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BBA - Proteins and Proteomics (v.1748, #1)
Effect of the polypeptide binding on the thermodynamic stability of the substrate binding domain of the DnaK chaperone by Naoki Tanaka; Shota Nakao; Jean Chatellier; Yasushi Tani; Tomoko Tada; Shigeru Kunugi (pp. 1-8).
The effect of polypeptide binding on the stability of the substrate binding domain of the molecular chaperone DnaK has been studied by thermodynamic analysis. The calorimetric scan of the fragment of the substrate binding domain DnaK384–638, consisting of a β-domain and an α-helical lid, showed two transitions centered at 56.2 and 76.0 °C. On the other hand, the thermal unfolding of the shorter fragment DnaK386–561, which lacks half of the α-helical lid, exhibited a single transition at 57.0 °C. Therefore, the transition of DnaK384–638 at 56.2 °C is mainly attributed to the unfolding of the β-domain. The calorimetric scan of DnaK384–638D526N showed that the unfolding of the β-domain was composed of two transitions. The polypeptide bound DnaK384–638 exhibited a symmetrical DSC peak at 58.6 °C, indicating that the substrate binding shifts the β-domain toward a single cooperative unit. A low concentration of GdnHCl (<1.0 M) induced a conformational change in the β-domain of DnaK384–638 without changes in the secondary structure. While the thermal unfolding of the β-domain of DnaK384–638 was composed of two transitions in the presence of GdnHCl, the β-domain of the substrate bound DnaK384–638 exhibited a single symmetrical DSC peak in the same condition. All together, our results indicate that complex between DnaK384–638 and substrate forms a rigid conformation in the β-domain.
Keywords: Abbreviations; SBD; substrate binding domain; GdnHCl; guanidine hydrochloride; RCMLA; reduced and carboxylmethylated α-lactalbuminMolecular chaperone; DnaK; Substrate binding domain; DSC; Thermal unfolding; Limited proteolysis
Characterization of a membrane-associated apoplastic lipoxygenase in Phaseolus vulgaris L. by Francesca Sicilia; Benedetta Mattei; Felice Cervone; Daniela Bellincampi; Giulia De Lorenzo (pp. 9-19).
An extracytoplasmic 86.7 kDa protein was isolated from intercellular washing fluids (IWF) of Phaseolus vulgaris etiolated hypocotyls. Micro sequencing of tryptic peptides of the 86.7 kDa protein revealed 100% identity with a bean lipoxygenase (LOX) protein fragment. Purified P87-LOX exhibited LOX activity characterized by an optimal pH of 6.0 and linolenic acid as an optimal substrate, and was classified as a 13-LOX with respect to its positional specificity of linoleic acid oxygenation. A protein identical to P87-LOX, as determined by MALDI-TOF analysis and biochemical characterization, was purified from hypocotyl microsomes. Immunoblot analysis showed that P87-LOX is present in plasma membrane-enriched fractions, from which it was solubilized using high ionic strength buffers. These observations suggest that P87-LOX is a peripheral protein associated to the apoplastic face of the plasma membrane.
Keywords: Abbreviations; FA; fatty acids; G6PDH; glucose-6-phosphate dehydrogenase; HR; hypersensitive response; IWF; intercellular washing fluids; LOX; lipoxygenaseApoplast; Membrane-associated protein; Lipoxygenase; Bean
Changing the metal ion selectivity of rabbit muscle enolase by mutagenesis: effects of the G37A and G41A mutations by Mary Judith Kornblatt (pp. 20-25).
During the reaction catalyzed by enolase, a mobile loop, residues 36–45, closes over the active site. In order to probe the role of this loop movement in catalysis, the glycines at positions 37 and 41 of rabbit muscle enolase (ββ) have been mutated to alanines. The mutant forms–G37A and G41A–of enolase are both active, but have altered selectivity for divalent cations. G37A, when assayed with Mg2+, has 12% the activity of the wild type. However, it is twice as active as wild type when assayed with Mn2+, Zn2+, or Co2+. G41A has 4% the activity of the wild type with Mg2+, is more active than wild type with Co2+, and slightly less active than wild type with Mn2+ and Zn2+. The kinetic isotope effect for both mutants is greater than that of the wild type with all 4 divalent cations. These results indicate that the flexibility of this loop has subtle effects on catalytic activity.
