The mechanism of the amyloidogenic conversion of T7 endonuclease I

Amyloid fibrils are associated with a range of human disorders. Understanding the conversion of amyloidogenic proteins from their soluble forms to amyloid fibrils is critical for developing effective therapeutics. Previously we showed that T7 endonuclease I forms amyloid-like fibrils. Here we study the mechanism of the amyloidogenic conversion of T7 endonuclease I. We show that T7 endonuclease I forms fibrils at pH 6.8, but not at pH 6.0 or 8.0. The amyloidogenicity at pH 6.8 is not correlated with thermodynamic stability, unfolding cooperativity, or solubility. Thermal melting experiments at various pH values show that the protein has a distinctive thermal transition at pH 6.8. The transition at pH 6.8 has a lower transition temperature than the unfolding transitions observed at pH 6.0 and 8.0 and leads to a beta-rich conformation instead of an unfolded state. Electron microscopy shows that the thermal transition at pH 6.8 results in fibril formation. The thermal transition at pH 6.8 leads to a protein state that is not accessible at pH 6.0 or 8.0, showing that the existence of the amyloidogenic conformation of T7 endonuclease I depends sensitively on solution conditions. Therefore, we propose that fibrillizing proteins need to be "prepared" for fibrillization. Preparation may consist of amino acid replacements or changing solution conditions and may require retention of some aspects of native structure. In this model, some amyloid-enhancing mutations decrease protein stability, whereas others have little effect.     

A partially unfolded structure of the alkaline-denatured state of pepsin and its implication for stability of the zymogen-derived protein

Pepsin, a gastric aspartic proteinase, is a zymogen-derived protein that undergoes irreversible alkaline denaturation at pH 6-7. Detailed knowledge of the structure of the alkaline-denatured state is an important step in understanding the mechanism of the formation of the active enzyme. An extensive analysis of the denatured state at pH 8.0 was performed using a variety of techniques including (1)H nuclear magnetic resonance spectroscopy and solution X-ray scattering. This analysis indicates that the denatured state under these conditions has a compact and globular conformation with a substantial amount of secondary and tertiary structures. The data suggest that this partially structured species has a highly folded region and a flexible region. The NMR measurements suggest that the folded region contains His53 and is located at least partly in the N-terminal lobe of the protein. The alkaline-denatured state experiences a further reversible denaturation step at higher pH or on heating; the midpoints of the unfolding transition are pH 11.5 (at 25 degrees C) and 53.1 degrees C (at pH 8.0), respectively. The present findings suggest that the proteolytic processing of pepsinogen has substantially modified the ability of the protein to fold, such that its folding process cannot progress beyond the partially folded intermediate of pepsin.     

Detection of Escherichia coli O157:H7 using immunomagnetic separation and absorbance measurement

An assay system for detection of Escherichia coli O157:H7 was developed based on immunomagnetic separation of the target pathogen from samples and absorbance measurement of p-nitrophenol at 400 nm from p-nitrophenyl phosphate hydrolysis by alkaline phosphatase (EC 3.1.3.1) on the "sandwich" structure complexes (antibodies coated onto micromagnetic beads--E. coli O157:H7-antibodies conjugated with the enzyme) formed on the microbead surface. The effects of immunoreaction time, phosphate buffer concentration, pH and temperature on the immunomagnetic separation of E. coli O157:H7 from samples were determined and the conditions used for the separation were 1-h reaction time, 1.0 x 10(-2) M PBS, pH 8.0 and 33 degrees C in this system. The effects of MgCl(2) concentration, Tris buffer concentration, pH and temperature on the activity of alkaline phosphatase conjugated on the immuno-"sandwich" structure complexes were investigated after immunomagnetic separation of the target pathogen and the conditions used for the enzymatic amplification were 1.0 x 10(-4) M MgCl(2), 1.0 M Tris buffer, pH 8.0, 28 degrees C and 30-min reaction time during the assay. The selectivity of the system was examined and no interference from the other pathogens including Salmonella typhimurium, Campylobacter jejuni and Listeria monocytogenes was observed. Its working range was from 3.2 x 10(2) to 3.2 x 10(4) CFU/ml, and the relative standard deviation was 2.5-9.9%. The total detection time was less than 2 h.     

