Binding of radiolabeled folate and 5-methyltetrahydrofolate to cow's milk folate binding protein at pH 7.4 and 5.0. Relationship to concentration and polymerization equilibrium of the purified protein

Binding of folate (pteroylglutamate) and 5-methyltetrahydrofolate, the major endogenous form of folate, to folate binding protein purified from cow's milk was studied at 7°C to avoid degradation of 5-methyltetrahydrofolate. Both folates dissociate rapidly from the protein at pH 3.5, but extremely slowly at pH 7.4, most likely due to drastic changes in protein conformation occurring after folate binding. Dissociation of 5-methyltetrahydrofolate showed no increase at 37°C suggesting that protein-bound-5-methyltetrahydrofolate is protected against degradation. Binding displayed two characteristics, positive cooperativity and a binding affinity that increased with decreasing concentrations of the protein. The binding affinity of folate was somewhat greater than that of 5-methyl tetrahydrofolate, in particular at pH 5.0. Ligand-bound protein exhibited concentration-dependent polymerization (8-mers formed at 13 M) at pH 7.4. At pH 5.0, only folate-bound forms showed noticeable polymerization. The fact that folate at pH 5.0 surpasses 5-methyltetrahydrofolate both with regard to binding affinity and ability to induce polymerization suggests that ligand binding is associated with conformational changes of the protein which favor polymerization.     

Peptide binding to HLA-DP proteins at pH 5.0 and pH 7.0: a quantitative molecular docking study

ABSTRACT: BACKGROUND: HLA-DPs are class II MHC proteins mediating immune responses to many diseases. Peptides bind MHC class II proteins in the acidic environment within endosomes. Acidic pH markedly elevates association rate constants but dissociation rates are almost unchanged in the pH range 5.0 - 7.0. This pH-driven effect can be explained by the protonation/deprotonation states of Histidine, whose imidazole has a pKa of 6.0. At pH 5.0, imidazole ring is protonated, making Histidine positively charged and very hydrophilic, while at pH 7.0 imidazole is unprotonated, making Histidine less hydrophilic. We develop here a method to predict peptide binding to the four most frequent HLA-DP proteins: DP1, DP41, DP42 and DP5, using a molecular docking protocol. Dockings to virtual combinatorial peptide libraries were performed at pH 5.0 and pH 7.0. RESULTS: The X-ray structure of the peptide - HLA-DP2 protein complex was used as a starting template to model by homology the structure of the four DP proteins. The resulting models were used to produce virtual combinatorial peptide libraries constructed using the single amino acid substitution (SAAS) principle. Peptides were docked into the DP binding site using AutoDock at pH 5.0 and pH 7.0. The resulting scores were normalized and used to generate Docking Score-based Quantitative Matrices (DS-QMs). The predictive ability of these QMs was tested using an external test set of 484 known DP binders. They were also compared to existing servers for DP binding prediction. The models derived at pH 5.0 predict better than those derived at pH 7.0 and showed significantly improved predictions for three of the four DP proteins, when compared to the existing servers. They are able to recognize 50% of the known binders in the top 5% of predicted peptides. CONCLUSIONS: The higher predictive ability of DS-QMs derived at pH 5.0 may be rationalised by the additional hydrogen bond formed between the backbone carbonyl oxygen belonging to the peptide position before p1 (p-1) and the protonated -nitrogen of His79beta. Additionally, protonated His residues are well accepted at most of the peptide binding core positions which is in a good agreement with the overall negatively charged peptide binding site of most MHC proteins.     

Phase behaviour of skin barrier model membranes at pH 7.4.

The main function of the skin is to protect the body against exogenous substances. The skin barrier is located in the outermost layer of the skin, the stratum corneum (SC). This layer consists of keratin enriched cells embedded in lipid lamellae that form the main barrier for diffusion of substances through the skin. The main lipid classes in this barrier are ceramides, cholesterol and free fatty acids. Cholesterol sulfate and calcium are also present in SC. Furthermore it has been suggested that a pH gradient exists. In a previous paper the effect of cholesterol sulfate and calcium on the lipid phase behaviour of mixtures prepared from cholesterol, ceramides and free fatty acids at pH 5 was reported (approximate pH at the skin surface). In the present study the phase behaviour of mixtures prepared from cholesterol, ceramides and free fatty acids prepared at pH 7.4 (the pH of viable cells) has been examined between 25 and 95 degrees C. Our studies reveal that a reversed hexagonal phase has been formed at elevated temperatures. Addition of calcium inhibits the formation of the reversed hexagonal phase, while cholesterol sulfate promotes the presence of the reversed hexagonal phase at increased temperatures. From our results we can conclude that the lipid mixtures prepared at pH 5 resemble more closely the lipid phase behaviour in intact SC than the lipid mixtures prepared at pH 7.4.     

