Molecular forms of AlpA and AlpB lytic endopeptidases from <Emphasis Type="Italic">Lysobacter</Emphasis> sp. Xl1: immunochemical determination of their intra- and extracellular localization

Homologous endopeptidases AlpA and AlpB are components of the secreted complex of lytic enzymes of the Gram-negative bacterium Lysobacter sp. ХL1. These enzymes are synthesized as precursors that consist of a signal peptide, propeptide, and proteolytically active mature part. To understand the topogenetic features of these proteins, bacterial cell fractions were investigated by a sensitive sandwich enzymelinked immunosorbent assay and immunoblot analysis with the use of monoclonal antibodies recognizing unique epitopes of proteins’ mature forms and their propeptides. Only mature forms of the enzymes, without propeptides, were shown to be released outside the cell into the environment. AlpA significantly exceeds AlpB in the production level at the early stationary growth stage. The AlpB precursor was revealed in the cytoplasmic and periplasmic fractions, and the AlpA precursor was found only in the cytoplasmic fraction. The periplasmic fraction was also found to contain the mature forms of both enzymes and their propeptides. These results indicate that AlpA and AlpB are released into the environment through different mechanisms. AlpA is translocated across the cell envelope without being interrupted in the periplasm. The homologous AlpB enzyme, on the contrary, accumulates in the periplasmic space and is captured by outer membrane vesicles in the process of their formation.     

Optimization of in vivo crosslinking technique for the study of AlpB-protein interactions in Lysobacter sp. XL1 cells

The bacterium Lysobacter species strain XL1 is known as a producer of extracellular lytic enzymes, which are capable of degrading cell wall components of other bacteria and simple eukaryotes. This ability determines the ecological, medical and agricultural relevance of Lysobacter sp. XL1. However, the molecular mechanism of secretion of lytic exoenzymes from Lysobacter cells is yet unknown, which in turn necessitates the search of protein–protein interactions that occur during exoenzyme secretion. The current paper is concerned with investigation of protein complexes that are likely formed during the secretion of AlpB lytic protease from the cells of Lysobacter sp. XL1. In this study, we have optimized the method of stabilization of protein complexes formed in the intact cells of Lysobacter sp. XL1 by using crosslinking reagent dithiobis(succinimidylpropionate) (DSP) and detected DSP-linked protein complexes by the monoclonal antibodies against AlpB propeptide.     

Folding pathway mediated by an intramolecular chaperone: the structural and functional characterization of the aqualysin I propeptide

Aqualysin I, a thermostable homologue of subtilisin, requires its propeptide (ProA) to function as an intramolecular chaperone (IMC). To decipher the mechanisms through which propeptides can initiate protein folding, we characterized ProA in terms of its sequence, structure and function. Our results show that, in contrast to ProS (propeptide of subtilisin), ProA can fold spontaneously, reversibly and cooperatively into a stable monomeric alpha-beta conformation, even when isolated from its cognate protease-domain. ProA displays an indiscernible amount of tertiary structure with a considerable solvent-accessible hydrophobic surface, but is not a classical molten-globule folding intermediate. Moreover, despite showing only 21 % sequence identity with ProS, ProA can not only inhibit enzymatic activity with a magnitude tenfold greater than ProS, but can also chaperone subtilisin folding, albeit with a lower efficiency. The structure of ProA complexed with subtilisin is different from that of isolated ProA. Hence, additional interactions seem necessary to induce ProA into a compact structure. Our results also suggest that: (a) propeptides that are potent inhibitors are not necessarily better IMCs; (b) propeptides within the subtilase family appear polymorphic and; (c) the intrinsic instability within propeptides may be necessary for rapid activation of the cognate protein.     

