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PubMed2001

Maternal serum placental leucine aminopeptidase (P-LAP)/oxytocinase and preterm delivery.

OBJECTIVE: To test the applicability of maternal serum placental leucine aminopeptidase (P-LAP) as a parameter for predicting preterm delivery. PATIENTS AND METHODS: Maternal serum P-LAP activity of 61 consecutive women with preterm labor at admission were assayed and examined to assess the relationship of levels to the incidence of preterm and term deliveries. RESULTS: When P-LAP activity of study patient serum was at or below the 10th percentile, we found a greater ratio of preterm delivery (P=0.0085) and an approximately 2.3-fold increase risk. The sensitivity, specificity, positive predictive value and negative predictive value were 29, 97, 89 and 62%, respectively. CONCLUSION: Maternal serum P-LAP activity decreases in cases with spontaneous preterm delivery and may be a clinically potential marker for some cases of preterm labor.

Adult↗
PubMed1976

Leucine aminopeptidase activity in maternal, cord blood and placenta of normal pregnancy and in pre-eclampsia.

1) Leucine aminopeptidase (LAP) was determined in maternal and fetal blood and in the placenta of mild and severe pre-eclamptic patients, then compared to the levels of normal pregnancy. 2) The enzyme activity increased significantly in severe pre-eclampsia as compared with the mild type and with normal pregnancy. 3) In mild pre-eclamptic patients LAP activity of maternal and cord blood did not increase significantly as compared with the normal cases but the placenta showed a slight decrease. 4) The enzyme level in cord blood did not increase markedly in pre-eclampsia.

Adult↗
PubMed1976

Bovine lens leucine aminopeptidase. A study of possible roles for the thiol groups in holo- and apoenzyme.

The apoenzyme of leucine aminopeptidase has to be prepared either under inert conditions or in the presence of 2-thioethanol. Due to the removal of essential zinc ions, buried -SH groups are exposed. These -SH groups show a higher reactivity than the six -SH groups accessible in the native zinc enzyme. During the incubation of the zinc enzyme with guanidine hydrochloride, up to 36 -SH groups are titratble in dependence on the concentration of guanidine hydrochloride. The -SH groups can be distinguished by their reactivity towards Ellman's reagent and can be differentiated into four classes with respect to their function.

Animals↗
PubMed1993

Inhibition of bovine lens leucine aminopeptidase by bestatin: number of binding sites and slow binding of this inhibitor.

Aminopeptidases catalyze the hydrolysis of amino acid residues from the amino terminus of peptide substrates. Their activity has been implicated in myriad fundamental biochemical and physiological processes, and alterations in aminopeptidase activity have been correlated with a variety of pathologies. Nevertheless, information about this group of proteases is less well developed. Bovine lens leucine aminopeptidase (blLAP) can be considered prototypical of many enzymes in this family of peptidases. It shows common features of (1) requiring divalent metal ions for activity, (2) having a relatively large size, and (3) having slow, relatively tight binding of bestatin, a transition-state analog of the substrate PheLeu. Bovine lens LAP is the only bestatin-inhibitable aminopeptidase for which structural and mechanistic data are available. However, full exploitation of these data required knowledge of the number of inhibitor molecules bound per subunit. Independent direct binding experiments and kinetic determinations indicate that one bestatin is bound per subunit in blLAP. Ki and Ki* for formation of the initial and final complexes are approximately 1.1 x 10(-7) and 1.3 x 10(-9) M, respectively. The mode of binding is slow and competitive. The t1/2 for formation and deformation of the final enzyme-inhibitor complex is approximately 30 and 22 min, respectively, with 10(-8) M bestatin. To perform these measures, a new assay using physiological peptides (LeuGlyGly) as substrate was adapted. Taken together with prior NMR, photoaffinity labeling, and crystallographic data, these binding data allow us to propose a mechanism of the blLAP-catalyzed hydrolysis of peptides.

Aminopeptidases↗
PubMed1983

[Statistical analysis of urinary estriol (U-E3), serum leucine aminopeptidase (LAP) and heat stable alkaline phosphatase (HSAP) in toxemia].

