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At least 163 records · Page 9Linked to original sources
PubMed2000

Estimates of leucine aminopeptidase activity in different marine and brackish environments.

AIM: Leucine aminopeptidase (LAP), an enzyme involved in the decomposition of natural peptides, was measured in different marine and brackish ecosystems, together with some environmental and microbiological parameters. METHODS AND RESULTS: The fluorogenic compound L-leucine-7-amido-4-methyl coumarin was specifically used for the determination of this in situ activity. The enzyme data obtained from this comparative study highlighted the strong spatial and temporal variability of the distribution of LAP in aquatic ecosystems, which was sometimes related to the course of environmental variables such as salinity and organic carbon content. CONCLUSIONS: LAP assay has proved to be a rapid method providing useful information on the microbial metabolic processes involved in the mineralization of organic matter. SIGNIFICANCE AND IMPACT OF THE STUDY: The determination of the potential rates of extracellular enzyme activity is of great ecological importance to extend knowledge on the role played by bacteria in aquatic biogeochemical cycles.

Bacteria↗
PubMed1986

Determination of leucine aminopeptidase using phenylalanyl-3-thia-phenylalanine as substrate.

The peptide mimetic L-phenylalanyl-L-3-thiaphenylalanine has been shown to facilitate a sensitive and simple determination of leucine aminopeptidase. A colorimetric assay, employing Ellman's reagent to detect the thiophenol released upon hydrolysis of the dipeptide, has been developed. Under the experimental conditions employed the substrate has a Km of 0.054 mM and a kcat of 5800 min-1 and can distinguish sharply between leucine aminopeptidase and aminopeptidase M.

Aminopeptidases↗
PubMed2006

Reduced activity of the hypertension-associated Lys528Arg mutant of human adipocyte-derived leucine aminopeptidase (A-LAP)/ER-aminopeptidase-1.

The adipocyte-derived leucine aminopeptidase (A-LAP)/ER aminopeptidase-1 is a multi-functional enzyme belonging to the M1 family of aminopeptidases. It was reported that the polymorphism Lys528Arg in the human A-LAP gene is associated with essential hypertension. In this study, the role of Lys528 in the enzymatic activity of human A-LAP was examined by site-directed mutagenesis. Among non-synonymous polymorphisms tested, only Lys528Arg reduced enzymatic activity. The replacement of Lys528 with various amino acids including Ala, Met, His and Arg caused a significant decrease in the enzymatic activity. Molecular modeling of the enzyme suggested that Lys528 is located near the entrance of the substrate pocket. These results suggest that Lys528 is important for maximal activity of A-LAP by maintaining the appropriate structure of the substrate pocket of the enzyme. The reduced enzymatic activity of A-LAP may cause high blood pressure and the observed association between the polymorphism and hypertension.

Adipocytes↗
PubMed2006

Identification of glutamate residues important for catalytic activity of Bacillus stearothermophilus leucine aminopeptidase II.

Each of four conserved glutamate residues of Bacillus stearothermophilus leucine aminopeptidase II (BsLAPII) was replaced with aspartate, lysine, and leucine respectively by site-directed mutagenesis. The over-expressed wild-type and mutant enzymes were purified to homogeneity by nickel-chelate chromatography and the molecular mass of the subunit was determined to be 44.5 kDa by SDS-PAGE. The specific activity for the Glu-316 and Glu-340 mutants was completely abolished, while Glu-249 mutants showed comparable activity to that of the wild-type BsLAPII. Compared with the wild-type enzyme, the E250D and E250L mutant enzymes retained less than 18% of the enzyme activity and exhibited a dramatic decrease in the value of k (cat)/K (m). These observations indicate that Glu-250, Glu-316, and Glu-340 residues are critical for the catalytic activity of BsLAPII.

Amino Acid Sequence↗
PubMed1982

Sulfhydryl content of bovine eye lens leucine aminopeptidase. Determination of the reactivity of the sulfhydryl groups of the zinc metalloenzyme, of the enzyme activated by Mg2+, Mn2+, and Co2+, and of the metal-free apoenzyme.

