Electron transport through the salivary gland of the Lone Star tick

After forcing current (using 3.4 volts dc at pH 7.0) across isolated salivary glands of the Lone Star tick (Amblyomna americanum L.), reduction of Nile Blue A (red coloration) appeared on the outside of the alveoli within 20 sec. In freshin vitro gland preparations a change of pH from 6.0 to 7.0 to 8.0 caused little or no change in the emf at which the red color could be seen to form. However, in “aged” preparations it was observed that a pH of 8.0 caused an increase of 0.08 volts. It was concluded that charge carriers in the tissue are quite probably both ionic and electronic in character. However, in “aged” preparations the results imply the readily detectable presence of hydrogenic (protonic) transport of charge across the tissue.     

Absorbance signals from resting frog skeletal muscle fibers injected with the pH indicator dye, phenol red

Singly dissected twitch fibers from frog muscle were studied on an optical bench apparatus after micro-injection with the pH indicator dye, phenol red. Dye-related absorbances in myoplasm, denoted by A0(lambda) and A90(lambda), were estimated as a function of wavelength lambda (450 nm less than or equal to lambda less than or equal to 640 nm) with light polarized parallel (0 degrees) and perpendicular (90 degrees) to the fiber axis respectively. At all lambda, A0(lambda) was slightly greater than A90(lambda), indicating that some of the phenol red molecules were bound to oriented structures accessible to myoplasm. The phenol red "isotropic" signal, [A0(lambda) + 2A90(lambda)]/3, a quantity equal to the average absorbance of all the dye molecules independent of their orientation, had a spectral shape that was red-shifted by approximately 10 nm in comparison with in vitro dye calibration curves measured in 140 mM KCl. The red-shifted spectrum also indicates that some phenol red molecules were bound in myoplasm. A quantitative estimate of indicator binding was obtained from measurements of the dye's apparent diffusion constant in myoplasm, denoted by Dapp. The small value of Dapp, 0.37 x 10(-6) cm2 s-1 (at 16 degrees C), can be explained if approximately 80% of the dye was bound to myoplasmic sites of low mobility. To estimate the apparent myoplasmic pH, denoted by pHapp, the isotropic absorbance of phenol red was fitted by in vitro calibration spectra. pHapp was found to be independent of dye concentration (0.2-2 mM), but varied widely (range, 6.8-7.5; mean value, 7.17) among fibers judged from functional characteristics to be normal. When fibers were subjected to acid or alkaline loads by exposure to Ringer's solution containing, respectively, dissolved CO2 or NH3, the changes in pHapp were in agreement with those expected from pH micro-electrode studies. It is concluded that in spite of the several indications for the presence of bound phenol red inside muscle cells, the pHapp signal from the indicator is useful for monitoring changes in myoplasmic pH in response to physiological and pharmacological manipulations.     

Trichomonas vaginalis: identification of a phospholipase A-dependent hemolytic activity in a vesicular subcellular fraction

Trichomonad total extracts (TTE), or vesicular (P30) and soluble (530) subcellular fractions from 3 pathogenic Trichomonas vaginalis strains (GT-3. GT-13. and GT-15), lysed both human and Sprague-Dawley rat erythrocytes in a time- and dose-dependent manner. The entire hemolytic activity of TTE was located in P30, showing 2 peaks of maximum activity, one at pH 6.0 and another at pH 8.0. in the presence of 1 mM Ca2+. Hemolytic activity on rat erythrocytes was greater at pH 6.0 16.71 +/- 0.33 hemolytic units IHU]/mg/hr to 11.60 +/- 0.24 HU/mg/hr) than at pH 8.0 (3.81 +/- 0.30 HU/mg/hr to 5.75 +/- 0.65 HU/mg/hr). and it was greater than that on human red blood cells at pH 6.0 (2.67 +/- 0.19 HU/mg/hr to 4.08 +/- 0.15 HU/mg/hr) or pH 8.0 (2.24 +/- 0.0 9 HU/mg/hr to 2.81 +/- 0.06 HU/mg/hr). The alkaline and acidic hemolytic activity diminished (60-93% at pH 6.0 and 78-93% at pH 8.0) by the effect of 80 microM Rosenthal's inhibitor, which also inhibited 27-45% and 29-54% trichomonad alkaline and acidic phospholipase A activities, respectively. Vesicles, vacuoles, and hydrogenosomes were rich in P30. Trichomonas vaginalis has a hemolytic PLA, which could be involved in its cytopathogenic mechanism.     

