes increased mildly: c-AP (n = 18) was 81.6 +/- 24.4 U/L and LDH (n = 13) was 674.0 +/- 168.8 U/L. Increased c-AP and LDH levels in patients with measles and rubella presumably originate from the destruction of infected, activated lymphocytes, especially T lymphocytes.

Alanine Transaminase↗

PubMed1991

Differences in the leucine aminopeptidase activity in extracts from human prostatic carcinoma and benign prostatic hyperplasia.

Extracts of tissue showed that prostatic carcinomas contain less leucine aminopeptidase activity than benign prostatic hyperplasia. This is true when activity is expressed as specific activity (P = 0.0033), specific activity/% epithelium (P = 0.0007), activity/wet weight of tissue (P = 0.0028), or activity/wet weight of tissue/% epithelium (P = 0.0005). Almost all histochemically demonstrable activity is located in the epithelium. Enzymatic activities in extracts and in histochemical preparations showed similar differences between carcinoma and benign prostatic hyperplasia and were related (R = -0.38, P = 0.0400) to Gleason's grades. The transurethrally resected prostate cancers studied contained no well-differentiated tumors and a high proportion of poorly differentiated tumors. Histochemical activity is absent in most prostatic carcinomas and decreased in others. This observation is particularly interesting in view of the growing knowledge of tumor suppressor genes.

Aged↗

PubMed2001

Investigation of the applicability of a sequential digestion protocol using trypsin and leucine aminopeptidase M for protein identification by matrix-assisted laser desorption/ionization--time of flight mass spectrometry.

An investigation of the applicability of a sequential digestion procedure involving endo- and exoprotease digestion of proteins is reported. The procedure involves the digestion of a protein sample with trypsin, yielding peptide fragments characteristic of the protein. The resulting mixture of peptide fragments is then subjected to N-terminal sequencing with leucine aminopeptidase M (LAP), with matrix-assisted laser desorption ionization-time of flight mass spectrometric analysis of the various digestion products. Several proteins in solution, as well as gel extracted proteins were subjected to this sequential enzyme digestion procedure. The results of these experiments reveal that LAP will preferentially cleave specific peptides of the trypsin digested sample with high efficiency, while leaving other peptides undigested. Also, the length of the amino acid sequence tags that can be generated with this method is limited; the longest sequence tag generated from a single tryptic peptide was four amino acids, even though the digestion was allowed to proceed for long times. In the experiments, N-terminal digestion products were detected as early as two minutes, or as late as 90 minutes, following the addition of LAP to the sample. The method was shown to be effective for subpicomole starting quantities of protein, although with some loss in digestion efficiency at lower concentrations of starting material. This method is useful in providing additional sequence information to increase the level of confidence in protein identification, as illustrated in the identification of bacterial proteins fractionated by high-performance liquid chromatography. In some instances, this method can provide additional sequence information where post source decay and nanospray mass spectrometry failed to generate fragment ion spectra. This is illustrated in an example where the procedure was applied to a membrane protein, CD9, that had been isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Although the sequential digestion procedure requires more human intervention, it is a straightforward method and can be readily implemented.

Cell Line↗

PubMed1991

Synergistic inhibition of aminopeptidase B by penicillamine or cysteine and metallic salts.

The inhibition of aminopeptidase B by D- and L-penicillamine and D- and L-cysteine is reported. All these alpha-amino-beta-mercapto acids inhibit moderately this enzyme, with IC50 values in the 10(-4) range. This inhibition was not reversed by Zn2+ ions, but a synergistic effect was observed. A similar effect took place by the combined use of D-penicillamine and Fe2+, Ca2+ or Ni2+ salts, but not with copper salts. The zinc-D-penicillamine and zinc-D-cysteine complexes 3 and 4 show an inhibitory potency approximately 5-fold higher than that of the corresponding amino acid.

Aminopeptidases↗

PubMed1992

Synthesis and inhibitory activities against enkephalin degrading aminopeptidase of H-Trp(Nps)-Lys-OMe analogues bearing chelating groups.