Mechanism of energy coupling to entry and exit of neutral and branched chain amino acids in membrane vesicles of Streptococcus cremoris

The energetics of neutral and branched chain amino acid transport by membrane vesicles from Streptococcus cremoris have been studied with a novel model system in which beef heart mitochondrial cytochrome c oxidase functions as a proton-motive force (delta p) generating system. In the presence of reduced cytochrome c, a large delta p was generated with a maximum value at pH 6.0. Apparent H+/amino acid stoichiometries (napp) have been determined at external pH values between 5.5 and 8.0 from the steady state levels of accumulation and the delta p. For L-leucine napp (0.8) was nearly independent of the pH. For L-alanine and L-serine napp decreased from 0.9-1.0 at pH 5.5 to 0-0.2 at pH 8.0. The napp for the different amino acids decreased with increasing external amino acid concentration. At pH 6.0, first order rate constants for amino acid exit (kex) under steady state conditions for L-leucine, L-alanine, and L-serine were 1.1-1.3, 0.084, and 0.053 min-1, respectively. From the pH dependence of kex it is concluded that amino acid exit in steady state is the sum of two processes, pH-dependent carrier-mediated amino acid exit and pH-independent passive diffusion (external leak). The first order rate constant for passive diffusion increased with increasing hydrophobicity of the side chain of the amino acids. As a result of these processes the kinetic steady state attained is less than the amino acid accumulation ratio predicted by thermodynamic equilibrium. The napp determined from the steady state accumulation represents, therefore, a lower limit. It is concluded that the mechanistic stoichiometry (n) for L-leucine, L-alanine, and L-serine transport most likely equals 1.     

Inhibitory Effects of Phenolic Ecotoxicants on Photobacteria at Various pH Values

Kinetic characteristics of light emission by intact cells of photobacteria Photobacterium phosphoreum and Vibrio harveyi were studied (at pH 5.5, 7.0, and 8.0), as well as inhibitory effects of 2,4-di- and 2,4,5-triphenoxyacetic acids (2,4-D and 2,4,5-T), pentachlorophenol (PCP), and 2,6-dimethylphenol (2,6-DMP) (at the same pH values). The emission kinetics lacked a steady state, irrespective of pH. At pH 5.5, luminescence decayed exponentially in the 60-s range; at pH 7.0 and 8.0, a 5-min luminescence activation was observed. The respiratory activity of the cells decreased by more than an order of magnitude at pH 5.5 (compared to the levels observed at pH 7.0 and 8.0). The inhibitory effects of 2,4-D, 2,4,5-T, and PCP differed by one to two orders of magnitude, depending on pH. Maximum cell sensitivity to these compounds appeared at pH 5.5; minimum sensitivity, at pH 8.0. The effect of 2,6-DMP was pH-independent. The inhibitory effect was determined by the hydrophobicity of the molecule and pK values of the toxicants. At all pH values, substrate-depleted cells of photobacteria were more sensitive to chlorophenolic compounds than cells supplied with energy.     

Studies of the relationship between the catalytic activity and binding of non-substrate ligands by the glutathione S-transferases.

The dimeric enzyme glutathione S-transferase B is composed of two dissimilar subunits, referred to as Ya and Yc. Transferase B (YaYc) and two other transferases that are homodimers of the individual Ya and Yc subunits were purified from rat liver. Inhibition of these three enzymes by Indocyanine Green, biliverdin and several bile acids was investigated at different values of pH (range 6.0-8.0). Indocyanine Green, biliverdin and chenodeoxycholate were found to be effective inhibitors of transferases YaYc and YcYc at low (pH 6.0) but not high (pH 8.0) values of pH. Between these extremes of pH intermediate degrees of inhibition were observed. Cholate and taurochenodeoxycholate, however, were ineffective inhibitors of transferase YcYc at all values of pH. The observed differences in bile acids appeared to be due, in part, to differences in their state of ionization. In contrast with the above results, transferase YaYa was inhibited by at least 80% by the non-substrate ligands at all values of pH. These effects of pH on the three transferases could not be accounted for by pH-induced changes in the enzyme's affinity for the inhibitor. Thus those glutathione S-transferases that contain the Yc subunit are able to act simultaneously as both enzymes and binding proteins. In addition to enzyme structure, the state of ionization of the non-substrate ligands may also influence whether the transferases can perform both functions simultaneously.     

