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PubMed1977

Influence of fixation and buffer treatment on the release of enzymes from the plasma membrane.

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.

Adenosine Triphosphatases↗

PubMed1976

Effects of increased intracellular pH-buffering capacity on the light response of Limulus ventral photoreceptor.

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.

Animals↗

PubMed1978

Initial activity and inactivation of alkaline phosphatase in different lots of buffer.

Alkaline phosphatase activities were determined in six lots of 2-amino-2-methyl-1-propanol (AMP) and in six lots of diethanolamine (DEA) buffers without preincubation of the sample. There appeared to be differences between the lot numbers in both cases, resulting in a variation in initial activity. When serum samples are preincubated with buffer a loss of activity was observed in 4 out of the 6 AMP buffers. Four human isoenzymes showed varying inactivation during preincubation with AMP buffer. No loss of activity was observed when the preincubation was done with the six DEA buffers. These results indicate that the purity of the commercially-available buffers is quite unsatisfactory.

Alkaline Phosphatase↗

PubMed1979

Influence of zinc ions addition to different lots of 2-amino-2-methyl-1-propanol (AMP) buffer on the alkaline phosphatase activities.

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.

Alkaline Phosphatase↗

PubMed1978

Intracellular buffering.

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.

Acid-Base Equilibrium↗

PubMed1976

Effect of hydrogen ion buffers on photosynthetic oxygen evolution in the blue-green alga, Agmenellum quadruplicatum.

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.

Buffers↗

PubMed1977

[Influence of different fixation buffers on the quantitative cytoarchitecture of the cochlear spiral ganglion. A morphometric study (author's transl)].

The influence of two fixation buffers on the quantitative cytoarchitecture of the cochlear spiral ganglion in guinea pigs was evaluated morphometrically. After fixation with phosphate buffered 1.3% OsO4 granular spiral ganglion cells lost 45% of their average individual volume as compared to the volume after fixation with s-collidine buffered 1.3% OsO4. Using the two fixatives there was no significant difference of the volume proportion of cell nuclei, mitochondria, lysosomes and rough endoplasmic reticulum per unit volume cytoplasm of the granular spiral ganglion cells. The volume proportion of their ribosomes and their Golgi apparatus per unit volume cytoplasm increased 3.5fold after fixation with phosphate buffered OsO4. The volume density of the granular ganglion cells decreased by 30%, the volume density of the interganglionar space (= space between granular ganglion cells) showed an increase of 50% using phosphate buffer. Mostly the extracellular space was participating in this relative increase of the interganglionar space. As a result fixation of the spiral ganglion for morphometric studies should be performed using s-collidine buffered OsO4. The morphometric findings underline the presumption of semicompact myelin being a fixation artefact.

Animals↗

PubMed1975

[Year-round buffering of cattle feed mixture and its effect on metabolism and productivity].

Commercial feed mixture was buffered with a 2% and 3% admixture of bentonite buffer in two beef cattle herds in the course of one year. The mixtures were fed on a continuous basis. The two-per-cent buffer concentration was tested in 110 test animals with 104 control animals and the three-per-cent concentration in 50 test animals with 50 controls. Throughout the trial the over-all health condition remained unchanged, the hematocrit and hemoglobin values were balanced in both groups. The biochemical indices were better in the test groups: hypocalcemia improved (in the controls it grew worse), magnesiemia was slightly increased, the inorganic serum factor did not go beyond physiological limits, and acidosis did not occur (as distinct from the control animals). The levels of transaminases (GOT, GPT), glutamic acid dehydrogenase, total serum protein, alkaline phosphatase as well as ammonia and urea in blood serum were at physiological values with po-differences within groups. In the case of the three-per-cent buffer concentration the daily gains were higher by 0.073 kg, and in the two-per-cent concentration by 0.058 kg, in the test animals. The average annual gain was higher by 25.5 kg, and by 18.3 kg, respectively. With respect to the price of buffer and to the efficiency of the animals tested, the economic indices of feed mixture buffering are highly effective.