Keywords: Abbreviations; PGA; 2-phosphoglyceric acid; PEP; phosphoenolpyruvate; PEG; polyethylene glycol; CD; circular dichroism; k.i.e.; kinetic isotope effectEnolase; Mutagenesis; Divalent cation
Characterization and kinetic analysis of enzyme–substrate recognition by three recombinant lactococcal tripeptidases by Sumiko Mori; Satoru Nirasawa; Shiro Komba; Takafumi Kasumi (pp. 26-34).
Tripeptidases from Lactococcus lactis subsp. lactis (L9PepTR), L. lactis subsp. cremoris (L6PepTR), and L. lactis subsp. hordniae (hTPepTR) were cloned, overexpressed, purified, and characterized. Although these enzymes contained three to seven naturally occurring amino acid differences, both metal-binding and catalytic sites were highly conserved. The kcat values of hTPepTR were approximately 1.5- to 2-fold higher than those of L9PepTR, while, for L6PepTR, they were approximately 0.8- to 1.4-times the L9PepTR values. The Km of tripeptidase from subsp. lactis (L9PepTR) was considerably larger when glycine was the amino acid located at both the N- and C-terminus of the peptide substrate. In addition, the Km values of L9PepTR increased in the following order for YGG, LGG, FGG, SGG, and α-aminoisobutyrylglycylglycine, while the kcat/ Km decreased in the same order. These results suggest that the dipole moment and steric hindrance of the N-terminal amino acid side chain may be the most important factors controlling substrate specificity.
Keywords: Tripeptidase; Recombinant PepT; Substrate specificity; Kinetic analysis; Lactococcus lactis
Cloning and expression of ostrich trypsinogen: an avian trypsin with a highly sensitive autolysis site by Borbála Szenthe; Carminita Frost; László Szilágyi; András Patthy; Ryno Naudé; László Gráf (pp. 35-42).
One of ostrich ( Struthio camelus) trypsinogen genes was cloned from pancreatic cDNA. Its amino acid sequence compared to known trypsin sequences from other species shows high identity and suggests that it is a member of the phylogenetically anionic trypsinogen I subfamily. After cytoplasmic over expression in Escherichia coli and renaturation, the activation properties of ostrich trypsinogen were studied and compared to those of human trypsinogen 1 (also called as human cationic trypsinogen). Ostrich trypsinogen undergoes bovine enterokinase activation and autoactivation much faster than human trypsinogen 1 and exhibits on a synthetic substrate a somewhat higher enzymatic activity than the latter one. The most interesting property of ostrich trypsin is its relatively fast autolysis that can be explained via a mechanism different from the common mechanism for rat and human 1 trypsins. The latter proteases have a site, Arg117–Val118, where the autolysis starts and then goes on in a zipper-like fashion. This is absent from ostrich trypsin. Instead it has a couple of cleavage sites within regions 67–98, including two unusual ones, Arg76–Glu77 and Arg83–Ser84. These appear to be hydrolysed fast in a non-consecutive manner. Such an autolysis mechanism could not be inhibited by a single-site mutation which in humans is proposed to lead to pancreatitis.
Keywords: Abbreviations; N; -CBZ-GPR-pNA; N; -benzyloxycarbonyl-Gly-Pro-Arg-; para; -nitroanilide; oligodT; oligonucleotide containing only thymine specific for the poly-adenine tail of the mRNA; LB; Lauria-Bertani broth; MUB; 4-methylumbelliferone; MUGB; 4-methylumbelliferyl-4-guanidinobenzoate; HuTg-1; human trypsinogen 1; HuTr-1; human trypsin 1; IPTG; isopropyl-thio-galactose; GdnHCl; guanidine-hydro-chloride; EDTA; ethylene diamine tetraacetic acid; PCR; polymerase chain reaction; STI-agarose; soybean trypsin inhibitor linked to agarose; Tris–HCl; α,α,α-Tris–(hydroxymethyl)-methylamine hydrochloride; SDS; sodium dodecyl sulfate; PAGE; polyacrylamide gel electrophoresis; ctg; chymotrypsinogen; sp; serin protease; tg; trypsinogen; tr; trypsin; NH; 4; (HCO; 3; ); ammonium–hydrogen–carbonateOstrich; Trypsinogen; Autolysis
Processing of chicken progastrin at post-Phe bonds by an aspartyl protease by Hanne Jensen; Kenji Yamamoto; Jens R. Bundgaard; Jens F. Rehfeld; Anders H. Johnsen (pp. 43-49).
