Development of leucine aminopeptidase activity during daylily flower growth and senescence

The possibility that exopeptidases, i.e. aminopeptidases and carboxypeptidases, in addition to the previously studied endopeptidase might also be developmentally regulated in daylily petals was examined. The level of leucine aminopeptidase and endopeptidase activities changed after the flower was fully open while that of carboxypeptidase activity remained relatively unchanged throughout senescence. Leucine aminopeptidase activity seemed to increase after the flower was fully open and peaked several hours earlier than endopeptidase did. Taken together, it is postulated that leucine aminopeptidase might play a role in protein turnover during flower opening and in the initiation of protein hydrolysis associated with petal senescence while the endopeptidase could be responsible for the breakdown of the bulk of proteins at the later stages. The drop in leucine aminopeptidase activity associated with the onset of daylily petal senescence was effectively halted by a cycloheximide treatment of cut daylily flowers for 24 h which was previously shown to prolong the vase life of the flowers and prevent protein loss from the petals. Apart from both being developmentally regulated in daylily petals, the leucine aminopeptidase activity and the previously studied endopeptidase are different in several aspects. They appear to have different pH optima, 8 for leucine aminopeptidase and 6.2 for endopeptidase. Unlike the endopeptidase activity, no new leucine aminopeptidase isozymes appeared during petal senescence, and the leucine aminopeptidase did not appear to belong to the cysteine class of proteolytic enzymes.     

Distribution of amino acids in the cellular fractions ofStaphylococcus aureus

WhenStaphylococcus aureus cells were labeled with a single radioactive amino acid for 20 minutes, the highest activity, except for alanine, leucine, and glycine, was found in the free pool. Significant amounts of the above amino acids and also valine and methionine were incorporated into the protein — cell wall fraction.Cells previously labeled with a single amino acid underwent a net loss of radioactivity when transferred to buffer, glucose, or complete medium. An exception was glycine. The greatest loss in activity occurred in the free pool.While some amino acids (alanine, cystine) were transferred from the free pool to the protein — cell wall fraction under all conditions tested, others (glutamic acid, proline) were transferred only under conditions of growth.Cells labeled with certain single amino acids and then transferred to a complete medium lost a significant portion of the label. The most extreme case noted was proline, but other amino acids also effluxed from the cell under these conditions.     

Mammalian lens dipeptidyl aminopeptidase III

An intracellular exopeptidase identified as dipeptidyl aminopeptidase III (DAP III) was found to be abundant in the bovine lens. The enzyme contained in aqueous extracts exhibited a marked preference, compared to other dipeptidyl-β-naphthylamides, for the release of Arg-Arg from Arg-Arg-2-NNap at the optimum pH 9.0 and 37°. The K_m for this substrate was estimated to be 2.83 × 10^?5M. Lens DAP III was inhibited by EDTA, p-chloromercuriphenyl sulfonate, and puromycin. Lens aminopeptidase activities measured at pH 7.5 on the β-naphthylamides of leucine, alanine, and arginine, included for comparison, suggested that not only is leucine aminopeptidase abundant, but also other aminopeptidases that appear to include alanine aminopeptidase and aminopeptidase B.     

Purification and properties of an intracellular leucine aminopeptidase from the fungus,Penicillium citrinum strain IFO 6352

An intracellular leucine aminopeptidase (LAP) fromPenicillium citrinum (IFO 6352) was purified to homogeneity using three successive purification steps. The enzyme has a native molecular mass of 63 kDa using HPLC gel filtration analysis and a molecular mass of 65 kDa when using SDS-polyacrylamide gel electrophoresis. This monomeric aminopeptidase showed maximum enzyme activity at pH 8.5. An optimum temperature was 45–50°C whenl-Leu-p-nitroanilide (pNA) was the substrate, and enzyme activity drastically decreased above 60°C. The Michaelis-Menten constants forl-Leu-pNA andl-Met-pNA were 2.7 mM and 1.8 mM, respectively. When the enzyme reacted with biosynthetic methionyl human growth hormone, it showed high specificity for N-terminal methionine residue and recognized a stop sequence (Xaa-Pro). The aminopeptidase was inactivated by EDTA or 1,10-phenanthroline, indicating that it is a metallo-exoprotease. Enzyme activity was restored to 90% of maximal activity by addition of Co²⁺ ions. The activity of EDTA-treated enzyme was restored by addition of Zn²⁺, but reconstitution with Ca²⁺, Mg²⁺ or Mn²⁺ restored some enzyme activity. It is likely that Co²⁺ ions play an important role in the catalysis or stability of thePenicillium citrinum aminopeptidase, as zinc plays a similar function in other leucine aminopeptidases.     

