Properties of methemoglobin reductase and kinetic study of methemoglobin reduction

A soluble erythrocyte cytochrome b5 was purified as the substrate of methemoglobin reductase and an electron carrier to methemoglobin. The isoelectric point of this protein was at pH 4.3, and E0' was -0.010 at pH 7.0.. The Km value of the enzyme for this protein was 1 x 10(-4) M, and the turnover number (k5) was 3.4 x 10(4) min-1, with NADH as an electron donor at pH 7.0. The optimum pH of the enzyme was pH 4.6 for ferricyanide and pH 5.5 for cytochrome b5, with a shoulder of activity at pH 7 to 9 for both substrates. The rate equation which represents the reduction of either methemoglobin or cytochrome c was obtained as a function of methemoglobin or cytochrome c, methemoglobin reductase, and cytochrome b5 by considering the E . S complex for both reductase and cytochrome b5, and the rate constants involved were determined. The rate constants between methemoglobin and reduced cytochrome b5 (k1, M-1 min-1) were 1.6 x 10(4), 3.1 x 10(6), and 4.1 x 10(6) at pH 7.0, pH 5.2, and pH 5.0, respectively. The rate constants between the reduced enzyme and oxidized cytochrome b5 (k'3, M-1 min-1) were 4.3 x 10(8), 12 x 10(8), and 9.3 x 10(8) at pH 7.0, pH 5.2, and pH 5.0, respectively. The rate constant between reduced hemoglobin and oxidized cytochrome b5 (k2) was 35 M-1 min-1 at pH 7.0. The theoretical Km for methemoglobin was 2.1 M at an infinite enzyme concentration at pH 7.0     

Avian adenovirus induces ion channels in model bilayer lipid membranes

The action of duck egg drop syndrome 1976 (EDS-76) adenovirus on model bilayer lipid membranes (BLM) has been investigated on planar egg phosphatidylcholine membranes and small unilamellar vesicles. It was found that the adenovirus formed channels in planar BLM in a pH-dependent manner. The addition of EDS-76 to planar BLM at pH 5 induced voltage-independent channel activity of about 60 pS conductivity after a lag phase. At pH 3, EDS-76 induced irregular spikes of current across the planar BLM which disappeared after several minutes. The adenovirus also was able to induce pH-dependent leakage of calcein-loaded liposomes. EDS-76 did not induce channel activity in planar BLM or liposome leakage at neutral pH.     

Plasma ascorbate and vitamin E levels in Hong Kong Chinese

Escherichia coli grown at pH 5.0 became acid-tolerant (acid-habituated) but, in addition, neutralized medium filtrates from cultures of E. coli grown to log-phase or stationary-phase at pH 5.0 (pH 5.0 filtrates) induced acid tolerance when added to log-phase E. coli growing at pH 7.0. In contrast, filtrates from pH 7.0-grown cultures were ineffective. The pH 5.0 filtrates were inactivated by heating in a boiling water-bath but there was less activity loss at 75 degrees C. Protease also inactivated such filtrates, which suggested that a heat-resistant protein (or proteins) in the filtrates was essential for the induction of acid tolerance. Filtrates from cells grown at pH 5.0 plus phosphate or adenosine 3':5'-cyclic monophosphate (cAMP) were much less effective in inducing acid tolerance, while the conversion of pH 7.0-grown log-phase cells to acid tolerance by pH 5.0 filtrates was inhibited by cAMP and bicarbonate. It seems likely that the acid tolerance response (acid habituation) involved the functioning of the extracellular protein(s) as protease reduces tolerance induction if added during acid habituation. Most inducible responses are believed to involve the functioning of only intracellular reactions and components; the present results suggest that this is not the case for acid habituation, as an extracellular protein (or proteins) is needed for induction.     