Prohormones mature to biologically active peptide hormones through posttranslational modifications, which include endoproteolytic cleavages. Cleavages at mono- and dibasic sites are well characterized, and several of the responsible prohormone convertases have been identified. There is, however, evidence that endoproteolytic maturation occurs also at other sites. Among these, post-Phe cleavage occurs in the maturation of chicken progastrin, where the processing to gastrin-30 has been examined in detail. In this study we have characterized an endoprotease of the aspartic acid protease family in chicken and human tissue capable of cleaving at the Phe site. Enzymatic activity was monitored by radioimmunoassays using antibodies specific for the N- and C-termini exposed after cleavage. Analysis showed that only pepstatin, a specific inhibitor of aspartic proteases, inhibited the enzyme. The pH optimum of the enzyme ranged from pH 2 to pH 5. Amino acid substitution from Phe to Ala in the substrate completely abolished enzyme activity. The endoproteolytic activity was identified in chicken antrum and pectoral muscle as well as human cardiac and prostate extracts, suggesting that the enzyme has widespread biological functions. Experiments using recombinant cathepsin D and E indicated that neither is responsible for the endoproteolytic cleavage of chicken progastrin at post-Phe bonds.
Keywords: Chicken; Gastrin; Posttranslational; Regulatory peptide; Aspartyl protease
Differential scanning calorimetry of the irreversible denaturation of Rapana thomasiana (marine snail, Gastropod) hemocyanin by Krassimira Idakieva; Katja Parvanova; Svetla Todinova (pp. 50-56).
The thermal denaturation of the hemocyanin from gastropod Rapana thomasiana (RtH) at neutral pH was studied by means of differential scanning calorimetry (DSC). The denaturation was completely irreversible as judged by the absence of any endotherm on rescanning of previously scanned samples. Two transitions, with apparent transition temperatures ( Tm) at 83 and 90 °C, were detected by DSC using buffer 20 mM MOPS, containing 0.1 M NaCl, 5 mM CaCl2 and 5 mM MgCl2, pH 7.2. Both Tm were dependent on the scanning rate, suggesting that the thermal denaturation of RtH is a kinetically controlled process. The activation energy ( EA) of 597±20 kJ mol−1 was determined for the main transition (at 83 °C). EA for the second transition was 615±25 kJ mol−1. The Tm and Δ Hcal values for the thermal denaturation of RtH were found to be independent of the protein concentration, signifying that the dissociation of the protein into monomers does not take place before the rate-determining state of the process of thermal unfolding.
Keywords: Abbreviations; Hc; hemocyanin; FU; functional unit; RtH; Rapana thomasiana; hemocyanin; RtH1 and RtH2; structural isoforms of RtH; DSC; differential scanning calorimetry; TRIS; tris (hydroxymethyl) aminomethane; EDTA; ethylenediaminetetraacetic acid; MOPS; 3-[; N; -Morpholino]propanesulfonic acid; HEPES; N; -[2-Hydroxyethyl]piperazine-; N′; -[2-ethanesulfonic acid]; PIPES; piperazine-; N; ,; N′; -bis[2-ethanesulfonic acid]Hemocyanin; Mollusca; Rapana thomasiana; Differential scanning calorimetry; Irreversible transition
Characterization of recombinant human protein C inhibitor expressed in Escherichia coli by Sophie M. Réhault; Margareta Zechmeister-Machhart; Yolanda M. Fortenberry; Julia Malleier; Nikki M. Binz; Scott T. Cooper; Margarethe Geiger; Frank C. Church (pp. 57-65).
