Acid phosphatase and leucine aminopeptidase isozymes as biochemical markers of homogeneity in oil seed rape androgenetic lines

Extracts of cotyledons of Brassica napus plants (seed progenies of doubled haploid plants) were separated by electrophoresis on polyacrylamide gels and stained for acid phosphatase (ACP-E.C. 3.1.3.2.) and leucine aminopeptidase (LAP-E.C. 3.4.11.1.) enzymes to investigate the possibility of utilising isozymes as markers of homogeneity (purity) of plant populations. One zone of activity for acid phosphatase and two zones of activity for leucine aminopeptidase were identified on gels, some variation in isozyme patterns occurred in several androgenetic lines. This method is appropriate and consistent for testing the homogeneity of breeding lines-progenies of double haploid (D.H.) plants.     

Ecoenzymatic Stoichiometry in Relation to Productivity for Freshwater Biofilm and Plankton Communities

The degradation of detrital organic matter and assimilation of carbon (C), nitrogen (N), and phosphorus (P) by heterotrophic microbial communities is mediated by enzymes released into the environment (ecoenzymes). For the attached microbial communities of soils and freshwater sediments, the activities of β-glucosidase, β-N-acetylglucosaminidase, leucine aminopeptidase, and phosphatase show consistent stoichiometric patterns. To determine whether similar constraints apply to planktonic communities, we assembled data from nine studies that include measurements of these enzyme activities along with microbial productivity. By normalizing enzyme activity to productivity, we directly compared the ecoenzymatic stoichiometry of aquatic biofilm and bacterioplankton communities. The relationships between β-glucosidase and α-glucosidase and β-glucosidase and β-N-acetylglucosaminidase were statistically indistinguishable for the two community types, while the relationships between β-glucosidase and phosphatase and β-glucosidase and leucine aminopeptidase significantly differed. For β-glucosidase vs. phosphatase, the differences in slope (biofilm 0.65, plankton 1.05) corresponded with differences in the mean elemental C:P ratio of microbial biomass (60 and 106, respectively). For β-glucosidase vs. leucine aminopeptidase, differences in slope (0.80 and 1.02) did not correspond to differences in the mean elemental C:N of biomass (8.6 and 6.6). β-N-Acetylglucosaminidase activity in biofilms was significantly greater than that of plankton, suggesting that aminosaccharides were a relatively more important N source for biofilms, perhaps because fungi are more abundant. The slopes of β-glucosidase vs. (β-N-acetylglucosaminidase + leucine aminopeptidase) regressions (biofilm 1.07, plankton 0.94) corresponded more closely to the estimated difference in mean biomass C:N. Despite major differences in physical structure and trophic organization, biofilm and plankton communities have similar ecoenzymatic stoichiometry in relation to productivity and biomass composition. These relationships can be integrated into the stoichiometric and metabolic theories of ecology and used to analyze community metabolism in relation to resource constraints.     

Streptomyces rimosus extracellular proteases 3. Isolation and characterization of leucine aminopeptidase

Summary A leucine aminopeptidase was purified to homogeneity fromStreptomyces rimosus culture filtrates, which are waste broth of oxytetracycline bioproduction process. Purification procedure includes ultrafiltration and chromatography on CM-Sephadex, AH-Sepharose and FPLC Mono S column. The enzyme is a monomer with molecular weight of 27,500 Daltons and pI of 7.3, stable in broad pH range and up to 70°C. It is a metallo enzyme dependent on Ca²⁺ ions for its full activity. By its specificity it is a true aminopeptidase active on amino acid amide, arylamide, peptide and ester bonds. The hydrolysing activity shows preference for leucine at the N-terminal position of substrates, also acts on aromatic acids and methionine, but does not release glycine, proline, acidic amino acids orD-amino acid residues.     

Plant-Induced Hatching of Eggs of the Soybean Cyst Nematode Heterodera glycines

Root diffusate from soybean plants caused greater hatching of Heterodera glycines eggs during vegetative growth of the host, but the activity declined with plant senescence. Chelation of the root diffusate with ethylenediamine tetraacetic acid (EDTA) significantly increased hatching activity for H. glycines eggs. Diffusate from leafless plants caused little hatching, whereas treatment of intact plants with the growth regulators gibberellin and kinetin had no effect on the hatching activity of root diffusate. Treating H. glycines eggs with zinc chloride and root diffusate reduced egg hatching from zinc chloride alone. Levels of zinc in the root diffusate were insufficient to induce egg hatch, based on analysis by atomic absorption spectrophotometry. The enzymatic activity of leucine aminopeptidase in H. glycines eggs was not altered by treatment with chelated or nonchelated root diffusate.     