Human polymorphonuclear leukocytes aminopeptidases

In human polymorphonuclear leukocytes a methionine, leucine, arginine, phenylalanine and alanine aminopeptidase activities were detected, both in cytosol and secondary granules. All activities were EDTA sensitive and their pH optima were in the range of pH 6.5 to 8.6. In the cytosol two enzymes could be distinguished, broad substrate specificity aminopeptidase of pH 4.7-4.9 and a chloride dependent arginine aminopeptidase of pI 5.3-5.5. The granules contain aminopeptidase of pI 4.0-4.6 and of pI 9.8-10.2, different from those in the cytosol. Among them broad specificity aminopeptidases and possibly specific methionine and leucine aminopeptidases could be discerned.     

Intracellular aminopeptidases inStreptomyces lividans 66

Summary We have investigated the aminopeptidase activities present inStreptomyces lividans strains. The majority of these activities proved to be intracellular with multiple active species. Two aminopeptidase P genes were identified to be responsible for the ability to hydrolyze amino terminal peptide bonds adjacent to proline residues. Two other broad spectrum aminopeptidases were found to display homology at both the DNA and protein levels. One showed significant homology to PepN proteins, particularly around the putative zinc-binding residues which are important for catalysis. The second broad spectrum activity was not analyzed in detail but showed a different spectrum of substrate specificity to that of PepN.     

Induction of synchronous differentiation (encystment) in Physarum polycephalum myxamoebae

Encystment of Physarum polycephalum myxamoebae, grown under nearly identical physiological conditions as plasmodia is induced by transfer to a salts medium containing 0.5 M mannitol or mannose. After 24 h induction approximately 50% of amoebae had differentiated to cells which were identified to be young cysts by light and electron microscopy. Several other polyols, sugars, biogenic amines, and a starvation period from 24 h to one week caused no reproducible cyst formation. In contrast to the formation of dormant forms in the plasmodial stage of the life cycle, the induction of cysts and their germination to amoebae are not inhibited neither by actinomycin C nor by cycloheximide. In addition, the isoenzyme spectra of aminopeptidases and acid proteases remain nearly identical in growing and differentiating amoebae.Abbreviations SD semi-defined BSS basal salts solution The investigation is a part of the Ph. D. thesis of A. Haars, Göttingen, 1976     

A lysosomal aminopeptidase isozyme in differentiating yeast cells and protoplasts

Summary Cells of Saccharomyces cerevisiae that have been growing exponentially for many generations contain low activities of lysosomal enzymes. In contrast to such fully adapted cells, differentiating or resting cells contain comparatively high activities of these enzymes. Thus, the digestive enzymes seem to be involved in the process of biochemical differentiation.One of the four aminopeptidase isozymes present in extracts from yeast cells is localized in the vacuoles. This lysosomal enzyme can be separated from the other aminopeptidases by Sephadex G-150 gel filtration. Its specific activity is about 4 times higher in stationary cells than in exponentially growing cells.Upon incubating protoplasts in a buffered sorbitol medium the activities of proteases and RNase increase significantly. A corresponding increase of lysosomal aminopeptidase activity occurs in the absence of glutamic acid or casein hydrolysate. Cycloheximide and actinomycin D inhibit the increase of lysosomal hydrolase activities in differentiating protoplasts. The observed changes of enzyme activities are probably due to induced synthesis of the respective proteins.The present work has been supported by the Swiss National Science Foundation.     

Proteolytic enzymes in Chlamydomonas I. A survey on the aminopeptidase pattern in asynchronous vegetative cells of Chlamydomonas reinhardii

The supernatant of a crude extract from vegetative cells ofChlamydomonas reinhardii contains three different types of aminopeptidases.They are similar in their substrate specificities to the relativealanine specific aminopeptidases, the relative leucine specificaminopeptidases and the specific proline iminopeptidases describedin many other systems. Relative alanine specific aminopeptidasewhich also cleaves N-terminal Lys and Leu residues has a molecularweight of 92,000 daltons and is inhibited by zinc and manganeseions.Relative leucine specific aminopeptidase shows high activitywith N-terminal Phe besides Leu, and is capable of cleavingTyr, Pro, and to a minor degree Ala. It has a molecular weightof 76,000 daltons. No effects on its activity were detectedin the presence of divalent cations or chelating agents. Theiminopeptidase specifically splits N-terminal Pro and has amolecular weight of about 255,000 daltons. All the enzymes showoptimal activity at pH 8.0–8.5. The two aminopeptidases can be separated from the iminopeptidaseby ammonium sulfate solubilization and from each other by subsequentfractionation on DEAE-cellulose. Relative leucine specific activityappeared as a single enzyme in all the fractionation techniquesused, but it gave two distinct bands when crude extracts wererun on native polyacrylamide gels. Therefore, this enzyme mayexist in multiple molecularforms. (Received October 17, 1978; )     

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.     

