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PubMed1976

Genetic control and distribution of leucine aminopeptidase in the cultivated chenopods (Chenopodium) and related weed taxa.

Isozymes of leucine aminopeptidase (LAP) in leaf tissue of the cultivated chenopods (Chenopodium quinoa and C. nuttalliae) and their sympatric weedy relatives (C. hircinum and C. berlandieri) can be electrophoretically resolved into a sum total of five anodally migrating bands. These are the products of two unlinked gene loci which apparently reflect genetic duplication via allotetraploidy. Accessions from the Andean and Mexican areas of cultivation are characterized by a lack of electrophoretically detectable variation. Andean weed and cultigen accessions appear to be genetically identical at both Lap loci, as do weed and cultigen material from Mexico. The two cultigens, and their sympatric weeds, can be differentiated by variation at the Lap-B locus, whereas they are monomorphic at Lap-A. This locus is, however, highly polymorphic in weedy C. berlandieri populations of western North America.

Alleles↗

PubMed2003

Overexpression, purification, and characterization of the recombinant leucine aminopeptidase II of Bacillus stearothermophilus.

For expression of Bacillus stearothermophilus NCIB 8924 leucine aminopeptidase II (LAP II) in Escherichia coli regulated by a T5 promoter, the gene was amplified by polymerase chain reaction and cloned into expression vector pQE-32 to generate pQE-LAPII. The His(6)-tagged enzyme was overexpressed in IPTG-induced E. coli M15 (pQE-LAPII) as a soluble protein and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 425 U/mg protein with a final yield of 76%. The subunit molecular mass of the purified protein was estimated to be 44.5 kDa by SDS-PAGE. The temperature and pH optima for the purified protein were 60 degrees C and 8.0, respectively. Under optimal condition, the purified enzyme showed a marked preference for Leu- p-nitroanilide, followed by Arg- and Lys-derivatives. The His(6)-tagged enzyme was stimulated by Co(2+) ions, but was strongly inhibited by Cu(2+) and Hg(2+) and by the chelating agents, DTT and EDTA. The EDTA-treated enzyme could be reactivated with Co(2+) ions, indicating that it is a cobalt-dependent exopeptidase. Taking the biochemical characteristics together, we found that the recombinant LAP II exhibits no important differences from those properties described for the native enzyme.

Anilides↗

PubMed1984

A domain-specific marker for the hepatocyte plasma membrane. II. Ultrastructural localization of leucine aminopeptidase to the bile canalicular domain of isolated rat liver plasma membranes.

Leucine aminopeptidase (LAP) is an integral membrane glycoprotein localized to the apical membrane domain of intestinal and kidney epithelial cells. By indirect immunofluorescence, we have shown that antibodies raised against rat intestinal LAP recognized a similar protein concentrated in the bile canalicular (BC) domain of the hepatocyte in situ (Roman, L.M., and A.L. Hubbard, 1983, J. Cell Biol., 96:1548-1558). We have extended this localization to the ultrastructural level. When a saponin-permeabilized, agarose-embedded plasma membrane (PM) fraction was incubated with affinity-purified anti-LAP, 85% of the protein A-gold particles associated with the three recognizable PM domains were present in the BC. The levels of labeling on the other two domains (sinusoidal and lateral) did not exceed that observed with nonimmune controls. The concentration of LAP in the BC domain in isolated PM sheets prompted us to use this antigen for the affinity isolation of BC membrane (Roman, L.M., and A.L. Hubbard, 1984, J. Cell Biol., 98:1497-1504, companion paper).

Animals↗

PubMed1967

Alkaline phosphatase and leucine aminopeptidase association in plasma of the chicken.

Two electrophoretic vari ants of leucine aminopeptidase show direct association with two genetically controlled forms of alkaline phosphatase. Treatment of plasma with neuraminidase converted the faster-migrating form of both enzymes to slower-moving forms, but plasmas with slowermigrating forms were unaffected by this treatment. The two forms of the enzymes may be due to the presence or absence of a single gene controlling the attachment of sialic acid to the enzyme molecules.

