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PubMed1996

Leucine aminopeptidase enzymuria: quantification of renal tubular damage following extracorporeal shock wave lithotripsy.

To evaluate the extent of renal parenchymal injury following high energy shock wave (HESW) application we measured the urine levels of two renal tubular brush border enzymes: leucine aminopeptidase (LAP) and gamma-glutamyltranspeptidase (GGT) the day before, 24 hours and 7 days after extracorporeal shock wave lithotripsy (ESWL) in 23 patients. All patients had caliceal stones and were treated with a Dornier MPL 9000 lithotriptor under sedoanalgesia. Creatinine concentration of each sample together with total shock wave effect (TSWE) values were also assessed and comparatively evaluated with enzymuria levels. Our results indicated a statistically significant rise in urinary excretion of both enzymes after 24 hours following lithotripsy (p < 0.05). All these values returned to normal limits within 7 days after ESWL (p > 0.05). Transient tubular damage due to HESW was found to be related to TSWE values assessed in our group.

Adolescent↗

PubMed1999

Vaccination with cathepsin L proteinases and with leucine aminopeptidase induces high levels of protection against fascioliasis in sheep.

The potential of different parasite proteinases for use as vaccine candidates against fascioliasis in sheep was studied by vaccinating animals with the cathepsin L proteinases CL1 and CL2 and with leucine aminopeptidase (LAP) purified from adult flukes. In the first trial, sheep were immunized with CL1 or CL2 and the mean protection levels obtained were 33 and 34%, respectively. Furthermore, a significant reduction in egg output was observed in sheep vaccinated either with CL1 (71%) or with CL2 (81%). The second trial was performed to determine the protective potential of the two cathepsin L proteinases assayed together, as well as in combination with LAP, and of LAP alone. The combination of CL1 and CL2 induced higher levels of protection (60%) than those produced when these enzymes were administered separately. Those sheep that received the cocktail vaccine including CL1, CL2, and LAP were significantly protected (78%) against metacercarial challenge, but vaccination with LAP alone elicited the highest level of protection (89%). All vaccine preparations induced high immunoglobulin G titers which were boosted after the challenge infection, but no correlations between antibody titers and worm burdens were found. However, the sera of those animals vaccinated with LAP contained LAP-neutralizing antibodies. Reduced liver damage, as assessed by the level of the liver enzyme gamma-glutamyl transferase, was observed in the groups vaccinated with CL1, CL2, and LAP or with LAP alone.

Animals↗

PubMed2005

Construction and one-step purification of Bacillus kaustophilus leucine aminopeptidase fused to the starch-binding domain of Bacillus sp. strain TS-23 alpha-amylase.

The starch-binding domain of Bacillus sp. strain TS-23 alpha-amylase was introduced into the C-terminal end of Bacillus kaustophilus leucine aminopeptidase (BkLAP) to generate a chimeric enzyme (BkLAPsbd) with raw-starch-binding activity. BkLAPsbd, with an apparent molecular mass of approximately 65 kDa, was overexpressed in Escherichia coli M15 cells and purified to homogeneity by nickel-chelate chromatography. Native PAGE and chromatographic analyses revealed that the purified fusion protein has a hexameric structure. The half-life for BkLAPsbd was 12 min at 70 degrees C, while less than 20% of wild-type enzyme activity retained at the same heating condition. Compared with the wild-type enzyme, the 60% decrease in the catalytic efficiency of BkLAPsbd was due to a 91% increase in K (m) value. Starch-binding assays showed that the K (d) and B (max) values for the fusion enzyme were 2.3 microM and 0.35 micromol/g, respectively. The adsorption of the crude BkLAPsbd onto raw starch was affected by starch concentration, pH, and temperature. The adsorbed enzyme could be eluted from the adsorbent by 2% soluble starch in 20 mM Tris-HCl buffer (pH 8.0). About 49% of BkLAPsbd in the crude extract was recovered through one adsorption-elution cycle with a purification of 11.4-fold.

Bacillus↗

PubMed1988

Steady-state kinetics of hydrolysis of dansyl-peptide substrates by leucine aminopeptidase.