Optical ellipsometry on the diffraction order of skinned fibers. pH-induced rigor effects.

The polarization properties of light diffracted from single-skinned fibers of skeletal muscles have been examined under conditions in which the bathing solution pH and the ionic strength are changed. For fibers in the relaxed state, we observe large decreases in both the total depolarization signal, r, and the total diffraction birefringence signal, delta nT, upon pH change from 7.0 to 8.0 at normal ionic strength. However, if the ionic strength is raised, then the r-value change as the pH changes from pH 7.0 to pH 8.0 is much smaller. If the rigor state is achieved at pH 8.0, and 0 mM ATP under either of the ionic strength conditions, the fiber can still be stretched. Rigor stiffness for this state is only approximately 20% that of the value of the stiffness at pH 7.0 rigor. Electron micrographs obtained under this pH 8.0 rigor state show that the overlap region can be decreased upon stretching the fiber, signifying a different kind of weaker-binding rigor state. Optically, the weaker-binding rigor state has a lower depolarization signal and larger form birefringence than the strong-binding rigor state. To convert from one type of rigor state (pH 7.0) to the other rigor state (pH 8.0), or vice versa, the fiber must first be relaxed. Apparently, either of the rigor states can block the full impact of the pH effect.     

Variations in the oxidation-reduction behavior of liganded species of Pseudomonas cytochrome oxidase

In an effort to determine the steady-state redox properties of Pseudomonas aeruginosa cytochrome cd1, changes in absorption spectra after the addition of excess reductant (ascorbate, ferrous ethylenediaminetetraacetic acid) were monitored for degassed unliganded enzyme and samples in the presence of CO and CN- at pH 6.0, 8.0, or 10.0. Plots of [c2+]/[c3+] vs. [d2+]/[d3+] indicate that a "pseudoequilibrium" was reached for all samples at pH 8.0. Calculated values of delta Ed-c, the difference in reduction potential between the heme c and heme d moieties, at pH 8.0 were -25 +/- 5 (unliganded), -10 +/- 5 (enzyme-CO), and -25 +/- 5 mV (enzyme-CN). Relative rates of heme c and heme d reduction were found to be dependent upon type of ligand, reductant, and pH. Evidence for a cooperative heme c-heme d interaction is discussed.     

The relation of glutathione reductase and diaphorase activity of glutathione reductase from Saccharomyces cerevisiae

Glutathione reductase from S. cerevisiae (EC 1.6.4.2) catalyzes the NADPH oxidation by glutathione in accordance with a "ping-pong" scheme. The catalytic constant kcat) is 240 s-1 (pH 7.0, 25 degrees C); kcat for the diaphorase reaction is 4-5 s-1. The enzyme activity does not change markedly at pH 5.5-8.0. At pH less than or equal to 7.0, NADP+ acts as a competitive inhibitor towards NADPH and as a noncompetitive inhibitor towards glutathione. NADP+ increases the diaphorase activity of the enzyme. The maximal activity is observed, when the NADP+/NADPH ratio exceeds 100. At pH 8.0, NADP+ acts as a mixed type inhibitor during the reduction of glutathione. High concentrations of NADP+ also inhibit the diaphorase activity due to the reoxidation of the reduced enzyme by NADP+ at pH 8.0. The redox potential of glutathione reductase calculated from the inhibition data is--306 mV (pH 8.0). Glutathione reductase reduces quinoidal compounds in an one-electron way. The hyperbolic dependence of the logarithm of the oxidation constant on the one electron reduction potential of quinone is observed. It is assumed that quinones oxidize the equilibtium fraction of the two-electron reduced enzyme containing reduced FAD.     