The levels of urinary estriol (U-E3), serum leucine aminopeptidase (LSP) and heat stable alkaline phosphatase (HSAP) were determined in toxemic and normal pregnancies. The significance of differences was established by weekly estimation of the parameters for both groups after the 28th week of gestation. Assayed values were classified into 4 types (type I-IV) by the mean values for 3 serial 24h assays and 3 patterns (pattern a-c) observed with these three determinations. The distribution rate for each type and pattern was examined according to the following: Severe toxemia, low birth weight, meconium staining at parturition and low apgar score groups. For U-E3, the incidence of the low value type (type IV) and the descending pattern (pattern c) both increased linearly in all the above groups. These findings suggest the clinical applicability of U-E3 as a reasonable indicator for evaluating intrauterine fetal well being because of the close correlation to the listed conditions. Thus these biochemical parameters should be considered in the antepartum fetal management of third trimester toxemic pregnancies.

Alkaline Phosphatase↗
PubMed1976

[Behavior of leucine aminopeptidase from bovine eye lens in guanidine hydrochloride. Dissociation and reassociation].

The dissociation and reassociation behavior of the hexameric leucine aminopeptidase (LAP) in solutions of 0.5 to 6 M guanidine-HC1 (Gu-HC1) was investigated by means of thin layer chromatography on Sephadex G-200 superfine. Up to 0.5 M Gu - hc1 the hexameric LAP-structure remains intact. In 0.75 to 2 M Gu - HC1 the enzyme dissociates nearly completely into its half-molecules (LAP-trimers, MW 160000 +/- 10,000). In 2.5 and 2.75 M Gu - HC1 a mixture of LAP-monomers (subunits) (MW 60000 +/- 5000) and trimers was to be found. Treatment with beta-mercapto ethanol increases the portion of monomers. Only monomers occurred in 3 to 6 M Gu - HC1. Parallel to the dissociation, a loss of the essential zinc (half life: 10.5 and 3.5 hours in 1 M and 4 M Gu - HC1 resp.) and a decrease of the enzymatic activity (30 min after treatment with 1 M and 4 M Gu - HC1 50% and 1% resp. residual activity) of the observed. On the other hand, the enzyme was activated by dilute solutions of Gu - HC1 (0.1 to 0.3 M: max. increase 50%). Removal of the denaturant causes reassociation of all types of fragments to hexameric LAP which was indistinguishable from the native LAP in Ouchterlony immune diffusion test. The reassociation was accompanied by a small increase in activity only.

Animals↗
PubMed2000

Potent new leucine aminopeptidase inhibitor of novel structure synthesised by a modified Wadsworth-Emmons (Horner) Wittig procedure.

The use of a leucine-derived alpha-keto-beta-aldehyde (glyoxal) as a substrate in the Horner-Emmons (Wadsworth) Wittig reaction has enabled the synthesis of (Z)-7-methyl-5(S)-amino-4-oxo-methyl-oct-2-eneoate. This novel compound is a potent inhibitor (Ki = 76 nM) of leucine aminopeptidase and provides an interesting new template for the development of metallopeptidase inhibitors.

Chemistry, Organic↗
PubMed1998

A family with high serum leucine aminopeptidase activity derived from a novel variant CD13.

We investigated a family in which some individuals showed extremely high serum leucine aminopeptidase (LAP) (EC 3.4.11.2) activity, mainly derived from a variant CD13. The isoelectric points of the variant and normal CD13 were 3.3 and 4.1, respectively, and both points converged at 4.4 after treatment with neuraminidase, indicating that more sialic acids are bound to the variant CD13 than normal. The molecular masses of both CD13s were 144 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After treatment with neuraminidase, N-glycosidase, and O-glycosidase, apparent molecular masses of the variant and normal CD13 were 106 kDa and 100 kDa, respectively, suggesting that the variant CD13 contains a longer peptide than normal. This is the first case of familial high serum LAP activity in which the origin could be demonstrated by anti-CD13 monoclonal antibodies to be a variant CD13 inherited in an autosomal dominant mode. The isoelectric point of the LAP activity after neuraminidase treatment was different from that previously reported.

Adult↗
PubMed1983

Identification and properties of the cell membrane bound leucine aminopeptidase interacting with the potential immunostimulant and chemotherapeutic agent bestatin.

Bestatin was found to be a competitive inhibitor (with respect to the Leu-NA substrate) not only of the isolated microsomal and cytosolic leucine aminopeptidases (Leu-APm and Leu-APc) but also of the aminopeptidases (APs) present in membrane preparations (from mouse liver) and on the cell surface of L5178Y cells. Kinetic parameters indicate that cellular AP is identical to Leu-APm. To rule out the possibility that AP-B is involved in the inhibition reactions, comparable studies with amastatin were performed. Electrophoretical studies revealed the solubilized cell membrane bound AP to co-migrate with Leu-APm in polyacrylamide gels. The activity of the separated membrane AP was inhibited by bestatin in situ. The cell membrane bound AP activity was found to be lowest in lymphocytes, higher in tumor cells and highest in bone marrow cells and macrophages. Using synchronized L5178Y cells, the AP activity changes during the cell division cycle; the lowest activity was determined during the G1-phase and 35% higher values were measured during the S/G2-phase. The fluctuation of the cell surface associated AP activity parallels with changes in the number of binding sites for bestatin.