Stopped-flow fluorescence experiments have been carried out at 23 degrees C to study the hydrolysis of Leu-Gly-NHNH-Dns [Dns = 5-(dimethylamino)naphthalene-1-sulfonyl] and Leu-Gly-NH(CH2)2NH-Dns by porcine kidney cytosol leucine aminopeptidase (LAP). Experiments have been performed with LAP species containing Mg(II), Mn(II), Ni(II), Cu(II), Zn(II), and no metal ion at the regulatory metal binding site. The fluorescence changes observed on hydrolysis of these dansyl substrates by LAP arise from changes in the concentration of substrate. Several kinetic relationships have been developed that allow the steady-state kinetic parameters for these reactions to be determined from the stopped-flow fluorescence traces. When any of the five metal ions are bound at the regulatory site, kcat and KM are both raised to approximately the same extent with the result that the maximum increase observed for kcat/KM is only approximately twofold. The effects of these metal ions on kcat, KM, and kcat/KM observed for these substrates differ markedly from those for less physiologically relevant substrates, such as Leu-p-nitroanilide, that do not have amino acids on both sides of the scissile bond. This suggests that earlier conclusions regarding the effect of the regulatory metal ion on the activity of LAP may have been misleading and casts doubt as to whether the term "regulatory site" has validity in the context of LAP-catalyzed reactions under physiological conditions.

Algorithms↗

PubMed2005

Placental leucine aminopeptidase might regulate the effects of oxytocin with resolution in endothelial cells.

BACKGROUND: Oxytocin (OT) is known to cause vascular relaxation via nitric oxide (NO) production and proliferation of endothelial cells. Previously we revealed that human umbilical vascular endothelial cells (HUVECs) expresses placental leucine aminopeptidase (P-LAP)/insulin regulated aminopeptidase (IRAP), which becomes translocation to the cell surface on activation of oxytocin receptor (OTR). However, the physiological roles of P-LAP in HUVECs have not been elucidated. MATERIAL/METHODS: Actions of OT on HUVECs transfected with a copy of the human P-LAP gene were therefore examined with a focus on changes in parameters linked to OTR such as calcium mobilization, NO production and cell proliferation. RESULTS: Cell surface P-LAP activity was significantly elevated (approximately 3 fold) in P-LAP overexpressing-HUVECs and overexpression of P-LAP resulted in remarkable inhibition of OT effects on HUVECs such as cell proliferation, [Ca2+]i, and NO production. CONCLUSIONS: These findings suggested that P-LAP on the plasma membrane in HUVECs regulates the effects of OT with resolution around OTR.

Calcium↗

PubMed1987

Identification of the FTBL protein of Sumner and Dounce as a leucine aminopeptidase.

The crystalline beef liver protein of Sumner and Dounce (A. L. Dounce, P. Z. Allen, and G. A. Mourtzikos (1978) Arch. Biochem. Biophys. 188, 251-265) termed FTBL (football) protein because of the shape of its crystals, has been identified as a crystalline leucine aminopeptidase (LAP), on the basis of its high specific LAP activity and coincidence of its N terminal amino acid sequence (30 amino acids) with that of beef eye lens LAP. Amino acid analyses of the two proteins are also in reasonable agreement when based on the exact monomer molecular weight of beef eye lens protein obtained by the van Loon group ((1982) J. Biol. Chem. 257, 7077-7081). Our previously published monomer molecular weight of the FTBL protein was 25% too high, leading to the erroneous conclusion that the beef liver FTBL-LAP protein was a tetramer rather than a hexamer, as found by the van Loon group for beef lens LAP. The present report, taken together with our first paper on the FTBL protein establishes that the FTBL-LAP protein has been isolated from beef kidney and beef spleen as well as from beef liver. We now find that the properties of FTBL-LAP protein indicate that it is the same protein as beef eye lens LAP. The cellular and intracellular distributions of the FTBL-LAP protein have been considered in our first publication on the FTBL protein.

Amino Acid Sequence↗

PubMed1992

Three-dimensional structural resemblance between leucine aminopeptidase and carboxypeptidase A revealed by graph-theoretical techniques.