Changes in phenol red absorbance in response to electrical stimulation of frog skeletal muscle fibers

Intact single twitch fibers from frog muscle were stretched to long sarcomere length, micro-injected with the pH indicator dye phenol red, and activated by action potential stimulation. Indicator-related absorbance changes (denoted by delta A0 and delta A90) were measured with 0 degree and 90 degrees polarized light (oriented, respectively, parallel and perpendicular to the fiber axis). Two components of delta A were detected that had generally similar time courses. The "isotropic" component, calculated as the weighted average (delta A0 + 2 delta A90)/3, had the wavelength dependence expected for a change in myoplasmic pH. If calibrated in pH units, this signal's peak amplitude, which occurred 15-20 ms after stimulation, corresponded to a myoplasmic alkalization of average value 0.0025 +/- 0.0002 (+/- SEM; n = 9). The time course of this change, as judged from a comparison with that of the fibers' intrinsic birefringence signal, was delayed slightly with respect to that of the myoplasmic free [Ca2+] transient. On average, the times to half-peak and peak of the phenol red isotropic signal lagged those of the birefringence signal by 2.4 +/- 0.2 ms (+/- SEM; n = 8) and 8.4 +/- 0.5 ms (+/- SEM; n = 4), respectively. The other component of the phenol red signal was "dichroic," i.e., detected as a difference (delta A0-delta A90 greater than 0) between the two polarized absorbance changes. The wavelength dependence of this signal was similar to that of the phenol red resting dichroic signal (Baylor and Hollingworth. 1990. J. Gen. Physiol. 96:449-471). Because of the presence of the active dichroic signal, and because approximately 80% of the phenol red molecules appear to be bound in the resting state to either soluble or structural sites, the possibility exists that myoplasmic events other than a change in pH underlie the phenol red isotropic signal.     

A modification of the Pseudomonas selective medium, CFC, that allows differentiation between meat pseudomonads and Enterobacteriaceae

Certain members of the family Enterobacteriaceae developing on beef steaks packaged in modified atmospheres may grow on the medium CFC (Cephaloridine-Fucidin-Cetrimide), which is selective for pseudomonads. The addition of arginine (1% w/v) and the pH indicator phenol red (0.002% w/v) to this medium differentiated between the two groups. The pseudomonads produced ammonia from arginine, whereas Enterobacteriaceae generally did not use this substrate. The alkaline drift in pH in and around pseudomonad colonies gave a pink colouration with phenol red.     

Glutaminyl cyclases unfold glutamyl cyclase activity under mild acid conditions

N-terminal pyroglutamate (pGlu) formation from glutaminyl precursors is a posttranslational event in the processing of bioactive neuropeptides such as thyrotropin-releasing hormone and neurotensin during their maturation in the secretory pathway. The reaction is facilitated by glutaminyl cyclase (QC), an enzyme highly abundant in mammalian brain. Here, we describe for the first time that human and papaya QC also catalyze N-terminal glutamate cyclization. Surprisingly, the enzymatic Glu(1) conversion is favored at pH 6.0 while Gln(1) conversion occurs with an optimum at pH 8.0. This unexpected finding might be of importance for deciphering the events leading to deposition of highly toxic pyroglutamyl peptides in amyloidotic diseases.     