Animals↗
PubMed1987

The behaviour of leucine aminopeptidase towards thionopeptides.

Thionoleucine S-anilide (Leut-anilide), Leut-Gly-OEt and Leut-Phe-OMe were synthesized and shown to be competitive inhibitors of leucine aminopeptidase from pig kidney. The kinetics of inhibition were determined in the presence of leucine 4-methylcoumarin-7-amide as substrate. Although the compounds showed only moderate inhibitory potency, it was found that all were resistant to hydrolysis by the enzyme, in contrast with the reported behaviour of some thionopeptide analogues of substrates for other Zn2+-peptidases such as carboxypeptidase A and angiotensin-converting enzyme.

Kinetics↗
PubMed2006

Identification and characterisation of a leucine aminopeptidase from the hard tick Haemaphysalis longicornis.

Aminopeptidases responsible for blood digestion have yet to be identified in haematophagous ticks. We report here the cloning and molecular characterisation of a cDNA encoding leucine aminopeptidase, a member of the M17 cytosolic aminopeptidase family, from the hard tick Haemaphysalis longicornis (HlLAP). Endogenous HlLAP was detected in the soluble fraction of adult tick extracts by immunoblotting. Immunohistochemical studies demonstrated that endogenous HlLAP expression mainly took place in the cytosol of midgut epithelial cells. Furthermore, expression of HlLAP was induced by a blood-feeding process. A functional recombinant HlLAP expressed in Escherichia coli efficiently hydrolyses synthetic substrates for aminopeptidase, a leucyl (with the Km value 0.19 +/- 0.011 mM and Vmax value 157.2 +/- 3.17 nmol/min/mgprotein) and a methionyl substrate (with the Km value 0.12+/-0.0052 mM and Vmax value 171.9 +/- 2.31 nmol/min/mgprotein). Enzyme activity was found to be optimum at pH 8 and 35 degrees C. The recombinant HlLAP enzyme activity was strongly dependent on metal divalent cations, Mn2+, and was inhibited by bestatin. These results indicate that HlLAP play an important role for host's blood digestion process.

Amino Acid Sequence↗
PubMed1987

Leucine aminopeptidase in intracytoplasmic membranes of Acinetobacter calcoaceticus.

Cells of Acinetobacter calcoaceticus strain 69-V contain an aminopeptidase that cleaves L-leucine amide, leucylglycine or leucine hydrazide with high efficiency. Leucine 4-nitroanilide and hydrazide are hydrolyzed to less than 0.1% and 1%, resp. of leucine amide. Grown on acetate-NH4+ medium the activity of the enzyme in the cytoplasm is increased 5-fold compared with cells grown on a casamino acid medium or on yeast extract. In these cases the specific activity of the unpurified enzyme is about 5 nkat/mg for the cytoplasmic and membrane-bound enzyme species as well. Up to 30% of the aminopeptidase activity were found mainly in intracytoplasmic membranes, less in cytoplasmic membranes and only traces in outer membranes, presumably as contaminations. It is solubilized by detergents but not by high salt concentrations. An addition of antipain or Z-Ala2-Phe-CH3 before cell rupture did not change the distribution of the enzyme. A mixture of EDTA and 1.10-phenanthroline diminished the membrane-bound enzyme from 11.4% to 4.3% and leupeptin or E-64 increased it to 20%. The enzyme is regarded as leucine aminopeptidase (LAP) bound to intracytoplasmic membranes.

Acinetobacter↗
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↗
PubMedDate not supplied

[X-ray structural study of leucine aminopeptidase with a resolution of 4 Angstroms].

An X-ray crystallographic structure determination has been carried out on bovine lens leucine aminopeptidase at 4.0 A resolution by using a combination of isomorphous replacement and solvent flattening. The two heavy atom derivatives used were obtained by soaking crystals in ethyl mercury chloride, which bound at four sites, and phenyl mercury acetate, which bound at one site in the monomer. The electron density map reveals that the enzyme hexameric oligomer, arranged in 32 symmetry, has a triangular barrel appearance and dimensions, of height 88 A and maximal width 118 A in barrel equatorial plane. Each subunit in an elongated ellipsoid of approximate length 92 A. Subunits contacts have been described. From an analysis of the map each subunit appears to contain some 36% alpha-helix and is organized into two distinct globular domains. Direct location of zinc cluster and competitive inhibitor binding site are presented.