Penetrating atherosclerotic aortic ulcer: documentation by transesophageal echocardiography

The effect of medium pH on the activity of cultured human osteoblasts was investigated in this study. Osteoblasts derived from explants of human trabecular bone were grown to confluence and subcultured. The first-pass cells were incubated in Hepes-buffered media at initial pHs adjusted from 7.0 to 7.8. Osteoblast function was evaluated by measuring lactate production, alkaline phosphatase activity, proline hydroxylation, DNA content, and thymidine incorporation. Changes in medium pH were determined from media pHs recorded at the beginning and end of the final 48 h incubation period. As medium pH increased through pH 7.6, collagen synthesis, alkaline phosphatase activity, and thymidine incorporation increased. DNA content increased from pH 7.0 to 7.2, plateaued from pH 7.2 to 7.6, and increased again from pH 7.6 to 7.8. The changes in the medium pH were greatest at pHs 7.0 and 7.8, modest at pHs 7.4 and 7.6, and did not change at 7.2, suggesting that the pHs are migrating towards pH 7.2. Lactate production increased at pH 7.0 but remained constant from 7.2 to 7.8. These results suggest that in the pH range from 7.0-7.6 the activity of human osteoblasts increases with increasing pH, that this increase in activity does not require an increase in glycolytic activity, and that pH 7.2 may be the optimal pH for these cells.     

Blood pressure control during eye surgery under local anesthesia using pulse oximetry

The purpose of this study was to compare the surface pH level of three different type sealers after mixing at various time intervals in vitro. The cements were mixed according to the manufacturer's instructions. They were incubated to set in 100% humidity at 37 degrees C for 1 h, 24 h, 5 days, 8 days, 2 wk, 3 wk, 4 wk, 5 wk, and 7 wk. pH was calculated by a Twin pH meter. The pH levels of the three sealers were different at various time intervals (p < 0.0001). The resin-based cement had a acid pH level (pH < 7.0). The calcium hydroxide-based cement showed a higher alkalinity pH level (pH > 7.0). The zinc oxide-eugenol-based cement showed a similar pH level to the calcium hydroxide cement at the end of the measurement. We postulated that, in endodontic therapy when those healing is needed, the alkaline-based sealer is the choice.     

A generic drug primer: regulatory aspects and scientific concepts

Organisms of Escherichia coli 1829 become alkali sensitized on transfer from pH 7.0 to pH 5.5 but they also secrete extracellular agents which induce alkali sensitivity when added (in neutralized filtrates) to organisms growing at pH 7.0. In contrast, filtrates from cultures grown at pH 7.0 have no effect. Filtrates were inactivated by protease but not by heat treatment in a boiling water-bath, suggesting that a very heat-stable protein is involved in alkali sensitivity induction. A heat-stable low molecular weight component (or components) may also be needed for induction, or the induction protein itself may be of low molecular weight. Strains with lesions in hns, fur or himA produced almost inactive filtrates and it therefore appears that H-NS, Fur and IHF are involved in synthesis of the induction components. As the presence of protease during incubation at pH 5.5 totally abolished alkali sensitization of strain 1829 while inhibition of sensitization induction occurred if the induction components were removed by filtration or dialysis during pH 5.5 incubation, it is proposed that the extracellular induction components (EICs) are essential for the original sensitization response. These results suggest that sensitization induction occurs by a different mechanism to that which is believed to occur for most bacterial inducible response systems; these are claimed to involve exclusively intracellular reactions and components whereas the present response involves functioning of extracellular components.     

Purification and characterization of an oxygen-sensitive, reversible 3,4-dihydroxybenzoate decarboxylase from Clostridium hydroxybenzoicum

A 3,4-dihydroxybenzoate decarboxylase (EC 4.1.1.63) from Clostridium hydroxybenzoicum JW/Z-1T was purified and partially characterized. The estimated molecular mass of the enzyme was 270 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a single band of 57 kDa, suggesting that the enzyme consists of five identical subunits. The temperature and pH optima were 50 degrees C and pH 7.0, respectively. The Arrhenius energy for decarboxylation of 3,4-dihydroxybenzoate was 32.5 kJ . mol(-1) for the temperature range from 22 to 50 degrees C. The Km and kcat for 3,4-dihydroxybenzoate were 0.6 mM and 5.4 x 10(3) min(-1), respectively, at pH 7.0 and 25 degrees C. The enzyme optimally catalyzed the reverse reaction, that is, the carboxylation of catechol to 3,4-dihydroxybenzoate, at pH 7.0. The enzyme did not decarboxylate 2-hydroxybenzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, 2,3-dihydroxybenzoate, 2,4-dihydroxybenzoate, 2,5-dihydroxybenzoate, 2,3,4-trihydroxybenzoate, 3,4,5-trihydroxybenzoate, 3-F-4-hydroxybenzoate, or vanillate. The decarboxylase activity was inhibited by 25 and 20%, respectively, by 2,3,4- and 3,4,5-trihydroxybenzoate. Thiamine PPi and pyridoxal 5'-phosphate did not stimulate and hydroxylamine and sodium borohydride did not inhibit the enzyme activity, indicating that the 3,4-dihydroxybenzoate decarboxylase is not a thiamine PPi-, pyridoxal 5'-phosphate-, or pyruvoyl-dependent enzyme.     