Alkaline Phosphatase↗

PubMed1986

Quantitative immunoferritin localization of leucine aminopeptidase on canine hepatocyte cell surface.

Rabbit monospecific antibody against canine kidney leucine aminopeptidase (LAP) (EC.3.4.11.2) specifically immunoprecipitated kidney and also liver LAP activity from corresponding plasma membrane preparations previously solubilized with Triton X-100. Immunological specificity of the antibody was also shown by immunoblotting of LAP from canine and rat liver plasma membranes and by electrophoretic analyses of the precursor forms in MDCK cells. Canine liver was pre-fixed by perfusion with 0.6% glutaraldehyde and the dissociated liver cells were prepared without losing their polarized structure (22). They were incubated with ferritin antibody conjugates against canine kidney LAP at the saturation level, and the distribution of ferritin particles on the three surface domains of the hepatocytes was investigated quantitatively by counting ferritin particles on the cross-sectional profiles of these surfaces. Our analysis clearly indicated that LAP exists only on the bile canalicular surface, and no significant number of ferritin particles was detected either on the sinusoidal front or on the lateral surface. Ferritin particles were distributed homogeneously both on the microvillar and intermicrovillar regions. All the bile canalicular surface domains of all the hepatocytes were heavily labeled with ferritin particles, without exception.

Animals↗

PubMed1989

Serum leucine aminopeptidase estimation: a sensitive prognostic indicator of invasiveness in breast carcinoma.

Serum Leucine Aminopeptidase (LAP) was studied in 25 patients of breast tumors, and compared with 50 age matched controls. Clinical staging was done in 13 cases of breast carcinoma. Serum LAP was increased but not significantly in stage I (P less than 0.1), while in stage II & III, significant increase was observed (P less than 0.05), but a sharp peak was noted in stage IV with metastasis in liver (P less than 0.001). Serum LAP values were correlated with histological sub-types of breast carcinoma. Highest serum LAP values were observed in filtrating duct carcinoma with productive fibrosis (P less than 0.01), followed by medullary carcinoma and the enzyme values were slightly decreased in intraductal carcinomas.

Adenocarcinoma, Scirrhous↗

PubMedDate not supplied

[Isolation and various properties of leucine aminopeptidase from biological fluids of patients with multiple sclerosis].

Specific form of leucine aminopeptidase (which was distinct from other forms of the enzyme found in blood) was characterized by its physico-chemical properties--pH optimum, substrate specificity, electrophoretic mobility and molecular mass. The enzyme was isolated from biological fluids of patients with multiple sclerosis. Free and bound forms of the enzyme were detected in blood and cerebrospinal fluid. Estimation of the bound enzyme activity has a diagnostic significance.

Electrophoresis, Polyacrylamide Gel↗

PubMedDate not supplied

[Determination of multiple forms of leucine aminopeptidase in blood plasma].

The methods of evaluation of different forms of leucine aminopeptidase (LAP) activity in human serum were elaborated. It was shown, that there are some forms of LAP in serum which has different pH optimum and different sensitivity to L-methionine and other substrates. It was found, that these forms of LAP have different activity in the blood serum of adults and infants. The conclusion was drawn about diagnostic value of LAP forms in the infants with periventricular brain damages.

Aging↗

PubMedDate not supplied

[Activity of serum leucine aminopeptidase lamp in normal and pathologic pregnancies].

The activity of the enzyme leucine aminopeptidase (LAmP) in serum was examined in 305 pregnant women (174 women with normal pregnancy, and 131 women with disturbed pregnancy). The activity of this enzyme increases during the normal development of pregnancy. In disturbed pregnancies, the LAmP activity is lower as compared to normal pregnancies. A peculiar case: toxemia of pregnancy, where the activity of the enzyme is considerably increased. This fact may be compared with the increase of LAmP activity in cases of liver damage. The LAmP activity test in pregnancy is reliable and can be performed practically in every laboratory, thus deserving its place among the many tests meant to assess the normal function of the foeto-placental unit. This test may also help in the diagnostic of some specific pregnancies such as twin pregnancy, hydatidiform mole and extra-uterine pregnancy.