Using 3-D searching techniques based on algorithms derived from graph theory we have established a striking structural similarity between the structure of bovine carboxypeptidase A and that of the C-terminal domain of bovine leucine aminopeptidase. There is no significant sequence homology between the aminopeptidases and the carboxypeptidases but the strong structural relationship detected in this complex fold suggests that there may be a very remote divergent evolutionary relationship between these two enzyme classes.

Animals↗

PubMed1982

Simultaneous determinations of plasma oxytocin and serum placental leucine aminopeptidase (P-LAP) during late pregnancy.

1. Plasma oxytocin concentrations were measured by radioimmunoassay in 231 normal pregnant patients. The mean level of the maternal plasma oxytocin increases gradually with advancing pregnancy, culminating in a marked increase during the last two months. 2. Serum placental leucine aminopeptidase (P-LAP) activities and oxytocinase were measured serially in 78 obstetrically normal pregnant patients during late pregnancy. The daily mean P-LAP activity rises progressively during late pregnancy, reaching a relatively high level at 11 days prior to the onset of labor, then fluctuates slightly until the onset of labor. 3. Simultaneous serial measurements of plasma oxytocin levels and serum P-LAP activities in 9 normal pregnant women during late pregnancy show that the onset of labor is preceded by either an increase in the oxytocin level or a decrease in the P-LAP activity.

Adult↗

PubMedDate not supplied

[Effect of genotypes of unspecific esterases and leucine aminopeptidase loci on the temporal dynamics of morphological variation in the black sea mussel Mytilus galloprovincialis Lam].

In the Black Sea mussel Mytilus galloprovincialis Lam., the dependence of variation in 21 morphological characters of the shell on the genotype at the polymorphic loci encoding unspecific esterases and leucine aminopeptidase was estimated by means of ANOVA in 12 samples differing in age and belonging to different generations. The genotype at these loci was found to have no constant influence on the dynamics of variation in any morphological character. The effect of the parameters of polymorphism was ambiguous: a factor having a significant effect on a certain character in one sample might have no such effect (or produce an effect on a different character) in another sample. A directional change in the significant effect of genotypes during a certain period of generation's lifetime was revealed. The dependence of the effect of polymorphism at the studied loci on ecological conditions is discussed.

Age Factors↗

PubMed2006

Binding structure of the leucine aminopeptidase inhibitor microginin FR1.

Natural bioactive compounds are of general interest for pharmaceutical research because they may serve as leads in drug development campaigns. Among them, microginins are linear peptides known to inhibit various exopeptidases. The crystal structure of microginin FR1 from Microcystis sp. bound to bovine lens leucine aminopeptidase was established at 1.73 Angstrom resolution. The observed binding structure could be beneficial for the design of potent aminopeptidase inhibitors.

Amino Acid Sequence↗

PubMed2005

Purification and characterization of hyperthermotolerant leucine aminopeptidase from Geobacillus thermoleovorans 47b.

A thermophilic bacterium, which we designated as Geobacillus thermoleovorans 47b was isolated from a hot spring in Beppu, Oita Prefecture, Japan, on the basis of its ability to grow on bitter peptides as a sole carbon and nitrogen source. The cell-free extract from G. thermoleovorans 47b contained leucine aminopeptidase (LAP; EC 3.4.11.10), which was purified 164-fold to homogeneity in seven steps, using ammonium sulfate fractionation followed by the column chromatography using DEAE-Toyopearl, hydroxyapatite, MonoQ and Superdex 200 PC gel filtration, followed again by MonoQ and hydroxyapatite. The enzyme was a single polypeptide with a molecular mass of 42,977.2 Da, as determined by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry, and was found to be thermostable at 90 degrees C for up to 1 h. Its optimal pH and temperature were observed to be 7.6-7.8 and 60 degrees C, respectively, and it had high activity towards the substrates Leu-p-nitroanilide (p-NA)(100%), Arg-p-NA (56.3%) and LeuGlyGly (486%). The K(m) and V(max) values for Leu-p-NA and LeuGlyGly were 0.658 mM and 25.0 mM and 236.2 micromol min(-1) mg(-1) protein and 1,149 micromol min(-1) mg(-1) protein, respectively. The turnover rate (k(cat)) and catalytic efficiency (k(cat)/ K(m)) for Leu-p-NA and LeuGlyGly were 10,179 s(-1) and 49,543 s(-1) and 15,470 mM(-1 ) s(-1) and 1981.7 mM(-1 ) s(-1), respectively. The enzyme was strongly inhibited by EDTA, 1,10-phenanthroline, dithiothreitol, beta-mercaptoethanol, iodoacetate and bestatin; and its apoenzyme was found to be reactivated by Co(2+) .