Studying factors involved in biogenesis of <Emphasis Type="Italic">Lysobacter</Emphasis> sp. XL1 outer membrane vesicles

The Gram-negative bacterium Lysobacter sp. XL1 produces outer membrane vesicles that are heterogeneous in size, density, and protein composition. One of the subpopulations is secretory vesicles for lytic protease L5 of Lysobacter sp. XL1 (Kudryakova et al. (2015) FEMS Microbiol. Lett., 362, fnv137). Protein L5 was assumed to influence biogenesis of these secretory vesicles that contain it. Using a Pseudomonas fluorescens Q2-87/B expression system, it was shown that the recombinant L5 protein may act as a factor of vesicle biogenesis. This points to a possible involvement of L5 protein in Lysobacter sp. XL1 vesicle biogenesis. Furthermore, it was established that the main phospholipid of Lysobacter sp. XL1 vesicles is cardiolipin, and vesicles are formed predominantly of outer membrane regions enriched with this phospholipid. This indicates that cardiolipin participates in biogenesis of all vesicle subpopulations in Lysobacter sp. XL1.     

Purification and Characterization of Intracellular Proteinases in Pleurotus ostreatus Fruiting Bodies

A serine proteinase (ProA, EC 3.4.22.9) and two metalloendopeptidases (ProB, EC 3.4.99.32 and ProC, 3.4.24.4), have been purified to homogeneity from the fruiting bodies of Pleurotus ostreatus. ProA is a serine proteinase with a mass of 30 kDa, which has amidolytic and esterolytic activities besides proteolysis and catalyzes preferential cleavage of the peptide bonds involving the carboxyl groups of hydrophobic amino acid residues in oxidized bovine insulin B chain. The N-terminal amino acid sequence was VTQTNAPWGLSRL.

ProB is a zinc-enzyme with a mass of 18 kDa, which is devoid of lysine, and its N-terminal sequence was ATFVGCSATRQ. The enzyme is inactivated completely by EDTA and 1,10-phenanthroline, and Zn²⁺-depleted ProB can regain the activity with Zn²⁺, Co²⁺, or Mn²⁺. Specific cleavage of Pro₂₉-LYS₃₀ in oxidized bovine insulin B chain, preferential generation of lysylpeptides from proteins, and a high susceptibility of polylysine suggest that ProB splits specifically the peptide bonds involving the α-amino group of lysyl residues.

ProC is a metalloendopeptidase of a mass of 42.5 kDa, and Zn²⁺ was the most effective divalent metal ion to activate the EDTA-inactivated enzyme.     

Structural Studies of Component of Lysoamidase Bacteriolytic Complex from <Emphasis Type="Italic">Lysobacter</Emphasis> sp. XL1

The lysoamidase bacteriolytic complex (LBC) comprising five enzymes (L1–L5) is secreted into the culture liquid by gram-negative bacterium Lysobacter sp. XL1. The medicinal agent lysoamidase has a broad-antimicrobial spectrum. Bacteriolytic protease L1 belongs to the LBC. Recombinant L1 protease of Lysobacter sp. XL1 was expressed, purified to homogeneity and crystallized. The X-ray structure of L1 at 1.35 Å resolution has been determined using the synchrotron data and the molecular replacement method. L1 protease is a thermostable whose thermal unfolding proceeds in one step without forming stable intermediates. Structural information concerning L1 will contribute to the development of new-generation antimicrobial drugs, whose application will not be accompanied by the selection of resistant microorganisms.     

Functional Analysis of the Cucumisin Propeptide as a Potent Inhibitor of Its Mature Enzyme