The reactivity of the sulfhydryl groups of leucine aminopeptidase from bovine eye lens has been studied by carboxymethylation with iodoacetate of the native enzyme (Zn2+-Zn2+), of the enzyme activated with magnesium (Zn+-Mg2+) or manganese (Zn2+-Mn2+), the enzyme incubated with cobalt (CO2+-Co2+), and the metal-free apoenzyme. The reactivity of the sulfhydryl groups was also determined in the presence of denaturing agents with or without reducing agents. All seven half-cystines per leucine aminopeptidase subunit are in the sulfhydryl form. In the native and the Zn2+-Mn2+ enzyme, only one of the cysteines (residue 344) reacts readily with iodoacetate. In the Zn2+-Mg2+ enzyme, two cysteines react (residues 344 and 412) and in the Co2+-incubated enzyme only one (residue 412). The metal-free apoenzyme has at least three reactive cysteines (residues 344, 412, and 429). The data suggest that sulfhydryl groups are involved in the binding of metal ions.

Amino Acid Sequence↗
PubMed1975

Purification and partial characterization of barley leucine aminopeptidase.

A peptidase acting on Leu-Gly-Gly and Leu-Tyr at pH 8 to 10 was purified about 670-fold from germinated grains of barley (Hordeum vulgare L.). Gel electrophoretic analyses indicated a purity of about 90%. The purified enzyme is remarkably similar to mammalian leucine aminopeptidases (EC 3.4.1.1) both in chemical and in enzymatic properties. It has a sedimentation constant of 12.7S and a molecular weight of about 260,000. The enzyme has a high activity on leucine amide and di- and tripeptides with N-terminal leucine or methionine; leucyl-beta-naphthylamide, in contrast, is hydrolyzed very slowly. The enzyme also liberates N-terminal amino acids from the insulin B chain. The pH optima for the hydrolysis of different substrates depend on the buffers used; highest reaction rates are generally obtained at pH 8.5 to 10.5. Mg(2+) and Mn(2+) ions stabilize (and probably activate) the enzyme. In contrast to mammalian leucine aminopeptidases, the barley enzyme is inactivated in the absence of reducing sulfydryl compounds.

Journal Article↗
PubMed1996

Possible effects of placental leucine aminopeptidase on the regulation of brain-gut hormones in the fetoplacental unit.

The hydrolysis of somatostatin by human placental subcellular fractions and pregnancy sera was studied in the presence of selective inhibitors and the antibody against pregnancy serum oxytocinase (placental leucine aminopeptidase; EC3.4.11.3) by measuring the released amino acids by high-performance liquid chromatography. We also studied the degradation of other brain-gut hormones, such as glucagon, growth hormone, growth hormone releasing factor, and insulin, in the human placenta and found that the human placenta degrades somatostatin, glucagon, and growth hormone releasing factor, but not insulin and growth hormone. The degradation velocity of somatostatin was ten times greater than that of growth hormone releasing factor in placental microsomal fractions. Our data suggest that the stimulatory control by growth hormone releasing factor is dominant in the fetal growth hormone secretion. Our data also identified the somatostatin-degrading protease in human placenta using placental leucine aminopeptidase. It is known that the mean somatostatin levels in the umbilical artery are about 2.5-fold higher than those in the umbilical vein. Our data on somatostatin levels in umbilical artery and vein of intrauterine growth retardation human fetuses showed that the ratio umbilical artery/vein is around 1. Since insulin is known to be the primary hormone regulating the ratio of fetal growth, our data suggest that the degradation of somatostatin in the placenta is decreased and that elongation of somatostatin effects may result in the inhibition of insulin secretion in the intrauterine growth retardation fetus.

Amino Acid Sequence↗
PubMed1976

[Affinity labeling of leucine aminopeptidase with new substrate analog inhibitors].

