Transmembrane pH gradient and membrane potential in Clostridium acetobutylicum during growth under acetogenic and solventogenic conditions

The proton motive force and its electrical and chemical components were determined in Clostridium acetobutylicum, grown in a phosphate-limited chemostat, using [14C]dimethyloxazolidinedione and [14C]benzoic acid as transmembrane pH gradient (delta pH) probes and [14C]triphenylmethylphosphonium as a membrane potential (delta psi) indicator. The cells maintained an internal-alkaline pH gradient of approximately 0.2 at pH 6.5 and 1.5 at pH 4.5. The delta pH was essentially constant between pH 6.5 and 5.5 but increased considerably at lower extracellular pH values down to 4.5. Hence, the intracellular pH fell from 6.7 to 6.0 as the external pH was lowered from 6.5 to 5.5 but did not decrease further when the external pH was decreased to 4.5. The transmembrane electrical potential decreased as the external pH decreased. At pH 6.5, delta psi was approximately -90 mV, whereas no negative delta psi was detectable at pH 4.5. The proton motive force was calculated to be -106 mV at pH 6.5 and -102 mV at pH 4.5. The ability to maintain a high internal pH at a low extracellular pH suggests that C. acetobutylicum has an efficient deacidification mechanism which expresses itself through the production of neutral solvents.     

Transmembrane pH gradient and membrane potential in Clostridium acetobutylicum during growth under acetogenic and solventogenic conditions.

The proton motive force and its electrical and chemical components were determined in Clostridium acetobutylicum, grown in a phosphate-limited chemostat, using [14C]dimethyloxazolidinedione and [14C]benzoic acid as transmembrane pH gradient (delta pH) probes and [14C]triphenylmethylphosphonium as a membrane potential (delta psi) indicator. The cells maintained an internal-alkaline pH gradient of approximately 0.2 at pH 6.5 and 1.5 at pH 4.5. The delta pH was essentially constant between pH 6.5 and 5.5 but increased considerably at lower extracellular pH values down to 4.5. Hence, the intracellular pH fell from 6.7 to 6.0 as the external pH was lowered from 6.5 to 5.5 but did not decrease further when the external pH was decreased to 4.5. The transmembrane electrical potential decreased as the external pH decreased. At pH 6.5, delta psi was approximately -90 mV, whereas no negative delta psi was detectable at pH 4.5. The proton motive force was calculated to be -106 mV at pH 6.5 and -102 mV at pH 4.5. The ability to maintain a high internal pH at a low extracellular pH suggests that C. acetobutylicum has an efficient deacidification mechanism which expresses itself through the production of neutral solvents.     

Lowering of cytoplasmic pH is essential for growth of Streptococcus faecalis at high pH.

The growth of Streptococcus faecalis at high pH was significantly stimulated by carbonate. In the absence of added carbonate the cells were unable to grow at a pH above 9.5, but in media containing 50 mM HCO3- they grew even at pH 10.5. Both rate and yield of growth at pH 9.5 were significantly stimulated by as little as 5 mM carbonate. The cytoplasmic pH in growing cells was maintained at about 7.8 to 8.2, whereas the medium pH ranged from 8.4 to 9.5. Nigericin and gramicidin D, ionophores which conduct protons, blocked growth at pH 9.5 but not at pH 7.5. These results indicate that lowering of the cytoplasmic pH is essential for the growth of this organism at high pH.     

Effect of pH and ionic strength on the catalytic and allosteric properties of native and chemically modified ox liver mitochondrial glutamate dehydrogenase.