Purification and characterization of NAD-dependent n-butanol dehydrogenase from solvent-tolerant n-butanol-degrading Enterobacter sp. VKGH12

The solvent-tolerant bacterium Enterobacter sp. VKGH12 is capable of utilizing n-butanol and contains an NAD+-dependent n-butanol dehydrogenase (BDH). The BDH from n-butanol-grown Enterobacter sp. was purified from a cell-free extract (soluble fraction) to near homogeneity using a 3-step procedure. The BDH was purified 15.37-fold with a recovery of only 10.51, and the molecular mass estimated to be 38 kDa. The apparent Michaelis-Menten constant (Km) for the BDH was found to be 4 mM with respect to n-butanol. The BDH also had a broad range of substrate specificity, including primary alcohols, secondary alcohols, and aromatic alcohols, and exhibited an optimal activity at pH 9.0 and 40oC. Among the metal ions studied, Mg2+ and Mn2+ had no effect, whereas Cu2+, Zn2+, and Fe2+ at 1 mM completely inhibited the BDH activity. The BDH activity was not inhibited by PMSF, suggesting that serine is not involved in the catalytic site. The known metal ion chelator EDTA had no effect on the BDH activity. Thus, in addition to its physiological significance, some features of the enzyme, such as its activity at an alkaline pH and broad range of substrate specificity, including primary and secondary alcohols, are attractive for application to the enzymatic conversion of alcohols.     

Evidence for inhibitory SH groups in the thiol activated K:Cl cotransporter of low K sheep red blood cells

The monofunctional thiol reagents N-ethylmaleimide (NEM) and methyl methanethiosulfonate (MMTS) stimulate ouabain resistant (OR) electroneutral K:Cl cotransport in LK sheep red blood cells at low, but not at high concentrations. Diamide (DM), on the other hand, only stimulates OR K:Cl flux (Lauf, P.K., J. Memb. Biol. 101: 179–188, 1988). The DM stimulated K:Cl cotransport was decreased toward the control value prior to DM stimulation when NEM or MMTS were added, subsequently. The inhibitory effect was dependent on the compound's concentration and exposure time and, in the case of MMTS, was reversed upon addition of dithiothreitol (DTT). The inhibition was more prominent when NEM treatment was performed at pH 8.0 and disappeared at pH 6.0. In contrast the NEM stimulatory effect was most effective when the pH of NEM treatment was 6.0 (Bauer, J. & Lauf, P.K., J. Memb. Biol. 73: 257–261, 1983). The results suggest the existence of additional, however, inhibitory thiol groups in the already thiol-activated K:Cl cotransporter, with a different pKa value and a lower affinity for NEM or MMTS as compared to the stimulatory thiol groups. Like the activating thiols, the inhibitory sulfhydryls appeared to be inaccessible to non-penetrating thiol reagents and hence, must be located deeper within the red cell membrane.     

Maintenance of internal pH and an electrochemical gradient in Trypanosoma brucei

The internal pH value (pHi) of the long-slender bloodstream form of Trypanosoma brucei was estimated from the distribution of 14C-labeled 5,5-dimethyl-2,4-oxazolidinedione or 14C-labeled methyl amine between the intracellular space of the cells and the medium. The pHi of T. brucei remained relatively constant at 7.0-7.2 throughout an extracellular pH (pHo) range of 6.0-8.0. The maintenance of an internal pH more acidic than the environment appears to be a unique feature. Preincubation of T. brucei with carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or CCCP + valinomycin had no appreciable effect on the delta pH across the T. brucei membrane when the external pH was 8.0. However, when the external pH was 6.0, CCCP abolished the observed delta pH. Nigericin significantly dissipated the delta pH across the T. brucei membrane at all pHo values. These data suggest that under physiological conditions, the maintenance of a delta pH across the bloodstream-form T. brucei membrane may be by a mechanism other than an energy-dependent gradient, whereas an energy-dependent pump may be needed for maintaining the pHi in an acidic environment. The electrical potential (delta psi) across the trypanosomal plasma membrane was also estimated using the lipophilic cation, [3H]tetraphenyl-phosphonium bromide. It appears dependent on both the external pH and the external salt conditions. Under ionic conditions similar to the host bloodstream, it ranges from -76 to -160 mV over an external pH range of 6.0 to 8.0, with an estimated value of -155.5 +/- 0.7 at the physiological pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