Bacillaceae↗

PubMed2003

Possible role of placental leucine aminopeptidase in the antiproliferative effect of oxytocin in human endometrial adenocarcinoma.

PURPOSE: Oxytocin (OT) was reported to inhibit the proliferation of various neoplastic tissues and cells, however, the regulation system remains unclear. This study examined the role of OT and its regulatory ability in endometrial adenocarcinoma. EXPERIMENTAL DESIGN: To investigate the possible function of placental leucine aminopeptidase (P-LAP) in endometrial adenocarcinoma, we transfected P-LAP cDNA into A-MEC cells, showing the lowest enzyme activity of P-LAP. Also we examined P-LAP protein expression in human endometrial adenocarcinoma. RESULTS: We demonstrated the presence of P-LAP, which is identical to cystine aminopeptidase as oxytocinase, in human endometrial adenocarcinoma tissues and found that the expression of P-LAP increase with advances in the grade. Exposure of endometrial adenocarcinoma cell lines to OT caused dose- and time-dependent inhibition of growth. Treatment with 10(-7) M OT for 72 h reduced cell growth by 62, 25, and 30% in A-MEC, HEC1A, and Ishikawa cells, respectively. P-LAP-transfectant cells not only partially recovered from OT-induced growth inhibition but also showed a higher growth rate than parental cells under condition without OT. An OT receptor antagonist and a protein kinase A inhibitor blocked OT-induced growth inhibition in A-MEC and A-MEC-pc cells but not in A-MEC-LAP cells. CONCLUSIONS: These findings suggested that P-LAP might be functionally positive on carcinoma cell growth by degrading suppressive peptides such as OT.

Adenocarcinoma↗

PubMed2005

Possible involvement of adipocyte-derived leucine aminopeptidase via angiotensin II in endometrial carcinoma.

OBJECTIVE: It has recently been appreciated that a local autocrine or paracrine renin-angiotensin system (RAS) may exist in a number of tissues. Angiotensin II (AngII) is a potent RAS-derived vasoconstrictor peptide, and it is involved in tumor angiogenesis. We have cloned human adipocyte-derived leucine aminopeptidase (A-LAP), which degrades Ang II. This study investigated whether the expression of A-LAP, Ang II, angiotensin type I receptor (AT1R) and vascular endothelial growth factor (VEGF) correlates with clinicopathologic factors and prognosis in patients with endometrial endometrioid adenocarcinoma. METHODS: Histologic sections of formalin-fixed, paraffin-embedded specimens from 94 primary endometrial carcinomas were stained for A-LAP, AngII, AT1R and VEGF using each antibody. Disease-free survival (DFS) and other clinicopathologic characteristics were analyzed according to the intensity of each staining. RESULTS: Of 94 cases, 91 (96.8%) showed specific A-LAP immunostaining. A-LAP expression demonstrated negative correlations with myometrial invasion (p = 0.01) and vascular infiltration (p = 0.01). Of 94 cases, 77 (81.9%) showed specific AngII immunostaining. We found a positive correlation between AngII expression and surgical stage (p = 0.01). Of 94 cases, 56 (59.6%) showed specific AT1R immunostaining and 73 (77.7%) specific VEGF immunostaining. We found a positive correlation between VEGF expression and lymph node metastasis (p = 0.05). AngII and AT1R expression predicted a significantly poorer prognosis. Contrarily, A-LAP expression indicated a significantly more favorable prognosis in endometrial endometrioid adenocarcinoma patients. Multivariate analysis demonstrated that A-LAP expression (odds ratio, 0.12; 95% confidence interval, 0.025-0.618; p = 0.01) was an independent prognostic factor. CONCLUSIONS: In this study, we demonstrated the existence of local RAS and A-LAP in endometrial endometrioid adenocarcinoma as prognostic predictors of clinical outcome. These findings suggest that the assessment of RAS and A-LAP status provides clinically useful prognostic information in patients with endometrial carcinoma.