Adolescent↗

PubMed2004

Inactivation of Bacillus stearothermophilus leucine aminopeptidase II by hydrogen peroxide and site-directed mutagenesis of methionine residues on the enzyme.

Leucine aminopeptidases (LAPs) are exopeptidases that remove the N-terminal L-leucine from peptide substrates. Oxidative stability assay showed that the recombinant Bacillus stearothermophilus LAP II (rLAPII) was sensitive to oxidative damage by hydrogen peroxide at the elevated temperature. The H2O2-treated enzyme experienced obvious changes in the secondary structure when the oxidant concentration increased to 300 mM. To investigate the role of methionine residues on the oxidative inactivation, each of the five methionine residues in the rLAPII was replaced with leucine by site-directed mutagenesis. The mutant enzymes with an apparent Mr of approximately 44.5 kDa were overexpressed in Escherichia coli and were purified to homogeneity by nickel-chelate chromatography. The specific activities for Met82Leu, Met88Leu, Met254Leu, and Met382Leu were similar to that of the wild-type enzyme, whereas a reduced activity was observed in Met136Leu. The 50% decrease in the catalytic efficiency (kcat/Km) for Met136Leu was caused by 47% decrease in kcat value. As compared with the wild-type enzyme, all mutant proteins were more sensitive to the oxidant, implying that the methionine residues of B. stearothermophilus LAP II are important for the protection of the enzyme from oxidative inactivation.

Amino Acid Substitution↗

PubMed1990

Intestinal leucine aminopeptidase and alkaline phosphatase: genetic regulation and development in mice.

Intestinal and serum leucine aminopeptidase (LAP) and alkaline phosphatase (AKP) were characterized by electrophoresis for eight inbred strains of laboratory mice. Intestinal LAP and AKP of adult mice were expressed concordantly within strains, as banded or diffuse, and concordantly for rate of migration within strains that had diffuse isozymes. All strains, except DD/S, had a single band of serum LAP and a single, diffuse zone of serum AKP. DD/S had a double band of serum LAP as well as isozymes of intestinal LAP and AKP unlike those of other strains. All strains displayed similar, neuraminidase-sensitive isozymes of intestinal LAP and of AKP prior to weaning, but after weaning there was marked sensitivity to neuraminidase only in DD/S. In interstrain crosses, banded/diffuse, migration rate, and neuraminidase sensitivity were inherited as independent autosomal traits, with indications of variable penetrance and genetic interaction.

Aging↗

PubMed1978

A novel fluorometric ultramicro determination of serum leucine aminopeptidase using a coumarine derivative.

A simple and rapid rate assay of serum leucine aminopeptidase is described, using a novel fluorogenic substrate, 7-L-leucyl-4-methylcoumarinylamide. The reaction is initiated by adding 10 microliter of serum, and the fluorescence development for 1 min due to the 7-amino-4-methylcoumarin liberated at 37 degrees C is followed directly on a recorder. The proposed method is proved to be free from error due to the adsorption of the substrate dye to serum albumin and to be applicable to hyperbilirubinemic sera by simple correction. The values obtained by this method showed good correlation with those obtained by the conventional method of Goldbarg and Rutenberg (Goldbarg, J.A. and Rutenberg, A.M. (1958) Cancer 11, 283-291).

Coumarins↗

PubMed1993

Electrophoretically mediated microanalysis of leucine aminopeptidase in complex matrices using time-resolved laser-induced fluorescence detection.