PH-dependence of detergent-induced hemolysis and vesiculation of erythrocytes

The influence of pH of the medium on the parameters of detergent-induced fast hemolysis and vesiculation of human erythrocytes was studied. In the range of pH 6.3-7.2 neither the extent nor the rate of the vesiculation induced by 25 microM sodium dodecyl sulfate (SDS) changed. However, a decrease of pH from 8.0 to 5.8 strongly modified both the extent and the rate of the hemolysis induced by SDS. Within the range of pH 8.0-6.4, the effect can be ascribed to the increase of the positive charge of the membrane. This could lead to the accumulation of the membrane-bound anion detergent and, hence, to the change of the hemolysis parameters. Non-charged detergent Triton X-100 did not display any pH-dependence. At pH between 6.4 and 5.8 the extent and rate of hemolysis changed in a complicated manner. The kinetic curves of hemolysis could be approximated by a single exponential within the pH range between 8.0 and 7.2. Upon further reduction of pH, a second exponential component, with a larger time constant, appeared in the kinetic curves. At 5.8 < pH < 7.2, the contribution of the "fast" hemolysis dropped virtually to zero, with pK about 6.0. This points to a structural transition of the membrane, possibly involving histidine. We suggest that the parameters of the detergent-induced hemolysis are sensitive to the changes of the charge and structural state of erythrocyte membrane.     

Effect of actin on the tryptic digestion of myosin subfragment 1 in the weakly attached state.

The structure of myosin subfragment 1 (S1) in the weakly attached complex with actin was studied at three specific sites, at the 50-kDa/20-kDa and 27-kDa/50-kDa junctions, and at the N-terminal region, using tryptic digestion as a structure-exploring tool. The structure of S1 at the vicinity of the 50-kDa/20-kDa junction is pH dependent in the weakly attached state because the tryptic cleavage at this site was fully protected by actin at pH 6.2, but the protection was only partial at pH 8.0. Since the actin protection is complete in rigor at both pH values, the results indicate that the structure of S1 at the 50-kDa/20-kDa junction differs in the two states at pH 8.0, but not at pH 6.2. Actin restores the ADP-suppressed tryptic cleavage after Lys213 at the 27-kDa/50-kDa junction in the strongly attached state, but not in the weakly attached state, which indicates structural difference between the two states at this site. ATP and ADP open a new site for tryptic cleavage in the N-terminal region of the S1 heavy chain between Arg23 and Ile24. Actin was found to suppress this cleavage in both weakly and strongly attached states, which shows that, in the vicinity of this site, the structure of S1 is similar in both states. The results indicate that the binding of S1 to actin induces localized changes in the S1 structure, and the extent of these changes is different in the various actin-S1 complexes.     

Inhibition of sarcoplasmic reticulum Ca2+-ATPase by Mg2+ at high pH

Steady state turnover of Ca2+-ATPase of sarcoplasmic reticulum has generally been reported to have a bell-shaped pH profile, with an optimum near pH 7.0. While a free [Mg2+] of 2 mM is optimal for activity at pH 7.0, it was found that this level was markedly inhibitory (K1/2 = 2 mM) at pH 8.0, thus accounting for the generally observed low activity at high pH. High activity was restored at pH 8.0 using an optimum free [Mg2+] of 0.2 mM. The mechanism of the Mg2+-dependent inhibition at pH 8.0 was probed. Inhibition was not due to Mg2+ competition with Ca2+ for cytoplasmic transport sites nor to inhibition of formation of steady state phosphoenzyme from ATP. Mg2+ inhibited (K1/2 = 1.8 mM) decay of steady state phosphoenzyme; thus, the locus of inhibition was one of the phosphoenzyme interconversion steps. Transient kinetic experiments showed that Mg2+ competitively inhibited (Ki = 0.7 mM) binding of Ca2+ to lumenal transport sites, blocking the ability of Ca2+ to reverse the catalytic cycle to form ADP-sensitive, from ADP-insensitive, phosphoenzyme. The data were consistent with a hypothesis in which Mg2+ binds lumenal Ca2+ transport sites with progressively higher affinity at higher pH to form a dead-end complex; its dissociation would then be rate-limiting during steady state turnover.     