With the aim of increasing the inhibitory potency of the analgesic dipeptide H-Trp(Nps)-Lys-OMe against enkephalin-degrading aminopeptidases, the following derivatives bearing chelating groups at the N-terminus have been synthesized: Ac-Trp(Nps)-Lys-OMe (3), HS(CH2)nCO-Trp(Nps)-Lys-OMe [n = 1 (4), n = 2 (5)], MeOCO(CH2)n-Trp(Nps)-Lys-OMe [n = 1 (6), n = 2 (7)] and analogues in which the N alpha-amino group has been replaced by a methoxycarbonyl group (8) and a bidentate hydroxamate function (9), respectively. The inhibitory activities of all these compounds and the S-protected derivatives EtNHCOS(CH2)nCO-Trp(Nps)-Lys-OMe [n = 1 (16), n = (17)] against the mentioned enzyme, isolated from rat striatum, are compared with those of the parent dipeptide 2 and bestatin. All the new derivatives showed, in general, inhibitory potencies of the same order of magnitude as compound 2.

Aminopeptidases↗

PubMed1993

Ketomethylenebestatin: synthesis and aminopeptidase inhibition.

The synthesis of (6R,5S,2RS)-6-amino-5-hydroxy-2-isobutyl-4-oxo-7- phenylheptanoic acid (9), a carbaanalogue of the aminopeptidase (AP) inhibitor bestatin (1) is described. This synthesis was carried out by a malonic ester alkylation with the suitably protected halomethyl ketone of (2S,3R)-AHPBA*), followed by a second alkylation with isobutyl bromide of the resulting 4-ketodiester, and subsequent decarboxylation and deprotection. The inhibitory potencies of the 1:1 diastereomeric mixture 9 against AP-B, AP-M and Leu-AP were approximately 10-fold lower than those of bestatin.

Aminopeptidases↗

PubMed2006

Aminopeptidase p mediated detoxification of organophosphonate analogues of sarin: mechanistic and stereochemical study at the phosphorus atom of the substrate.

The activity of the aminopeptidase P from Escherichia coli in hydrolyzing a series of organophosphonate sarin analogues (1-6) was evaluated. The enzymatic rates of hydrolysis for methylphosphonate 1 with a methoxy group attached to the phosphorus center were 7- to 15-fold higher than those for the corresponding analogues 2-6. Double mutant R153W/R370L was able to hydrolyze the S(p) enantiomer of racemic 1 at a considerable rate. This mutant allowed the preparation of the R(p) isomer of the sarin analogue 1. All the mutants, R370L, R153A, W88L, R153L/R370L, and R153W/R370L, preferred the formation of (S(p))-8 to that of the corresponding (R(p))-8 enantiomer and displayed a better enantiomeric excess of products, by 1.4- to 2-fold as compared to the wild-type enzyme. Enzymatic hydrolysis of O,O-diisopropyl-p-nitrophenyl phosphate (9) in H(2) (18)O led to the formation of the (18)O-labeled O,O-diisopropyl phosphate product and confirmed that the catalytic reaction starts with cleavage of the P--O bond. From chemical and kinetic studies, the utilization of an optically pure S(p) enantiomer of O-methyl-p-nitrophenyl methylphosphonothioate (S(p))-MNMPT, 7) has demonstrated that the enzymatic reaction proceeds through a displacement mechanism and generates a chiral product in situ with an inversion of stereochemical configuration at the phosphorus atom. The results also lead to the conclusion that alteration of the active site through site-directed mutagenesis can result in a preference for (S(p))-MNMPT (7) rather than the R(p) isomer.

Aminopeptidases↗

PubMed2003

Stereoselective synthesis of 1-aminoalkanephosphonic acids with two chiral centers and their activity towards leucine aminopeptidase.

The stereoselective synthesis of 1-amino-2-alkylalkanephosphonic acids, namely, compounds bearing two chiral centers, was achieved by the condensation of hypophosphorous acid salts of (R)(+) or (S)(-)-N-alpha-methylbenzylamine with the appropriate aldehydes in isopropanol. Simultaneous deprotection and oxidation by the action of bromine water provided equimolar mixtures of the RS:RR and SR:SS diastereomers of desired acids. They appeared to act as moderate inhibitors of kidney leucine aminopeptidase with potency dependent on the absolute configuration of both centers of chirality.

2-Propanol↗

PubMed1995

Flow cytometric determination of aminopeptidase activities in viable cells using fluorogenic rhodamine 110 substrates.

