Leucine aminopeptidase crystals from the bovine pancreas

Crystals of leucinaminopeptidase from bovine pancreas were obtained. Space group (P6322), parameters of the cell a-b-132 A, c = 122 A and distribution of spot intensities on precessional X-ray patterns were in full agreement with corresponding parameters for leucinaminopeptidase crystals from bovine eye lens. A conclusion is drawn about similarity between the spatial structures of leucinaminopeptidase from bovine pancreas and eye lens.     

Leucine aminopeptidase activity in pain-induced emotional stress

It is established that leucine aminopeptidase activity during emotional-algesic stress increases in the brain hemispheres, left ventricle and liver of the rats as compared to that of intact animals. Maximum activation of the enzyme in the brain and liver is detected for the first two days after the stress while in the heart - for the whole period of the total stress damages volume development. A conclusion is drawn that the shifts observed in leucine aminopeptidase activity during emotional-algesic stress affect the methionine-enkephalin and leucin-enkephalin ratio.     

Leucine aminopeptidase in extracts of swine muscle

1. Leucine aminopeptidase (EC 3.4.1.1) has been demonstrated in swine muscle at a level of activity one-fifth that of the swine kidney. 2. The enzyme has been purified 110-fold by precipitation with ammonium sulphate, heat treatment and chromatography on Sephadex G-100. 3. The enzyme is heat-stable, but is rapidly inactivated below pH7. It requires Mg(2+) or Mn(2+) for activity. The Michaelis constant for leucine amide with Mg(2+)-activated enzyme is 5.0x10(-3)m. 4. Muscle leucine aminopeptidase is very similar to the kidney enzyme.     

Leucine aminopeptidase and hatching of Schistosoma mansoni eggs

Leucine aminopeptidase (LAP) activity has been measured in extracts of eggs, miracidia, cercariae and adult worms of Schistosoma mansoni. Activity measured at pH 7.2 using L-leu-7-amino-4-trifluoro-methylcoumarin as substrate is 6- to 17-fold greater in eggs than in other life stages. LAP activity is also high in soluble egg antigen preparations and in hatching fluid. The release of LAP from eggs parallels hatching, and inhibitors of LAP also inhibit hatching. The possible role of LAP in the hatching process of S. mansoni eggs is discussed.     

Leucine aminopeptidase as an echo-enzyme of polymorphonuclear neutrophils

Intact polymorphonuclear neutrophils were modified chemically by a poorly permeable reagent, diazotized sulfanilic acid, and the changes in the activity of 5'-nucleotidase, alkaline phosphodiesterase, and leucine aminopeptidase were examined. Among three plasma membrane enzymes, 5'-nucleotidase activity was hardly detected in the human neutrophils. The activity of alkaline phosphodiesterase was observed in all the neutrophils examined, but was not inhibited by diazotized sulfanilic acid in the guinea-pig neutrophils. On the other hand, the activity of leucine aminopeptidase was not only found but also inhibited by diazotized sulfanilic acid without the inhibition of lactate dehydrogenase, a cytosol enzyme, in all the neutrophils, suggesting that leucine aminopeptidase is located generally on the plasma membrane as an ecto-enzyme in the neutrophils.     

Leucine aminopeptidase as an ecto-enzyme of polymorphonuclear neutrophils

Intact polymorphonuclear neutrophils were modified chemically by a poorly permeable reagent, diazotized sulfanilic acid, and the changes in the activity of 5′-nucleotidase, alkaline phosphodiesterase, and leucine aminopeptidase were examined. Among three plasma membrane enzymes, 5′-nucleotidase activity was hardly detected in the human neutrophils. The activity of alkaline phosphodiesterase was observed in all the neutrophils examined, but was not inhibited by diazotized sulfanilic acid in the guinea-pig neutrophils. On the other hand, the activity of leucine aminopeptidase was not only found but also inhibited by diazotized sulfanilic acid without the inhibition of lactate dehydrogenase, a cytosol enzyme, in all the neutrophils, suggesting that leucine aminopeptidase is located generally on the plasma membrane as an ecto-enzyme in the neutrophils.     

Leucine aminopeptidase and ferricyanide reductase activities in radish microsomes

A NADH-ferricyanide reductase activity found in radish microsomes isolated from germinated seeds has been shown to be stimulated by pCMB and pCMBS which are both strong nactivators of many plant proteolytic enzymes. In the same preparation a leucine aminopeptidase was found while endoprotease and carboxypeptidase activities were not detected using exogenous substrates. The aminopeptidase, highly active at the same optimal pH-condition of FeCN reductase, was stimulated by CoCl₂ and non-polar detergents (Triton X-100 and Brij 35). It was inhibited by sulphydryl reagents. By gel filtration of microsomal detergent extract two peaks of activity were separated: red I coeluted with LeuAPase and red II, free of aminopeptidase. Red I, a protein, was inhibited by sulphydral reagents and stimulated by duroquinone. Red II, stimulated by pCMB, is not a protein because of the small size and the noninfluence of heating treatment on catalytic activity.     