Animals↗
PubMed1977

Alkaline phosphatase, leucine aminopeptidase, and alanine aminotransferase activities with obstructive and toxic hepatic disease in cats.

The activities of serum alkaline phosphatase (serum ALP), leucine aminopeptidase (serum LAP), and alanine aminotransferase (serum ALT) were determined in 15 cats before and after treatment by 3 methods: common bile duct occlusion, left hepatic duct(s) occlusion, and carbon tetrachloride administration. Significant increases in serum ALP, LAP, and ALT activities occurred in all cats in the 3 groups. Sustained mean increases of ninefold in ALP and 13-fold in LAP occurred in the cats with common bile duct occlusion. Lesser mean increases of these enzymes (fourfold) occurred in the cats with partial biliary occlusion. Transient mean increases (100-fold) in ALT occurred in the carbon tetrachloride-treated cats. Urine ALP excretion was measured in 3 cats with common bile duct occlusion. There was no significant difference between rates of urine ALP excretion before and after common bile duct occlusion. Specific ALP activities of hepatic extracts from normal cats and biliary-obstructed cats were compared. Mean specific activity was onefold higher in liver from cats with common bile duct occlusion of 21 days' duration. The findings in the present studies were interpreted to indicate that serum ALP and LAP are useful to detect biliary occlusive disease in cats and, in conjunction with serum ALT, may be used to differentiate primary hepatodegenerative disease and biliary occlusive disease.

Alanine Transaminase↗
PubMed1993

Isolation of bovine kidney leucine aminopeptidase cDNA: comparison with the lens enzyme and tissue-specific expression of two mRNAs.

Aminopeptidases catalyze the hydrolysis of amino acid residues from the amino terminus of peptide substrates. Leucine aminopeptidase (LAP) from bovine lens is the best characterized aminopeptidase and the only LAP for which the amino acid sequence was determined by protein sequencing. Using this sequence information, we isolated a bovine kidney LAP cDNA and compared its deduced amino acid sequence to the published amino acid sequence for bovine lens LAP. Overall, the sequences are highly conserved. However, several differences are observed. The kidney LAP cDNA indicates a 26 amino acid extension at the amino terminus which is not found in the mature purified lens LAP. The cDNA also indicates an additional octapeptide in the C-terminal region which was not indicated in the published lens LAP amino acid sequence but which was required for best fit of crystallographic data regarding bovine lens LAP. Several other single amino acid changes were also noted. Levels of LAP transcripts were examined in bovine lens and kidney tissue as well as in cultured lens cells. Lens epithelial tissue showed only one LAP transcript (2.4 kb) whereas two transcripts (2.0 and 2.4 kb) were observed in cultured lens cells derived from epithelial tissue and in kidney tissue. Using Northern blot analysis, we correlated LAP mRNA levels with previously determined changes of LAP activity in aging lens tissue and in progressively passaged lens epithelial cells which were used to simulate aging in vitro. No differences were found in LAP mRNA levels in epithelial tissue from old and young lenses.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1984

Immunologic characterization of human seminal leucine aminopeptidase (LAP) and its medicolegal use.

A new method for identification of seminal stains is described, based on the immunologic demonstration of leucine aminopeptidase (LAP), which is extremely abundant in human semen and specific for the prostate as well as semen. An antiserum against human seminal plasma was obtained by repeated immunization of rabbits with seminal plasma and Freund's adjuvant. Ouchterlony's double immunodiffusion test and Culliford's precipitin electrophoresis were performed to demonstrate specific proteins of seminal plasma. LAP activity was visualized with L-leucyl-beta-naphthylamide as substrate and with Fast Garnet GBC as coupler. The immunologic analysis of LAP produced two precipitin lines with enzyme activity. One was observed in kidney, jejunum, pancreas, prostate, as well as in semen, and was completely absorbed with kidney homogenates. The other was found only in semen and the prostate and was not absorbed with kidney homogenates. When the anti-seminal plasma serum absorbed with the kidney was used, the semen-specific LAP could be demonstrated by precipitin electrophoresis only in seminal stains stored for up to 2 months, whereas it was not demonstrated in stains from other human body fluids. By means of precipitin electrophoresis the detection of the semen-specific LAP was possible at semen dilutions of up to 1:32. The method described here greatly enhances the value of semen identification and is quite recommendable for the examination of stains in medico-legal practice.

Body Fluids↗