Cucumisin is a subtilisin-like serine protease (subtilase) that is found in the juice of melon fruits (Cucumis melo L.). It is synthesized as a preproprotein consisting of a signal peptide, NH₂-terminal propeptide, and 67-kDa protease domain. We investigated the role of this propeptide (88 residues) in the cucumisin precursor. Complementary DNAs encoding the propeptides of cucumisin, two other plant subtilases (Arabidopsis ARA12 and rice RSP1), and bacterial subtilisin E were expressed in Escherichia coli independently of their mature enzymes. The cucumisin propeptide strongly inhibited cucumisin in a competitive manner with a K_i value of 6.2 ± 0.55 nm. Interestingly, cucumisin was also strongly inhibited by ARA12 and RSP1 propeptides but not by the subtilisin E propeptide. In contrast, the propeptides of cucumisin, ARA12, and RSP1 did not inhibit subtilisin. Deletion analysis clearly showed that two hydrophobic regions, Asn³²–Met³⁸ and Gly⁹⁷–Leu¹⁰³, in the cucumisin propeptide were important for its inhibitory activity. Site-directed mutagenesis also confirmed the role of a Val³⁶-centerd hydrophobic cluster within the Asn³²–Met³⁸ region in cucumisin inhibition. Circular dichroism spectroscopy revealed that the cucumisin propeptide had a secondary structure without a cognate protease domain and that the thermal unfolding of the propeptide at 90 °C was only partial and reversible. A tripeptide, Ile³⁵-Val³⁶-Tyr³⁷, in the Asn³²–Met³⁸ region was thought to contribute toward the formation of a proper secondary structure necessary for cucumisin inhibition. This is the first report on the function and structural information of the propeptide of a plant serine protease.     

Trypsin inhibitors from ridged gourd (Luffa acutangula Linn.) seeds: Purification,properties, and amino acid sequences

Two trypsin inhibitors, LA-1 and LA-2, have been isolated from ridged gourd (Luffa acutangula Linn.) seeds and purified to homogeneity by gel filtration followed by ion-exchange chromatography. The isoelectric point is atpH 4.55 for LA-1 and atpH 5.85 for LA-2. The Stokes radius of each inhibitor is 11.4 å. The fluorescence emission spectrum of each inhibitor is similar to that of the free tyrosine. The biomolecular rate constant of acrylamide quenching is 1.0×10⁹ M^?1 sec^?1 for LA-1 and 0.8 × 10⁹ M^?1 sec^?1 for LA-2 and that of K₂HPO₄ quenching is 1.6×10¹¹ M^?1 sec^?1 for LA-1 and 1.2×10¹¹M^?1 sec^?1 for LA-2. Analysis of the circular dichroic spectra yields 40%α-helix and 60%Β-turn for La-1 and 45%α-helix and 55%Β-turn for LA-2. Inhibitors LA-1 and LA-2 consist of 28 and 29 amino acid residues, respectively. They lack threonine, alanine, valine, and tryptophan. Both inhibitors strongly inhibit trypsin by forming enzymeinhibitor complexes at a molar ratio of unity. A chemical modification study suggests the involvement of arginine of LA-1 and lysine of LA-2 in their reactive sites. The inhibitors are very similar in their amino acid sequences, and show sequence homology with other squash family inhibitors.     

Effect of calcium on differentiation of Friend leukemia cells

Induction of hemoglobin synthesis of Friend leukemia cells is inhibited by changing the ratio between internal and external Ca²⁺ concentrations. The concentration ratio can be successfully manipulated by the addition of the growth medium of (1) Ca²⁺ channel blocker D600 (90 nM-4 × 10² nM), (2) Ca²⁺ ionophore A23187 (1 × 10²–2 × 10² nM), and (3) EGTA at molar concentrations comparable to the Ca²⁺ concentration of the medium formulation (3 × 10² μM). The observations suggest that a specific ratio between intra- and extracellular Ca²⁺ is required for erythroid differentiation to proceed.     