Substrate analogous compounds differing both in type and reactivity (diazonium group, chloro and mo ethyl ketone groups) and in the position of the reactive group at the inhibitor molecule were studied for their adequacy for affinity labelling of leucine amino-peptidase. The chloro methyl ketone derivatives of amino acids with free alpha-amino group are competitive inhibitors. Also, the more reactive bromine compound PheCh2br (Ki 1.2 mM) compared with the PheCH2C1 (Ki 0.3 mM) fails to give an irreversible inactivation of leucine amino-peptidase. The dipeptide derivatives Leu-PheCH2C1 and Phe-LeuCH2C1 also inhibit the enzyme activity up to 65%, but they are split at the peptide bond under reactivation of the enzyme. p-Diazophenylalanine methylketone Phe (pN+/2)CH3 and the two dipeptides Phe(pN+/2)-Phe and Phe(pN+/2)-Phe(pN+/2) with N-terminal diazonium groups afford an irreversible inactivation of leucine aminopeptidase in a time- and concentration-dependent reaction. The Phe-Phe(pN+/2) reactive only C-terminally, is less effective; the inhibition is partly reversible. The inactivation is strongly reduced by the competitive inhibitor Thr(but)-Phe-Pro. These effects are discussed with regard to the specific site of attack of the inhibitors in the active binding centre of leucine aminopeptidase. The synthesis of the three parasubstituted amino derivatives of phenylalanyl-phenylalanine Phe(pNH2)-Phe, Phe-Phe(pNH2) and Phe(pNH2)-Phe(pNH2) and their selective conversion to the respective diazonium peptides, retaining the aliphatic alpha-amino group, are discussed.

Affinity Labels↗
PubMed1985

FACS quantitation of leucine aminopeptidase and acid phosphatase on tumor-associated macrophages from metastatic and nonmetastatic mouse mammary tumors.

Macrophages were isolated by adherence from tumors produced by a number of murine mammary carcinoma lines and were examined by fluorescence-activated cell sorting for quantitation of leucine aminopeptidase and acid phosphatase. The tumors included three lines, 66, 67, and 168, which were originally derived from a single, spontaneously arising tumor in a BALB/cfC3H mouse and two other lines, D2A1 and D2F2, which were derived from a single tumor arising from the transplantable hyperplastic alveolar nodule line, D2. These five lines differ from one another in a number of characteristics, including the ability to metastasize spontaneously to the lung from subcutaneous implants and to form experimental metastases in lungs following intravenous injection. Line 67 is nonmetastatic under both circumstances, whereas lines 66, D2A1, and D2F2 are metastatic under the same conditions. Intermediate to these is line 168, which is nonmetastatic from the subcutaneous site but capable of colonizing the lung with an efficiency similar to 66 when injected IV. Tumor-associated macrophages (TAM) from lines 66, D2A1, and D2F2 contained the greatest amounts of leucine aminopeptidase (LAP) and those from line 67 the least, with TAM from 168 being intermediate. Conversely, the TAM from line 67 had the greatest amounts of acid phosphatase (APTase) and those from line 168 the least. In addition to differences among tumors in enzyme levels of the adherent TAM, the precentages of TAM that were adherent were also different among the tumors. Only 12% of TAM from line 67 were recovered in the adherent fraction as opposed to 35-38% of TAM from lines 66 and 168. These results confirm and extend our previous findings that TAM from metastatic tumors have increased levels of LAP compared to TAM from nonmetastatic tumors. They also demonstrate noncoordinate expression of LAP and APTase in TAM, and illustrate how a population of TAM can be homogeneous for one enzyme and heterogeneous for another. Furthermore, the difference in the percentage of macrophages that are adherent between metastatic and nonmetastatic tumors is another indication both of the heterogeneous nature of TAM and of the role of a tumor in determining the type of host infiltrate with which it is associated.

Acid Phosphatase↗
PubMed1985

A new variant of serum leucine aminopeptidase in the mouse: its development and possible regulation.