Variations des zymogrammes du tube digestif et du muscle chez Cyclonassa neritea en fonction de la température d''acclimatation

Soluble proteins, esterases 2C, acid phosphatases of the digestive gland and foot muscle of Cyclonassa neritea, were compared using polyacrylamide gradient gels. α-Glucosidases, alkaline phosphatases, l-leucine aminopeptidase and peptidase were studied from digestive gland extracts. Molecular weights of isoenzymes were evaluated with 5000 d accuracy. Variation in activity of the most important isoenzymes of each enzyme under the influence of acclimation temperature was measured. In both muscle and digestive gland, the concentration of soluble proteins is stable. Through the whole acclimation temperature range, esterase activity per mg protein decreased with increased temperature. l-Leucine aminopeptidase activity decreases steadily from 10 to 25°, even though the two alkaline phosphatase isoenzyme activities increase. The other enzymes have their maximum activities at 20°.     

Biochemical Differences Between Crop Tissue and Crop Milk of Pigeons (Columba livia)

Biochemical differences between crop tissue (CT) and crop milk (CM) of pigeons were evaluated in terms of activity of certain enzymes, content of protein and nucleic acids and mitogenisity in vitro. Whereas the activities of aspartate transaminase, alanine transaminase, maltase, trehalase and cellobiase were significantly higher in CT, those of alkaline phosphatase, leucine aminopeptidase, gamma glutamyl transpeptidase, acid phosphatase and lactase were greater in CM. The CM also showed significantly higher levels of protein, DNA and RNA than CT. In contrast, the in vitro mitogenic effect of CT was greater than that of CM, and this property was enhanced during the breeding period. The biochemical composition of crop milk did not differ significantly in the first 4 days of secretion. It appears that certain factors responsible for growth stimulation accumulate in the crop tissue of pigeons during incubation.     

EXTRACELLULAR ENZYMES OF THE MICROCYSTIS AERUGINOSA PCC 7813 STRAIN ARE INHIBITED IN THE PRESENCE OF HYDROQUINONE AND PYROGALLOL,ALLELOCHEMICALS PRODUCED BY AQUATIC PLANTS1

Several cyanobacterial species have a high potential to dominate in marine environments and freshwater reservoirs, and the ecological and physiological reasons for this phenomenon are not understood comprehensively. In this study, the ability of a Microcystis aeruginosa Kütz. strain to produce free dissolved enzymes was documented. We have observed that this highly toxic strain releases alkaline phosphatase, leucine aminopeptidase, and β‐glucosidase into the ambient environment. Additionally, the inhibitory activity of selected phenols produced by aquatic plants on the activity of these enzymes was analyzed. The investigated compounds, pyrogallol and, to a lesser degree, hydroquinone, decreased the activity of extracellular enzymes produced by M. aeruginosa, with leucine aminopeptidase being the most sensitive to the inhibitors. The noncompetitive character of enzymatic inhibition suggests that the polyphenols produced by aquatic plants are able to influence the activity of different extracellular or membrane‐bound enzymes.     

Isoenzyme patterns of pathogenic and nonpathogenic thermophilic Naegleria strains by isoelectric focusing

Pernin P. 1984. Isoenzyme patterns of pathogenic and nonpathogenic thermophilic Naegleria strains by isoelectric focusing. International Journal for Parasitology14: 459–465. The isoenzymatic patterns of different strains of Naegleria were studied by isoelectric focusing (I.E.F.) on polyacrylamide gels for seven enzymatic activities (leucine amino peptidase; lactate dehydrogenase; glucose 6 phosphate dehydrogenase; propionyl esterase; glucose phosphate isomerase; malate dehydrogenase; acid phosphatase), two of which (lactate dehydrogenase and glucose 6 phosphate dehydrogenase) were being investigated for the first time. The three pathogenic N. fowleri strains share a common pattern for most of the enzymes tested except for glucose 6 phosphate dehydrogenase, and thus form a very homogeneous species, while thermophilic nonpathogenic strains show more heterogeneity particularly for leucine amino peptidase and glucose 6 phosphate dehydrogenase.I.E.F. must be considered as a supplementary and rapid method for the identification of N. fowleri and as a powerful tool to demonstrate the complexity of different genera of free-living amoebas.     