Alanine Transaminase↗

PubMedDate not supplied

Buffer catalysis of the racemization reaction of some 5-phenylhydantoins and its relation to the in vivo metabolism of ethotoin.

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.

Hydantoins↗

PubMed1977

Influence of inorganic phosphate and organic buffers on cephalosporin production by Streptomyces clavuligerus.

A high concentration of potassium phosphate (75--100 mM) stabilized pH and supported extensive growth of Streptomyces clavuligerus in a chemically defined medium; such a concentration also inhibited cephalosporin production. Although Tris buffer was found to have detrimental effects on growth and antibiotic production, 3-(N-morpholine)-propane sulfonate (MOPS) or 2-(NP-morpholine)-ethane sulfonate (MES) buffer provided a nontoxic buffering system. In the presence of MOPS buffer, cephalosporin production was optimal at 25 mM phosphate, whereas higher concentrations of phosphate progressively inhibited antibiotic production up to 85% without modifying the pH pattern. MOPS buffer can be used to conduct fermentations at a relatively constant pH value in shake flasks.

Alkanesulfonates↗

PubMed1976

Photophosphorylation as a function of illumination time. II. Effects of permeant buffers.

(1) The amounts of orthophosphate, bicarbonate and tris (hydroxymethyl)-aminomethane found inside the thylakoid are almost exactly the amounts predicted by assuming that the buffers equilibrate across the membrane. Since imidazole and pyridine delay the development of post-illumination ATP formation while increasing the maximum amount of ATP formed, it follows that such relatively permeant buffers must also enter the inner aqueous space of the thylakoid. (2) Photophosphorylation begins abruptly at full steady-state efficiency and full steady-state rate as soon as the illumination time exceeds about 5 ms when permeant ions are absent or as soon as the time exceeds about 50 ms if valinomycin and KC1 are present. In either case, permeant buffers have little or no effect on the time of illumination required to initiate phosphorylation. A concentration of bicarbonate which would delay acidification of the bulk of the inner aqueous phase for at least 350 ms has no effect at all on the time of initiation of phosphorylation. In somewhat swollen chloroplasts, the combined buffering by the tris(hydroxymethyl) aminomethane and orthophosphate inside would delay acidification of the inside by 1500 ms but, even in the presence of valinomycin and KC1, the total delay in the initiation of phosphorylation is then only 65 ms. Similar discrepancies occur with all of the other buffers mentioned. (3) Since these discrepancies between internal acidification and phosphorylation are found in the presence of saturating amounts of valinomycin and KC1, it seems that photophosphorylation can occur when there are no proton concentration gradients and no electrical potential differences across the membranes which separate the medium from the greater part of the internal aqueous phase. (4) We suggest that the protons produced by electron transport may be used directly for phosphorylation without even entering the bulk of the inner aqueous phase of the lamellar system. If so, phosphorylation could proceed long before the internal pH reflected the proton activity gradients within the membrane.

Adenosine Triphosphate↗

PubMed1979

Intrinsic electric fields and proton diffusion in immobilized protein membranes. Effects of electrolytes and buffers.

We present a theory for proton diffusion through an immobilized protein membrane perfused with an electrolyte and a buffer. Using a Nernst-Planck equation for each species and assuming local charge neutrality, we obtain two coupled nonlinear diffusion equations with new diffusion coefficients dependent on the concentration of all species, the diffusion constants or mobilities of the buffers and salts, the pH-derivative of the titration curves of the mobile buffer and the immobilized protein, and the derivative with respect to ionic strength of the protein titration curve. Transient time scales are locally pH-dependent because of protonation-deprotonation reactions with the fixed protein and are ionic strength-dependent because salts provide charge carriers to shield internal electric fields. Intrinsic electric fields arise proportional to the gradient of an "effective" charge concentration. The field may reverse locally if buffer concentrations are large (greater to or equal to 0.1 M) and if the diffusivity of the electrolyte species is sufficiently small. The "ideal" electrolyte case (where each species has the same diffusivity) reduces to a simple form. We apply these theoretical considerations to membranes composed of papain and bovine serum albumin (BSA) and show that intrinsic electric fields greatly enhance the mobility of protons when the ionic strength of the salts is smaller than 0.1 M. These results are consistent with experiments where pH changes are observed to depend strongly on buffer, salt, and proton concentrations in baths adjacent to the membranes.