Label-free detection of enhanced saccharide binding at pH 7.4 to nanoparticulate benzoboroxole based receptor units

Nanoparticles modified with either 6-amino-1-hydroxy-2,1-benzoxaborolane (3-aminobenzoboroxole) or 3-aminophenylboronic acid were prepared by nucleophilic substitution of a styrene-co-DVB-co-vinylbenzylchloride latex (25 nm). Isothermal titration calorimetry (ITC) was used as a label-free detection method for the analysis of the binding between monosaccharides and these two differently derivatized nanoparticle systems at pH 7.4. Because ITC reveals, thermodynamical parameters such as the changes in enthalpy ΔH, free energy ΔG, and entropy ΔS, possible explanations for the higher binding constants can be derived in terms of entropy and enthalpy changes. In case of the modified nanoparticles, the free energy of binding is dominated by the entropy term. This shows that interfacial effects, besides the intrinsic affinity, lead to a higher binding constant compared with the free ligand. The highest binding constant was found for fructose binding to the benzoboroxole modified nanoparticles: Its value of 1150 M(-1) is twice as high as for the free benzoboroxole and five times as high as with phenylboronic acid or 3-aminophenylboronic acid. In contrast to the binding of fructose to free boronic acids, which is an enthalpically driven process, the binding of fructose to the modified nanoparticles is dominated by the positive entropy term.     

Enhancement of Electrochemical Differentiation Ability of Nucleobases in Phosphate Buffer Solution at pH 7.4

The electrochemical behavior of nucleobases has been studied in 0.1 M phosphate buffer solution at pH 7.4, using a bare graphite electrode. Guanine and adenine produced well-defined oxidation peaks at about +0.63 and +0.91 V at 100 mV/s, respectively. Nucleobases exhibit an irreversible and hybrid-controlled electrochemical process, including adsorption and diffusion. The nucleobase oxidation peaks shift due to the selective interactions of nucleobases with each other. The oxidation peaks for three different pyrimidine bases, uracil, cytosine, and thymine, can be clearly identified at +1.26, +1.41, and +1.32 V, respectively. These differences in the electrochemical behavior among nucleobases can be attributed to their different chemical structures.     

Mapping human brain activity in vivo.

A wide range of structural and functional techniques now exists to map the human brain in health and disease. These approaches span the gamut from external tomographic imaging devices (positron-emission tomography, single photon-emission computed tomography, magnetic resonance imaging, computed tomography), to surface detectors (electroencephalography, magnetoencephalography, transcranial magnetic stimulation), to measurements made directly on the brain''s surface or beneath it (intrinsic signal imaging, electrocorticography). The noninvasive methods have been combined to provide unique and previously unavailable insights into the macroscopic organization of the functional neuroanatomy of human vision, sensation, hearing, movement, language, learning, and memory. All methods have been applied to patients with neurologic, neurosurgical, and psychiatric disease and have provided a rapidly expanding knowledge of the pathophysiology of diseases such as epilepsy, cerebrovascular disease, neoplasms, neurodegenerative diseases, mental illness, and addiction states. In addition, these new methods have become a mainstay of preoperative surgical planning and the monitoring of pharmacologic or surgical (transplantation) interventions. Most recently, the ability to observe the reorganization of the human nervous system after acute injury, such as occurs with cerebral infarction or head trauma, or in the course of a progressive degenerative process such as Alzheimer''s or Parkinson''s disease, may provide new insights and methods in the rapidly expanding field of neurorehabilitation. Our newfound ability to generate maps and databases of human brain development, maturation, skill acquisition, aging, and disease states is both an exciting and formidable task.     

High activity of human butyrylcholinesterase at low pH in the presence of excess butyrylthiocholine.