Inorganic phosphate, a strong activator of glutamate dehydrogenase at pH 8.0–9.0, is an inhibitor at pH 6.0–7.6. The extent of inhibition increases with the decrease of pH. The same effect is shown by other electrolytes, including Tris-hydroxymethyl-aminomethane and NaCl.The combined effect of pH and ionic strength also alters the allosteric characteristics of the enzyme. Lowering the pH minimizes the activation by high concentrations of NAD; phosphate partially restores this activation. The allosteric activation by ADP disappears at pH around neutrality; in the pH range 6.0–7.0, ADP becomes a strong inhibitor, the inhibition being enhanced by the addition of ionic compounds. Similarly, the extent of allosteric inhibition by guanosine 5′-triphosphate (pyro) (GTP), which is maximal at pH 9.0, decreases at lower pH values and a slight activation is observed in the presence of electrolytes at pH 6.0.Glutamate dehydrogenase, selectively desensitized by dinitrophenylation in the presence of ADP, can be activated by ADP at pH 9.0, but is no longer inhibited by the same effector at pH 6.0, high salt concentration. The densensitized enzyme is not inhibited by GTP at pH 9.0, but is activated by this effector at pH 6.0 in the presence of ionic compounds. Conversely, GTP-protected dinitrophenylated glutamate dehydrogenase is desensitized only to the effect of the activating modifier, ADP at pH 9.0, GTP at pH 6.0, high salt concentration. These findings suggest that the conformation of each allosteric site of glutamate dehydrogenase is changed by pH and ionic strength so that it keeps its specificity for the ligand which brings about a given effect, activation or inhibition, independently from its chemical structure.     

Studies on Aspergillus Fumigatus; Properties of Intracellular Proteinase

Mycelial filtrates from Aspergillus fumigatus (AF) hydrolyzed protein substrate buffered at various pH values. Using casein as substrate there were distinct activity optima at pH 2.9, pH 6.2, and pH 10, with maximum activity at pH 6.2. Using haemoglobin as substrate there were activity optima at pH 3.6, pH. 4.6, and pH 10, with the biggest activity peak at pH 4.6. The pH stability at 4°G of the caseinase activity at pH 6.2 and pH 10 was strongest at pH 4, common to both, whereas the caseinase activity at pH 2.9 showed maximum pH stability at pH 6—7. The casein hydrolyzing activity at pH 2.9, pH 6.2, and pH 10 showed different optimum incubation temperatures and irregular heat inactivation. Normal rabbit serum inhibited the caseinase activity at pH 2.9 and pH 6.2 to some extent. The caseinase activity at pH 10 was almost completely inhibited. Antiserum against mycelial filtrate showed no definite inhibition beyond that exerted by normal serum. Following electrophoresis of antiserum, the presence of specific neutralizing antibodies against the casein precipitating enzyme of mycelial filtrate from AF could be established. Investigations of 14 AF strains showed immunological uniformity with respect to the casein precipitating enzyme.     

Effects of pH on tubulin-nucleotide interactions

Significant GTP-independent, temperature-dependent turbidity development occurs with purified tubulin stored in the absence of unbound nucleotide, and this can be minimized with a higher reaction pH. Since microtubule assembly is optimal at lower pH values, we examined pH effects on tubulin-nucleotide interactions. While the lowest concentration of GTP required for assembly changed little, GDP was more inhibitory at higher pH values. The amounts of exogenous GTP bound to tubulin at all pH values were similar, but the amounts of exogenous GDP bound and endogenous GDP (i.e., GDP originally bound in the exchangeable site) retained by tubulin rose as reaction pH increased. Endogenous GDP was more efficiently displaced by exogenous GTP than GDP at all pH values, but displacement by GTP was 10-15% greater at pH 6 than at pH 7. Dissociation constants for GDP and GTP were about 1.0 microM at pH 6 and 0.02 microM at pH 7. A small increase in the affinity of GDP relative to that of GTP occurs at pH 7 as compared to pH 6, together with a 50-fold absolute increase in the affinity of both nucleotides for tubulin at pH 7. The time courses of microtubule assembly and GTP hydrolysis were compared at pH 6 and pH 7. At pH 6, the two reactions were simultaneous in onset and initially stoichiometric. At pH 7, although the reactions began simultaneously, hydrolysis seemed to lag substantially behind assembly. Unhydrolyzed radiolabeled GTP was not incorporated into microtubules, however, indicating that GTP hydrolysis is actually closely coupled to assembly. The apparent lag in hydrolysis probably results from a methodological artifact rather than incorporation of GTP into the microtubule with delayed hydrolysis.     