Changes in bacterial glycolipids as an index of intestinal lactobacilli and epithelial glycolipids in the digestive tracts of mice after administration of penicillin and streptomycin

The major lipid constituent of symbiotic gram-positive bacteria in animals are phosphatidylglycerol, cardiolipin and dihexaosyl diglycerides (DH-DG), whose hydrophobic structures are characteristic of the environments, and the carbohydrate structures of DH-DGs are bacterial species-characteristic. Immunization of rabbits with intestinal lactobacilli generated antibodies against DH-DGs and their modified structures, among which Galα1-6-substituted DH-DG, i.e., Lactobacillus tetrahexaosyl diglyceride (LacTetH-DG), reacted with antibodies more intensely than DH-DG. Whereas, from the 16S-rRNA sequence, the intestinal lactobacilli in murine digestive tracts were revealed to be L. johnsonii, in which LacTetH-DG is present at the concentration of 2.2 ng per 1?×?10⁶ cells. To obtain more accurate estimates of intestinal lactobacilli in several regions of the digestive tract of mice, LacTetH-DG was detected by TLC-immunostaining with anti-Lactobacillus antisera, being found in the stomach, cecum and colon of normal breeding mice, 1.0?×?10⁹, 3.5?×?10⁹ and 7.4?×?10⁹ cells, respectively. Administration of penicillin and streptomycin for 6 days resulted in a reduction in the number of intestinal lactobacilli, the levels being 0 %, 30 % and 4 % of the control ones in the stomach, cecum and colon, respectively, which was associated with the accumulation of the contents in the tracts from the stomach to the cecum and with diarrhea. In addition, a reduced amount of fucosyl GA1 (FGA1) and a compensatory increase in GA1 due to the reduced activity of α1,2-fucosyltransferase in the small intestine and the enhanced discharge of FGA1 into the contents occurred in mice, probably due to the altered population of bacteria caused by administration of penicillin and streptomycin.     

Structural polypeptides and products of late genes of bacteriophage Mu: Characterization and functional aspects

This paper describes the identification and functional role of late gene products of bacteriophage Mu, including an analysis of the structural proteins of the Mu virion.In vitro reconstitution of infectious phage particles has shown that four genes (E, D, I, J) control the formation of phage heads and that a cluster of eight genes (K, L, M, N, P, Q, R, S) controls the formation of phage tails.Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of Mu polypeptides synthesized in Escherichia coli minicells infected by Mu phages carrying amber mutations in various late genes has resulted in the identification of the products of gene C (15.5 × 10³M_r); H (64 × 10³M_r); F (54 × 10³M_r); G (16 × 10³M_r); L (55 × 10³M_r); N (60 × 10³M_r); P (43 × 10³M_r) and S (56 × 10³M_r). Minicells infected with λpMu hybrid phages and deletion mutants of Mu were used to identify polypeptides encoded by the V-β region of the Mu genome. These are the products of genes V, W or R (41.5 × 10³M_r, and 45 × 10³M_r); U (20.5 × 10³M_r) and of genes located in the β region (24 × 10³M_r (gpgin) and 37 × 10³M_r (possibly gpmom)).Analytical separation of the proteins of the Mu virion revealed that it consists of a major head polypeptide with a molecular weight of 33 × 10³, a second head polypeptide of 54 × 10³ (gpF) and two major tail polypeptides with molecular weights of 55 × 10³ and 12.5 × 10³ (gpL and gpY, respectively). In addition, there are five minor components of the tail (including gpN, gpS and gpU) and approximately seven minor components of the head structure of the virion (including gpH).     

Kinetics of the low pH decolorization of azurin

Reaction rates were measured for the low pH-induced Cu ligand modification of azurins from Pseudomonas aeruginosa and Alcaligenes faecalis. Loss of the intense absorption band at 625 nm obeyed a rate law:

where Az is the concentration of azurin in its native oxidized form possessing the 625 nm band. For Pseudomonas aeruginosa at 25°C, n = 1 and k = 4.0 × 10^-2 sec^-1 M^-1 in citrate buffer but 1.2 × 10^-2 sec^-1 M^-1 in phosphate buffer. For Alcaligenes faecalis, n = 3 and k = 1.5 × 10⁴ sec^-1 M^-3 in citrate and 2.6 × 10³ sec^-1 M^-3 in phosphate. In equilibrium experiments on Alcaligenes faecalis azurin in citrate buffer, the pH-dependent change to the low pH form exhibited an apparent transition pK of 3.1. The form of the rate law implies a mechanistic scheme that contains fast equilibrium protonation steps prior to a rate limiting ligand rearrangement.     

