Characterization of Aminopeptidase P from the Unicellular Cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC6803

The PepP protein has been purified in vitro and characterized for the first time. It is encoded by the sll0136 gene of the unicellular cyanobacterium Synechocystis sp. PCC6803. It is established that the PepP protein is a Mn²⁺-dependent Xaa-Pro-specific aminopeptidase. The protein in the reaction of hydrolysis of the fluorescent peptide Lys(N-Abz)-Pro-Pro-pNA has a maximal activity at pH 7.6 and 32°C.     

Effect of X-Prolyl Dipeptidyl Aminopeptidase Deficiency on Lactococcus lactis

The genetic determinant (pepXP) of an X-prolyl dipeptidyl aminopeptidase (PepXP) has recently been cloned and sequenced from both Lactococcus lactis subsp. cremoris (B. Mayo, J. Kok, K. Venema, W. Bockelmann, M. Teuber, H. Reinke, and G. Venema, Appl. Environ. Microbiol. 57:38-44, 1991) and L. lactis subsp. lactis (M. Nardi, M.-C. Chopin, A. Chopin, M.-M. Cals, and J.-C. Gripon, Appl. Environ. Microbiol. 57:45-50, 1991). To examine the possible role of the enzyme in the breakdown of caseins required for lactococci to grow in milk, integration vectors have been constructed and used to specifically inactivate the pepXP gene. After inactivation of the gene in L. lactis subsp. lactis MG1363, which is Lac^- and Prt^-, the Lac⁺ Prt⁺ determinants were transferred by conjugation by using L. lactis subsp. lactis 712 as the donor. Since growth of the transconjugants relative to the PepXP⁺ strains was not retarded in milk, it was concluded that PepXP is not essential for growth in that medium. It was also demonstrated that the open reading frame ORF1, upstream of pepXP, was not required for PepXP activity in L. lactis. A marked difference between metenkephalin degradation patterns was observed after incubation of this pentapeptide with cell extracts obtained from wild-type lactococci and pepXP mutants. Therefore, altered expression of the pepXP-encoded general dipeptidyl aminopeptidase activity may change the peptide composition of fermented milk products.     

Aminopeptidase activity from germinated jojoba cotyledons

One major and two minor aminopeptidase activities from germinated jojoba (Simmondsia chinensis) cotyledon extracts were separated by ammonium sulfate precipitation and chromatofocusing. None of the activities were inhibited by 1,10 phenanthroline.

The major aminopeptidase, purified 260-fold, showed a pH optimum of 6.9 with leucine-p-nitroanilide as substrate, a molecular weight estimated at 14,200 by electrophoretic analysis, and an isoelectric point of 4.5 according to the chromatofocusing pattern. Activity was inhibited by p-chloromercuribenzoate, slightly stimulated by 1,10 phenanthroline and 2-mercaptoethanol, and not influenced by Mg²⁺ or diethyl pyrocarbonate. Inhibition by p-chloromercuribenzoate was prevented by the presence of cysteine in the assay. Leucine-p-nitroanilide and leucine-β-naphthylamide were the most rapidly hydrolyzed of 11 carboxy-terminal end blocked synthetic substrates tested. No activity on endopeptidase or carboxypeptidase specific substrates was detected. The major aminopeptidase showed activity on a saline soluble, jojoba seed protein preparation and we suggest a possible physiological role for the enzyme in the concerted degradation of globulin reserve proteins during cotyledon senescence.

     

Aminopeptidase C of Aspergillus niger Is a Novel Phenylalanine Aminopeptidase

A novel enzyme with a specific phenylalanine aminopeptidase activity (ApsC) from Aspergillus niger (CBS 120.49) has been characterized. The derived amino acid sequence is not similar to any previously characterized aminopeptidase sequence but does share similarity with some mammalian acyl-peptide hydrolase sequences. ApsC was found to be most active towards phenylalanine β-naphthylamide (F-βNA) and phenylalanine para-nitroanilide (F-pNA), but it also displayed activity towards other amino acids with aromatic side chains coupled to βNA; other amino acids with nonaromatic side chains coupled to either pNA or βNA were not hydrolyzed or were poorly hydrolyzed. ApsC was not able to hydrolyze N-acetylalanine-pNA, a substrate for acyl-peptide hydrolases.     