Adipocytes↗

PubMed2004

Placental leucine aminopeptidase (P-LAP) expression is associated with chemosensitivity in human endometrial carcinoma.

OBJECTIVE: Although treatment for advanced or recurrent endometrial carcinoma has improved over recent years with the introduction of paclitaxel- and platinum-based chemotherapy, in most, the disease remains incurable because of resistance to chemotherapy. In the previous study, we have shown that placental leucine aminopeptidase (P-LAP) is associated with poor prognosis. The objective of this study was to determine whether P-LAP expression affects the chemosensitivity in endometrial carcinoma patients. METHODS: Here, we investigated the effect of P-LAP to response for paclitaxel and carboplatin in advanced and recurrence endometrial carcinoma. Furthermore, we transfected P-LAP cDNA into endometrial carcinoma cells (AMEC) and investigated cell growth and apoptosis by paclitaxel or carboplatin. RESULTS: In 15 of 17 patients, P-LAP was positive. Twelve of seventeen patients were evaluable for response. Among the eight patients strongly positive for P-LAP, only two patients (25%) showed PR. However, all four patients who were weakly positive for P-LAP showed either complete response (CR) or partial response (PR). P-LAP overexpressor (P-LAP2 and P-LAP8) and a vector control were used to assay chemosensitivity. P-LAP2 clone displayed a 1.7-fold increase in IC(50) against paclitaxel and carboplatin when compared with the vector control, and P-LAP8 clone displayed a 1.6-fold increased in IC(50) against paclitaxel and carboplatin when compared with V1. Compared to vector control cells, apoptotic effect by carboplatin treatment was clearly inhibited in P-LAP2 and P-LAP8 cells. Carboplatin, 10(-6) M, induced the 12.5-fold rate of apoptosis compared to that without treatment at 48 h in vector control cells. However, in P-LAP2 and P-LAP8 clones, 10(-6) M carboplatin induced only 3.2- and 5.1-fold rates of apoptosis, respectively, compared to that of without treatment. CONCLUSIONS: P-LAP was suggested to be involved in reducing chemosensitivity and may be a therapeutic target in endometrial carcinoma.

Aged↗

PubMed1993

Value of serum alkaline phosphatase, aminotransferases, gamma-glutamyl transferase, leucine aminopeptidase, and bilirubin in the distinction between benign and malignant diseases causing jaundice and cholestasis: results from a prospective study.

A prospective study of 187 patients with jaundice and 33 patients with unjaundiced cholestasis was carried out to evaluate the value of serum bilirubin, alkaline phosphatase (ALP), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), gammaglutamyl transferase (GGT), and leucine aminopeptidase (LAP) in the differential diagnosis between benign and malignant diseases causing jaundice and/or cholestasis. In the patients with malignant disease (n = 60), the mean serum bilirubin and ALP concentrations were significantly higher (p < 0.001) than in the patients with benign disease (n = 160). Serum LAP, ASAT, ALAT, or GGT levels did not show any significant differences. A stepwise discriminant analysis was carried out to evaluate the value of laboratory tests in predicting malignancy. The discrimination function is DF = bilirubin x 0.71 + ALP x 0.58 + ASAT x -0.24 + ALAT x 0.18 + LAP x 0.08 + GGT x -0.22. When the discriminant function was considered as a diagnostic score (DS), the sensitivity of it in detecting malignancy was 58% with a specificity of 89% and an efficiency of 81%. The DS of serum bilirubin and ALP reached the sensitivity of 61% with a 87% specificity and an efficiency of 79%. The post-test probability of malignant disease calculated by in this test combination was 69%. The LR+ was 4.8 and LR- 0.44. In conclusion, serum bilirubin and alkaline phosphatase seem to be the most potential tests of these laboratory tests in distinguishing benign and malignant causes of jaundice and/or cholestasis, high levels being suggestive of malignant disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1986

Inhibition of leucine aminopeptidase (LAP) activity in the small intestines of rats exposed to dietary cadmium.