Chymotrypsin inhibitor from potatoes: interaction with target enzymes

The interactions of chymotrypsin, subtilisin and trypsin with a low MW proteinase inhibitor from potatoes were investigated. The K_i value calculated for the binding of inhibitor to chymotrypsin was 1.6 ± 0.9 × 10^?10M, while the second-order rate constant for association was 6 × 10⁵ M^?1/sec. Although binding was not observed to chymotrypsin which had been treated with diisopropyl fluorophosphate or with l-tosylamide-2-phenylethyl chloromethyl ketone, the 3-methylhistidine-57 derivative bound inhibitor with a K_i value of 9.6 × 10^?9 M. The inhibitor also exhibited a tight association with subtilisin (K_i < 4 × 10^?9 M). In contrast, little inhibition of trypsin was observed, and this was believed to be due to low levels of a contaminant in our preparations. No evidence for reactive site cleavage was observed after incubation of the inhibitor with catalytic amounts of chymotrypsin or subtilisin at acid pH.     

Occurrence in vivo of "hidden breaks" at specific sites of 26 S ribosomal RNA of Musca carnaria.

A fragmentation process occurs in 26 S ribosomal RNA of mature cytoplasmic ribosomes of Musca carnaria. It consists of the sequential appearance of three “hidden breaks” that fragment 26 S rRNA (M_r = 1.42 × 10⁶) into four pieces with approximate molecular weights of 0.68 × 10⁶, 0.35 × 10⁶, 0.29 × 10⁶ and 0.096 × 10⁶, respectively. This fragmentation was not observed in 17 S rRNA (M_r = 0.74 × 10⁶).Extremely mild treatment of newly assembled ribosomes with pancreatic RNAase reproduces the 26 S rRNA fragmentation phenomenon in vitro in the same way as it occurs in vivo.This evidence is discussed in relation to the secondary structure of 26 S rRNA and its binding with specific ribosomal proteins.     

Bovine toxoplasmosis: experimental infections.

Three calves dosed per os with 0.75 × 10⁶, 0.75 × 10⁶ and 1.5 × 10⁶Toxoplasma oocysts developed high titres of Toxoplasma antibodies as measured by the indirect fluorescent antibody test and the dye test. Toxoplasma gondii was reisolated from 2 of these animals. Two calves given 1 × 10³ tissue cysts and 2 × 10⁶ tachyzoites intramuscularly did not develop such high fitres, but T. gondii was reisolated from the calf injected with tachyzoites.Of 4 pregnant cows given 7 × 10⁴, 7 × 10⁴ and 1.6 × 10⁵ oocysts and 1.5 × 10² tissue cysts only 2 developed significant levels of Toxoplasma antibodies. There was no evidence of Toxoplasma infection in the calves born by these cows.It was concluded that cattle do not readily acquire persistent T. gondii infections, and this may be due, in part at least, to early elimination of Toxoplasma from their tissues.     

Three Antigenic Regions in p17 of Human Immunodeficiency Virus Type 1 (HIV-1) Revealed by Mouse Monoclonal Antibodies and Human Antibodies in HIV-1 Carrier Sera

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (K_A = 1.9 × 10⁵?1.4 × 10⁸ M^?1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (K_A = 2.0 × 10⁵?1.8 × 10⁷ M^?1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.     