Aminopeptidases (AP) are ubiquitously occurring, nonspecific exopeptidases involved in protein degradation. They cleave the N-terminal amino acid of peptides and occur in practically all mammalian cells and tissues. Physiological and pathological processes such as metastasis of tumors and inflammation have been thought to involve changes in AP activities. Determination of AP (EC 3.4.11.X) activity in viable cells by flow cytometry was the subject of this study because of its general biological and clinical interest. Different bis-substituted rhodamine 110 (R110) peptide derivatives were synthesised and used as AP- and exopeptidase (EC 3.4.13.X-EC 3.4.14.X) substrates for flow cytometric measurements. Intracellular AP activities in viable lympho-, mono-, granulo-, and thrombocytes were detected by fluorescence increase from R110 following intracellular substrate cleavage. Eukaryotic-AP do not cleave D-amino acids and hence NH2(D-Leu)2R110 substrate served as negative control. Specific substrate cleavage by AP is shown by complete inhibition of flFLuorescence generation following preincubation of cells with leucine-chloromethylketone inhibitor. R110 AP- and exopeptidase substrates are suitable indicators for coupled endopeptidase reactions due to their rapid cleavage and largely pH independent generation of intracellular fluorescence.

Amino Acids↗

PubMed1993

Characterization and purification of T lymphocyte aminopeptidase B: a putative marker of T cell activation.

We have previously described that human T lymphocytes express membrane-associated peptidase activities (Mari et al., EMBO J., 1992, 11:3875). We show in this report that intact Jurkat T cells readily cleaved H-Arg-paranitroanilide, an aminopeptidase B (AP-B) substrate. The identification of the hydrolyzing activity as AP-B was confirmed by its sensitivity to both arphamenine B and bestatin in the nanomolar range. Significant AP-B activity was released in the supernatant upon incubation of intact T lymphocytes at 37 degrees C. However, AP-B activity was found mainly in the cytosolic fraction of Jurkat T cells. Cytosolic T cell AP-B was purified to homogeneity and exhibited a molecular mass of 72 kDa. Purified AP-B cleaved N-terminal basic amino acid-containing peptides such as thymopentin (H-Arg-Lys-Asp-Val-Tyr-OH), indicating that it might play a role in the regulation of the concentration of important soluble mediators of T cell activation. A rabbit polyclonal antibody was shown to recognize AP-B as assessed by both immunoprecipitation and Western blot experiments. Finally, we found that AP-B was up-regulated during activation of normal and leukemic T lymphocytes.

Aminopeptidases↗

PubMed1993

A mouse aminopeptidase N is a marker for antigen-presenting cells and appears to be co-expressed with major histocompatibility complex class II molecules.

To analyze the expression of mouse aminopeptidase N (APN) on the cells of the immune system a panel of rat monoclonal antibodies against mouse intestinal APN was generated. These antibodies were used to affinity purify functional mouse APN from both intestine and kidney, and by flow cytometry to examine the APN expression of the cells of the mouse immune system. An APN closely related, perhaps identical, to the intestinal APN was expressed on a subpopulation of spleen cells and stimulated peritoneal exudate cells, primarily representing antigen-presenting cells, such as B cells, macrophages, dendritic cells, and veiled cells. In contrast this APN expression could not be detected on thymocytes or spleen T cells. As a corollary, APN was expressed on monocyte, macrophage, and B lymphoma cell lines, but not on T hybridoma or thymoma cell lines. The expression of APN showed a striking correlation with the MHC class II expression in all the cell populations studied. This apparent co-expression suggests a role for APN in antigen processing.

Aminopeptidases↗

PubMed1992

Chicory seed lot variety identification by leucine-aminopeptidase and esterase zymogram analysis.

A method of identifying closely related chicory varieties has been developed using native polyacrylamide gel electrophoresis and subsequent leucine aminopeptidase and esterase staining of bulked seed sample extracts. The variety zymograms are analyzed according to a mobility-density scheme. This quick, easy and low-cost method can be a useful tool for chicory breeders.

Electrophoresis, Polyacrylamide Gel↗

PubMed1996

Purification of circulating liver plasma membrane fragments using a monoclonal antileucine aminopeptidase antibody.