Aminopeptidase Activity in Marine Chroococcoid Cyanobacteria

Synechococci are important primary producers in the ocean and can also utilize some components of the dissolved organic matter (DOM). The readily utilizable DOM in seawater is mainly polymeric (e.g., protein, polysaccharide) or phosphorylated and requires hydrolysis prior to uptake. We examined whether synechococci express ectoenzymes to hydrolyze DOM components and considered the possible significance of ectohydrolases for Synechococcus ecology and organic matter cycling in the sea. Five strains of non-nitrogen-fixing synechococci in axenic cultures were tested for enzyme activities with fluorogenic substrates. All strains show ectocellular aminopeptidase activity, but other enzymes were undetectable. The aminopeptidase level was in the range determined for five marine heterotrophic bacterial isolates tested for comparison. Aminopeptidase was not secreted into the medium; the majority (74%; tested in WH 7803) was cell surface bound, and a small fraction was periplasmic. The periplasmic activity was not released by cold osmotic shock of WH 7803. Phenylmethylsulfonyl fluoride and EDTA, inhibitors of serine and metalloproteases, strongly or completely inhibited WH 7803 aminopeptidase. The enzyme seemed constitutive; per-cell activity did not change during incubations in unenriched seawater, bovine serum albumin, or nitrate-replete mineral medium. In natural planktonic assemblages in the Southern California Bight, aminopeptidase activity was correlated with Synechococcus abundance as well as the abundance of other bacteria. Ectocellular aminopeptidase may be common in marine synechococci and play roles in their nitrogen nutrition, particularly in low-nitrate and low-light environments. Since synechococci are much less abundant than heterotrophic bacteria in seawater, the impact of Synechococcus aminopeptidase on proteolysis in the sea is likely to be episodic and restricted to specialized microenvironments.     

Inactivation of sll0136 gene in Synechocystis sp. PCC 6803 results in the disturbance in protein biogenesis of photosynthetic complexes

The genome of cyanobacterium Synechocystis sp. PCC 6803 contains the sll0136 (pepP) gene encoding the putative homolog of proline aminopeptidase PII (AMPPII) of the heterotrophic bacterium Escherichia coli. AMPPII is known to cleave the N-terminal amino acid residue of peptides and proteins only in the case of a penultimate proline position. The Synechocystis sp. PCC 6803 insertion mutant with inactivated pepP gene is characterized by the reduced content of phycobiliproteins and also proteins of photosystem II, which may be related to the reduced synthesis or stability of corresponding proteins. A possible involvement of PepP in biogenesis of proteins of the photosynthetic apparatus is discussed.     

Ultrasound Treatment for Harvesting an Aminopeptidase from Lactic Acid Bacteria and Quantitation of the Enzyme by Enzyme-Linked Immunosorbent Assays

Ultrasound treatment of Lactococcus lactis subsp. cremoris AM2 was optimized to release a maximum amount of intracellular aminopeptidase without modifying the antigenicity of the enzyme. The cells were sonicated three times for 30 s at 23 W. Antibodies produced against the aminopeptidase purified from L. lactis subsp. cremoris AM2 enabled us to use immunoblotting to detect the enzyme in the lysates of all of the lactococci tested but not in the lysates of Leuconostoc strains, lactobacilli, and Streptococcus salivarus subsp. thermophilus. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantify the purified aminopeptidase; the detection limit was 4 ng/ml. The aminopeptidase in the supernatant obtained after the ultrasound treatment of strain AM2 cells was detected with the ELISA starting with a total protein concentration of 200 ng/ml. The proportion of equivalent purified aminopeptidase in the supernatant of L. lactis subsp. cremoris AM2 was about 2% of the total protein. Similarly, the aminopeptidase was quantified in different lactococci; the percentages varied between 0.16 and 2%, depending on the strain. The aminopeptidase content in a mixture of several lactic bacteria was also determined with the sandwich ELISA.     