Influence of pH on the appearance of active peptides in the course of peptic hydrolysis of bovine haemoglobin

Influence of pH on the appearance of active peptides in peptic hydrolysis of bovine haemoglobin was studied in a homogenous phase system. Six active peptides were studied: three hemorphins: LVVH-7 (beta 31-40), VVH-7 (beta 32-40), VVH-4 (beta 32-37), one bradykinin-potentiating peptide (alpha 110-125), one antibacterial peptide (alpha 1-23), and neokyotorphin (alpha 137-141). The influence of pH was investigated in the course of the hydrolysis of haemoglobin by pepsin at 23 degrees C in acetate buffer at pH 3.5, pH 4.5, and pH 5.5. The hydrolysis of haemoglobin was studied in the presence or absence of urea. The haemoglobin hydrolysis at pH 4.5 is taken as a reference. Two different mechanisms of hydrolysis were observed: "one by one" for native haemoglobin hydrolysis at pH 4.5 and 5.5, and "zipper" for denatured haemoglobin at pH 3.5, pH 4.5, and pH 5.5, and native haemoglobin at pH 3.5. Whatever the pH and medium, a selectivity change by the pepsin was noticed. In the presence of urea, there are two phenomena: some peptides are preferentially produced at pH 3.5 and other peptides at pH 5.5, which seems to favour one particular site of pepsin that is cut. In the absence of urea, these active peptides reached a higher concentration at pH 3.5. In order to prepare these six active peptides, it is suitable to hydrolyse haemoglobin in the absence of urea at pH 3.5 (this pH denatures haemoglobin) where a "zipper" mechanism is obtained, and the peptide quantity is more significant at pH 3.5 than at pH 4.5.     

A proton-translocating ATPase regulates pH of the bacterial cytoplasm

Regulatory mechanisms of cytoplasmic pH in Streptococcus faecalis with no respiratory chain were investigated. In a mutant defective in cytoplasmic alkalization conducted by a proton-translocating ATPase (H+-ATPase), the cytoplasmic pH is approximately 0.4 to 0.5 pH units lower than the medium pH, at pH 5.5 to 9.0. The cytoplasmic pH of the wild-type strain was always higher than that of the mutant at a pH below 8 and was the same as that of the mutant at an alkaline pH over 8. Thus, the cytoplasmic pH is regulated only by the cytoplasmic alkalization, and there is no regulation at alkaline pH in S. faecalis. A generation of the protonmotive force conducted by the H+-ATPase depended on the cytoplasmic pH rather than the medium pH, and the generation decreased rapidly when the cytoplasmic pH was increased over 7.7. The decrease at alkaline pH was not caused by increases in the rate of proton influx. These results suggest that cytoplasmic alkalization is diminished when alkaline pH of the cytoplasm is over 7.7, because of a low activity of proton extrusion by the H+-ATPase, and consequently, the cytoplasmic pH is regulated at about 7.7. The cytoplasmic pH was regulated at a high level in cells that had a high level of H+-ATPase. I conclude that in S. faecalis, the cytoplasmic pH is regulated by H+-ATPase.     