Studies on the histochemical “Leucine aminopeptidase” reaction

Summary This paper summarizes the basic characteristics of the reactants involved in the usual LAP (naphthylamidase) reaction and the tetrazonium dye DBB coupling. In the subsequent experimental part the adsorption characteristics of the reactants, their diffusion rates and their behaviour towards lipids are studied in different model systems. Attention is called to the microcrystalline and insoluble nature of the final reaction product (FRP), and further to the very marked diffusion hindrance to this product in tissue sections. The quantitative aspects of the reaction pattern are considered.The findings lead to the suggestion of extended control experiments, in which the adsorption characteristics of DBB, the 2-Namine and the FRP are separately checked. These recommendations should be applied in cases where there is reason to doubt the correct localization of the FRP.The rate of coupling seems rapid enough to ensure a correct subcellular enzyme localization provided the PRP—the 2-Namine—is liberated and trapped in a compartment containing excess of DBB, i.e. in activated lysosomes and autophagosomes.The following Abbreviations will be used in the text LAP the so-called leucine Amino-peptidase reaction - DBB Diazo Blue B, Fast Blue B, tetrazotized diorthoanisidine (C.I. No. 37235); the amine content not stated (Sigma Comp.) - 1-NA 1-Naphthol - 2-NA 2-Naphthylamine - LNA Leucyl-2-naphthylamide - Arg-NA Arginine-2-naphthylamide - BANA Benzoyl-DL-arginine-2-naphthylamide - PRP The primary reaction product - FRP The final reaction product     

Leucine ureido derivatives as aminopeptidase N inhibitors using click chemistry. Part II

Aminopeptidase N (APN) has been proved to be deeply associated with cancer angiogenesis, metastasis and invasion. Therefore, APN gains increasing attention as a promising anti-tumor target. In the current study, we report the design, synthesis, biological evaluation and structure-activity relationship of one new series of leucine ureido derivatives containing the 1,2,3-triazole moiety. Among them, compound 31f was identified as the best APN inhibitor with IC₅₀ value being two orders of magnitude lower than that of the positive control bestatin. Compound 31f possessed selective cytotoxicity to several tumor cell lines over the normal cell line human umbilical vein endothelial cells (HUVECs). Notably, when combined with 5-fluorouracil (5-Fu), 31f exhibited synergistic anti-proliferation effect against several tumor cell lines. At the same concentration, 31f exhibited much better anti-angiogenesis activities than bestatin in the HUVECs capillary tube formation assay and the rat thoracic aorta rings test. In the in vitro anti-invasion assay, 31f also exhibited superior potency over bestatin. Moreover, considerable in vivo antitumor potencies of 31f alone or in combination with 5-Fu were observed without significant toxic signs in a mouse heptoma H22 tumor transplant model.     

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.     

Leucine aminopeptidase activity in the digestive tract of perch, Perca fluviatilis L.

The distribution of the enzyme leucine aminopeptidase (LAP, EC 3.4.1.1) in the digestive tract of perch, Perca fluviatilis L., was investigated histochemically. The enzyme was present in the mucosa of the entire intestine and was absent in the oesophagus, stomach and pyloric sphincter. In the intestine, the enzyme was localized in the supranuclear cytoplasm of the columnar cells and was strongest in the brush border area. Enzyme activity was also present in the lumen of the intestine. The activity of the enzyme in the intestine decreased slightly towards the rectum. In perch, the final digestion of peptides and aminopeptides is both extracellular and intracellular and takes place along the entire length of the intestine.     

A survey of proteinases active during meiotic development

Microsporogenesis in Lilium longiflorum Thunb. is a naturally synchronous process and affords a system in which to study stage-specific events of meiosis and anther development. Zymogram gel analyses were conducted with extracts from a variety of stages of anther development to identify proteinases which likely play roles in anther metabolism. These experiments revealed that several proteinases are present at different stages of anther development, and class-specific inhibitors were used to classify these enzymes. Proteolytic activities increased as anther development proceeded and these activities were temporally correlated with the apoptotic events which precede dehiscence, as well as with events crucial for the maturation of viable pollen. Received: 12 October 1999 / Accepted: 13 November 1999