Essential Role for an M17 Leucine Aminopeptidase in Encystation of Acanthamoeba castellanii

Encystation of Acanthamoeba leads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions such as starvation, low temperatures, and exposure to biocides. During encystation, a massive turnover of intracellular components occurs, and a large number of organelles and proteins are degraded by proteases. Previous studies with specific protease inhibitors have shown that cysteine and serine proteases are involved in encystation of Acanthamoeba, but little is known about the role of metalloproteases in this process. Here, we have biochemically characterized an M17 leucine aminopeptidase of Acanthamoeba castellanii (AcLAP) and analyzed its functional involvement in encystation of the parasite. Recombinant AcLAP shared biochemical properties such as optimal pH, requirement of divalent metal ions for activity, substrate specificity for Leu, and inhibition profile by aminopeptidase inhibitors and metal chelators with other characterized M17 family LAPs. AcLAP was highly expressed at a late stage of encystation and mainly localized in the cytoplasm of A. castellanii. Knockdown of AcLAP using small interfering RNA induced a decrease of LAP activity during encystation, a reduction of mature cyst formation, and the formation of abnormal cyst walls. In summary, these results indicate that AcLAP is a typical M17 family enzyme that plays an essential role during encystation of Acanthamoeba.     

Aminopeptidase Co,a new yeast peptidase

A new aminopeptidase — aminopeptidase Co — has been detected in the yeast $\text{Saccharomyces}\text{cerevisiae}$. The enzyme is only active in the presence of Co²⁺ions. Zn^2+- and Mn²⁺ions are inhibitory. The enzyme activity is also inhibited by chelating agents. Of the p-nitroanilide derivatives tested only those containing basic amino acids are cleaved.     

Purification and Functional Characterisation of Rhiminopeptidase A,a Novel Aminopeptidase from the Venom of Bitis gabonica rhinoceros

Background

Snake bite is a major neglected public health issue within poor communities living in the rural areas of several countries throughout the world. An estimated 2.5 million people are bitten by snakes each year and the cost and lack of efficacy of current anti-venom therapy, together with the lack of detailed knowledge about toxic components of venom and their modes of action, and the unavailability of treatments in rural areas mean that annually there are around 125,000 deaths worldwide. In order to develop cheaper and more effective therapeutics, the toxic components of snake venom and their modes of action need to be clearly understood. One particularly poorly understood component of snake venom is aminopeptidases. These are exo-metalloproteases, which, in mammals, are involved in important physiological functions such as the maintenance of blood pressure and brain function. Although aminopeptidase activities have been reported in some snake venoms, no detailed analysis of any individual snake venom aminopeptidases has been performed so far. As is the case for mammals, snake venom aminopeptidases may also play important roles in altering the physiological functions of victims during envenomation. In order to further understand this important group of snake venom enzymes we have isolated, functionally characterised and analysed the sequence-structure relationships of an aminopeptidase from the venom of the large, highly venomous West African gaboon viper, Bitis gabonica rhinoceros.

Methodology and Principal Findings

The venom of B. g. rhinoceros was fractionated by size exclusion chromatography and fractions with aminopeptidase activities were isolated. Fractions with aminopeptidase activities showed a pure protein with a molecular weight of 150 kDa on SDS-PAGE. In the absence of calcium, this purified protein had broad aminopeptidase activities against acidic, basic and neutral amino acids but in the presence of calcium, it had only acidic aminopeptidase activity (APA). Together with the functional data, mass spectrometry analysis of the purified protein confirmed this as an aminopeptidase A and thus this has been named as rhiminopeptidase A. The complete gene sequence of rhiminopeptidase A was obtained by sequencing the PCR amplified aminopeptidase A gene from the venom gland cDNA of B. g. rhinoceros. The gene codes for a predicted protein of 955 amino acids (110 kDa), which contains the key amino acids necessary for functioning as an aminopeptidase A. A structural model of rhiminopeptidase A shows the structure to consist of 4 domains: an N-terminal saddle-shaped β domain, a mixed α and β catalytic domain, a β-sandwich domain and a C-terminal α helical domain.

Conclusions

This study describes the discovery and characterisation of a novel aminopeptidase A from the venom of B. g. rhinoceros and highlights its potential biological importance. Similar to mammalian aminopeptidases, rhiminopeptidase A might be capable of playing roles in altering the blood pressure and brain function of victims. Furthermore, it could have additional effects on the biological functions of other host proteins by cleaving their N-terminal amino acids. This study points towards the importance of complete analysis of individual components of snake venom in order to develop effective therapies for snake bites.     