Glucocorticoid modulation of lymphokine-induced macrophage proliferation

The ability of glucocorticoids to modify lymphokine-induced macrophage proliferation, an in vitro correlate of cellular immunity in the guinea pig, was investigated. Lymphocyte production of macrophage mitogenic factor (MMF) was decreased in the presence of physiological concentrations of glucocorticoids. Inhibition was concentration dependent (IC₅₀ of triamcinolone acetonide (TA): 2 × 10^?9M), glucocorticoid specific, and reversed by cortexolone. In contrast, pharmacological concentrations of glucocorticoids were necessary to inhibit macrophage proliferation induced by suboptimal dilutions of MMF. This inhibition was concentration dependent (IC₅₀ of TA: 4 × 10^?7M), glucocorticoid specific, and reversed by cortexolone. At supraoptimal dilutions of MMF, glucocorticoids caused a twofold potentiation of MMF-induced macrophage proliferation. Potentiation was concentration dependent (EC₅₀ of TA: 3 × 10^?8M), glucocorticoid specific, reversed by glucocorticoid antagonists, and occurred in the presence of indomethacin. Thus, glucocorticoids regulate both the initiation and effector phases of this in vitro model of delayed hypersensitivity. However, the results indicate that the major mechanism of glucocorticoid-mediated anti-inflammatory action occurs at the level of the MMF-producing lymphocyte rather than at the effector macrophage, as MMF-induced proliferation is likely controlled by opposing glucocorticoid-sensitive mechanisms.     

Simple Determination of Trace Amounts of Anionic Surfactants in River Water by Spectrophotometry Combined with Solid-phase Extraction

A simple and sensitive specrophotometric method combined with solid-phase extraction (SPE) for the simultaneous determination of sodium linear-dodecylbenzenesulfonate (DBS) and sodium dodecyl sulfate (SDS) is described. The C2 (ethyl group bonded silicagel) cartridge could be repeatedly used more than 500 times for SPE, and it enabled the anionic surfactants to be concentrated by 50-fold. The calibration graph for DBS was linear in the range from 1.6×10^?8 M to 5.0×10^?7 M and for SDS from 2.0×10^?9 M to 3.0×10^?7 M. The relative standard deviation (n=5) for 5.0×10^?7 M DBS was 3.1% and for 2.5×10^?7 M SDS was 1.7%. The proposed method was applied to the simultaneous determination of DBS and SDS in river-water samples.     

Complexes of antiparallel telomeric G-quadruplex d(TTAGGG)4 with carboxymethyl tetracationic porphyrins

Porphyrins are a chemical class that is widely used in drug design. Cationic porphyrins may bind to DNA guanine quadruplexes. We report the parameters of the binding of 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium) porphyrin (P1) and 5,10,15,20-tetrakis(N-etoxycarbonylmethyl-4-pyridinium) porphyrin (P2) to antiparallel telomeric G-quadruplex formed by d(TTAGGG)₄ sequence (TelQ). The binding constants (K _i ) and the number of binding sites (N _j ) were determined from absorption isotherms generated from the absorption spectra of complexes of P1 and P2 with TelQ. Compound P1 demonstrated a high affinity to TelQ (K _i = (40 ± 6) × 10⁶ M^?1, N ₁ = 1; K ₂ = (5.4 ± 0.4) × 10⁶ M^?1, N ₂ = 2). In contrast, the binding constants of P2-TelQ complexes (K ₁ = (3.1 ± 0.2) × 10⁶ M^?1, N ₁ = 1; K ₂ = (1.2 ± 0.2) × 10⁶ M^?1, N ₂ = 2) were one order of magnitude smaller than the corresponding values for P2-TelQ complexes. Measurements of the quantum yield and fluorescence lifetime of the drug’s TelQ complexes revealed two types of binding sites for P1 and P2 on the quadruplex oligonucleotide. We concluded that strong complexes can result from the interaction of the porphyrins with TTA loops whereas the weaker complexes are formed with G-quartets. The altered TelQ conformation detected by the circular dichroism spectra of P1-TelQ complexes can be explained by the disruption of the G-quartet. We conclude that peripheral carboxy groups contribute to the high affinity of P1 for the antiparallel telomeric G-quadruplex.