Buffers↗

PubMed1977

Changes in apparent pH on freezing aqueous buffer solutions and their relevance to biochemical electron-paramagnetic-resonance spectroscopy.

Changes in apparent pH occurring during fast freezing of aqueous buffer solutions and cooling to -196 degrees C were studied by various semiquantitative methods, including simple visual measurements of colour changes with pH indicators, as well as measurements of pH-dependent changes in the e.p.r. (electron paramagnetic resonance) spectra of solutions of three different metalloenzymes. It is concluded that apparent pH changes of up to about 3pH units may occur under particular conditions. Such changes were independent of the time taken to freeze the samples, when this was varied from about 3ms t0 20s, but were affected by the presence of some proteins in solution. Recommendations on the buffers that should be used to avoid such apparent pH changes in e.p.r. spectroscopy and other low-temperature biochemical work are made. Phosphate and pyrophosphate buffers, which gave large decreases (2-3 pH units), and Tris, which under some conditions gave increases of about the same magnitude, are to be avoided. Certain zwitterionic buffers such as Bicine [NN-bis-(2-hydroxyethyl)glycine] are satisfactory. Apparent pH effects were found to depend on buffer and protein concentration. It is therefore recommended that as a prelude to future detailed low-temperature biochemical work, appropriate tests with an indicator system should be performed.

Buffers↗

PubMed1979

Norepinephrine-stimulated fatty-acid release and oxygen consumption in isolated hamster brown-fat cells. Influence of buffers, albumin, insulin and mitochondrial inhibitors.

Brown fat cells isolated from adult golden hamsters have earlier been found to respond to addition of the physiological agonist norepinephrine with an increased rate of oxygen consumption and with fatty acid release. Working with these cells, we found the following. 1. The presence of albumin in the incubation medium (phosphate buffer) increases norepinephrine-induced fatty acid release and tends to stabilize the rate of oxygen consumption; bubbling of phosphate buffer with 5% CO2 in air has only a slight effect on fatty acid release. 2. In the presence of albumin, the norepinephrine-induced rate of oxygen consumption is also stable in bicarbonate buffer; it is higher than in the phosphate + CO2 buffer and the brown fat cells have a higher sensitivity to norepinephrine. 3. 20 mM phosphate (as e.g. present in a phosphate buffer) inhibits both fatty acid release and oxygen consumption. 4. Insulin inhibits the rate of oxygen consumption, but only at suboptimal concentrations of norepinephrine. 5. Atractylate inhibits submaximal norepinephrine-induced respiration, indicating that some oxidative phosphorylation takes place in norepinephrine-stimulated brown fat cells. 6. Fatty acid export from brown fat should be regarded as physiologically important.

Adipose Tissue, Brown↗

PubMed1976

The effect of carbon dioxide on the intracellular pH and buffering power of snail neurones.

1. Intracellular pH (pHi) was measured using pH-sensitive glass micro-electrodes. The effects on pHi of CO2 applied externally and HCO3-, H+ and NH4+ injected iontophoretically, were investigated. 2. The transport numbers for iontophoretic injection into aqueous micro-droples were found by potentiometric titration to be 0-3 for HCO3- and 0-94 for H+. 3. Exposure to Ringer, pH 7-5, equilibrated with 2-2% CO2 caused a rapid, but only transient, fall in pHi. Within 1 or 2 min pHi began to return exponentially to normal, with a time constant of about 5 min. 4. When external CO2 was removed, pHi rapidly increased, and then slowly returned to normal. The pHi changes with CO2 application or removal gave a calculated intracellular buffer value of about 30 m-equiv H+/pH unit per litre. 5. Injection of HCO3- caused a rise in pHi very similar to that seen on removal of external CO2. 6. The pHi responses to CO2 application, CO2 removal and HCO3- injection were slowed by the carbonic anhydrase inhibitor acetazolamide. 7. H+ injection caused a transient fall in pHi. In CO2 Ringer pHi fell less and recovered faster than in CO2-free Ringer. Calculation of the internal buffer value from the pHi responses to H+ and HCO3- injection gave very similar values. 8. The internal buffer value (measured by H+ injection) was greatly increased by exposure to CO2 Ringer. Acetazolamide reduced this effect of CO2, suggesting that the function of intracellular carbonic anhydrase may be to maximize the internal buffering power in CO2. 9. It was concluded that the internal HCO3- was determined primarily by the CO2 level and pHi, that internal HCO3- made a large contribution to the buffering power, and that after internal acidfication pHi was restored to normal by active transport of H+, OH- or HCO3- across the cell membrane. The active transport was much faster in CO2 than in CO2-free Ringer.

