[Histochemical demonstration of leucine-aminopeptidase activity in the normal human placenta].
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Aminopeptidases↗
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Results for “LEUCINE AMINOPEPTIDASE”
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Phosphorus amino acid analogues as inhibitors of leucine aminopeptidase.
A variety of phosphorus amino acid and dipeptide analogues have been synthesized and evaluated as inhibitors of the metalloenzyme leucine aminopeptidase from porcine kidney. Two phosphonate dipeptides were found to be modest inhibitors of the enzyme (8e, Ki = 58 microM; 8h, Ki = 340 microM). The phosphinic acid (17-OH) and phosphinamide (17-NH2) analogues related to bestatin were prepared by condensation of the phosphinate amino acid derivative 11, via a trivalent phosphonite ester 12, with leucine isocyanate derivatives 13. These compounds also proved to be unexceptional in their inhibition of LAP (17-O-, Ki = 56 microM; 17-NH2, Ki = 40 microM). A series of simple (alpha-aminoalkyl)phosphonic acid and -phosphinic acids were also evaluated, and the most potent inhibitors were found to be the phosphonic acid analogues of L-Leu and L-Phe [R)-3e, Ki = 0.23 microM; (R)-3h, Ki = 0.42 microM). Slow-binding behavior was observed for (R)-3e (kon = 400 +/- 55 M-1 s-1) and (R)-3h (kon = 445 +/- 50 M-1 s-1). The phosphinic acid analogues of Leu and Phe are 100-fold less potent than the phosphonate derivatives. The fact that tetrahedral phosphorus analogues are less potent inhibitors of LAP than they are of other zinc peptidases suggests that the mechanism of LAP may be fundamentally different than that of the latter enzymes.
Amino Acids↗
Leucine aminopeptidase activity in blood and hemopoietic organs of different vertebrates. A histochemical comparative study.
Leucine aminopeptidase distribution was examined in the cells of peripheral blood and hemopioetic organs of different Vertebrate classes using histoenzymatic methods. Various degrees of staining intensity were observed in leukocytes of the distinct Vertebrates: in particular, leukocytes of fresh water Osteichthyes and Mammals looked strongly positive, whereas leukocytes of Amphibians, Reptiles, and Birds were barely reactive without great differences among species. The observations should be related to enzyme molecular structure and to kinetics and substrate specificity.
Amphibians↗
Leucine aminopeptidase from swine kidney: purification, molecular weight, subunit and amino acid composition.
A homogeneous leucine aminopeptidase was obtained from mixed breed swine kidneys by means of chromatography on a special column. After coupling an inhibitor, N-sulfanilyl N'-butylcarbamide, to Sepharose 6B, the derivative did not absorb the enzyme, but absorbed a non-enzymatically active protein. The enzyme showed a single band on disc-gel electrophoresis. The molecular weight of the enzyme has 320,000 daltons. In 6 M guanidine solution containing 0.5% 2-mercaptoethanol at pH 8, the enzyme exhibited a molecular weight of 53,000 on equilibrium centrifugation. A similar value, 54,000, for the subunit of the enzyme was found on SDS-gel electrophoresis. The amino acid composition of the enzyme is also reported.
Amino Acids↗
Expression of adipocyte-derived leucine aminopeptidase in endometrial cancer. Association with tumor grade and CA-125.
Adipocyte-derived leucine aminopeptidase (A-LAP) is a novel zinc-metallopeptidase involved in angiotensin II (AngII) metabolism, cell migration and antigen presentation. These functions are implicated in the progression of cancer, whereas A-LAP expression and involvement have not been studied in any type of cancer. We investigated the expression of A-LAP in endometrial cancer as well as its association with angiogenesis and clinicopathological features. Immunohistochemical staining of 58 endometrial endometrioid adenocarcinoma specimens revealed that 37 were A-LAP immunoreactive. We also found that A-LAP staining correlated with histological tumor grade in a significant and reverse manner. In addition, serum CA-125 levels in patients with A-LAP positive cancers were significantly higher. However, contrary to our hypothesis that A-LAP suppresses angiogenic activity via AngII metabolism, A-LAP expression was not associated with the microvessel count determined by CD34 immunostaining. Our results suggest that A-LAP is involved in endometrial cancer cell growth and differentiation. However, further studies, especially of the biological roles of A-LAP, are required to confirm this notion.