Aluminium rhizotoxicity in maize grown in solutions with Al3+ or Al(OH)-4 as predominant solution Al species

The rhizotoxicity of aluminium at low-pH with Al(3+) and at high pH with Al(OH)-(4) as the main Al species was studied. Aluminium reduced root growth to similar levels at pH 8.0 and pH 4.3, although the mononuclear Al concentration at pH 8.0 was three times lower than at pH 4.3. Al contents of root apices were much higher at pH 8 than at pH 4.3. Callose was induced only marginally at pH 8 and the formation was confined to the epidermis, whereas it proceeded through the cortex with time at pH 4.3. Well-documented genotypical differences in callose formation and Al accumulation could not be found at pH 8. The largest fraction of the root-tip Al was recovered in the cell-wall fraction independent of the solution pH. A sequential extraction of isolated cell walls suggests that most of the cell-wall Al was precipitated Al(OH)(3) at pH 8.0. This can be explained by a drastic pH reduction in the root apoplastic sap to 6.2, whereas at bulk solution pH 4.3 it rose to 5.6. Al precipitation was also confirmed by the microscopic localization of Al. At pH 8, Al could mostly be found in the epidermis, but in the apoplast of the outer cortex at pH 4.3. It is proposed here that at pH 4.3, Al(3+) inhibits root growth through binding to sensitive binding sites in the apoplast of the epidermis and the outer cortex. At pH 8, Al(OH)(3) precipitation in the epidermis causes a mechanical barrier thus impairing the root-growth control of the epidermis.     

Growth and bioenergetics of alkaliphilic Bacillus firmus OF4 in continuous culture at high pH.

The effect of external pH on growth of alkaliphilic Bacillus firmus OF4 was studied in steady-state, pH-controlled cultures at various pH values. Generation times of 54 and 38 min were observed at external pH values of 7.5 and 10.6, respectively. At more alkaline pH values, generation times increased, reaching 690 min at pH 11.4; this was approximately the upper limit of pH for growth with doubling times below 12 h. Decreasing growth rates above pH 11 correlated with an apparent decrease in the ability to tightly regulate cytoplasmic pH and with the appearance of chains of cells. Whereas the cytoplasmic pH was maintained at pH 8.3 or below up to external pH values of 10.8, there was an increase up to pH 8.9 and 9.6 as the growth pH was increased to 11.2 and 11.4, respectively. Both the transmembrane electrical potential and the phosphorylation potential (delta Gp) generally increased over the total pH range, except for a modest fall-off in the delta Gp at pH 11.4. The capacity for pH homeostasis rather than that for oxidative phosphorylation first appeared to become limiting for growth at the high edge of the pH range. No cytoplasmic or membrane-associated organelles were observed at any growth pH, confirming earlier conclusions that structural sequestration of oxidative phosphorylation was not used to resolve the discordance between the total electrochemical proton gradient (delta p) and the delta Gp as the external pH is raised. Were a strictly bulk chemiosmotic coupling mechanism to account for oxidative phosphorylation over the entire range, the deltaGp/deltap ration (which would equal the H+/ATP ratio) would rise from about 3 at pH 7.5 to 13 at pH 11.2, dropping to 7 at pH 11.4 only because of the rise in cytoplasmic pH relative to other parameters. Moreover, the molar growth yields on malate were higher at pH 10.5 than at pH 7.5, indicating greater rather than lesser efficiency in the use of substrate at the more alkaline pH.     

Potassium extrusion by the moderately halophilic and alkaliphilic methanogen methanolobus taylorii GS-16 and homeostasis of cytosolic pH.

Methanolobus taylorii GS-16, a moderately halophilic and alkaliphilic methanogen, grows over a wide pH range, from 6.8 to 9.0. Cells suspended in medium with a pH above 8.2 reversed their transmembrane pH gradient (delta pH), making their cytosol more acidic than the medium. The decreased energy in the proton motive force due to the reversed delta pH was partly compensated by an increased electric membrane potential (delta psi). The cytosolic acidification by M. taylorii at alkaline pH values was accompanied by K+ extrusion. The cytosolic K+ concentration was 110 mM in cells suspended at pH 8.7, but it was 320 mM in cells suspended at neutral pH values. High external K+ concentrations (210 mM or higher) inhibited the growth of M. taylorii at alkaline pH values, perhaps by preventing K+ extrusion. Cells suspended at pH 8.5 and 300 mM external K+ failed to acidify their cytosol. The key observation indicative of the involvement of K+ transport in cytosolic acidification was that valinomycin (0.8 microM), a K+ uniporter, inhibited the growth of M. taylorii only at alkaline pH values. Experiments with resting cells indicated that at alkaline pH values valinomycin uncoupled catabolic reactions from ATP synthesis. Thus, K+/H+ antiport activity was proposed to account for the K+ extrusion and the uncoupling effect of valinomycin at alkaline pH values. Such antiport activity was demonstrated by the sharp drop in pH of the bulk medium of the cell suspension upon the addition of 0.1 M KCl. The antiporter appeared to be active only at alkaline pH values, which was in accordance with a possible role in pH homeostasis by M. taylorii growing at alkaline pH values.     