The serine protease inhibitor (serpin) protein C inhibitor (PCI; also named plasminogen activator inhibitor-3) regulates serine proteases in hemostasis, fibrinolysis, and reproduction. The biochemical activity of PCI is not fully defined partly due to the lack of a convenient expression system for active rPCI. Using pET-15b plasmid, Ni2+-chelate and heparin-Sepharose affinity chromatography steps, we describe here the expression, purification and characterization of wild-type recombinant (wt-rPCI) and two inactive mutants, R354A (P1 residue) and T341R (P14 residue), expressed in Escherichia coli. Wild-type rPCI, but not the two mutants, formed a stable bimolecular complex with thrombin, activated protein C and urokinase. In the absence of heparin, wt-rPCI-thrombin, -activated protein C, and -urokinase inhibition rates were 56.7, 3.4, and 2.3×104 M−1 min−1, respectively, and the inhibition rates were accelerated 25-, 71-, and 265-fold in the presence of 10 μg/mL heparin for each respective inhibition reaction. The stoichiometry of inhibition (SI) for wt-rPCI-thrombin was 2.0, which is comparable to plasma-derived PCI. The present report describes for the first time the expression and characterization of recombinant PCI in a bacterial expression system and demonstrates the feasibility of using this system to obtain adequate amounts of biologically active rPCI for future structure–function studies.
Keywords: Abbreviations; APC; activated protein C; IPTG; isopropyl-beta-; d; -thiogalactopyranoside; LB medium; l; -broth medium; Ni-NTA; nickel-nitrilotriacetic acid; rPCI; recombinant protein C Inhibitor; P1-rPCI; R355A rPCI; P14-rPCI; T341R rPCISerpin; Recombinant protein C inhibitor; Thrombin; Heparin; Purification
Compact molten globule-like state of hUBF HMG Box1 at extremely low pH by Xuecheng Zhang; Jiahai Zhang; Xuan Li; Junjie Xu; Hongda Huang; Quan Chen; Jihui Wu; Yunyu Shi (pp. 66-73).
Using far and near-UV CD, ANS fluorescence and 2D NMR spectroscopy, an acid-induced partly folded state (A state) at extremely low pH for hUBF HMG Box1 was identified and characterized. As compared to the native state (N), the A state has similar secondary structure, less compact pack with larger amounts of exposed hydrophobic surface, and narrower chemical shift dispersion in1H–15N HSQC spectrum, which implies that it is a molten globule (MG)-like species. On the other hand, substantial tertiary contacts and cooperative thermal denaturing transition indicate that the A state is closer–relative to the classic MG–to the native folded state. In addition, when the solution pH is adjusted to neutrality, the protein in the A state refolds to the native state easily. All these data suggest that the A state of hUBF HMG Box1 could represent a potential folding intermediate on protein folding pathway.
Keywords: Abbreviations; UV; ultraviolet; CD; circular dichroism; ANS; 1-anilino-8-aphthalene sulfonate; 2D NMR; two dimensional nuclear magnetic resonance; HSQC; heteronuclear single quantum coherence; MG; molten globule; GdnHCl; guanidine hydrochloride; C; m; concentration midpoint of detergent denaturation; T; m; temperature midpoint of thermal denaturation; ppm; parts per million; DMSO; dimethylsulfoxideAcid-induced state; Molten globule; Protein folding; hUBF HMG Box1
Unfolding and inactivation of cutinases by AOT and guanidine hydrochloride by Tomas Ternström; Allan Svendsen; Mikael Akke; Patrick Adlercreutz (pp. 74-83).