U-Shape Suppressive Effect of Phenol Red on the Epileptiform Burst Activity via Activation of Estrogen Receptors in Primary Hippocampal Culture

Phenol red is widely used in cell culture as a pH indicator. Recently, it also has been reported to have estrogen-like bioactivity and be capable of promoting cell proliferation in different cell lines. However, the effect of phenol red on primary neuronal culture has never been investigated. By using patch clamp technique, we demonstrated that hippocampal pyramidal neurons cultured in neurobasal medium containing no phenol red had large depolarization-associated epileptiform bursting activities, which were rarely seen in neurons cultured in phenol red-containing medium. Further experiment data indicate that the suppressive effect of the phenol red on the abnormal epileptiform burst neuronal activities was U-shape dose related, with the most effective concentration at 28 µM. In addition, this concentration related inhibitory effect of phenol red on the epileptiform neuronal discharges was mimicked by 17-β-estradiol, an estrogen receptor agonist, and inhibited by ICI-182,780, an estrogen receptor antagonist. Our results suggest that estrogen receptor activation by phenol red in the culture medium prevents formation of abnormal, epileptiform burst activity. These studies highlight the importance of phenol red as estrogen receptor stimulator and cautions of careful use of phenol red in cell culture media.     

The ionization behavior of retinoic acid in aqueous environments and bound to serum albumin.

The ionization behavior of retinoic acid (RA) in an aqueous phase and when bound to bovine serum albumin was studied. Titrations of RA in the various phases were followed by monitoring the red shift in the absorption maximum of RA that occurred upon deprotonation. The apparent pK of RA was dependent on the concentration of this compound. At the concentration range 6-20 microM, the pK of RA in water had a value of approximately 8.0. As the concentration was decreased in the range 1-6 microM, the value of the pK decreased continuously. The lowest pK observed was approximately 6.0. It was concluded that RA in an aqueous phase at concentrations in the microM range, forms micelles, and that the values of the pK of RA monomers and micelles in water are less than 6.0 and 8.0, respectively. The presence of 0.15 M NaCl caused a decrease in the pK of RA micelles and lowered the value of the CMC. Titration of RA in the presence of bovine serum albumin revealed the presence of a heterogeneous population comprised of three distinct microenvironments for RA associated with this protein. Two populations of RA were found to undergo complete titration in the pH range 4-8. A third population became apparent at pH greater than 9.5.     

A comparison between the responses of neutral red and acridine orange: acridine orange should be preferential and alternative to neutral red as a dye for the monitoring of contaminants by means of biological sensors

The acridine orange (AO) and neutral red (NR) dyes, commonly used as probes to measure the internal pH in acidic vesicles, are compared in this article. The comparison between the two dyes (arising from calculations taking into account their analytical constants) illustrated that the use of AO is preferential to that of NR because the AO response is sensitive over the whole pH range between 4.0 and 7.4, whereas the NR response is effective only between pHs 4.0 and 6.0. In addition, it became evident from the mitochondrial respiration response that NR, unlike AO, is a protonophore. When taken into consideration, these two properties suggest that AO is more suitable than NR as an indicator of toxicity measurements in water samples because the environmental toxic compounds induce pH changes in the acidic vesicles of biological structures that are used as environmental biosensors.     

