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Stationary phase cultures of a clonal line of rat astrocytes (C6) were maintained at pH values ranging from 6.0 to 8.4 using media buffered with various combinations of organic buffers or graded concentrations of bicarbonate ion at a constant CO2 tension. The accumulation of a soluble acidic protein unique to the nervous system (S-100) in media buffered with organic buffers was optimal in the pH range 6.4 to 6.8, significantly more acid than that optimal for cell growth (pH 7.0 to 7.8). Cells maintained in CO2-bicarbonate-buffered media exhibited a higher and less marked pH optimum for S-100 protein accumulation and a lower efficiency of accumulation of the protein. These data suggest that the organic buffer ions themselves, apart from their function as buffers, are influencing the accumulation of S-100. The specific activity (assayed at the enzymatic pH optimum) of a membrane-bound enzyme enriched in glial cells and myelin, 2',3'-cyclic nucleotide 3'-phosphohydrolase, was markedly pH-dependent. The optimal pH range was 6.4 to 6.7 in organic buffer controlled media. In CO2-bicarbonate controlled media the optimal pH range was only slightly higher (pH 6.6 to 7.0), but the specific activities were reduced relative to organic buffer-grown cells. The structural relationship of some of the aminoethanesulfonic acid buffers used in these experiments to certain compounds of neurochemical interest (such as taurine and alpha-flupenthixol) is noted.
1. The extent to which the cytoplasmic membrane of the Gram-positive bacterium Bacillus licheniformis formed inside-out vesicles was studied with the freeze-fracture technique. The membrane orientation appeared to be dependent on the buffer compositon as well as on the lysis procedure used. 2. By manipulating these conditions, membrane preparations were obtained with the percentage of inside-out vesicles varying from 15 to 80%. 3. More vesicles had the opposite orientation when the cells were lysed in potassium phosphate buffer than when they were lysed in sodium phosphate buffer. Tris-HCl buffer favoured the formation of inside-out vesicles more than phosphate buffer. 4. Lysis of protoplasts in hypotonic buffers resulted in more inside-out vesicles than did direct lysis of cells in hypotonic media. 5. In an attempt to explain the observed differences, experiments were performed in which the morphology of thin-sectioned lysing cells in sodium phosphate buffer was compared with that in potassium phosphate buffer. The results from these experiments indicate that the formation of inside-out vesicles is brought about by an effect on the membrane itself rather than on the cell wall, on the cell wall membrane association, or on the cytoplasm.
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 buffering capacity inside thylakoids is determined and the magnitude of flash-induced pH changes inside is calibrated in the pH range from 6.4 to 8.1. The work is based on flash-induced absorption changes of neutral red in a chloroplast suspension in which the outer phase is strongly buffered by bovine serum albumin. It is shown that neutral red is bound inside thylakoids. The binding can be described by a simple isotherm with an apparent Km = 4 microM and satruation at 1 neutral red per 17 chlorophylls. The apparent pK of neutral red is shifted from 6.6 in solution to 7.25 when bound inside. It is demonstrated that neutral red is a clean indicator of pH changes inside, i.e. when properly used it shows no response to other events. Although bound it reports pH changes which occur in the internal osmolar (aqueous) volume of thylakoids. This is obvious from the influence of chemically very different buffers on the magnitude of the absorption changes of neutral red. These act in a manner proportional to their calculated buffering capacity in aqueous solution. The intrinsic buffering capacity of the internal phase is determined with the aid of these buffers, at pH 7.2 it is between 0.8 and 1 mM (at 60 mosM). The absence of large variations in the buffering capacity in the range from pH 6.4 to 8.1 suggests that proteinaceous groups are involved in addition to the lipids which may dominate the buffering capacity at lower pH. The magnitude of the internal pH change is arrpox. 0.6 (at pH 7.3) under stimulation of both photosystems with a short xenon flash of light.
The demineralization of routine histological specimens in buffers of weakly ionized organic acids, unbuffered formic acid, and EDTA was investigated. The rate of demineralization was measured by a chemical method and from radiographs. Lactate-containing buffers and buffers of formic acid with its potassium salt were more rapid in effect than any other agent. Acidic buffers and unbuffered formic acid produced rapid diffuse demineralization with secondary precipitation of calcium salts. Preservation of dental enamel in such buffers resulted from the significantly slower rate of enamel demineralization than that for bone and dentine. In rapid demineralizing agents the secondary salts were quickly redissolved while in slow buffers these salts persisted. Multivalent ions such as citrate and maleate slowed the rate of demineralization, and a citrate-containing buffer was the slowest of all the agents tested. Demineralization in EDTA exhibited a different pattern with the establishment of a well-defined front of demineralization without apparent reprecipitation. EDTA attacked enamel, bone and dentine at the same rate. An attempt was made to relate the observed rates of demineralization to current theories of the demineralization process.