Cytochemical Method to Localize Acidic Nuclear Proteins

Fast green FCF was used to localize acidic nuclear proteins in sections of young flower buds of Limnophyton obtusifolium (L.) Miq. After extracting nucleic acids, the slides were stained at hydrogen ion concentrations ranging from pH 2.6 to 9.0. At pH 5.0 and 8.0 staining is confined to the nucleus with no cytoplasmic reaction. Staining intensity is greater at pH 5.0 than at pH 8.0. The proteins responding to fast green at pH 8.0 are basic proteins. The positive reaction at pH 5.0 is attributed to acidic nuclear proteins. These findings are confirmed by control preparations. Acetylated slides and slides treated with 0.25 N HCl were unstained at pH 8.0 but staining at pH 5.0 was undisturbed. Dilute alkali (0.003 N NaOH) reduced staining intensity at pH 5.0 but had no effect at pH 8.0. Methylated slides did not stain at pH 5.0, but at pH 8.0 staining was unaffected. Deamination blocked staining at both pH's. It is concluded that fast green at pH 5.0 specifically binds with acidic nuclear proteins.     

Tetramerization of the LexA repressor in solution: implications for gene regulation of the E.coli SOS system at acidic pH

Structural changes on LexA repressor promoted by acidic pH have been investigated. Intense protein aggregation occurred around pH 4.0 but was not detected at pH values lower than pH 3.5. The center of spectral mass of the Trp increased 400 cm(-1) at pH 2.5 relatively to pH 7.2, an indication that LexA has undergone structural reorganization but not denaturation. The Trp fluorescence polarization of LexA at pH 2.5 indicated that its hydrodynamic volume was larger than its dimer at pH 7.2. 4,4'-Dianilino-1,1'-binaphthyl-5,5'- disulfonic acid (bis-ANS) experiments suggested that the residues in the hydrophobic clefts already present at the LexA structure at neutral pH had higher affinity to it at pH 2.5. A 100 kDa band corresponding to a tetramer was obtained when LexA was subject to pore-limiting native polyacrylamide gel electrophoresis at this pH. The existence of this tetrameric state was also confirmed by small angle X-ray scattering (SAXS) analysis at pH 2.5. 1D 1H NMR experiments suggested that it was composed of a mixture of folded and unfolded regions. Although 14,000-fold less stable than the dimeric LexA, it showed a tetramer-monomer dissociation at pH 2.5 from the hydrostatic pressure and urea curves. Albeit with half of the affinity obtained at pH 7.2 (Kaff of 170 nM), tetrameric LexA remained capable of binding recA operator sequence at pH 2.5. Moreover, different from the absence of binding to the negative control polyGC at neutral pH, LexA bound to this sequence with a Kaff value of 1415 nM at pH 2.5. A binding stoichiometry experiment at both pH 7.2 and pH 2.5 showed a [monomeric LexA]/[recA operator] ratio of 2:1. These results are discussed in relation to the activation of the Escherichia coli SOS regulon in response to environmental conditions resulting in acidic intracellular pH. Furthermore, oligomerization of LexA is proposed to be a possible regulation mechanism of this regulon.     

Acid pH-induced conformational changes in bovine liver rhodanese.