Deoxyribonuclease I from rat urine: affinity purification, characterization, and immunochemical studies

Deoxyribonuclease I (DNase I) from rat urine was purified about 3,000-fold to apparent homogeneity with a 14% yield by affinity chromatography utilizing polyguanylic acid-agarose and DNA-cellulose. The purified enzyme preparation was found to contain no other detectable nucleases. Isoelectric focusing electrophoresis revealed that all six isoelectric forms of the enzyme had been purified, and the resulting bands all contained DNase I activity. Quantitative amino acid analysis and N-terminal amino acid sequencing were performed on the purified DNase I. The N-terminal sequence up to the 15th residue of the enzyme was identical to that of rat parotid DNase I. The enzyme was found to be a glycoprotein, containing 1 fucose, 10 galactose, 17 mannose, 12 glucosamine, and at least 3 sialic acid residues per molecule. The isoelectric multiplicity of the enzyme was partly due to differences in the sialic acid content of the isoforms. Gel filtration on Superose 12 and electrophoresis on sodium dodecyl sulfate polyacrylamide gels indicated an approximate molecular mass for DNase I of 32 kDa. The enzyme had an optimum pH of 6.5 and required divalent cations such as Ca2+ for its activity. Its activity was inhibited by 1 mM EDTA and EGTA, but not G-actin. An antibody against the purified enzyme was found to be monospecific against rat urine and the pure antigen, and completely blocked the activity of the purified enzyme.     

Differential effects of mucosal pH on human (Caco-2) intestinal epithelial cell motility, proliferation, and differentiation

Mucosal pH abnormalities are associated with anastomotic dehiscence, ischemia, and malignancy. We postulated that intraluminal pH influences intestinal epithelial motility, proliferation, and differentiation and studied extracellular pHo (7.0-8.5) effects on human (Caco-2) intestinal epithelial motility, proliferation, and differentiation. Mucosal healing was modeled by sheet migration and differentiation by alkaline phosphatase and dipeptidyl dipeptidase specific activity. In parallel differentiation and motility studies, we inhibited proliferation with mitomycin to dissociate indirect mitogenic effects. Intracellular pHi was quantitated using BCECF/AM at varying extracellular pHo and in migrating cells. Motility was maximal at pHo 7.6 and proliferation at 7.2. Each decreased with acidity and alkalinity. By contrast, brush border enzyme activity was lowest at pHo 7.0 and highest at pHo 8.5. pHi was highest at pHo 8.5. Migrating cell pHi was higher than static cell pHi. Thus, extracellular pHo deviations perturb Caco-2 pHi homeostasis and motility. Alkalinity promotes differentiation while acidity induces proliferation and limits differentiation.     

Stability of hemagglutinating activity of extracellular and intracellular forms of Japanese encephalitis virus exposed to acidic pH

The hemagglutinating (HA) activity of extracellular and intracellular forms of Japanese encephalitis (JE) virus was comparatively titrated by exposure to acidic pH below 7.0. A pH-dependent irreversible loss in titer was observed with the virus grown in both C6/36 and BHK 21 (BHK) cells maintained in the pH range of 5.8 to 7.0 for 10 min at 37 C. The HA activity of intracellular virus was relatively more stable than that of extracellular virus in the pH range of 5.8 to 6.4. Virion structural components, envelope glycoprotein (E), capsid (C), and membrane (M) proteins in extracellular virus and E, C, and the precursor form of M (prM) proteins in intracellular virus were detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. A panel of monoclonal antibody (mAb) directed for nine antigenic epitopes on the JE virus E protein molecule was used for the analysis of antigenic reactivity of E protein after treatment at pH 6.0. The reaction between the extracellular virus and three HA-inhibiting (HI) mAbs was significantly reduced after acid treatment; however, the antigenic reactivity of intracellular virus was much more stable with a 100- to 1,000-fold difference. Infectivity titers of extracellular and intracellular viruses in Vero cells were reduced by 1/24,100 and 1/21,666 after acidic treatment at pH 6.0. In contrast, the infectivity of intracellular viruses was more stable, with residual infectivity of 1/182 and 1/340 for BHK and C6/36 cell-grown virus, respectively. Acidic treatment of JE virus not only resulted in the irreversible loss of its HA activity but also affected the antigenic reactivity of HI epitopes on its E protein molecule.     