Butyrylcholinesterase is a serine esterase, closely related to acetylcholinesterase. Both enzymes employ a catalytic triad mechanism for catalysis, similar to that used by serine proteases such as alpha-chymotrypsin. Enzymes of this type are generally considered to be inactive at pH values below 5, because the histidine member of the catalytic triad becomes protonated. We have found that butyrylcholinesterase retains activity at pH 相似文献   

Short-chain organic acids at pH 5.0 kill Escherichia coli and Salmonella spp. without causing membrane perturbation

When strains of Escherichia coli K12 and Salmonella spp. were incubated with 0.5–0.7 mol/l formic or propionic acid at pH 5.0, propionic acid was more active than formic acid. It killed 90% of the cell population within 60 min compared with over 3 h for formic acid. Cell death was not associated with a reduction in culture turbidity or a loss of membrane integrity since morphologically normal membranes were observed by electron microscopy and only a small proportion of the cytoplasmic enzyme β-galactosidase leaked into the supernatant fluid of acid-treated E. coli K12 cultures.     

Heterogeneity of maize NAD-malate dehydrogenase: generation of multiple forms by incubation at pH 5.0.

Three isoenzymes of NAD-malate dehydrogenase are constitutive to many strains of maize. Two are mitochondrial and a third is cytosolic. Evidence presented here indicates that additional forms can be generated by pH 5.0 treatment of homogenates, although the purified isoenzymes are insensitive to the treatment.     

Functional equivalence of iron bound to human transferrin at low pH or high pH.

Human transferrin was labeled with 59Fe at one of its two metal-binding sites (designated A) at pH 6.0. 55Fe was then added to site B at pH 7.5. Both isotopes of iron were taken up in equal proportions by human reticulocytes. These experiments do not support the hypothesis that each binding site of transferrin has a different physiologic function.     

Acidic pH as a physiological regulator of human cathepsin L activity.

Human cysteine protease cathepsin L was inactivated at acid pH by a first-order process. The inactivation rate decreased with increasing concentrations of a small synthetic substrate, suggesting that substrates stabilize the active conformation. The substrate-independent inactivation rate constant increased with organic solvent content of the buffer, consistent with internal hydrophobic interactions, disrupted by the organic solvent, also stabilizing the enzyme. Circular dichroism showed that the inactivation is accompanied by large structural changes, a decrease in alpha-helix content being especially pronounced. The high activation energy of the reaction at pH 3.0 (200 kJ.mol-1) supported such a major conformational change occurring. The acid inactivation of cathepsin L was irreversible, consistent with the propeptide being needed for proper folding of the enzyme. Aspartic protease cathepsin D was shown to cleave denatured, but not active cathepsin L, suggesting a potential mechanism for in-vivo regulation and turnover of cathepsin L inside lysosomes.     

The partitioning of fatty acid and cholesterol between core and surfaces of phosphatidylcholine-triolein emulsions at pH 7.4

Fatty acids are important intermediate molecules in lipid metabolism. During lipolysis of intracellular lipid droplets or plasma triacylglycerol-rich lipoproteins, fatty acids are generated and may transiently accumulate. We therefore studied the distribution of both fatty acid and free cholesterol between the core and surface of phosphatidylcholine-triolein emulsions at pH 7.4. Nine emulsion systems containing 0.8 to 6.6% cholesterol and 0.16 to 1.02% oleic acid were formed, and core and surface phases were isolated. Phospholipid distributes only to the surface phase. The distribution coefficient of cholesterol surface to core was 23.9 +/- 3.6 S.D., i.e., there was approx. 24-times more cholesterol per unit mass in the surface than in the core phase. This distribution was unchanged by the presence of different quantities of fatty acid in the emulsion particles. The apparent distribution coefficient of fatty acid in surface to core was about 10 at low cholesterol contents and fell to about 7 at high cholesterol contents. However, when the apparent distribution coefficient of fatty acid was related only to the phospholipid component of the surface, the apparent distribution coefficient was constant at about 12.3 +/- 1.1 S.D. Since the fatty acid in the surface phase is about half ionized the true distribution coefficient of unionized fatty acids is about 6.2. The results indicate that fatty acids partition into the phospholipid domains of the surface and not into cholesterol domains and the distribution of fatty acids into surface phospholipid domain is not affected by cholesterol content.     