Serum leucine aminopeptidase (LAP) isozymes were compared in four strains of inbred mice during postnatal development, adult life, and pregnancy. In pregnancy, no changes in the maternal serum LAP pattern were observed, in contrast to human studies. One strain, DD/S, differs from the other three in serum LAP. Polymorphism in serum LAP has not been previously described in the mouse. Neonatal DD/S mice exhibit a single band of serum LAP upon starch gel electrophoresis; however, between 14 and 18 days of age, two distinct bands appear, which persist throughout adult life. In the strains C57BL/6J, BALB/cJ, and DBA/2J there is a single band of activity at all stages. Crosses and backcrosses between DD/S and C57BL/6J show that the double-band variant is inherited as an autosomal recessive. The variant is independent of both the supernatant malic enzyme (Mod-1) and the intestinal LAP (Lap-1) loci, which are known to be linked on chromosome 9. The serum LAP variant is linked to an intestinal alkaline phosphatase variant. The presence of a separate structural gene is suggested by the genetic independence of the serum LAP variant from Lap-1. Also, the two serum LAP bands of DD/S are not interconverted by treatment with neuraminidase, beta-mercaptoethanol, or heat or by mixing the sera of DD/S and C57BL/6J prior to electrophoresis. The level of serum LAP activity in DD/S is approximately twice that in C57BL/6J. While these observations imply two structurally distinct proteins, the absence of any trace of the second LAP band in the heterozygote strongly suggests that the LAP variant protein is not the result of a separate structural gene. Intestinal LAP in DD/S migrates with the same electrophoretic mobility as the serum LAP variant, implying that the variant might originate in the intestine and its appearance in the serum be modulated by some factor at an unlinked locus.

Age Factors↗
PubMed2000

Characterization of recombinant human adipocyte-derived leucine aminopeptidase expressed in Chinese hamster ovary cells.

Adipocyte-derived leucine aminopeptidase (A-LAP) is a recently identified novel member of the M1 family of zinc-metallopeptidases. Transfection of the A-LAP cDNA into COS-7 cells resulted in the secretion of the enzyme. In this study, recombinant A-LAP was expressed in Chinese hamster ovary cells, purified to homogeneity and its enzymatic properties were characterized. The purified enzyme was active towards a synthetic substrate, L-leucyl-p-nitroanilide, yielding a V(max) of 3.55 micromol/min/mg and a K(m) of 1.28 mM, and was shown to be a monomeric protein with molecular mass of 120 kDa in solution. By monitoring the sequential N-terminal amino acid liberation, it was found that the enzyme hydrolyzes a variety of bioactive peptides, including angiotensin II and kallidin. Immunohistochemical analysis indicated that the enzyme is expressed in the cortex of the human kidney, where tissue kallikrein is localized. Taken together, these results indicate that A-LAP possesses a broad substrate specificity towards naturally occurring peptide hormones and suggest that it plays a role in the regulation of blood pressure through the inactivation of angiotensin II and/or the generation of bradykinin in the kidney.

Adipocytes↗
PubMed1987

Circadian rhythm in gamma glutamyltranspeptidase and leucine aminopeptidase urinary activity in rats.

Urinary gamma glutamyltranspeptidase (GGT) and leucine aminopeptidase (LAP), renal tubular brush border enzymes, have been shown to be sensitive indicators of renal tubular functions. This study documents circadian rhythms in the urinary activity of GGT and LAP, statistically validated and quantified by the cosinor method, in 15 male Wistar rats standardized to a LD 12:12 illumination schedule (light from 0800 hr to 2000 hr) and fed ad libitum. The acrophase of the circadian rhythms in urinary GGT and LAP activity occurred at the end of the rest span of the animals: between 1730 and 1915 for GGT (depending on the mode of expression of the activity) and between 1700 and 1910 for LAP. Of striking resemblance in their timing, both these rhythms were also of large amplitude (about 50% of the mesor for urinary GGT activity and about 45% for LAP one). The circadian acrophases of urinary GGT and LAP activity led in timing the circadian rhythms in urine volume and creatinine excretion by about 13 hr. Such findings consistent with the circadian variations found by other investigators in GGT in kidney homogenates or in LAP in human urine thus reflect a periodicity in renal tubular function. The reasons for these circadian variations, still unknown at this time, are discussed. The influence recently demonstrated of the hormonal context on protein and enzyme synthesis at the tubule, and its phase relations to urinary enzyme excretion emphasize how much the circadian rhythm in urinary GGT and LAP activity is well included in the murine time structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1976

[Spectroscopic investigatons on the structural thermal stability of leucine aminopeptidase. ESR, CD and fluorescence studies].