Acetazolamide↗

PubMed1977

Cell envelope of Neisseria gonorrhoeae: relationship between autolysis in buffer and the hydrolysis of peptidoglycan.

Neisseria gonorrhoeae readily underwent autolysis when suspended in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer at alkaline pH values. Autolysis was inhibited by the addition of Mg2+ or other divalent cations. Autolysis was also suppressed at acid pH (pH 6.0). Suspension of cells in buffer was accompanied by the hydrolysis of peptidoglycan. The rate of peptidoglycan hydrolysis in HEPES buffer was maximal at pH 8.5 and was similar in the presence or absence of Mg2+. Therefore, divalent cation stabilization against autolysis is not mediated by inhibition of peptidoglycan hydrolysis. Peptidoglycan hydrolysis occurred in HEPES buffer (pH 6.0), but at a rate that was 50% of the maximum. Incubation of cells with chloramphenicol or rifampin before suspension in HEPES buffer (pH 8.5) partially prevented autolysis; under these conditions, peptidoglycan hydrolysis still occurred, but at a reduced rate. Old and new peptidoglycans were hydrolyzed at similar rates. Peptidoglycan hydrolysis results in solubilization of both the peptide and glycan moieties.

Buffers↗

PubMed1976

A search for the best buffer to use in assaying human lactate dehydrogenase with the lactate-to-pyruvate reaction.

Highly purified human lactate dehydrogenases I and V were assayed in 17 different buffers, at a variety of reaction pH's. Diethanolamine and 2-amino-2-methyl-1,3-propanediol provided the best measurements of the enzyme, assayed lactate-to-pyruvate. However, the commercial preparation of 2-amino-2-methyl-1,3-propanediol contained insoluble matter and was relatively expensive. All of the four buffers nowmost commonly used were found to present difficulties. Glycine and pyrophosphate were inhibotory tolactate dehydrogenase activity with increasing buffer concentration. 2-Amino-2-methyl-1-propanol had three major disadvantages: it is chemically unstable during reagent preparation; activity is dependent on buffer concentration; and the pH optima for isoenzymes I and V are vastly different. The pKa of tris(hydroxymethyl)aminomethane is 8.0 at 30 degrees C, whereas to measure total activity the reaction pH should be greater than 8.5; thus tris(hydroxymethyl)aminomethane has limited buffering capacity at the reaction pH.

Buffers↗

PubMed1978

D(-)-N-Methylglucamine buffer for pH 8.5 to 10.5.

A new amine buffer for the pH range 8.5 to 10.5 based on D(-)-N-methylglucamine and its hydrochloride is described. Important physical and chemical constants characterizing this buffer system have been determined: thermodynamic pKa = 9.52 (25 degrees C), buffer capacity = 0.055, dilution value pH 1/2 = -0.02, temperature coefficient dpKa/dT = 0.023, the heat of ionization delta H degrees = 36.93 kj mol-1. D(-)-N-Methylglucamine, which is available in highly purified form, is highly soluble in water. Thus one can prepare either buffer solutions or stable solid mixtures when combined with D(-)-N-methylglucaminium chloride. D(-)-N-Methylglucamine buffers are suitable for both biochemistry and pH control.

Buffers↗