Adipocytes↗
Immunochemical identification of the cell surface bound leucine aminopeptidase, the target enzyme for the immunostimulant bestatin.
The microbial product bestatin is known to inhibit soluble microsomal- and cytosolic leucine aminopeptidase (Leu-APm and Leu-APc) as well as aminopeptidase B (AP-B). To clarify which of these enzymes is the target for bestatin on the cell surface, indirect immunofluorescence studies with antisera raised against purified Leu-APm and AP-B were performed. These antibodies (anti-Leu-APm and anti-AP-B) were found to react with intracellularly localized Leu-APm and AP-B of ethanol-treated L5178y cells. Using non-treated L5178y cells fluorescence was detected only on the cell surface after incubation with anti-Leu-APm. To confirm the supposition that only Leu-APm is present on the cell surface, the AP from the cell membrane was solubilized and analyzed electrophoretically. Based on relative migration data it could be shown, that the cell surface is charged with Leu-APm and not with detectable amounts of Leu-APc or AP-B. Moreover, it could be demonstrated that the solubilized Leu-APm binds to [3H]bestatin.
Adjuvants, Immunologic↗
Histidines 345 and 378 of Bacillus stearothermophilus leucine aminopeptidase II are essential for the catalytic activity of the enzyme.
The conserved histidine residues, His-191, His-227, His-345, and His-378, in Bacillus stearothermophilus leucine aminopeptidase II (LAPII) were replaced with leucine by site-directed mutagenesis. The overexpressed wild-type and mutant enzymes have been purified by nickel-chelate chromatography and their molecular masses were approximately 44.5 kDa. Under assay conditions, no LAP activity was detected in H345L and H378L. Although the Km value for H191L increased more than 30% with respect to the wild-type LAPII, alteration in this residue did not lead to a significant change on the catalytic efficiency. The 39% decrease in Kcat/Km for H227L was partly caused by a 3.9-fold increase in Km value. Based on these results, it is suggested that His-345 and His-378 play a crucial role in the catalytic reaction of B. stearothermophilus LAPII.
Amino Acid Sequence↗
Assay of leucine aminopeptidase activity in vitro using large-pore reversed-phase chromatography with fluorescence detection.
A chromatographic method for determination of leucine aminopeptidase (LAP) activity in complex matrices is described. L-Leucine-beta-naphthylamide was used as the substrate and its hydrolytic product, beta-naphthylamine, was monitored by fluorescence at 280 nm excitation and 400 nm emission wavelengths. Under optimized conditions, the components in the incubation mixture were baseline separated and eluted out of a large-pore (300 angstroms) reversed-phase C4 column (RPC4) within 15 min with a non-linear gradient elution of methanol (0.05% (v/v) trifluoroacetic acid additive). The detection limit of the hydrolytic product reached 0.35 pmol at three time signal-to-noise (S/N) ratio with 5 microl sample injection. The method showed a wide dynamic range for quantitation of both the hydrolytic product (10 ng/ml to 80 microg/ml) and LAP (0.1-46.0 microg/ml) with correlation coefficient larger than 0.998 and reproducibility <3 and 10% R.S.D. (n=3), respectively. A fairly broad range of incubation time could be selected within 1 h. The LAP activities and concentrations in rabbit serum, tears, and mouse lens homogenates were determined to be 41.8 (0.3 mg/ml), 2.8 (40.0 microg/ml), and 1.6 pmol/(microl min) (17.5 microg/ml), respectively, with reproducibility of 2-9% R.S.D. (n=3) and intra- and inter-day variation for the retention time of the hydrolytic product being <1% R.S.D. (n=3). The results indicate that the present method is rapid and sensitive as compared to the conventional one.
Animals↗
Transition state analogue L-leucinephosphonic acid bound to bovine lens leucine aminopeptidase: X-ray structure at 1.65 A resolution in a new crystal form.