Adaptation of intestinal cell membrane enzymes to low temperatures in the Antarctic teleost Pagothenia bernacchii

The enzymatic activity (expressed as milliunits per milligram total proteins) of three intestinal brush-border membrane enzymes, leucine aminopeptidase, alkaline phosphatase and maltase, measured over a range of temperatures between 1.5 and 37 °C, has been found to be much higher in the Antarctic fish Pagothenia bernacchii than in the temperate fish Anguilla anguilla. To explain this experimental observation the apparent Michaelis-Menten constant, the maximal velocity, the activation energy values and the thermal stability of these three enzymes were measured. The apparent Michaelis-Menten constant values of leucine amino peptidase and alkaline phosphatase were different in the intestine mucosal homogenate of the two fish at each measured temperature (from a minimum of 2.5 to a maximum of 37 °C). However, the values found at 2.5 °C for the Antarctic species and 15 °C for the eel where comparable. Furthermore, its value was unchanged in eel intestine apical membranes, both in the presence and without enzyme lipid microenvironment. While the maximal enzymatic activities of the leucine aminopeptidase and maltase did not decrease without their enzyme lipid microenvironment, produced by treatment with Triton X-100, the impairment of alkaline phosphatase maximal activity cannot be significantly differentiated from a non-specific inhibitory effect of the detergent. The activation energy values of leucine amino peptidase, alkaline phosphatase and maltase were lower in the Antarctic fish (11.7, 5.6 and 11.8 kcal·mol^-1, respectively) than in the eel (13.6, 7.6 and 13.1 kcal·mol^-1, respectively). The thermal stability of alkaline phosphatase and maltase is different in Pagothenia bernacchii and Anguilla anguilla intestinal homogenate.Abbreviations BBM brush border membrane - E _a activation energy - EGTA ethyleneglycol-bis-(-amino ethylether)N, N-tetraacetic acid - HEPES 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethane sulphonic acid - Km_app apparent Michaelis-Menten constant - PMSF phenylmethyl-sulphonyl fluoride - TRIS TRIS (hydroxymethyl)-aminomethane     

Enzyme histochemistry and hormones of the developing gastrointestinal tract of the chick embryo

Summary During the differentiation of the gastrointestinal tract cell death occurs. In the present study the activity of lysosomal enzymes which may be associated with this process are investigated. These enzymes are leucine aminopeptidase, acid phosphatase and beta galactosidase. The action of glucagon on leucine aminopeptidase and acid phosphatase is reported.     

Serum Lactic Dehydrogenase,Leucine Aminopeptidase and 5-Nucleotidase Activity: Observations in Patients with Carcinoma of the Pancreas and Hepatobiliary Disease

Serum lactic dehydrogenase, leucine aminopeptidase, 5-nucleotidase and alkaline phosphatase activities were investigated in a number of diseases involving the hepatobiliary system.Leucine aminopeptidase was found to be a sensitive indicator of biliary obstruction, serum 5-nucleotidase slightly less sensitive, and alkaline phosphatase appreciably less sensitive. Leucine aminopeptidase and 5-nucleotidase activities were often increased by malignant infiltration of the liver and primary hepatic disease even in the absence of jaundice.Serum lactic dehydrogenase was frequently increased in primary hepatic disease and malignant disorders but was not apparently affected by bile duct obstruction per se. Thirty-five of 45 patients with proved malignancy had increased lactic dehydrogenase levels.The highest leucine aminopeptidase levels were encountered in carcinoma of the head of the pancreas. The frequent increase in both serum lactic dehydrogenase and leucine aminopeptidase activities in patients with carcinoma of the head of the pancreas suggests that these combined estimations are useful laboratory procedures in the diagnosis of malignant extrahepatic obstruction.     

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.     

Purification and Identification of a Novel Leucine Aminopeptidase from <Emphasis Type="Italic">Bacillus thuringiensis israelensis</Emphasis>

A novel leucine aminopeptidase was purified from a Bacillus thuringiensis israelensis (Bti) culture. The purification stages included heating the concentrated supernatant to 65°C for 90 min, anion-exchange chromatography by DEAE cellulose, and hydrophobic chromatography by phenyl Sepharose. The specific activity of leucine aminopeptidase after the hydrophobic chromatography increased by 215.5-fold and the yield was 16%. The molecular weight of the active enzyme was 59 kDa. Mass spectrometry analysis of the 59-kDa leucine aminopeptidase revealed that this protein has at least 41% homology with the cytosol leucine aminopeptidase produced by Bacillus cereus. Maximal leucine aminopeptidase activity occurred at 65°C, pH 10 toward leucine as the amino acid terminus. The enzyme was strongly inhibited by bestatin, dithiothreitol, and 1,10-phenanthroline, indicating that the enzyme might be considered as a metallo-aminopeptidase that has disulfide bonds at the catalytic site or at a region that influences its configuration. Examination of the purified leucine aminopeptidase’s effect on the activation of the protoxin Cyt1Aa from Bti revealed that when it acts synergistically with Bti endogenous proteases, it has only a minor role in the processing of Cyt1Aa into an active toxin.     