The effect of the concentration of red cell 2,3-diphosphoglycerate (2,3-DPG, 0.5-21 mumoles/g cells) on the buffering properties and on the slope of the relation between the extracellular and intracellular pH (deltapHi/deltapHe) of human blood was studied. The results were evaluated in connection with previous findings concerning the effect of 2,3-DPG on the Donnan ratio rH+ = H+e/H+i. deltapHi/deltapHe decreases with rising red cell 2,3-DPG content as well as with rising extracellular pH. deltapHi/deltapHe and rH+ can be related to each other by the empirical equation deltapHi/deltapHe = 1 + log rH+ = 1 + pHi - pHe. The validity of this equation appears to be restricted to conditions where the Donnan ratio rH+ is altered between 0.3 and 1 either by changes of the red cell concentration of buffering anions such as 2,3-DPG or by changes of the extracellular pH. As determined in suspensions of red cells with intact membranes, the 2,3-DPG-and pH-induced changes of deltapHi/deltapHe lead to proportional changes in the buffering power of the non-bicarbonate buffers of erythrocytes. Due to this effect the buffering power of suspensions of cells containing 5 times the normal concentration of the buffer 2,3-DPG is lower than that of cells with normal 2,3-DPG content (at extracellular pH values above 7). These findings demonstrate that the action of intracellular non-bicarbonate buffers in blood is effectively modulated by the physico-chemical properties of the red cell membrane.
The effect of pH on the activity of buffered sodium hypochlorite solution, and a buffered methanol/sodium hypochlorite mixture, against Bacillus subtilis spores was investigated. The best results, considering both sporicidal activity and stability, were achieved in the pH range 7.6--8.1. The sporicidal activity and stability of five alcohol/hypochlorite mixtures, each containing a different alcohol and buffered to pH 7.6 and of hypochlorite alone buffered to pH 7.6, were compared. The mixtures were marginally more sporicidal than hypochlorite alone when fresh but were much less stable. An unbuffered methanol/hypochlorite mixture, a methanol/hypochlorite mixture buffered to pH 7.6, and hypochlorite alond buffered to pH 7.6 were all found to be effective against six vegetative organisms and spores of B. subtilis and Clostridium sporogenes. By buffering alcohol/hypochlorite mixtures or hypochlorite solution alone in the pH range 7.6--8.1, high sporicidal activity can be achieved with low concentrations of alcohol and hypochlorite. Such formulations show promise for the disinfection of heat-sensitive medical equipment.
Substitution of synthetic buffers (Tris, TES, HEPES, or 3,3-dimethylglutarate) for CO2-bicarbonate buffer in Ringer solution perfusing the isolated in vitro frog spinal cord preparation altered membrane properties and reflex activity. Perfusion with Ringer solution gassed with O2 and containing synthetic bu,fers consistently produced a depolarization of motoneurons and dorsal root fibers, decreased the amplitude (and usually the duration) of ventral and dorsal root potentials, and had variable effects on motoneuron and dorsal root reflex discharges. With Tris-Ringer these discharges decreased in amplitude; with Ringer containing one of the other synthetic buffers, these discharges were augmented. All changes were reversible when the cord was returned to bicarbonate-buffered Ringer aerated with 95% O2/5% CO2. The use of a combined buffer system-one containing a synthetic buffer and bicarbonate-induced smaller or minimal changes in bioelectric activity. At present the data are insufficient to allow firm conclusions concerning the mechanisms underlying these results; but it is evident 1) that changes in PCO2 and bicarbonate concentration and 2) that the pharmacological properties of synthetic buffers are important variables.
1. Pretreatment of frozon cryostat sections with formaldehyde or calcium ions inhibits diffusion of the plasma membrane enzymes 5' nucleotidase, ATP-ase and alkaline phosphatase during incubation. 2. Treatment of fixed sections with different kinds of buffer at 37 degrees C induces diffusion of enzyme activity from the plasma membrane to other sites of the section and into the incubation medium. This buffer influence depends on temperature: at 4 degrees C only a slight diffusion occurs. Addition of phospholipase C, digitonin or taurocholate to the buffer opposes the buffer effect. 3. Pretreatment of frozen cryostat sections with a mixture of equal parts of chloroform and acetone give a good fixation of the plasma membrane enzymes 5'-nucleotidase, ATP-ase, alkaline phosphate and leucyl-beta-naphthylamidase. During this treatment the different kinds of lipids present in the membrane are ex-racted equally. After this fixation buffer treatment does not cause a visible diffusion of enzyme activity in the section. Only a slight diffusion (1 till 7 percent) into the buffer solution takes place. 4. The mentioned treatments open up possibilities to get insight into the membrane anchorage of plasma membrane enzymes.