Membrane-bound liver alkaline phosphatase (Mem-LiALP, EC 3.1.3.1) is a high-molecular-mass liver alkaline phosphatase (ALP) present in metastatic, infiltrative and cholestatic liver disease. Shedding of hepatocyte plasma membrane fragments (LiPMF) is thought to be responsible for the appearance of Mem-LiALP in the circulation. Several other membrane-bound enzymes, such as gamma-glutamyltransferase (gamma-GT), leucine aminopeptidase (LAP), and 5'-nucleotidase (5'-Nu) are present in the membrane of the shedded LiPMF. By means of immunohistochemical and immunoassay procedures, we presently show that AD-1, a specific monoclonal antibody originally produced against Mem-LiALP, reacts with LAP, a constituent of the human liver plasma membrane. Using AD-1 as an immunosorbant, we isolated circulating LiPMF from cholestatic sera to a high level of purity and separated it from other high-molecular-mass material, such as liver ALP or similar lipoprotein-X complexes. These purified membrane fragments retained their biochemical characteristics. Glycosyl-phosphatidylinositol anchor bearing liver ALP (Anch-LiALP) could be released from the LiPMF by Triton X-100. Whereas ALP was released upon treatment of AD-1 purified LiPMF with phospholipase C, phospholipase D only cleaved the glycosyl-phosphatidylinositol anchor following detergent solubilization of the enzyme. Serum LiPMF from patients with different kinds of cholestatic liver disease were bound onto AD-1 coated nitrocellulose disks and the activity of four membrane-bound enzymes (LAP, ALP, 5'Nu, gamma-GT) was analyzed. A considerable interindividual variation of enzyme activities was observed, suggesting some heterogeneity in the membrane composition of these fragments.

5'-Nucleotidase↗

PubMed2002

Identification of 33 polymorphisms in the adipocyte-derived leucine aminopeptidase (ALAP) gene and possible association with hypertension.

Adipocyte-derived leucine aminopeptidase (ALAP) inactivates angiotensin II and/or generates bradykinin in the kidney, suggesting a possible role for ALAP in the regulation of blood pressure. We considered the hypothesis that genomic variants of the ALAP gene are associated with hypertension or individual variations in blood pressure. We screened for mutations in the ALAP gene in 48 unrelated Japanese individuals and identified 33 polymorphisms including 15 novel polymorphisms. We then performed a two-stage analysis. In the first stage, the eight missense polymorphisms were evaluated for associations with blood pressure in 96 apparently healthy individuals. In the second stage, only the most promising polymorphisms were evaluated for association with essential hypertension in 143 hypertensive and 348 normotensive subjects. Among the eight missense polymorphisms, the Ile276Met and Lys528Arg polymorphisms showed significant association with blood pressure. Subsequent analysis confirmed association between the Lys528Arg polymorphism and essential hypertension. The estimated odds ratio for essential hypertension was 2.3 for presence of the Arg allele at codon 528, in comparison with presence of the Lys/Lys genotype (P = 0.004). These findings support involvement of ALAP in the regulation of blood pressure.

Adipocytes↗

PubMed2005

Novel inhibitors targeted to methionine aminopeptidase 2 (MetAP2) strongly inhibit the growth of cancers in xenografted nude model.

Inhibition of angiogenesis is emerging as a promising strategy for the treatment of cancer. In our study reported here, the effects of 4 highly potent methionine aminopeptidase 2 (MetAP2) inhibitors, IDR-803, IDR-804, IDR-805 and CKD-732 (designed by structure-based molecular modeling), on angiogenesis and tumor growth were assessed. Concentrations of these inhibitors as low as 2.5 nM were able to inhibit the growth of human umbilical vein endothelial cells (HUVEC) by as much as 50%, arresting growth in the G1 stage of mitosis. An intracellular accumulation of p21(WAF1/Cip1) protein was also observed. Furthermore, at higher concentrations (25 nM) of these 4 MetAP2 inhibitors, a significant induction of apoptosis was apparent in the same HUVEC cultures. As a result of these findings, the possible anticancer effects of these inhibitors were examined, utilizing the SNU-398 hepatoma cell line. Interestingly, pretreatment with these inhibitors led to an increased number of apoptotic cells of up to 60% or more, compared to untreated controls. Moreover, utilizing an in vivo xenografted murine model, these inhibitors suppressed the growth of engrafted tumor. In conclusion, these 4 inhibitory compounds potently exert an antiangiogenic effect to inhibit the growth of cancers in vivo and could potentially be useful for the treatment of a variety of cancers.