Purification and properties of an extracellular leucine aminopeptidase from the Bacillus sp. N2

A halophilic bacterium was isolated from fermented anchovy sauce and identified as Bacillus species. An extracellular leucine aminopeptidase from Bacillus sp. N2 was purified to homogeneity using four successive purification steps. The enzyme has a native molecular mass of about 57 000 Da using FPLC gel filtration analysis and a molecular mass of 58 000 Da using SDS-polyacrylamide gel electrophoresis. This monomeric leucine aminopeptidase showed maximum enzyme activity at pH 9·5. The optimum temperature was 50 °C when L -Leu- p -nitroanilide was the substrate. The leucine aminopeptidase was inactivated by 1,10-phenanthroline, dithiothreitol and sodium dodecyl sulphate. Enzyme activity was increased by addition of Co²⁺. It is likely that Co²⁺ plays an important role in the catalysis or stability of the Bacillus sp. N2 leucine aminopeptidase. Sodium chloride (0–4·5 mol l⁻¹) increased the hydrolytic activity towards L -Leu- p -nitroanilide. The N-terminal amino acid sequence was Glu-Arg-Glu-Leu-Pro-Phe-Lys-Ala-Lys-His-Ala-Tyr-Ser-Thr-Ile. The purified enzyme had a high specificity for L -Leu- p -nitroanilide.     

Progesterone induction of lysozyme and peptidase activities in the porcine uterus.

When nonpregnant ovariectomized gilts were treated daily for 15 days with progesterone or progesterone plus estradiol, there was a tenfold increase in the amount of secreted protein that could be flushed from their uteri compared with control animals administered either no steroid or only estradiol. This increase was due to the appearance of a number of new proteins not present in the controls. In addition to a purple-colored protein with acid phosphatase activity, which has been described previously, there were large increases in lysozyme and leucine aminopeptidase activity. All three enzymes appeared to be induced by progesterone. By continuing the progesterone treatments for periods up to 60 days, it was possible to recover very high levels of each of these enzymes from the uterine flushings of the pigs. Cathepsin activities (B₁, D, and E) were also found to increase as progesterone treatment was prolonged. The levels of the peptidases does not seem to be coordinated since their activities change relative to each other when different animals are examined. Acid phosphatase (due entirely to the purple protein), lysozyme, and leucine aminopeptidase activities are also detectable in allantoic fluid after Day 30 of pregnancy and reach a maximum between Days 60 and 80. It is suggested that these enzymes may be maternal in origin.     

Biochemical and conformational characterization of a leucine aminopeptidase from Geobacillus thermodenitrificans NG80-2

In order to search for valuable and extremely thermo-stable enzymes that could be used in the protein hydrolysis industry, the gene corresponding to a leucine aminopeptidase from Geobacillus thermodenitrificans NG80-2 (GtLAP) was cloned and expressed in E. coli. The recombinant enzyme was purified, and its characteristics were examined. Meanwhile, potential applications of GtLAP in the hydrolysis of anchovy proteins were also investigated. GtLAP was overexpressed in IPTG-induced E. coli BL21 (pET28a-LAP) as a soluble protein, and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 125?±?8.75 U/mg proteins. The molecular mass of GtLAP was estimated to be 55?kDa by SDS-PAGE analysis. The optimal reaction temperature and pH of GtLAP were 70?°C and 8.0, respectively. Under optimal conditions, GtLAP showed a marked preference for Leu-p-nitroanilide, followed by Met- and Phe-derivatives. Activity of GtLAP was strongly stimulated by Ni²⁺ ions, but was strongly inhibited by Hg²⁺. Conformational studies via circular dichroism spectroscopy indicated that various factors could influence the secondary structure of GtLAP to various extents and further induce changes in enzymatic activity. Results of hydrolytic experiment showed that combining GtLAP with endogenous enzymes could significantly increase the degree of hydrolysis to anchovy proteins and concentrations of free amino acids in hydrolysates. In this regard, GtLAP could potentially be used in the protein hydrolysis industry.     