Influence of phosphate and pH on myofibrillar ATPase activity and force in skinned cardiac trabeculae from rat

1. The effects of inorganic phosphate (Pi) and pH on maximal calcium-activated isometric force and MgATPase activity were studied in chemically skinned cardiac trabeculae from rat. ATP hydrolysis was coupled enzymatically to the breakdown of NADH, and its concentration was determined photometrically. Measurements were performed at 2.1 microns sarcomere length and 20 degrees C. ATPase activity and force were also determined when square-wave-shaped length changes were applied, with a frequency of 23 Hz and an amplitude of 2.5%. 2. At pH 7.0 without added Pi, the average isometric force (+/- S.E.M.) was 51 +/- 3 kN m-2 (n = 23). The average isometric ATPase activity was 0.43 +/- 0.02 mM s-1 (n = 23). During the changes in length ATPase activity increased to 152 +/- 3% of the isometric value, while the average force level decreased to 48 +/- 2%. 3. Isometric force gradually decreased to 31 +/- 2% of the control value when the Pi concentration was increased to 30 mM. Isometric ATPase activity, however, remained constant for Pi concentrations up to 5 mM and decreased to 87 +/- 3% at 30 mM Pi. When Pi accumulation inside the preparation due to ATP hydrolysis was taken into account, a linear relationship was found between isometric force and log [Pi]. The decrease in relative force was found to be 44 +/- 4% per decade. 4. During the length changes, ATPase activity and average force showed, apart from the increase in ATPase activity and decrease in average force, the same dependence on Pi as the isometric values. Stiffness, estimated from the amplitude of the force responses during the length changes, decreased in proportion to isometric force when the Pi concentration was increased. The changes in the shape of the force responses due to the repetitive changes in length as a function of the Pi concentration were relatively small. These results suggest that the effect of Pi on the transitions which influence ATP turnover is rather insensitive to changes in cross-bridge strain. 5. Isometric force, normalized to the control value at pH 7.0, increased gradually from 54 +/- 1% at pH 6.2 to 143 +/- 10% at pH 7.5. ATPase activity remained practically constant for pH values from 6.8 to 7.2 but decreased to 80 +/- 1% at pH 6.2 and to 83 +/- 5% at pH 7.5. ATPase activity during the length changes was reduced more than the isometric ATPase activity when pH was lowered.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation, purification, and characterization of a neutral Mg(2+)-dependent deoxyribonuclease of the Colorado potato beetle Leptinotarsa decemlineata Say

A neutral Mg(2+)-dependent deoxyribonuclease from the Colorado potato beetle was isolated and characterized in physicochemical terms. An electrophoretically homogeneous preparation of the enzyme was obtained using salt fractionation, Sephadex G-100 gel filtration, and subsequent preparative isoelectrofocusing in an Ultrodex layer. The molecular weight of the purified DNase preparation (with a purification degree of 104) and its isoelectric point were 100 kD and 9.1, respectively. The enzyme activity was maximal at pH 7.2 and 46 degrees C in the presence of 10 mM Mg2+. The DNase of the Colorado beetle preferentially hydrolysed denatured DNA via the endonuclease pathway, degrading the substrate to oligonucleoside-3'-phosphates. As far as the physical and chemical properties are concerned, this Colorado beetle DNase seems different from previously investigated DNases of other insect species.     