We present a comparative analysis of the unfolding and inactivation of three cutinases in the presence of guanidine hydrochloride (GdnHCl) and bis(2-ethylhexyl) sodium sulfosuccinate (AOT). Previous investigations have focused on the cutinase from Fusarium solani pisi (FsC). In addition to FsC, the present study includes the cutinase from Humicola insolens (HiC) and a mutant variant of HiC (μHiC) with increased activity and decreased surfactant sensitivity. Equilibrium and time-resolved denaturation by AOT were studied in aqueous solution and reverse micelles, and were compared with GdnHCl denaturation. The far-UV CD and fluorescence denaturation profiles obtained in the aqueous solutions of the two denaturants coincide for all three cutinases, indicating that unfolding is a co-operative two-state process under these conditions. In reverse micelles, the cutinases unfold with mono-exponential rates, again indicating a two-state process. The free energy of denaturation in water was calculated by linear extrapolation of equilibrium data, yielding very similar values for the three cutinases with averages of −11.6 kcal mol−1 and −2.6 kcal mol−1 for GdnHCl and AOT, respectively. Hence, the AOT denatured state (DAOT) is less destabilised than the GdnHCl denatured state (DGdnHCl), relative to the native state in water. Far-UV CD spectroscopy revealed that DAOT retains some secondary structure, while DGdnHCl is essentially unstructured. Similarly, fluorescence data suggest that DAOT is more compact than DGdnHCl. Activity measurements reveal that both DAOT and DGdnHCl are practically inactive (catalytic activity <1% of that of the native enzyme). The fluorescence spectrum of DAOT in reverse micelles did not differ significantly from that observed in aqueous AOT. NMR studies of DAOT in reverse micelles indicated that the structure is characteristic of a molten globule, consistent with the CD and fluorescence data.
Keywords: Abbreviations; CD; circular dichroism; UV; ultraviolet; GdnHCl; guanidine hydrochloride; AOT; bis(2-ethylhexyl) sodium sulfosuccinate; SDS; sodium dodecyl sulfate; PNPB; p; -nitrophenylbutyrate; PNPV; p; -nitrophenylvalerate; FsC; Fusarium solani pisi; cutinase; HiC; Humicola insolens; cutinase; μHiC; H. insolens; cutinase mutant; D; denatured protein; N; native proteinCutinase; AOT; Denaturant; Detergent; Reverse micelle; Protein folding
The binding of 1,8 ANS congeners to I-FABP and comparison of some hypotheses about ANS' spectral sensitivity to environment by William R. Kirk (pp. 84-93).
The ANS congeners 1-anilinonaphthalene and 1-amino,8-sulfonato naphthalene were investigated as analogs of 1,8 anilinonaphthalene sulfonate. Like 1,8 ANS, they also bind to I-FABP, and the fully-bound spectra reveal interesting similarities and differences with respect to ANS binding. The nature of these differences suggests that certain hypotheses in the literature about ANS photophysics ought to be revised. The conceptual decomposition of energetic effects in the thermodynamics of ANS binding proposed earlier [1] [W. Kirk, E. Kurian, F. Prendergast, Characterization of the sources of protein–ligand affinity: 1-sulfonato-8-(1′)anilinonaphthalene binding to intestinal fatty acid binding protein. Biophys. J. (1996) 70 69–83.] is extended further in this report.
Keywords: Anilinonaphthalene; Aminonaphthalene sulfonate; 1,8 ANS congeners; Stretched exponentials
Log P effect of organic solvents on a thermophilic alcohol dehydrogenase by Hidehiko Hirakawa; Noriho Kamiya; Yutaka Kawarabayashi; Teruyuki Nagamune (pp. 94-99).
An alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix was activated by water-miscible organic solvents. This activation was influenced by the kind and the concentration of the added organic solvents. The kcat was increased by a factor of over ten when the mole fraction of acetonitrile was 0.1. This effect was large when organic solvents with large log P values were added. In fact, the kcat showed a strong positive correlation with the log P value of the mixed solvent at a constant mole fraction of water, while it was not affected by the kind of organic solvents added. Both the activation enthalpy and the entropy decreased with an increase in log P. The contribution of the activation enthalpy to the free energy of activation was larger than that of the activation entropy, and the free energy of activation decreased with an increase in log P.
Keywords: Alcohol dehydrogenase; Aeropyrum pernix; Log; P; Water mole fraction; Activation by organic solvents; Water miscible
A comparison of solution conformation and hydrodynamic properties of equine, porcine and rabbit serum albumin using viscometric measurements by Karol Monkos (pp. 100-109).