Male and female rats, 8 weeks old, were exposed for 40 days to a sole source of drinking water containing 0.2 g cadmium/l. Similar control rats received deionised water over the same period. Post mortem, selected areas of small intestinal tissue from each rat were examined histologically and assayed for cadmium burdens and leucine aminopeptidase (LAP) activity. A significant inhibition of LAP activity occurred in the duodenums of experimental rats, whilst in the distal ileums LAP activity was slightly higher than that of control rats. Cadmium burdens, however, showed a small increase in ileums as compared to duodenums.

Animals↗

PubMed2006

Dietary insulin affects leucine aminopeptidase, growth hormone, insulin-like growth factor I and insulin receptors in the intestinal mucosa of neonatal pigs.

BACKGROUND: Recent studies suggest that milk-borne insulin may regulate the development of the gastrointestinal tract in neonatal mammals. OBJECTIVES: To explore the mechanism by which milk-borne insulin affects gastrointestinal tract development, we examined the effect of dietary insulin on the expression levels of leucine aminopeptidase (LAP) and insulin-like growth factor I (IGF-I), as well as its effect on growth hormone (GH), IGF-I and insulin receptors in the small intestinal mucosa of neonatal pigs. METHODS: Five piglets were anesthetized and sampled within 2-4 h after birth. They were not allowed to suckle and were used as newborn controls (group N). Ten other piglets from 5 litters were randomly divided into group M (n=5), which was fed cow's milk, and group MI (n=5), which was fed cow's milk and insulin (2.5 mg/l). Piglets in groups M and MI were artificially fed for 3 days and then sampled. Total RNA in their intestinal mucosa was extracted with Tripure reagents (Roche, USA). Reverse transcription PCR (RT-PCR) was used to semi-quantify mRNA levels of target genes and 18S rRNA was used in an RT-PCR system as an internal control. PCR products were loaded onto a 9% nondenaturing polyacrylamide gel. The gel was stained by silver staining agents. Digital photos were taken and the strength of the band areas was quantified using software. RESULTS: The results showed that the DNA contents and LAP activity in the small intestines of the piglets in group MI were higher (p<0.05) than in the piglets in group N. Compared with group M, piglets in group MI exhibited significantly increased expression levels of both insulin and GH receptor in the ileum, and LAP in the jejunum (p<0.05); IGF-I receptor expression levels in both the jejunum and ileum were significantly decreased (p<0.01 and p<0.05, respectively), while IGF-I expression was unchanged (p>0.05). CONCLUSION: Collectively, dietary insulin increased mRNA levels of insulin and GH receptor, which could help explain the effect of dietary insulin on receptor-mediated postnatal development of the small intestine. Dietary insulin suppressed IGF-I receptor expression, which may be the result of negative feedback caused when insulin binds to IGF-I receptors.

Animal Feed↗

PubMed1992

Induction of leucine aminopeptidase by interferon-gamma. Identification by protein microsequencing after purification by preparative two-dimensional gel electrophoresis.

The protein previously called "Mr approximately 50/pI approximately 6.9," which we observed to be induced by the immunoregulatory cytokine interferon (IFN)-gamma in human fibroblasts, was purified from a total cell lysate by preparative two-dimensional gel electrophoresis and identified by partial amino-terminal sequencing as leucine aminopeptidase (LAP), a 53-kDa cytosolic exopeptidase. Induction of LAP protein by IFN-gamma, confirmed by immunoblotting with an antiserum raised against bovine lens LAP, is a consequence of induction of LAP mRNA and occurs in all four human cell lines examined: HS153 fibroblasts, ACHN renal carcinoma, A549 lung carcinoma, and A375 melanoma. Induction of LAP mRNA is a secondary response to IFN-gamma, blocked by inhibition of protein synthesis with cycloheximide.

Amino Acid Sequence↗