The enzyme rhodanese is greatly stabilized in the range pH 4-6, and samples at pH 5 are fully active after several days at 23 degrees C. This is very different from results at pH greater than 7, where there is significant loss of activity within 1 h. A pH-dependent conformational change occurs below pH 4 in a transition centered around pH 3.25 that leads slowly to inactive rhodanese at pH 3 (t 1/2 = 22 min at pH3). The inactive rhodanese can be reactivated by incubation under conditions required for detergent-assisted refolding of denatured rhodanese. The inactive enzyme at pH 3 has the maximum of its intrinsic fluorescence spectrum shifted to 345 nm from 335 nm, which is characteristic of native rhodanese at pH greater than 4. At pH 3, rhodanese shows increased exposure of organized hydrophobic surfaces as measured by 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid binding. The secondary structure is maintained over the entire pH range studied (pH 2-7). Fluorescence anisotropy measurements of the intrinsic fluorescence provide evidence suggesting that the pH transition produces a state that does not display greatly increased average flexibility at tryptophan residues. Pepsin digestibility of rhodanese follows the pH dependence of conformational changes reported by activity and physical methods. Rhodanese is resistant to proteolysis above pH 4 but becomes increasingly susceptible as the pH is lowered. The form of the enzyme at pH 3 is cleaved at discrete sites to produce a few large fragments. It appears that pepsin initially cleaves close to one end of the protein and then clips at additional sites to produce species of a size expected for the individual domains into which rhodanese is folded. Overall, it appears that in the pH range between pH 3 and 4, titration of groups on rhodanese leads to opening of the structure to produce a conformation resembling, but more rigid than, the molten globule state that is observed as an intermediate during reversible unfolding of rhodanese.     

Viral aggregation: buffer effects in the aggregation of poliovirus and reovirus at low and high pH.

The effects of the buffer employed in maintaining a given pH value were tested on the aggregation of two viruses, poliovirus and reovirus. Poliovirus was found to aggregate at pH values of 6 and below, but not at pH 7 or above, except in borate buffer. Reovirus aggregated at pH 4 and below, but was found to aggregate only in acetate or tris(hydroxymethyl)aminomethane-citrate buffers at pH 5. Other buffers tested for aggregation of reovirus at pH 5 (succinate, citrate, and phosphate-citrate) induced little aggregation. No significant aggregation was found for reovirus at pH 6 and above. For both viruses, the most effective aggregation was induced by buffers having a substantial monovalently charged anionic component, such as acetate at pH 5 and 6 or citrate at pH 3. Cationic buffers at low pH, such as glycine, were generally weaker in aggregating ability than anionic buffers at the same pH. These results, when correlated with the isoelectric point of the viruses (poliovirus at pH 8.2; reovirus at pH 3.9) indicated that both viruses aggregated strongly when their overall charge was positive, but only under certain circumstances when their overall charge was negative. Although reovirus aggregated massively at its isoelectric point, poliovirus remained dispersed at its isoelectric point. The conclusion can be drawn that those pH and buffer conditions which induced aggregation of one virus do not necessarily induce it in another.     