This paper presents the results of viscosity determinations on aqueous solutions of equine, porcine and rabbit serum albumin over a wide range of concentrations and at temperatures ranging from 5 °C to (42–45) °C. The results are compared with human and bovine serum albumin previously studied. Viscosity–temperature dependence is discussed on the basis of the modified Arrhenius formula. The effective specific volume, the activation energy and entropy of viscous flow for all investigated albumins are compared. Viscosity–concentration dependence, in turn, is discussed on the basis of Mooney equation. Based on the assumption that theoretical and experimental values of Simha factor – at high temperature limit – are equal to each other, the hydrodynamic volume of the studied albumins has been calculated. The numerical values of a self-crowding factor were also obtained. At low concentration limit, the numerical values of the intrinsic viscosity and of Huggins coefficient were compared.
Keywords: Albumin; Activation energy; Effective specific volume; Hydrodynamic volume; Intrinsic viscosity; Huggins coefficient
The first archaeal agmatinase from anaerobic hyperthermophilic archaeon Pyrococcus horikoshii: cloning, expression, and characterization by Shuichiro Goda; Haruhiko Sakuraba; Yutaka Kawarabayasi; Toshihisa Ohshima (pp. 110-115).
Agmatinase is one of the key enzymes in the biosynthesis of polyamines such as putrescine and sperimidine from arginine in microorganisms. The gene (PH0083) encoding the putative agmatinase of hyperthermophilic archaeon Pyrococcus horikoshii was identified based on the genome database. The gene was cloned and expressed, and the product was mainly obtained as inactive inclusion body in Escherichia coli. The inclusion body was dissolved in 6 M guanidine–HCl and successively refolded to active enzyme by the dilution of the denaturant. The enzyme exclusively catalyzed the hydrolysis of agmatine, but not arginine. This indicates that PH0083 codes agmatinase. The enzyme required divalent cations such as Co2+, Ca2+ and Mn2+ for the activity. The highest activity was observed under fairly alkaline conditions, like pH 11. The purified recombinant enzyme consisted of four identical subunits with a molecular mass of 110–145 kDa. The enzyme was extremely thermostable: the full activity was retained on heating at 80 °C for 10 min, and a half of the activity was retained by incubation at 90 °C for 10 min. From a typical Michaelis–Menten type kinetics, an apparent Km value for agmatine was determined to be 0.53 mM. Phylogenic analysis revealed that the agmatinase from P. horikoshii does not belong to any clusters of enzymes found in bacteria and eukarya. This is the first description of the presence of archaeal agmatinase and its characteristics.
Keywords: Abbreviations; DMSO; dimethyl sulfoxide; GuHCl; guanidine hydrochlorideArchaea; Agmatinase; Pyrococcus horikoshii; Polyamine; Agmatine
Comparative study of the stability of poplar plastocyanin isoforms by A. Shosheva; A. Donchev; M. Dimitrov; G. Kostov; G. Toromanov; V. Getov; E. Alexov (pp. 116-127).
The stability of the two isoforms of poplar plastocyanin (PCa and PCb) was studied with differential scanning calorimetry (DSC) technique. It was shown that the thermal unfolding of both isoforms is an irreversible process with two endothermic and one exothermic peaks. The melting temperature of PCb was found to be 1.3±0.2 K degrees higher than of PCa, which indicates that PCb is more stable. The enthalpy of unfolding was estimated from the heat capacity curves and was found to be significantly higher for PCb at salt concentration I=0.1 M. In addition, PCb unfolding enthalpy and melting temperature are much more sensitive to the changes in the salt concentration as found in the experiments done at different ionic strength. The experiments were complemented with numerical calculations. The salt effect on the stability was modeled using the X-ray structure of PCa and a homology modeled structure of PCb. It was found, in agreement with the experimental data, that the stability of PCb changes by 4.7 kJ more than PCa, as the salt concentration increases from zero to 0.1 M. Thus, the differences in only 12 amino acid positions between “a� and “b� isoforms result in a measurable difference in the folding enthalpy and a significant difference in the salt dependence. The optimization of the electrostatic energies of PCa and PCb were studied and it was shown that PCb is better electrostatically optimized.