Aspects of a possible involvement of hydrogen ions in the electrophysiological responses to light of Limulus ventral photoreceptors were investigated. A 1 M solution of either a zwitter-ionic pH buffer or a weakly-buffering control substance was pressure injected through a micropipette into a ventral photoreceptor cell. To estimate the amount injected, 35SO4 was included in the solution. Membrane currents induced by light flashes were measured by a voltage-clamp technique. The buffer-filled micropipette passed current and a 3M KCl filled micropipette monitored membrane voltage. The sensitivity (peak light-induced current/stimulus energy) was measured, after dark adaptation, before and after injection. Injections of buffers, pH 6.3-7.2, to intracellular concentrations of at least 40-200 mM produced only a small mean decrease in sensitivity, approximately equal to that caused by injections of control substances. Excitation, therefore, apparently is not mediated by a change in intracellular pH. Buffers with pH values 5.4-8.4 were also injected. The time to peak of the response depended on pH, being shortened by up to 20% at pH values below 7.7 and lengthened at higher pH values. The time to peak of the response appeared to be shortened by an increase in intracellular pH-buffering capacity even when there was no change in intracellular pH.
The influence of Zn2+ on the inactivation of alkaline phosphatase [orthophosphoric monoester phosphohydrolase (alkaline optimum), EC 3.1 3.1] in serum during preincubation with 2-amino-2-methyl-1-propanol (AMP) buffers was investigated. Addition of Zn2+ to the buffer before preincubation increases the enzyme activity. An optimum Zn2+ concentration different for each lot of AMP buffer can be found, at which the enzyme activities are restored to a level equal to activities measured without preincubation. There is a relation between the inactivating properties of the different AMP buffers and the amount of Zn2+ needed to prevent this inactivation. Since Zn2+ chelating substituted diamines are held responsible for the inactivation by removing Zn2+ from the enzyme, we assume that the addition of Zn2+ to the buffer prevents this removal. As Zn2+ itself is an inhibitor of the enzyme, the addition of both too much or too little Zn2+ results in lower enzyme activities after preincubation with AMP buffer.
In the quantification of buffering the distinct roles of bicarbonate and of other buffers must be taken into account. The determination of the total non-bicarbonate buffer value, betaa, in intact tissues is complicated by active pH regulation and by heterogeneity of cytoplasm with respect to betaa, while heterogeneity with respect to pH in vivo could cause errors in estimates made with homogenates. Available estimates of betaa are discussed, as are the individual contributions of proteins, dipeptides and phosphates. A high betaa is appropriate in cells which sometimes have high rates of glycolysis, or which buffer extracellular fluid, but non-protein buffer concentrations can be well below the limits imposed by osmolarity, perhaps because buffering can upset ionic gradients.
The photosynthetic oxygen evolution capacity of Agmenelium quadruplication suspended in four hydrogen ion buffers (pH 7.4, 0.05 M) and its synthetic marine growth medium was measured with an oxygen electrode. High rates of oxygen evolution were obtained in the growth medium and N-tris(hydroxymethyl)-methylglycine (Tricine) buffer. Compared to oxygen evolution in the growth medium, rates in phosphate buffer and N-tris(hydroxymethyl)-2-aminoethanesulphonic acid (TES) buffer were sometimes reduced by up to 30% and rates in tris (hydroxymethyl) amino-methane (Tris) were consistently reduced by 50%. An incubation-rinsing procedure caused inhibition of oxygen evolution in TES, phosphate, and Tris by 50 to 100%. Oxygen evolution could be restored to cells rinsed in TES or phosphate by resuspension in growth medium or in buffer plus magnesium and calcium ions. Bezoquinone-supported oxygen evolution was not affected by rinsing with any buffer tested except Tris. Ferricyanide was photoreduced at a low rate by cells rinsed in Tes but at a high rate in TES plus magnesium and calcium ions. We interpreted our results to mean that, in Agmenellum quadruplicatum, inhibition of photosynthetic oxygen evolution by Tris occurs at the level of photosystem 2 while the effects of TES and phosphate are on electron-transport occurring after the rate-limiting reaction.
Evidence is presented to show that an optical isomer of 5-phenylhydantoin is subject to racemization (interconversion) in different buffer systems. With phosphate buffers in the pH range of 6.0-7.5, it appears that the buffer-catalyzed racemization reaction is due solely to catalysis by divalent phosphate (general base catalysis). Other buffers studied include arsenate, imidazole, triethanolamine, and pyrophosphate. When 5-phenylhydantoin, the N-de-ethylated metabolite of ethotoin, was administered to dogs in an earlier investigation, the observation was made that somewhat more than the theoretical quantity (50 mole percent of the dose) of the substances recovered from urine had the R-configuration. The principal metabolite was (R)-(-)-2-phenylhydantoic acid, formed stereo-specifically in a ring-opening reaction of (R)-5-phyenylhydantoin by dihydropyrimidinase (EC 3.5.2.2). The results of the present in vitro study support the hypothesis that in vivo the interconversion of the optical isomers of 5-phenylhydantoin can be catalyzed by buffering components of the mammalian physiological system, and that the catalytic activities of the endogenous buffer components can account for the racemization and ultimate metabolism of the (S)-isomer of 5-phenylhydantoin by dihydropyrimidinase.