Dimethyl-and monomethyloxyluciferins as analogs of the product of the bioluminescence reaction catalyzed by firefly luciferase

The absorption and fluorescence spectra of dimethyloxyluciferin (DMOL) and monomethyloxyluciferin (MMOL) were studied at pH 3.0-12.0. In the range of pH 3.0-8.0, the fluorescence spectrum of DMOL exhibits a maximum at lambda(em) = 639 nm. At higher pH values an additional emission maximum appears at lambda(em) = 500 nm (wavelength of excitation maximum lambda(ex) = 350 nm), which intensity increases with time. It is shown that this peak corresponds to the product of DMOL decomposition at pH > 8.0. The absorption spectra of MMOL were studied in the range of pH 6.0-9.0. At pH 8.0-9.0, the absorption spectrum of MMOL exhibits one peak at lambda(abs) = 440 nm. At pH 7.3-7.7, an additional band appears with maximum at lambda(abs) = 390 nm. At pH 6.0-7.0 two maxima are observed, at lambda(abs) = 375 and 440 nm. The fluorescence spectra of MMOL (pH 6.0-9.7, lambda(ex) = 440 or 375 nm) exhibit one maximum. It is shown that decomposition of DMOL and MMOL in aqueous solutions results in products of similar structure. DMOL and MMOL are rather stable at the pH optimum of luciferase. It is suggested that they can be used as fluorescent markers for investigation of the active site of the enzyme.     

Some Properties of Acid Protease from the Thermophilic Fungus, Penicillium duponti K1014

A purified acid protease from a true thermophilic fungus, Penicillium duponti K1014, was most active at pH 2.5 for milk casein and at pH 3.0 for hemoglobin. The enzyme was stable at a pH range of 2.5 to 6.0 at 30 C for 20 h. The acid protease retained full activity after 1 h at 60 C at a pH range between 3.5 and 5.5. At the most stable pH of 4.5, more than 65% of its activity remained after heat treatment for 1 h at 70 C. These thermal properties show the enzyme as a thermophilic protein. The enzyme activity was strongly inhibited by sodium lauryl sulfate and oxidizing reagents such as potassium permanganate and N-bromosuccinimide. No inhibition was caused by chelating reagents, potato inhibitor, and those reagents which convert sulfhydryl groups to mercaptides. Reducing reagents showed an activating effect. The enzyme showed the trypsinogen-activating property at an acidic pH range; optimal trypsinogen activation was obtained at a pH of approximately 3.0. The isoelectric point of the enzyme was estimated to be pH 3.89 by disk electrofocusing. By using gel filtration, an approximate value of 41,000 was estimated for the molecular weight.     

Some physicochemical properties of human milk bile-salt activated lipase

Influence of pH, deoxycholate and denaturing reagents on human milk bile-salt activated lipase (EC 3.1.1.3) has been studied. It appears that pH between 5.0-8.0 has no significant effect on the secondary structure of this lipase, but its higher order structures are affected. Lipase-dependent 8-anilino-1-naphthalene sulphonate fluorescence has revealed that the deoxycholate activated form of lipase has a surface rich in hydrophobic amino acid residues. Circular dichroism and second derivative electronic absorption spectroscopic observations have also provided an evidence for deoxycholate-induced alterations in the surface conformation of this lipase.     

Metal ion chelating peptoids with potential as anti-oxidants: complexation studies with cupric ions

The cupric ion binding characteristics of the chelator EDTA bis (ethyl tyrosinate) are reported. Potentiometric studies in aqueous solutions over the pH range of 2.0-12.0 allowed identification and quantification of the species in solution. The principal species CuA predominates over the physiological pH range of 4.0-8.0 pH units. The logarithm of the stability constant (log beta(pqr)) for this species is 16.43. The cupric ion binding characteristics were further assessed using electronic absorption spectroscopic investigations. These results support the use of this chelator as a metal binding anti-oxidant.     