The three-dimensional structure of bovine lens leucine aminopeptidase (blLAP) complexed with L-Leucinephosphonic acid (LeuP) has been determined by molecular replacement using the structure of native blLAP as a starting model. Cocrystallization of the enzyme with the inhibitor yielded a new crystal form of space group P321 which has cell dimensions a = 130.4 A and c = 125.4 A. Refinement of the model against data from 7.0 to 1.65 A resolution resulted in a final structure with a crystallographic residual of 0.160 (R(free) = 0.191). The N-terminal amino group of LeuP is coordinated to Zn-489, one phosphoryl oxygen atom bridges both metal ions, and another phosphoryl oxygen atom is coordinated to Zn-488. The side chain of Arg-336 interacts with the inhibitor via three water molecules. LeuP resembles the presumed tetrahedral gem-diolate transition state after direct attack of a water or hydroxide ion nucleophile on the scissile peptide bond. On the basis of the LeuP binding mode and the previous structural and biochemical data, three plausible reaction pathways are evaluated. The two-metal ion mechanisms discussed herein share as common features a metal-bound hydroxide ion nucleophile and polarization of the carbonyl group by the zinc ions. Possible catalytic roles of Arg-336 and Lys-262 in the direct or indirect (through H2O) protonation of the leaving group, in the stabilization of a zinc-bound OH- nucleophile and in the stabilization of the negatively charged intermediate, are discussed. A site 3 metal ion approximately 12 A away from the active site 2 zinc ion probably serves a structural role.
Amino Acid Sequence↗
[Leucine aminopeptidase (arylamidase) activity in the serum. Determination using leucine-p-nitroanilide as substrate].
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Adolescent↗
L-leucinthiol - a potent inhibitor of leucine aminopeptidase.
L-leucinthiol (2-amino-4-methyl-1-pentanethiol) was designed as an inhibitor of leucine aminopeptidase by analogy with sulfhydryl inhibitors of other zinc-containing peptidases. It was synthesized from L-leucinol and shown to be a potent competitive inhibitor of the microsomal aminopeptidase from porcine kidney (Ki = 2.2 X 10(-8) M). The results suggest that the mechanism of aminopeptidase may be similar to that of other metalloproteases.
Animals↗
Spectroscopic and thermodynamic characterization of the E151D and E151A altered leucine aminopeptidases from Aeromonas proteolytica.
Previous kinetic characterization of the glutamate 151 (E151)-substituted forms of the leucine aminopeptidase from Aeromonas proteolytica (Vibrio proteolyticus; AAP) has provided critical evidence that this residue functions as the general acid/base. The close proximity of similar glutamate residues to the bridging water/hydroxide of the dinuclear active sites of metalloenzymes (2.80 and 3.94 angstroms in carboxypeptidase G2 and 3.30 and 3.63 angstroms in AAP), suggests it may also be involved in stabilizing the active-site metal ions. Therefore, the structural perturbations of the dinuclear active site of AAP were examined for two E151-substituted forms, namely E151D-AAP and E151A-AAP, by UV-vis and electron paramagnetic resonance (EPR) spectroscopy. UV-vis spectroscopy of Co(II)-substituted E151A-AAP did not reveal any significant changes in the electronic absorption spectra. However UV-vis spectra of mono- and dicobalt(II) E151D-AAP exhibited a lower molecular absorptivity compared to AAP (23 and 43 M(-1) cm(-1) vs. 56 and 109 M(-1) cm(-1) for E151D-AAP and AAP, respectively) suggesting both Co(II) ions reside in distorted octahedral coordination geometry in E151D-AAP. EPR spectra of [Co_(E151D-AAP)], [ZnCo(E151D-AAP)], and [(CoCo(E151D-AAP)] were identical, with g(perpendicular) = 2.35, g(parallel) = 2.19, and E/D = 0.19, similar to [CoCo(AAP)]. On the other hand, the EPR spectrum of [Co_(E151A-AAP)] was best simulated assuming the presence of two species with (i) g(x,y) = 2.509, g(z) = 2.19, E/D = 0.19, A = 0.0069 cm(-1) and (ii) g(x,y) = 2.565, g(z) = 2.19, E/D = 0.20, A = 0.0082 cm(-1) indicative of a five- or six-coordinate species. Isothermal titration calorimetry experiments revealed a large decrease in Zn(II) affinities, with K(d) values elevated by factors of approximately 850 and approximately 24,000 for the first metal binding events of E151D- and E151A-AAP, respectively. The combination of these data indicates that E151 serves to stabilize the dinuclear active site of AAP.
Aeromonas↗
Relationship between the changes in maternal serum placental leucine aminopeptidase (P-LAP) activity and umbilical artery waveforms in normal pregnancy.
The relation between placental leucine aminopeptidase (P-LAP) activity in maternal sera and umbilical artery waveforms (systolic/diastolic ratio, S/D) obtained by pulsed Doppler has been examined by cross-sectional study in 26 normal pregnancies during weeks 26-38. A negative correlation was seen to exist suggesting that P-LAP may have a role in the regulation of uteroplacental blood flow.