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris Wg2

An aminopeptidase was purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that included diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, gel filtration, and high-performance liquid chromatography over an anion-exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 95,000. The aminopeptidase was capable of degrading several peptides by hydrolysis of the N-terminal amino acid. The peptidase had no endopeptidase or carboxypeptidase activity. The aminopeptidase activity was optimal at pH 7 and 40°C. The enzyme was completely inactivated by the p-chloromecuribenzoate mersalyl, chelating agents, and the divalent cations Cu²⁺ and Cd²⁺. The activity that was lost by treatment with the sulfhydryl-blocking reagents was restored with dithiothreitol or β-mercapto-ethanol, while Zn²⁺ or Co²⁺ restored the activity of the 1,10-phenantroline-treated enzyme. Kinetic studies indicated that the enzyme has a relatively low affinity for lysyl-p-nitroanilide (K_m, 0.55 mM) but that it can hydrolyze this substrate at a high rate (V_max, 30 μmol/min per mg of protein).     

Aminopeptidase activity in the hemolymph and body tissues of the pulmonate gastropod Biomphalaria glabrata

The levels of aminopeptidase activity in the whole hemolymph, serum, hemocytes, headfoot, and visceral mass of Biomphalaria glabrata were determined. The highest enzyme level occurs in the serum and the lowest in the hemocytes. Both the headfoot and visceral mass include subequal levels of aminopeptidase activity. From our data, it now appears possible that the serum aminopeptidase in B. glabrata, which has an open circulatory system, could have originated in hemocytes as well as in other tissues. The biologic function of serum aminopeptides is uncertain; however, because of the known chemical function of this enzyme, it could serve to degrade foreign proteins in serum prior to phagocytosis.     

Purification and Characterization of a Prolyl Aminopeptidase from Debaryomyces hansenii

A prolyl aminopeptidase (PAP) (EC 3.4.11.5) was isolated from the cell extract of Debaryomyces hansenii CECT12487. The enzyme was purified by selective fractionation with protamine and ammonium sulfate, followed by two chromatography steps, which included gel filtration and anion-exchange chromatography. The PAP was purified 248-fold, with a recovery yield of 1.4%. The enzyme was active in a broad pH range (from 5 to 9.5), with pH and temperature optima at 7.5 and 45°C. The molecular mass was estimated to be around 370 kDa. The presence of inhibitors of serine and aspartic proteases, bestatin, puromycin, reducing agents, chelating agents, and different cations did not have any effect on the enzyme activity. Only iodoacetate, p-chloromercuribenzoic acid, and Hg²⁺, which are inhibitors of cysteine proteases, markedly reduced the enzyme activity. The K_m for proline-7-amido-4-methylcoumarin was 40 μM. The enzyme exclusively hydrolyzed N-terminal-proline-containing substrates. This is the first report on the identification and purification of this type of aminopeptidase in yeast, which may contribute to the scarce knowledge about D. hansenii proteases and their possible roles in meat fermentation.     

Denaturation of Either Manduca sexta Aminopeptidase N or Bacillus thuringiensis Cry1A Toxins Exposes Binding Epitopes Hidden under Nondenaturing Conditions

The effect of polypeptide denaturation of Bacillus thuringiensis Cry1A toxins or purified Manduca sexta 120-kDa aminopeptidase N on the specificities of their interactions was investigated. Ligand and dot blotting experiments were conducted with ¹²⁵I-labeled Cry1Ac, Cry1Ac mutant ⁵⁰⁹QNR-AAA⁵¹¹ (QNR-AAA), or 120-kDa aminopeptidase N as the probe. Mutant QNR-AAA does not bind the N-acetylgalactosamine moiety on the 120-kDa aminopeptidase. Both ¹²⁵I-Cry1Ac and ¹²⁵I-QNR-AAA bound to 210- and 120-kDa proteins from M. sexta brush border membrane vesicles and purified 120-kDa aminopeptidase N on ligand blots. However, on dot blots ¹²⁵I-QNR-AAA bound brush border vesicles but did not bind purified aminopeptidase except when aminopeptidase was denatured. In the reciprocal experiment, ¹²⁵I-aminopeptidase bound Cry1Ac but did not bind QNR-AAA. ¹²⁵I-aminopeptidase bound Cry1Ab to a limited extent but not the Cry1Ab domain I mutant Y153D or Cry1Ca. However, denatured ¹²⁵I-aminopeptidase detected each Cry1A toxin and mutant but not Cry1Ca on dot blots. The same pattern of recognition occurred with native (nondenatured) ¹²⁵I-aminopeptidase probe and denatured toxins as the targets. The broader pattern of toxin-binding protein interaction is probably due to peptide sequences being exposed upon denaturation. Putative Cry toxin-binding proteins identified by the ligand blot technique need to be investigated under native conditions early in the process of identifying binding proteins that may serve as functional toxin receptors.     