Amino acid residues complexed with eosin 5-isothiocyanate in band 3 protein of the human erythrocyte

The amino-acid residues of band 3 protein taking part in the ionic interaction with an anion-transport inhibitor, eosin 5-isothiocyanate (EITC), were determined by pH titration. The plots of the absorbance of EITC-ghost system against pH reveal five equilibria, at pH 3.7, 6.4, 8.0, 11.0 and 13.1. Since the three equilibria, 3.7, 8.0 and 11.0, are representative of the EITC molecule, the others, 6.4 and 13.1, may be due to the interaction of EITC molecules with histidine and arginine residues, respectively. The same experiment using a reconstituted system of band 3-lipid vesicles gave results in good agreement with the EITC-ghost system. The intensity of the induced CD band at 530 nm of EITC molecules bound to ghosts was decreased by preincubation with arginine-specific reagents, phenylglyoxal and 1,2-cyclohexanedione, or histidine-specific reagents, diethylpyrocarbonate (DEPC) and p-diazobenzene sulfonate. The repression effects by these chemical modifiers were evaluated by measuring the concentrations which elicit 50% reduction. The histidine-specific reagents repressed the CD of EITC more effectively than the arginine-specific reagents. Furthermore, it was found that DEPC effectively inhibited the sulfate efflux from intact erythrocytes. These results suggest that the histidine residues participate in the anion-transport system of human red cells.     

Proton release during the reductive half-reaction of D-amino acid oxidase

Changes in the net protonation of D-amino acid oxidase during binding of competitive inhibitors and during reduction by amino acids have been monitored using phenol red as a pH indicator. At pH 8.0, no uptake or release of protons from solution occurs upon binding the inhibitors benzoate, anthranilate, picolinate, or L-leucine. The Kd values for both picolinate and anthranilate were determined from pH 5.4 to 9.0. The results are consistent with a single group on the enzyme having a pK of 6.3 which must be unprotonated for tight binding, as is the case with benzoate binding (Quay, S., and Massey, V. (1977) Biochemistry 16, 3348-3354) and with tight binding of the inhibitor form with an unprotonated amino group. Upon reduction of the enzyme by amino acid substrates, two protons are released to solution. The first is released concomitantly with reduction to the reduced enzyme-imino acid charge transfer complex. The second is released only upon dissociation of the charge transfer complex to free reduced enzyme and imino acid. The first proton is assigned as arising from the amino acid group and the second from the amino acid alpha-hydrogen. These results are consistent with the flavin in reduced D-amino acid oxidase being anionic.     

Estrogenic activity of phenol red in rat anterior pituitary cells in culture

The estrogenic activity of phenol red, a pH indicator widely used in cell culture media, was studied in rat anterior pituitary cells. After 72 hours of incubation with 40 microM phenol red, a 40-50% increase in prolactin cell content and a 100% stimulation of luteinizing hormone-releasing hormone induced luteinizing hormone release was observed. Both effects could be completely reversed by simultaneous incubation with the antiestrogen LY156758. In the rat uterine [3H] estradiol binding assay, phenol red showed a significant displacement at concentrations above 10 microM while its concentration in the commonly used culture media is about 40 microM. From the present results, we conclude that phenol red acts as a weak estrogen in normal tissues and that its estrogenic activity should be taken into account in studies using estrogen-sensitive cell or tissue cultures.     

Application of thermostable xylanase of Dictyoglomus sp. in enzymatic treatment of kraft pulps

Enzymatic treatment of pine and birch kraft pulps with a xylanase preparation from a thermophilic anaerobic bacterium Dictyoglomus sp. strain B1 was studied in order to improved pulp bleachability. Maximal solubilization of pulp xylan was obtained at 90°C and pH 6.0–7.0. The enzyme was also active in the alkaline pH range; at pH 9.0 xylan hydrolysis was decreased by only 18% from the maximum at pH 7.0. The positive effect of xylanase pretreatment at 80°C and pH 6.0 or 8.0 on bleachability of pine kraft pulp was demonstrated. The brightness was increased by two ISO units in one-stage peroxide delignification, which corresponds well to values obtained with other enzymes at lower temperatures and pH values. Thus, the Dictyoglomus xylanase is well suited for pulp treatments at elevated temperatures in neutral and alkaline conditions.Correspondence to: M. Rättö