Leucine aminopeptidase, a clinically significant enzyme, was assayed in complex biological samples using a new technique termed electrophoretically mediated microanalysis. The assay was performed in capillary electrophoresis columns using time-resolved laser-induced fluorescence detection. Human serum, human urine, and Escherichia coli supernatant samples were assayed using this method. Results for serum and urine were within the ranges of expected values found in the literature. A low concentration of 6 x 10(-13) M enzyme in buffer was detected using this method. A detection limit (3 sigma) of 400 enzyme molecules in buffer was determined.

Buffers↗

PubMed2005

A water sluice is generated in the active site of bovine lens leucine aminopeptidase.

Nature has provided the binuclear zinc based active site of bovine lens leucine aminopeptidase (blLAP) with two water channels: one for substrate docking and a much smaller one (function unknown) above Zn1. In addition, Zn1 possesses an unusual pentacoordinate geometry with a loosely bound carbonyl ligand (Ala333). Extensive DFT calculations on a model of the active site provide first mechanistic implications for these structural features. The weakly bound carbonyl ligand is capable of functioning as a "traffic cop" to direct water molecules coming from the small channel into the heart of the active site. A water sluice is thus generated that is capable of repeatedly providing a series of nucleophilic active "Zn-OH" functionalities.

Animals↗

PubMed1990

Molecular structure of leucine aminopeptidase at 2.7-A resolution.

The three-dimensional structure of bovine lens leucine aminopeptidase (EC 3.4.11.1) complexed with bestatin, a slow-binding inhibitor, has been solved to 3.0-A resolution by the multiple isomorphous replacement method with phase combination and density modification. In addition, the structure of the isomorphous native enzyme has been refined at 2.7-A resolution, and the current crystallographic R factor is 0.169 for a model that includes the two zinc ions and all 487 amino acid residues comprising the asymmetric unit. The enzyme is physiologically active as a hexamer, which has 32 symmetry and is triangular in shape with a triangle edge length of 115 A and maximal thickness of 90 A. The monomers are crystallographically equivalent and each is folded into two unequal alpha/beta domains connected by an alpha-helix to give a comma-like shape with approximate maximal dimensions of 90 x 55 x 55 A3. The secondary structural composition is 40% alpha-helix and 19% beta-strand. The N-terminal domain (160 amino acids) mediates trimer-trimer interactions and does not appear to participate directly in catalysis. The C-terminal domain (327 amino acids) is responsible for catalysis and binds the two zinc ions, which are 2.88 A apart. The pair of metal ions is located near the edge of an eight-stranded, saddle-shaped beta-sheet. One zinc ion is coordinated by carboxylate oxygen atoms of Asp-255, Asp-332, and Glu-334 and the carbonyl oxygen of Asp-332. The other zinc ion is coordinated by the carboxylate oxygen atoms of Asp-255, Asp-273, and Glu-334. The active site also contains two positively charged residues, Lys-250 and Arg-336. The six active sites are themselves located in the interior of the hexamer, where they line a disk-shaped cavity of radius 15 A and thickness 10 A. Access to this cavity is provided by solvent channels that run along the twofold symmetry axes.

Amino Acid Sequence↗

PubMed2004

Expression of placental leucine aminopeptidase is associated with a poor outcome in endometrial endometrioid adenocarcinoma.