Leucine aminopeptidase from Arabidopsis thaliana. Molecular evidence for a phylogenetically conserved enzyme of protein turnover in higher plants.

Leucine aminopeptidases are exopeptidases which are presumably involved in the processing and regular turnover of intracellular proteins; however, their precise function in cellular metabolism remains to be established. Towards this goal, a full-length complementary DNA encoding a plant leucine aminopeptidase was isolated from a cDNA library of Arabidopsis thaliana and sequenced. The nucleotide sequence showed 49.5% identity to the Escherichia coli xerB-encoded leucine aminopeptidase. Sequence analysis revealed that the cDNA encodes a polypeptide of 520 amino acids with a calculated molecular mass of 54,506 Da. The C-terminal part (amino acids 200-520) of the deduced amino acid sequence showed 43.8% sequence identity to the xerB-encoded leucine aminopeptidase and 42.6% sequence identity to the amino acid sequence of bovine lens leucine aminopeptidase (EC 3.4.11.1). No sequence similarity (not even over short sequence elements) was observed with any other known peptidase or proteinase sequence. The cDNA was expressed as a fusion protein from the lacZ promoter in E. coli. Enzymatic analysis proved that the cloned cDNA encoded an active leucine aminopeptidase. The properties of this enzyme, including metal requirements, inhibitor sensitivity, pH optimum and the remarkable temperature stability, are very similar to those reported for leucine aminopeptidases from other tissues. Amino acids involved in metal and substrate binding in bovine lens aminopeptidase are completely conserved in the plant enzyme as well as in the XerB protein. Our results show that leucine aminopeptidases form a superfamily of highly conserved enzymes, spanning the evolutionary period from the bacteria to animals and higher plants. This is the first aminopeptidase cloned from a plant.     

Proteinases in growth and spore formation of Bacillus thuringiensis]

Some serine proteases and leucine aminopeptidases were detected inside and outside the cells during the analysis of three crystalline and two acrystalline strains of Bac. thuringiensis var. galleriae. The data obtained on the protease formation during growth and sporulation and the level of their activity are indicative of intracellular proteases involvement in spore- and crystal formation. The enzymes isolated from the culture medium do not probably take part in these processes. The intracellular enzymes may account for the different crystal protein composition of various strains due to limited proteolysis of crystal proteins in the course of biosynthesis.     

Purification and Some Properties of Two Aminopeptidases from Sardines

Two fish aminopeptidases designated as aminopeptidases I and II were purified by DEAE-cellulose chromatography, gel filtration on Sephadex G-200, and isoelectric focusing. The final preparations of enzymes I and II were judged nearly homogenous by polyacrylamide gel I, electrophoresis. The molecular weights of enzymes I and II were determined by gel filtration to be 370,000 and 320,000, respectively. The isoelectric points were 4.1 (I) and 4.8 (II), Both enzymes were inhibited by EDTA and activated by Co⁺⁺. Bestatin could inhibit enzyme I but not enzyme II. Enzymes I and II rapidly hydrolyzed not only synthetic substrates containing alanine or leucine but also di-, tri-, and tetra-alanine. Judged from all of these properties, sardine aminopeptidases resemble human alanine aminopeptidase. Enzyme I retained more than 70% of its original activity in 15% NaCl, suggesting the enzyme participates in hydrolyzing fish proteins and peptides during fish sauce production.     

Sensitivity of D-beta-hydroxybutyrate dehydrogenase to proteolytic enzymes; influence of phospholipids

Controlled proteolysis of D-beta-hydroxybutyrate dehydrogenase in different forms were carried out using several proteases with different and well known specificities. The results obtained were the following: Purified apoBDH (phospholipid-free) was rapidly and strongly inactivated by all proteases tested except leucine aminopeptidase , in contrast with non-membrane enzymes which were unaffected by all proteases. BDH activity was completely preserved when proteases were incubated with either native BDH (membrane linked) or reconstituted BDH with reactivating-phospholipids (lecithins of total mitochondrial phospholipids), while non-reactivating-phospholipids gave no protection against proteases. C-terminal part of the enzyme was found to be essential for enzymatic activity while the N-terminal aminoacid is N-substituted. Controlled proteolysis whatever the protease used (except leucine aminopeptidase ) was followed by strong inactivation of the enzyme.     