Acidic pH and detergents enhance in vitro conversion of human brain PrPC to a PrPSc-like form

In the presence of a low concentration of denaturants or detergents, acidic pH triggers a conformational transition of alpha-helices into beta-sheets in recombinant prion protein (PrP), likely mimicking some aspects of the transformation of host-encoded normal cellular PrP (PrP(C)) into its pathogenic isoform (PrP(Sc)). Here we observed the effects of acidic pH and guanidine hydrochloride (GdnHCl) on the physicochemical and structural properties of PrP(C) derived from normal human brain and determined the ability of the acid/GdnHCl-treated PrP to form a proteinase K (PK)-resistant species in the absence and presence of PrP(Sc) template. After treatment with 1.5 m GdnHCl at pH 3.5, PrP(C) from normal brain homogenates was converted into a detergent-insoluble form similar to PrP(Sc). Unlike PrP(Sc), however, the treated brain PrP(C) was protease-sensitive and retained epitope accessibility to monoclonal antibodies 3F4 and 6H4. Brain PrP(C) treated with acidic pH/GdnHCl acquired partial PK resistance upon further treatment with low concentrations of sodium dodecyl sulfate (SDS). Formation of this PrP(Sc)-like isoform was greatly enhanced by incubation with trace quantities of PrP(Sc) from Creutzfeldt-Jakob disease brain. Acid/GdnHCl-treated brain PrP may constitute a "recruitable intermediate" in PrP(Sc) formation. Further structural rearrangement seems essential for this species to acquire PK resistance, which can be promoted by the presence of a PrP(Sc) template.     

Cathepsin H activity in the human brain and human brain neoplasms

The human brain cathepsin H is shown to be a specific cysteine aminopeptidase with the optimum activity at pH 6.0. Human brain tumours of neuroectodermal (astrocytomas and glioblastomas) and epithelial (meningiomas) origin were used to study the cathepsin H activity in the malignant brain tissue. A significant increase in the aminopeptidase cathepsin H activity was found in malignant human brain tumours as compared to benign tumours and normal brain tissues.     

Characterization of a hyaluronic acid-binding protein from sheep brain comparison with human brain hyaluronectin.

1. A hyaluronic acid (HA)-binding glycoprotein from sheep brain was characterized. 2. The specific affinity for HA was shown in vitro by high performance liquid chromatography, polyacrylamide gel electrophoresis and ELISA methods. 3. The KD for high molecular weight HA was 5.4 10(-9) M at 37 degrees C and lower than 10(-10) M at 4 degrees C. 4. No link protein was found and HA molecules could bind up to 10 times their weight of the glycoprotein. 5. The specific site for interaction was the HA-derived decasaccharide HA10. 6. The protein is composed of one polypeptidic chain. Tryptophan and lysine play a prominent role in the conformation of the binding site to HA. 7. Enzyme analysis indicated that the protein different forms are due to differences in glycosylation and that N- and O-linkages coexist in the molecules. 8. Immunohistochemistry localized the glycoprotein at the nodes of Ranvier and at the periphery of neurons. The perineuronal labeling was seen around all neurons studied in the cerebellum whereas it was almost undetectable in the cerebral hemispheres. 9. HA is not saturated by hyaluronectin (HN) in the sheep nervous system. 10. The glycoprotein is largely similar to human brain HN, and different from the hyaluronate-binding protein characterized in the cartilage.     

Penicillinase-producing Neisseria gonorrhoeae: a molecular comparison of 5.3-kb and 7.4-kb beta-lactamase plasmids.

Restriction endonuclease analysis and heteroduplex studies indicate that the only difference between the 5.3-kilobase (kb) and 7.4-kb plasmids from beta-lactamase-producing Neisseria gonorrhoeae is that the latter is the 5.3-kb plasmid with a 2.1-kb insertion. The insertion is bounded by inverted repeats of approximately 300 base pairs. Several plasmids from beta-lactamase-producing N. gonorrhoeae isolated at different times and in different countries were compared. Nine 5.3-kb plasmids were examined and found to be indistinguishable, as were sixteen 7.4-kb plasmids.     

Tolerance to acid in pH 5.0-grown organisms of potentially pathogenic Gram-negative bacteria

S.A. NOJOUMI, D.G. SMITH AND R.J. ROWBURY. 1995. A wide range of potentially pathogenic species of Gram-negative bacteria were far more resistant to extreme acidity (pH 2.0–3.5) when cultured at pH 5.0 (habituated to acid) than after pH 7.0 culture. The differences were particularly great for Citrobacter spp., Enterobacter spp., Klebsiella spp. and for Vibrio parahaemolyticus ; substantial habituation was also observed for Proteus mirabilis and Aeromonas formicans but the effect was less marked for Serratia marcescens and Acinetobacter calcoaceticus . Growth at pH 5.0 was substantially poorer than at pH 7.0 for most of the above species and also for Salmonella typhimurium and Salm. enteritidis but phosphate markedly enhanced growth at pH 5.0 for many of these species without affecting growth at pH 7.0.