Determination by photoreduction of flip-flop kinetics of spin-labeled stearic acids across phospholipid bilayers

Spin-labeled stearic acid derivatives (N-DS) can be used to determine the rate at which lipid-derived drugs can cross a phospholipid bilayer (flip-flop). The flip-flop rate of N-DS (where N=5, 6, 7, 9, 10, 12, 16), was measured using vectorial photoreduction of nitroxides to their corresponding hydroxylamine by FMN, a charged, membrane-impermeable flavin, by hydrogen atom transfer from EDTA. From the time difference in the photoreduction rates of N-DS located in the outer and inner half of the bilayer, the flip-flop rate of N-DS across the bilayer can be determined. The results show that at pH 8.0 or lower, the photoreduction of 5-DS on one side of the membrane by FMN is slower than the flip-flop rate of 5-DS across phospholipid bilayers. For 5-DS at pH 7.0, this rate is at least 33.8+/-4.24 s or faster. Stearic acids with the spin label at different positions along the acyl chain (N=5, 6, 7, 9, 10, 12) have similar flip-flop rates in the liposomes at pH 7.0 although 16-DS is slower, probably due to the inaccessibility of the nitroxide moiety to FMN. It is most likely that the fast distribution of 5-DS in cells is due to the fast movement of acidic form, but not the salt form, of 5-DS across membrane bilayers. The oxazolidine (nitroxide moiety) does not seem to affect the pKa ( approximately 8.3) of stearic acid at air-water interface. Thus, N-DS are good probes for studying the distribution kinetics of stearic acid derivatives in biological systems.     

The effect of environmental pH on the proton motive force of Helicobacter pylori

BACKGROUND & AIMS: Responses of the proton motive force (the driving force for protons) in Helicobacter pylori to varying medium pH may explain gastric colonization. The aim of this study was to determine the effect of external pH (pHout) on the proton motive force, the sum of the pH gradient, and the potential difference across the bacterial membrane. METHODS: Intracellular pH (pHin) was measured by bis-carboxyethyl-carboxy fluorescein fluorescence and transmembrane potential difference (PD) by fluorescent quenching of 3,3'-dipropyl thiadicarbocyanine iodide at differing pHout and was correlated with survival. RESULTS: PD was -131 +/- 0.36 mV (n = 3), and pHin was about 8.4 at loading pHout 7.0. PD increased as pHout was increased from 4.0 to 8.0, giving a constant proton motive force of about -220 mV. Outside these limits, PD collapsed irreversibly to zero. Addition of 5 mmol/L urea to weak buffer at pH 3.0 or 3.5 prevented irreversible collapse of PD by elevation of pHout caused by NH3 production. Urea addition to weak buffer at pH 7.0 collapsed the PD as urease activity increased the pHout to about 8.4. Survival was also limited to this range of pHout. CONCLUSIONS: H. pylori survives over the range of pHout where it maintains a proton motive force. The effect of urease activity on pHout, while allowing gastric survival in acidic media, may limit survival in nonacidic media.     

Polyomavirus large T antigen binds cooperatively to its multiple binding sites in the viral origin of DNA replication

Polyomavirus large T antigen binds to multiple 5'-G(A/G)GGC-3' pentanucleotide sequences in sites 1/2, A, B, and C within and adjacent to the origin of viral DNA replication on the polyomavirus genome. We asked whether the binding of large T antigen to one of these sites could influence binding to other sites. We discovered that binding to origin DNA is substantially stronger at pH 6 to 7 than at pH 7.4 to 7.8, a range often used in DNA binding assays. Large T antigen-DNA complexes formed at pH 6 to 7 were stable, but a fraction of these complexes dissociated at pH 7.6 and above upon dilution or during electrophoresis. Increased binding at low pH is therefore due at least in part to increased stability of protein-DNA complexes, and binding at higher pH values is reversible. Binding to fragments of origin DNA in which one or more sites were deleted or inactivated by point mutations was measured by nitrocellulose filter binding and DNase I footprinting. The results showed that large T antigen binds cooperatively to its four binding sites in viral DNA, suggesting that the binding of this protein to one of these sites stabilizes its binding to other sites via protein-protein contacts. Sites A, B, and C may therefore augment DNA replication by facilitating the binding of large T antigen to site 1/2 at the replication origin. ATP stabilized large T antigen-DNA complexes against dissociation in the presence, but not the absence, of site 1/2, and ATP specifically enhanced protection against DNase I digestion in the central 10 to 12 bp of site 1/2, at which hexamers are believed to form and begin unwinding DNA. We propose that large T antigen molecules bound to these multiple sites on origin DNA interact with each other to form a compact protein-DNA complex and, furthermore, that ATP stimulates their assembly into hexamers at site 1/2 by a "handover" mechanism mediated by these protein-protein contacts.     