OBJECTIVE: Placental leucine aminopeptidase (P-LAP) is a cell surface aminopeptidase (oxytocinase). We cloned P-LAP cDNA and found a widespread tissue distribution of P-LAP. Since P-LAP can degrade several small peptide hormones such as oxytocin, this enzyme may affect many cellular functions of carcinoma cells as well as normal cells. This study investigated whether the expression of P-LAP correlates with clinicopathologic factors and prognosis in patients with endometrial endometrioid adenocarcinoma. METHODS: Histologic sections of formalin-fixed, paraffin-embedded specimens from 99 primary endometrial carcinomas were stained for P-LAP using polyclonal P-LAP antibody. Disease-free survival and other clinicopathologic characteristics were analyzed according to the intensity of P-LAP staining. RESULTS: Of 99 cases, 69 (69.7%) showed specific P-LAP immunostaining. We found a positive correlation between the expression of P-LAP and histological grade (p < 0.01), surgical stage of the disease (p = 0.02), myometrial invasion (p = 0.01), lymph node involvement (p < 0.01), and vascular infiltration (p < 0.01). In patients who had strongly positive P-LAP staining, the disease-free interval was significantly lower than in patients who had negative or weakly positive P-LAP staining (p < 0.01). Multivariate analysis demonstrated that strongly immunoreactive P-LAP (odds ratio, 12.8; 95% confidence interval, 2.84-58.8; p < 0.01) and surgical stage (odds ratio, 8.78; 95% confidence interval, 2.77-27.8; p < 0.01) are independent prognostic factors. CONCLUSIONS: This study suggests P-LAP as an independent prognosticator of clinical outcome in patients with endometrial carcinoma. Therefore, assessment of the P-LAP status provides clinically useful prognostic information in patients with endometrial carcinoma.

Analysis of Variance↗

PubMed1985

[The significance of serum leucine aminopeptidase (P-LAP) determination in the gynecological malignancies].

We determined Placental-Leucine Aminopeptidase (P-LAP) activity, one of the oncodevelopmental antigens, in sera and in tissues of patients with gynecological cancers. The incidence of P-LAP activity and clinical usefulness of the determination of serum P-LAP activity were studied. The mean level in healthy non-pregnant sera used as controls was 6.0 +/- 2.4mg/dl/h. The mean level of P-LAP activity in patients with benign tumors such as myoma uteri and benign ovarian tumor did not increase in comparison with the controls. The mean level of P-LAP activity in patients with malignant tumor increased with advancing stages, and especially in advanced cervical and ovarian cancer, serum P-LAP levels were significantly higher than in the controls. Serum P-LAP activity correlated with the serum ferritin concentration (r=0.613), but not with the serum alpha-fetoprotein and serum carcinoembryonic antigen concentration. Serial measurements of serum P-LAP activities in patients with gynecological cancer showed that serum P-LAP activity might reflect the progress of cervical and ovarian cancer. Tissue P-LAP activities in 29 ovarian cancers were compared with those in normal tissues. Tissue P-LAP activities in 26 cases out of 29 increased to twice as high as the mean activities in 10 normal ovaries. Our present results suggest the possibility of using P-LAP activity as one of tumor markers for gynecological malignant tumors.

Adult↗

PubMed1994

Functional analysis of a leucine aminopeptidase from Solanum tuberosum L.

A protein encoded by a potato cDNA homologous to a leucine aminopeptidase (LAP) from bovine lens (Hildmann et al. 1992) was expressed in Escherichia coli cells and biochemically characterized by hydrolysis of leucine p-nitroanilide. Activity was highest under alkaline conditions with an optimum at about pH 10. Maximal activities were measured at 65 degrees C. Apart from leucine p-nitroanilide the enzyme could also efficiently hydrolyze the p-nitroanilides of arginine and methionine. Complete inhibition of the enzyme was achieved by incubating bacterial extracts with bestatin and EDTA, which classifies the enzyme as a metalloprotease belonging to the same group as the homohexameric LAPs from mammals. Protein blots showed low constitutive expression of the LAP in all organs of potato plants: buds, flowers, tubers, roots and leaves. An increase in steady-state protein that was paralleled by an increase in total LAP activity was observed in leaf extracts after supplying jasmonic acid via the petioles. Plants containing the cDNA in antisense orientation behind the constitutive Cauliflower Mosaic Virus 35S promoter showed nearly complete reduction of the corresponding mRNA in leaves. However, in these plants LAP activities were only decreased by about 20% as compared to non-transgenic potato plants, while after feeding with jasmonic acid the activity of transgenic plants was reduced to about 5% of that of non-transgenic plants also induced by jasmonic acid. There was no phenotypic difference between wild-type and LAP antisense plants.

Amino Acid Sequence↗