The thermal behaviour of leucine aminopeptidase (LAP, EC: 3.4.11.1) from bovine eye lens has been investigated in the temperature region 20--70 degrees C by spin-labelling of SH-groups (ESR), by CD and by fluorescence of tryptophane residues. Enzymatic activity of LAP was compared with spectroscopic data in this temperature region. From 20-60 degrees C the structural parts (alpha, beta, random coil) estimated from CD spectra remain unchanged. Within 20-55 degrees C no irreversible exposure of tryptophane residues takes place. In both types of spin-labelled LAP the strong immobilizing environment of the label retains its highly ordered structure up to 55 degrees C. Reversible changes of mobility and polarity of the environment of the label induced by temperature within 20-50 degrees C do not reduce the enzymatic activity and are regarded as local loosening of ordered structure. At 65 degrees C strong precipitation occurs. From 55 degrees C to 65 degrees C tryptophane residues are irreversibly exposed. The highly ordered environment of the label is destroyed about 55 degrees C, and a considerable amount of spin label molecules is reduced at the NO group by exposed SH groups. The above mentioned local loosening of structure becomes irreversible at 60 degrees C. The environment of both labels dominating above 60 degrees C is highly mobile and strongly polar and represents an extensively unfolded conformation. Until 60 degrees C no essential disordering of protein structure leading to a decrease of enzymatic activity occurs. Above 60 degrees C a sharp breakdown of ordered structures takes place, which is accompanied by a strong diminution of enzymatic activity.

Animals↗
PubMed1985

Leucine aminopeptidase of neutrophils and lymphocytes in patients with cancer.

The leucine aminopeptidase activity has been determined by using the cytochemical method of Burston and Folk in peripheral blood neutrophils and lymphocytes of 45 patients with various malignancies. Lung cancer, carcinoma of the stomach and cancer of the colon was diagnosed in 24, 16, and 5 patients, respectively. Patients with metastases showed a significantly higher activity of the enzyme if compared with that in the control group of healthy subjects and patients without metastases. The percentage of enzyme-positive lymphocytes was elevated significantly in patients with metastases whereas a total percentage of lymphocytes with regard to differential leukocyte count was diminished both in patients with and without metastases. The absolute count of neutrophils was elevated both in patients with and without metastases. The authors discuss the significance of their observation with regard to the antitumor cytotoxic effect of neutrophils and lymphocytes.

Colonic Neoplasms↗
PubMed1983

[Mechanism of oxygen exchange in the amide group of substrates during their hydrolysis catalyzed by leucine aminopeptidase and pepsin].

Oxygen exchange in the amide group of leucine amide catalyzed by leucine aminopeptidase, and in leucyltyrosine amide catalyzed by porcine pepsin, was found to proceed mainly by the transfer of the leucyl residue onto the ammonia or tyrosine amide which are formed during the hydrolysis. Thus oxygen exchange in the non-hydrolyzed substrate can not be a proof of the tetrahedral intermediate formation in the course of the catalysis by proteolytic enzymes.

Amides↗
PubMed2006

Residues threonine 346 and leucine 352 are critical for the proper function of Bacillus kaustophilus leucine aminopeptidase.

The importance of Thr-346 and Leu-352 residues in Bacillus kaustophilus leucine aminopeptidase (BkLAP) was explored by site-directed mutagenesis. The impact of substitutions at these positions was evaluated with His6-BkLAP fusion proteins expressed in Escherichia coli. Substitution of Thr-346 with Tyr, Arg, and Leu, respectively, resulted in a dramatic reduction in LAP activity. A complete loss of activity was observed in L352E and L352R variants with the exception of L352 V, which retained approximately 60% of the wild-type activity. Zinc content analysis and protein modeling suggested that Thr-346 and Leu-352 of BkLAP play a role in maintaining the coordination environment for the zinc-binding residues.

Amino Acid Sequence↗