Kinetic and Structural Properties of NADP-Malic Enzyme from Sugarcane Leaves

Oligomeric structure and kinetic properties of NADP-malic enzyme, purified from sugarcane (Saccharam officinarum L.) leaves, were determined at either pH 7.0 and 8.0. Size exclusion chromatography showed the existence of an equilibrium between the dimeric and the tetrameric forms. At pH 7.0 the enzyme was found preferentially as a 125 kilodalton homodimer, whereas the tetramer was the major form found at pH 8.0. Although free forms of l-malate, NADP⁺, and Mg²⁺ were determined as the true substrates and cofactors for the enzyme at the two conditions, the kinetic properties of the malic enzyme were quite different depending on pH. Higher affinity for l-malate (K_m = 58 micromolar), but also inhibition by high substrate (K_i = 4.95 millimolar) were observed at pH 7.0. l-Malate saturation isotherms at pH 8.0 followed hyperbolic kinetics (K_m = 120 micromolar). At both pH conditions, activity response to NADP⁺ exhibited Michaelis-Menten behavior with K_m values of 7.1 and 4.6 micromolar at pH 7.0 and 8.0, respectively. Negative cooperativity detected in the binding of Mg²⁺ suggested the presence of at least two Mg²⁺ - binding sites with different affinity. The K_a values for Mg²⁺ obtained at pH 7.0 (9 and 750 micromolar) were significantly higher than those calculated at pH 8.0 (1 and 84 micromolar). The results suggest that changes in pH and Mg²⁺ levels could be important for the physiological regulation of NADP-malic enzyme.     

培养介质pH和EGTA对水霉(Saprolegnia ferax)菌丝细胞肌动蛋白的分布与结构的影响

菌丝在pH 5.0—8.0介质中维持顶端生长,Rhodamin-phalloidin荧光探针显示在菌丝顶端都存在F-actin的“帽子”结构;加入EGTA到培养介质中不影响菌丝的顶端生长和actin的“帽子”结构。值得注意的是:菌丝的Rhodamin-phalloidin荧光强度大小与菌丝顶端生长速率成正比;在含有或不含有EGTA的pH5.0培养条件下,菌丝的生长速率均很低,且后部颗粒状的荧光斑点消失;在pH 3.0-4.0培养介质中菌丝生长停止,不但F-actin“帽子”结构消失,整个菌丝荧光也变得非常微弱无法观察,提示酸性pH可引起F-actin的解聚,从而导致生长速率下降甚至生长停止。     

X-ray structures of Aerococcus viridans lactate oxidase and its complex with D-lactate at pH 4.5 show an alpha-hydroxyacid oxidation mechanism

l-Lactate oxidase (LOX) belongs to a family of flavin mononucleotide (FMN)-dependent α-hydroxy acid-oxidizing enzymes. Previously, the crystal structure of LOX (pH 8.0) from Aerococcus viridans was solved, revealing that the active site residues are located around the FMN. Here, we solved the crystal structures of the same enzyme at pH 4.5 and its complex with d-lactate at pH 4.5, in an attempt to analyze the intermediate steps. In the complex structure, the d-lactate resides in the substrate-binding site, but interestingly, an active site base, His265, flips far away from the d-lactate, as compared with its conformation in the unbound state at pH 8.0. This movement probably results from the protonation of His265 during the crystallization at pH 4.5, because the same flip is observed in the structure of the unbound state at pH 4.5. Thus, the present structure appears to mimic an intermediate after His265 abstracts a proton from the substrate. The flip of His265 triggers a large structural rearrangement, creating a new hydrogen bonding network between His265-Asp174-Lys221 and, furthermore, brings molecular oxygen in between d-lactate and His265. This mimic of the ternary complex intermediate enzyme-substrate-O₂ could explain the reductive half-reaction mechanism to release pyruvate through hydride transfer. In the mechanism of the subsequent oxidative half-reaction, His265 flips back, pushing molecular oxygen into the substrate-binding site as the second substrate, and the reverse reaction takes place to produce hydrogen peroxide. During the reaction, the flip-flop action of His265 has a dual role as an active base/acid to define the major chemical steps. Our proposed reaction mechanism appears to be a common mechanistic strategy for this family of enzymes.     