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.     

Purification and Characterization of an Arginine Aminopeptidase from Lactobacillus sakei

An arginine aminopeptidase (EC 3.4.11.6) that exclusively hydrolyzes basic amino acids from the amino (N) termini of peptide substrates has been purified from Lactobacillus sakei. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included hydrophobic interaction, gel filtration, and anion-exchange chromatography. This procedure resulted in a recovery rate of 4.2% and a 500-fold increase in specific activity. The aminopeptidase appeared to be a trimeric enzyme with a molecular mass of 180 kDa. The activity was optimal at pH 5.0 and 37°C. The enzyme was inhibited by sulfhydryl group reagents and several divalent cations (Cu²⁺, Hg²⁺, and Zn²⁺) but was activated by reducing agents, metal-chelating agents, and sodium chloride. The enzyme showed a preference for arginine at the N termini of aminoacyl derivatives and peptides. The K_m values for Arg-7-amido-4-methylcoumarin (AMC) and Lys-AMC were 15.9 and 26.0 μM, respectively. The nature of the amino acid residue at the C terminus of dipeptides has an effect on hydrolysis rates. The activity was maximal toward dipeptides with Arg, Lys, or Ala as the C-terminal residue. The properties of the purified enzyme, its potential function in the release of arginine, and its further metabolism are discussed because, as a whole, it could constitute a survival mechanism for L. sakei in the meat environment.     

Studies on parasitic castration: Aminopeptidase activity levels and protein concentrations in Ilyanassa obsoleta (mollusca) parasitized by larval trematodes

Specimens of the estuarine prosobranch Ilyanassa obsoleta collected in the vicinity of Charleston, South Carolina, during June, July, and August 1981, were found to be parasitized by larvae of the digeneans Cercaria dipterocerca, Himasthla quissetensis, Lepocreadium setiferoides, Microbilharzia variglandis, Microphalloides nassicola, Stephanostomum tenue, and Zoogonus lasius. The aminopeptidase activity levels and the total protein concentrations of the hemocytes and sera of these infected snails were determined. It was ascertained that snails parasitized by larvae of L. setiferoides had significantly higher levels of hemocytic aminopeptidase activity and hemocytic total protein concentrations than noninfected snails. Furthermore, snails parasitized by L. setiferoides larvae had significantly higher hemocytic levels of aminopeptidase activity than snails parasitized by larvae of S. tenue, Z. lasius, M. nassicola, and H. quissetensis.     

Aminopeptidase P from bovine lung: solubilization,properties, and potential role in bradykinin degradation

Aminopeptidase P was solubilized from bovine lung by sodium deoxycholate extraction of salt-washed, delipidated lung acetone powders. Hydrolysis of the standard aminopeptidase P substrate, Gly-Pro-Hyp, as well as cleavage of Arg-Pro-Pro and the Arg¹-Pro² bond of bradykinin, co-eluted from a Mono Q anion exchange column and demonstrated identical inhibitory profiles suggesting that all activities were functions of the same enzyme. The metal chelator, 1,10-phenanthroline, completely inhibited activity suggesting that aminopeptidase P is a metallopeptidase. 2-Mercaptoethanol was both a potent and specific inhibitor of the enzyme (at 4 mM). A variety of other peptidase inhibitors showed either no effect or failed to completely inhibit even at high concentrations. The inhibitory profile and substrate specificity differ considerably from previous reports claiming to study the properties of this enzyme. Evidence is provided that aminopeptidase P may have an important role in the pulmonary degradation of the potent vasoactive peptide, bradykinin.Abbreviations X--NA aminoacyl--naphthylamide - DFP diisopropylphosphofluoridate - HPLC high performance liquid chromatography - APP aminopeptidase P - APM aminopeptidase M - DAP IV dipeptidyl-aminopeptidase IV