Adult↗
Effects of fish meal as source of protein on intestinal maltase activity and intestinal and renal leucine aminopeptidase activity of growing rats.
The effect of high-protein fish meal on maltase and leucine aminopeptidase (LAPase) activities of the intestinal mucosa as well as the renal LAPase activity was studied. Four groups of female Wistar rats, weighing between 40-60 g, were fed diets with a 4 or 12% protein content of dry matter for 25 days. The protein source was casein for the control groups and fish meal derived from Coryphaenoides rupestris for the test groups. The results show a decrease (p less than 0.005) in intestinal maltase and LAPase activities and renal LAPase activity in animals fed with 12% of fish meal protein compared to those fed with casein, while the rats fed 4% of fish meal protein showed a decrease in intestinal maltase activity and no significant difference in LAPase activity compared with the control group. These results seem to indicate that the intestinal maltase is influenced by the quality and quantity of dietary protein, while the intestinal and renal LAPase activity is only changed by the quality of protein.
Animals↗
Serum leucine aminopeptidase profile in cancers of gastro-intestinal tract with special reference to hepatic metastasis.
Serum leucine aminopeptidase (LAP) was estimated in 30 cases of gastro-intestinal cancers, and compared with 50 age and sex matched controls. Highly significant increase of serum LAP was seen in 7 patients with hepatic metastasis (p less than 0.001) from adenocarcinoma of stomach, colon and rectum. The enzyme values showed a highly significant increase in carcinoma of colon, when compared with different anatomical sites of the gastro-intestinal tract (p less than 0.001). However, in adenocarcinoma of stomach and rectum, significantly increased level of serum LAP was observed (p less than 0.01) which contrasted sharply with the normal enzyme values in squamous cell carcinoma of oesophagus and anal canal.
Female↗
Inactivation of oxytocin and angiotensin II by placental leucine aminopeptidase (P-LAP).
The inactivation of oxytocin and angiotensin II by retroplacental serum and placental leucine aminopeptidase (P-LAP), which had been highly purified from retroplacental serum, was demonstrated using biological methods.
Angiotensin II↗
Leucine aminopeptidases: the ubiquity of LAP-N and the specificity of LAP-A.
The wound-induced leucine aminopeptidase (EC 3.4.11.1) genes, LapA1 and LapA2, from tomato (Lycopersicon esculentum Mill.) were isolated and characterized. The genes were organized in a tandem array with approximately 6 kb separating their coding regions. Quantitation of LapA mRNA levels in conjunction with nuclear run-on experiments indicated that LapA genes were primarily under transcriptional control after wounding and infection with Pseudomonas syringae pv. tomato. In contrast, actin genes were down-regulated after pathogen infection. The sequences of the LapA1 and LapA2 5'-flanking regions were determined and several potential regulatory motifs were identified. Ribonuclease protection studies revealed that LapA1 and LapA2 had short 18-bp 5'-untranslated regions (UTR), both genes were expressed after wounding, and LapA1 mRNAs were 3.3-fold more abundant than LapA2 transcripts. While the region surrounding LapA1 was conserved, the 3'-UTRs and 3'-flanking regions of LapA2 had diverged in two inbred tomato lines. The accumulation of LapA mRNAs and of LAP-A (acidic pI), LAP-N (neutral pI) and LAP-related proteins were examined in two monocot and five dicot species. The LAP-N and 66- and 77-kDa LAP-related proteins were detected in healthy and wounded leaves of all plants examined. The LAP-A proteins were only detected in nightshade and their accumulation was distinct from that observed in tomato.
3' Untranslated Regions↗
Placental and non-placental leucine aminopeptidases during normal pregnancy.
1. A simple and rapid method discriminating between placental and non-placental leucine aminopeptidases (LAP) existing in pregnancy serum is described, which includes technics of heat inactivation at 60 degrees for 30 minutes and inhibition by 0.02 M L-methionine. 2. Serial determinations of serum total, heat-stable and L-methionine-insensitive LAP activities during normal pregnancy were carried out. 3. The results were ascertained by disc electrophoresis and it was found that the heat-stable enzyme corresponds to normal serum LAP; the L-methionine-insensitive enzyme to pregnancy serum LAP of placental origin; the sum of these two to total serum LAP.
Female↗