Enzymatic analysis of Blomia tropicalis and Blomia kulagini (Acari: Echimyopodidae) allergenic extracts obtained from different phases of culture growth

The majority of important allergenic extracts from arthropods present enzymatic activity. This activity has been studied particularly in Dermatophagoides house dust mites because of its implication in the stability and immunogenicity of extracts used as tools for the diagnosis and specific treatment of allergic diseases. Extracts from cultures of Blomia tropicalis [van Bronswijk (1973a, b). Acarologia 15:477–489, 490–505] and Blomia kulagini (Zakhvatkin 1936) were used to study enzymatic profiles during three growth periods of the mite population: latency phase, maximum mite concentration during exponential growth, and drop stage. The activities of 19 enzymes were analyzed using the Api Zym system. The results show a large variety of enzymes. Some enzymatic activity was found to be (almost) exclusively attributable to mites. The activity levels of proteases, glycosidases and lipases overlapped with the growth curve. Only phosphatase activity showed no significant change during mite growth when compared with the culture medium. We suggest that the glycosidases (β-galactosidase, β-glucuronidase, β-N-acetylglucosaminidase, α-mannosidase and α-fucosidase) and proteases (leucine aminopeptidase and trypsin) may constitute suitable parameters for inclusion in the quality control process for the production of allergenic mite extracts, and may help define a new index for conducting environmental controls.     

The ecology and electrophoretic analysis of the damselfly,Argia vivida hagen,living in a geothermal gradient

The ecology and electrophoretic properties of a damselfly, Argia vivida Hagen, inhabiting a geothermal gradient were studied. Monthly sampling of five sites revealed nymphal colonization along a 15–40°C thermal gradient; greatest densities occurred between 15–27°C. An electrophoretic analysis of proteins suggest that nymphs were adapted to a wide range of temperatures which was evidenced by differential activity of four enzyme systems (glucose-6-phosphate dehydrogenase, lactate dehydrogenase, leucine aminopeptidase, and tetrazolium oxidase). Evidence suggests the nymphs acclimated to different temperatures by altering the structure of important isozymes and expressed certain genetic features characteristic of individuals naturally found at a given temperature.Published with the approval of the Director of the University of Idaho Agriculture Experiment Station as Research Paper No. 78612.     

Aminopeptidase from Sphingomonas capsulata

A novel aminopeptidase with unique substrate specificity was purified from a culture broth of Sphingomonas capsulata. This is the first reported aminopeptidase to demonstrate broad substrate specificity and yet release glycine and alanine with the highest efficacy. On a series of pentapeptide amides with different N-terminal amino acids, this enzyme efficiently releases glycine, alanine, leucine, proline, and glutamate with the lowest turnover value of 370 min(-1) for glutamate. At pH 7.5 (pH optimum) and 25 degrees C, the kinetic parameters for alanine para-nitroanilide were found to be k(cat) = 7600 min(-1) and K(m) = 14 mm. For alanine beta-naphthylamide, they were k(cat) = 860 min(-1) and K(m) = 6.7 mm. Polymerase chain reaction primers were designed based upon obtained internal sequences of the wild type enzyme. The subsequent product was then used to acquire the full-length gene from an S. capsulata genomic library. An open reading frame encoding a protein of 670 amino acids was obtained. The translated protein has a putative signal peptide that directs the enzyme into the supernatant. A search of the amino acid sequence revealed no significant homology to any known aminopeptidases in the available data bases.     

Enzymatic digestibility of peptides cross-linked by ionizing radiation

p6gestibility by proteolytic enzymes of peptides cross-linked by ionizing radiation was investigated. Small peptides of alanine and phenylalanine were chosen as model compounds and aminopeptidases and carboxypeptidases were used as proteolytic enzymes. Peptides exposed to gamma-radiation in aqueous solution were analysed by high-performance liquid chromatography before and after hydrolysis by aminopeptidase M, leucine aminopeptidase, carboxypeptidase A and carboxypeptidase Y. The results obtained clearly demonstrate the different actions of these enzymes on cross-linked aliphatic and aromatic peptides. Peptide bonds of cross-linked dipeptides of alanine were completely resistant to enzymatic hydrolysis whereas the enzymes, except for carboxypeptidase Y, cleaved all peptide bonds of cross-linked peptides of phenylalanine. The actions of the enzymes on these particular compounds are discussed in detail.