Kinetic studies on thermal denaturation of C-phycocyanin

The kinetics of thermal denaturation of a biliprotein, C-phycocyanin (C-PC) isolated from Spirulina platensis were studied at different pH values, ranging from 4.0 to 8.0. The denaturation of C-PC follows the first order kinetics and rate constant at pH 5.0 and temperature 55 degrees C is found to be 4.37 x 10(-5) s(-1), which increases to 5.46 x 10(-1) s(-1) at pH 7.0. The denaturation rate is much higher at 65 degrees C and pH 7.0 (7.96 x 10(-4)), as compared to at pH 5.0 (1.46 x 10(-4)). The thermal stability of C-PC is more at pH 5.0, as compared to other pH values. The observed differences in entropy values at pH 5.0, as compared to other pH values indicate a considerably close fit structure of the protein at pH 5.0, which increases the stability of native structure, even at higher temperature (65 degrees C).     

On the nature of the unfolded intermediate in the in vitro transition of the colicin E1 channel domain from the aqueous to the membrane phase.

The transition of the colicin E1 channel polypeptide from a water-soluble to membrane-bound state occurs in vitro at acid pH values that are associated with an unfolded channel structure whose properties qualitatively resemble those of a "molten globule," or "compact unfolded," intermediate state. The role of such a state for activity was tested by comparing the pH dependence of channel-induced solute efflux and the amplitude of the near-UV CD spectrum. The requirement of a partly unfolded state for activity was shown by the coincidence of the onset of channel activity measured for 4 different lipid compositions with the decrease in near-UV CD amplitude as a function of pH. Tertiary constraints on the 3 tryptophans of the colicin channel, assayed by the amplitude of the near-UV CD spectrum, are retained over the pH range 3-4 where channel activity could be measured and, as well, at pH 2. In addition, the tryptophan fluorescence emission spectrum is virtually unchanged over the pH range 2-6. The temperature independence of the near-UV spectrum at pH 3-6 up to 70 degrees C implies that the colicin E1 channel polypeptide is more stable than that of colicin A. A transition between 53 and 58 degrees C in the amplitude of the near-UV CD is consistent with preservation of part of the hydrophobic core in a destabilized state at pH 2. Thus, the unfolded state associated with colicin activity at acidic pH has the properties of a "compact unfolded" state, having some, but not all of the properties of a "molten globule."(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of liming a neutral soil on the uptake of manganese by plants

Summary The uptake of manganese by oats in a manganese-deficient soil was increased on raising the pH from 7.0 to 8.0 with Ca(OH)₂. This effect occurred in the absence of added manganese as well as when manganese was added to the soil as -MnO₂. When the soil was initially sterilized the uptake of manganese at pH 7.0 was greatly increased at both pH's, but the uptake at pH 8.0 was now a smaller than at pH 7.0.     

Proton-coupled structural changes upon binding of carbon monoxide to cytochrome cd1: a combined flash photolysis and X-ray crystallography study

We have investigated dynamic events after flash photolysis of CO from reduced cytochrome cd(1) nitrite reductase (NiR) from Paracoccus pantotrophus (formerly Thiosphaera pantotropha). Upon pulsed illumination of the cytochrome cd(1)-CO complex, at 460 nm, a rapid (<50 ns) absorbance change, attributed to dissociation of CO, was observed. This was followed by a biphasic rearrangement with rate constants of 1.7 x 10(4) and 2.5 x 10(3) s(-1) at pH 8.0. Both parts of the biphasic rearrangement phases displayed the same kinetic difference spectrum in the region of 400-660 nm. The slower of the two processes was accompanied by proton uptake from solution (0.5 proton per active site at pH 7.5-8.5). After photodissociation, the CO ligand recombined at a rate of 12 s(-1) (at 1 mM CO and pH 8.0), accompanied by proton release. The crystal structure of reduced cytochrome cd(1) in complex with CO was determined to a resolution of 1.57 A. The structure shows that CO binds to the iron of the d(1) heme in the active site. The ligation of the c heme is unchanged in the complex. A comparison of the structures of the reduced, unligated NiR and the NiR-CO complex indicates changes in the puckering of the d(1) heme as well as rearrangements in the hydrogen-bonding network and solvent organization in the substrate binding pocket at the d(1) heme. Since the CO ligand binds to heme d(1) and there are structural changes in the d(1) pocket upon CO binding, it is likely that the proton uptake or release observed after flash-induced CO dissociation is due to changes of the protonation state of groups in the active site. Such proton-coupled structural changes associated with ligand binding are likely to affect the redox potential of heme d(1) and may regulate the internal electron transfer from heme c to heme d(1).