Keywords: Plastocyanin; Polymorphism; Calorimetry; Folding; Thermal denaturation; Electrostatic; Delphi; Side chain replacement
A survey for phosphoglucose isomerase with lysyl aminopeptidase activity in Vibrionaceae and non- Vibrio pathogens by Gary P. Richards; Salina Parveen (pp. 128-133).
Phosphoglucose isomerase (PGI) with a novel lysyl aminopeptidase (LysAP) activity was recently purified and characterized from Vibrio vulnificus. We showed that it cleaves the amino-terminal lysyl residue from des-Arg10-kallidin to produce des-Arg9-bradykinin, suggesting that it plays a role in virulence. A survey was conducted to determine the presence of this potential virulence-enhancing enzyme among twenty-three halotolerant human and fish pathogens from eleven species within the Vibrionaceae family, including V. vulnificus, V. parahaemolyticus, V. cholerae, Aeromonas hydrophila, and Plesiomonas shigelloides. In addition, fourteen species of non-Vibrionaceae pathogens were screened for LysAP activity. Cell lysates were partially purified by anion exchange chromatography and fractions were screened for LysAP and isomerase activities. PGI-LysAP activity was detected in chromatographic fractions from all the Vibrio species tested, but was not detected in any of the non-Vibrionaceae pathogens. Levels of isomerase and LysAP activity correlated ( R2=0.92) for nine strains of V. vulnificus. Since the Vibrionaceae represent an important family of human and fish pathogens, our identification of PGI-LysAP activity in a broad array of vibrios may lead to the development of improved analytical methods for their identification as well as interventions to reduce the high morbidity and mortality associated with some Vibrionaceae infections in clinical, veterinary, and aquaculture settings.
Keywords: Abbreviations; APW; alkaline peptone water; ATCC; American Type Culture Collection; CDC; Centers for Disease Control and Prevention; CU; calculated units; CV; column volume; FDA; United States Food and Drug Administration; l; -Lys-AMC; l; -lysyl-7-amino-4-methylcoumarin; LysAP; lysyl aminopeptidase; PGI; phosphoglucose isomerase; PGI-LysAP; phosphoglucose isomerase with a lysyl aminopeptidase activity; RT; room temperature; tdh; thermostable direct hemolysin gene; trh; thermostable related hemolysin gene; TSA; tryptic soy agar; TSA-N; tryptic soy agar containing 1% NaCl; TSB; tryptic soy broth; TSB-N; tryptic soy broth containing 1% NaCl; USDA; United States Department of Agriculture; USUHS; Uniformed Services University of the Health SciencesProtease; PGI; Glucose-6-phosphate isomerase; Phosphohexose isomerase; Lysyl aminopeptidase; Kinin; Vibrio
The target of RNAIII-activating protein (TRAP) from Staphylococcus aureus: purification, crystallization and preliminary X-ray analysis by Young-Hyun Han; Yang-Gyun Kim; Dong Young Kim; Sung Chul Ha; Neratur K. Lokanath; Kyeong Kyu Kim (pp. 134-136).
The target of the RNAIII-activating protein (TRAP) is a 21 kDa protein in which phosphorylation is activated by the RNAIII-activating protein (RAP), which causes an increase in RNAII and RNAIII synthesis and the production of the virulence factors. In an attempt to examine the structural role of TRAP in the signal transduction pathway, TRAP from Staphylococcus aureus was overexpressed, purified and crystallized using PEG 8000 and 5% Jeffamine M600 (pH 7.0), as precipitants by hanging-drop vapour diffusion methods at 287 K. The crystals belong to the orthorhombic space group, P212121, with unit cell parameters of a=39.68, b=50.41, c=85.45 Å. There is one monomer of TRAP per crystallographic asymmetric unit with a crystal volume per protein mass ( VM) of 2.06 Å3 Da−1 and a solvent content of 40.3%. A complete data set diffracting to 1.9 Å resolution was collected from a single crystal at 100 K using a synchrotron-radiation source.
Keywords: Target of RNAIII-activating protein (TRAP); Crystallization; Staphylococcus aureus; Quorum sensing