pH dependence of the acetylcholine receptor channel: a species variation

The effects of pH changes on the miniature endplate current (mepc) and on endplate current fluctuations (acetylcholine [ACh] noise) were examined at the neuromuscular junction in vitro in two species of frogs. In Rana pipiens the relationship between the decay time constant of the mepc (tau') and pH had a symmetrical bell shape; the value of tau' being largest at pH 7 and decreasing at more acid or more alkaline pH. In acid pH the mepc amplitude (A) decreased relative to its value at pH 7, and in alkaline pH A increased. In Rana ridibunda a narrower and asymmetric bell-shaped dependence of tau' on pH, having a maximum of pH 5.5, was found. The mepc amplitude was again reduced in acid pH but had a peak at pH 5.5. Also, its value at pH 9 was larger than at pH 7. These results were obtained with a number of different buffers and were not found to be sensitive to the nature of the buffer chosen. By performing ACh-noise analysis we found that in Rana pipiens at acid pH (5.5-5.0), the single channel conductance (gamma) and the single channel open time (tau) were significantly reduced relative to their value at pH 7. However, in Rana ridibunda at acid pH (5.4) gamma was unchanged and tau was markedly increased relative to their values at pH 7. The results can be explained quantitatively by electrostatic interaction between two fixed and titratable ionic groups and a mobile charge in the receptor molecule. The model fits the data for groups having pKs approximately 4.8 and approximately 9.8 for Rana pipiens and approximately 4.6 and approximately 6.3 for Rana ridibunda. The groups can be tentatively identified as amino acid residues; glutamic or aspartic and lysine or tyrosine for Rana pipiens; glutamic or aspartic and histidine for Rana ridibunda. The difference in the fitted values of the other model parameters for these two species can be attributed to differences in the spatial configuration of the charged groups.     

Inhibitory effects of phenolic ecotoxicants on photobacteria at various pH values

Kinetic characteristics of light emission by intact cells of the photobacteria Photobacterium phosphoreum and Vibrio harveyi at pH 5.5, 7.0, and 8.0 were studied as well as specific features of 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. Nonstationarity of emission kinetics was observed at all the pH values studied. Exponential luminescence decay in a 60-sec range was observed at pH 5.5; a 5-min luminescence activation, at pH 7.0 and 8.0. The cell respiratory activity drops by over one order of magnitude at pH 5.5 compared with the activities at pH 7.0 and 8.0. The inhibitory effects of 2,4-D, 2,4,5-T, and PCP differ by one-two orders of magnitude depending on pH. The maximal cell sensitivity to these compounds appears at pH 5.5; the minimal, at pH 8.0. The effect of 2,6-DMP is independent of pH. As is demonstrated, it is hydrophobicity of the molecule and pK values of the toxicants that determine the inhibitory effect. Characteristic of the substrate-starved photobacterial cells are higher sensitivity to chlorophenolic compounds compared with the cells provided with high energy supply at all the pH values.     

Viral aggregation: buffer effects in the aggregation of poliovirus and reovirus at low and high pH.

The effects of the buffer employed in maintaining a given pH value were tested on the aggregation of two viruses, poliovirus and reovirus. Poliovirus was found to aggregate at pH values of 6 and below, but not at pH 7 or above, except in borate buffer. Reovirus aggregated at pH 4 and below, but was found to aggregate only in acetate or tris(hydroxymethyl)aminomethane-citrate buffers at pH 5. Other buffers tested for aggregation of reovirus at pH 5 (succinate, citrate, and phosphate-citrate) induced little aggregation. No significant aggregation was found for reovirus at pH 6 and above. For both viruses, the most effective aggregation was induced by buffers having a substantial monovalently charged anionic component, such as acetate at pH 5 and 6 or citrate at pH 3. Cationic buffers at low pH, such as glycine, were generally weaker in aggregating ability than anionic buffers at the same pH. These results, when correlated with the isoelectric point of the viruses (poliovirus at pH 8.2; reovirus at pH 3.9) indicated that both viruses aggregated strongly when their overall charge was positive, but only under certain circumstances when their overall charge was negative. Although reovirus aggregated massively at its isoelectric point, poliovirus remained dispersed at its isoelectric point. The conclusion can be drawn that those pH and buffer conditions which induced aggregation of one virus do not necessarily induce it in another.     

The Viscometric Behavior of a Natto Mucin in Solution

The 2% solution of a natto mucin behaved as a thixotropic flow at pH 5.7, although the flow of the same concentration was apparently Newtonian at pH 4.3. In the 6% solution the viscometric behavior at pH 4.3 was almost the same as that at pH 5.7. The results can be explained in terms of the conformational change of the molecule according to the pH change; that is, the mucin molecule is randomly coiled at pH 5.7 and a rod-like molecule at pH 4.3. The conformational change was shown by means of the electron microscope.