Partial characterization of the proteolytic enzymes in the gut of the banana weevil, Cosmopolites sordidus, and effects of soybean Kunitz trypsin inhibitor on larval performance

The proteolytic enzymes in the gut of the banana weevil, Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae), have been characterized. Both larvae and adults rely on a complex proteolytic system based on at least cathepsin D‐, cathepsin B‐, trypsin‐, chymotrypsin‐, leucine aminopeptidase‐, carboxypeptidase A‐, and carboxypeptidase B‐like activities. All endoproteolytic activities were higher in the anterior section of the gut, whereas the exopeptidases were evenly distributed in the anterior and middle sections, and almost no activity was detected in the posterior section. Gelatin‐containing gels confirmed the spatial organization of the proteolytic digestive process. According to this proteolytic profile, the STI (soybean Kunitz trypsin inhibitor) was tested in vivo to establish its potential as a resistance factor against C. sordidus. Newly hatched larvae fed on diets containing 0.2% (w/w) STI experience lower survival rates and display significant reductions in larval growth. Biochemical analysis carried out on guts of larvae reared on STI‐treated diet showed a reduction of trypsin‐like activity compared to that from larvae fed on control diet. This decrease was compensated with an induction of cathepsin B, whereas cathepsin D, chymotrypsin, and leucine aminopeptidase were not affected. These results are discussed as a basis for selecting appropriate inhibitors to obtain transgenic banana and plantain plants with enhanced resistance to this pest.     

Symbiotic interactions between arbuscular mycorrhizal (AM) fungi and male papaya plants: Its status,role and implications

Experiments were conducted to study the arbuscular mycorrhizal (AM) status and its role in P-uptake through assay of root phosphatases activities in four varieties of male Carica papaya L. viz. CO-1, CO-2, Honey Dew and Washington during flowering stages. In the present study, mean total root colonization of AM fungi recorded peak increase in flowering stage-II while mean root phosphatase (acid and alkaline) activities recorded peak increase in flowering stage-I. Unlike root colonization and root phosphatase activities, spore density did not exhibit any definite patterns and recorded a narrow range of fluctuation during different flowering stages of male C. papaya. The study brought out the fact that root colonization and spore density of AM fungi along with root phosphatase activities varied significantly within the four varieties of male C. papaya plants during each flowering stage. The study also recorded consistently higher acid root phosphatase activity than alkaline root phosphatase activity under P-deficient, acidic soil conditions during all flowering stages of male C. papaya plants. Studies revealed that the root colonization of AM fungi influenced root phosphatase activities (acid and alkaline) positively and significantly during all flowering stages of male C. papaya plants. A total of twelve species of AM fungi belonging to five genera viz. Acaulospora, Dentiscutata, Gigaspora, Glomus, and Racocetra were recovered from the rhizosphere of male C. papaya plants.     

Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques: A combined histochemical and biochemical investigation

Summary The reliability of enzyme histochemical observations of activities of acid hydrolases was investigated with a combined histochemical and biochemical study. Specimens of m. soleus, m. plantaris, m. gastrocnemius and diaphragm of normal and of vitamin E deficient rabbits were used. For the histochemical investigation, activity and localization of acid phosphatase, -glucuronidase, leucine aminopeptidase and E₆₀₀ resistant non-specific aryl-esterase were examined with semipermeable membrane techniques. For the biochemical investigation, activity of acid phosphatase, -glucuronidase, cathepsin D, acid maltase and neutral maltase was determined.By means of statistical calculations the enzyme activities demonstrated with histochemical techniques were compared with the enzyme activities determined with biochemical techniques.In the present communication the histochemical findings are reported and discussed. From the histochemical findings it appeared that activity of the acid hydrolases investigated is strongly increased in both a granular and a diffuse pattern in skeletal muscle of vitamin E deficient rabbits. The statistical calculations of the histochemical findings clearly reveal that the increased activity of one acid hydrolase was highly significantly paralleled by an increased activity of a second acid hydrolase. Moreover the probability that the activity of all other histochemically studied acid hydrolases was significantly increased was rather high.The increase in activity of the acid hydrolases studied was the same in muscles with an aerobic or an anaerobic metabolism. Moreover there was no difference in activity and localization of the acid hydrolases in aerobic type I and anaerobic type II fibres.The localization of acid phosphatase and -glucuronidase activity in muscle fibres mostly coincided. In cases where these enzymes were localized both centrally and in the subsarcolemnal areas of the muscle fibres, the activity of E₆₀₀ resistant naphtholesterase was usually, and the activity of leucine aminopeptidase was exclusively located in the subsarcolemnal areas. All of the examined acid hydrolases were found to be present in the inflammatory exudate and in the connective tissue.This study was partly extracted from the Ph. D. thesis of D.E. Israël (1977).