Specific protection by Na+ or Li+ of the F1F0-ATPase of Propionigenium modestum from the reaction with dicyclohexylcarbodiimide

Incubation of the purified F1F0-ATPase of Propionigenium modestum with dicyclohexylcarbodiimide (DCCD) led to inactivation of the enzyme in a strongly pH-dependent manner. Rapid inactivation occurred at pH 5-7, while the increase of the pH from 7 to 9 resulted in a continuous reduction of the inactivation rate. In the presence of Na+ ions, the ATPase was specifically protected from inactivation by DCCD. The protective effect of Na+ was most pronounced at pH 9.0 and less significant at pH 7.0. In addition to Na+, Li+ also protected the ATPase from inactivation by DCCD, but approximately 10 times higher concentrations were required for the same effect. Similarly, the Na+ concentration causing half-maximal stimulation of ATPase activity was about 10 times below the Li+ concentration required for the same activation. It is concluded from these results that a binding site is present for Na+ or Li+ on the enzyme with an about 10 times lower affinity for the latter alkali ion, which when occupied stimulates ATPase activity and protects it from inactivation by DCCD. Inactivation of ATPase activity by DCCD correlated well with a specific labeling of subunit c of the enzyme in the presence of the [14C]DCCD derivative. Like ATPase inactivation, the labeling was promoted by more acidic pH values and inhibited by Na+ ions. We suggest from these data that the DCCD-reactive amino acid residue of subunit c (most likely Glu-65) must be protonated for the reaction with the carbodiimide and provides the Na(+)-binding site in its deprotonated state. Dissociation of the carboxylic acid (at high pH) and binding of Na+ ions to the carboxylate thus abolish the reactivity toward DCCD.     

Relative stability of triplexes containing different numbers of T.AT and C+.GC triplets

We have used DNase I footprinting to compare the stability of parallel triple helices containing different numbers of T.AT and C+. GC triplets. We have targeted a fragment containing the 17mer sequence 5'-AGGAAGAGAAAAAAGAA with the 9mer oligonucleotides 5'-TCCTTCTCT, 5'-TTCTCTTTT and 5'-TTTTTTCTT, which form triplexes at the 5'-end, centre and 3'-end of the target site respectively. Quantitative DNase I footprinting has shown that at pH 5.0 the dissociation constants of these oligonucleotides are 0.13, 4.7 and >30 microM respectively, revealing that increasing the proportion of C+.GC triplets increases triplex stability. The results suggest that the positive charge on the protonated cytosine contributes to triplex stability, either by a favourable interaction with the stacked pisystem or by screening the charge on the phosphate groups. In the presence of a naphthylquinoline triplex binding ligand all three oligonucleotides bind with similar affinities. At pH 6.0 these triplexes only form in the presence of the triplex binding ligand, while at pH 7.5 footprints are only seen with the oligonucleotide which generates the fewest number of C+.GC triplets (TTTTTTCTT) in the presence of the ligand.     

Salivary deoxyribonuclease I polymorphism separated by polyacrylamide gel-isoelectric focusing and detected by the dried agarose film overlay method

Isoelectric focusing of whole saliva samples on polyacrylamide gels (pH 3.5-5), followed by dried agarose film overlay detection, was employed to determine the type of salivary deoxyribonuclease I (DNase I). Since this detection method had not only a high sensitivity, but also a high band resolution, it was possible to determine DNase I types from saliva samples of 2-5 microL. Pretreatment of saliva samples with neuraminidase simplified the isozyme pattern and enhanced the sensitivity. The DNase I types in all 30 saliva samples showed a good correlation with the types found in the corresponding blood, semen, and urine samples. This preliminary trial indicates that DNase I typing from saliva samples is a new and promising method for individualization of casework samples in the field of forensic biology.