Aminopeptidases↗

PubMed1995

Yeast aminopeptidase I is post-translationally sorted from the cytosol to the vacuole by a mechanism mediated by its bipartite N-terminal extension.

Transport of aminopeptidase I (API) to the vacuole appears to be insensitive to blockage of the secretory pathway. Here we show that the N-terminal extension of the 61 kDa precursor of API (pAPI) is proteolytically processed in two sequential steps. The first step involves proteinase A (PrA) and produces a 55 kDa unstable intermediate (iAPI). The second step involves proteinase B (PrB) and converts iAPI into the 50 kDa stable, mature enzyme (mAPI). Reversion of the cup1 growth phenotype by a pAPI-CUP1 chimera indicates that pAPI is transported to the vacuole by a post-translational mechanism. Deletion of the first 16 amino acids results in accumulation of the truncated protein in the cytosol, indicating that pAPI is actively transported to the vacuole. The chimera pAPI-myc, constructed by fusing a myc tag to the C-terminus of pAPI, was exploited to dissect the mechanism of pAPI transport. Cell fractionation studies show the presence of iAPI-myc and mAPI in a fraction of vacuoles purified by density centrifugation. This and the sequential conversion of pAPI-myc into iAPI-myc and mAPI lacking the myc tag is consistent with insertion of pAPI into the vacuolar membrane through its N-terminal extension. The specific mechanism of API sorting demonstrates a new pathway of protein transport in vacuolar biogenesis.

Amino Acid Sequence↗

PubMed2005

Evidence of a dominant negative mutant of yeast methionine aminopeptidase type 2 in Saccharomyces cerevisiae.

Eukaryotic methionine aminopeptidase type 2 (MetAP2, MetAP2 gene (MAP2)), together with eukaryotic MetAP1, cotranslationally hydrolyzes initiator methionine from nascent polypeptides when the side chain of the second residue is small and uncharged. In this report, we took advantage of the yeast (Saccharomyces cerevisiae) map1 null strain's reliance on MetAP2 activity for the growth and viability to provide evidence of the first dominant negative mutant of eukaryotic MetAP2. Replacement of the conserved His(174) with alanine within the C-terminal catalytic domain of yeast MetAP2 eliminated detectable catalytic activity against a peptide substrate in vitro. Overexpression of MetAP2 (H174A) under the strong GPD promoter in a yeast map1 null strain was lethal, whereas overexpression under the weaker GAL1 promoter slightly inhibited map1 null growth. Deletion mutants further revealed that the N-terminal region of MetAP2 (residues 2-57) is essential but not sufficient for MetAP2 (H174A) to fully interfere with map1 null growth. Together, these results indicate that catalytically inactive MetAP2 is a dominant negative mutant that requires its N-terminal region to interfere with wild-type MetAP2 function.

Aminopeptidases↗

PubMed1984

Formation of desTyr dynorphins 5-17 by a purified cytosolic aminopeptidase of rat brain.

An aminopeptidase purified to homogeneity from cytosol of rat brain cleaved dynorphins having 5-17 residues and selected proenkephalins at the Tyr-Gly bond only to release Tyr and the desTyr fragments. The enzyme protein consisted of a single polypeptide chain of Mr 103,000 and was inhibited by puromycin, bestatin, and chelating reagents to yield Ki in the micromolar range. Hydrolysis of Leu-2-naphthylamide was inhibited by Dyn 1-5 competitively (Ki, 18 microM); the Km for Dyn 1-5, the best substrate of the series, was 63.8 microM (Kcat/Km ratio 580 mM-1 min-1). Rates of N-tyrosyl release decreased with peptide size; the presence of Arg in position 6 led to 50% loss for Dyn 1-6, and the C-terminal extensions of Dyn 1-13 or 1-17 to a 98% loss in activity as compared to the pentapeptide. Rapid degradation of small peptides is consistent with a paracrine (neurotransmitter) role as compared to the postulated precursor or exocrine roles for the dynorphins with 13 residues or more.

Aminopeptidases↗