Alteration of the anterior acrosome of motile bovine spermatozoa by fructose and hydrogen ion concentration

Storing cauda epididymal spermatozoa in seminal plasma or in defined media at 1 x 10(9) spermatozoa/ml for 24 h at 4 degrees C caused swelling of the apical ridge on motile spermatozoa (SAR) provided concentrations of fructose in the range normally found in seminal plasma or comparable levels of glucose were present. Evaluation of these conditions indicated that, with glycolysable sugars in the media, pH dropped from 6.6-6.7 to 5.7-6.0. Most of the pH decrease occurred during the first 2 h of slow cooling from 37 to 4 degrees C. pH decrease was undoubtedly due to sperm organic acid production which overwhelmed the relatively weak buffering capacity of the defined media and/or seminal plasma. Inducing pH decreases with HCl in fructose-free conditions, and using NaOH to prevent a pH decrease when fructose was included in media, demonstrated that exposing spermatozoa to pH values of 5.7-6.0 and not a specific response to fructose was the major cause of SAR.     

Effect of ionophores and pH on growth of Streptococcus bovis in batch and continuous culture.

Batch cultures (pH 6.7) of Streptococcus bovis JB1 were severely inhibited by 1.25 and 5 microM lasalocid and monensin, respectively, even though large amounts of glucose remained in the medium. However, continuous cultures tolerated as much as 10 and 20 microM, respectively, and used virtually all of the glucose. Although continuous cultures grew with high concentrations of ionophore, the yield of bacterial protein decreased approximately 10-fold. When pH was decreased from 6.7 to 5.7, the potency of both ionophores increased, but lasalocid always caused a larger decrease in yield. The increased activity of lasalocid at pH 5.7 could largely be explained by an increased binding of the ionophore to the cell membrane. Because monensin did not show an increased binding at low pH, some other factor (e.g., ion turnover) must have been influencing its activity. There was a linear increase in lasalocid binding as the concentration increased, but monensin binding increased markedly at high concentrations. Based on the observations that (i) S. bovis cells bound significant amounts of ionophore (the ratio of ionophore to cell material was more important than the absolute concentration), (ii) batch cultures responded differently from continuous cultures, and (iii) pH can have a marked effect on ionophore activity, it appears that the term "minimum inhibitory concentration" may not provide an accurate assessment of microbial growth inhibition in vivo.     

Characteristics of the interaction of calcium with casein submicelles as determined by analytical affinity chromatography

Interaction of calcium with casein submicelles was investigated in CaCl2 and calcium phosphate buffers and with synthetic milk salt solutions using the technique of analytical affinity chromatography. Micelles that had been prepared by size exclusion chromatography with glycerolpropyl controlled-pore glass from fresh raw skim milk that had never been cooled, were dialyzed at room temperature against calcium-free imidazole buffer, pH 6.7. Resulting submicelles were covalently immobilized on succinamidopropyl controlled-pore glass (300-nm pore size). Using 45Ca to monitor the elution retardation, the affinity of free Ca2+ and calcium salt species was determined at temperatures of 20 to 40 degrees C and pH 6.0 to 7.5. Increasing the pH in this range or increasing the temperature strengthened the binding of calcium to submicelles, similar to previous observations with individual caseins. However, the enthalpy change obtained from the temperature dependence was considerably greater than that reported for alpha s1- and beta-caseins. Furthermore, the elution profiles for 45Ca in milk salt solutions were decidedly different from those in CaCl2 or calcium phosphate buffers and the affinities were also greater. For example, at pH 6.7 and 30 degrees C the average dissociation constant for the submicelle-calcium complex is 0.074 mM for CaCl2 and calcium phosphate buffers, vs 0.016 mM for the milk salt solution. The asymmetric frontal boundaries and higher average affinities observed with milk salts may be due to binding of calcium salts with greater affinity in addition to the binding of free Ca2+ in these solutions.     

pH and temperature resolve the kinetics of two pools of calcium bound to the sarcoplasmic reticulum Ca2+ -ATPase

Calcium bound to the sarcoplasmic reticulum Ca2+ -ATPase was removed by chelating free calcium ion with EGTA. The kinetic calcium binding reaction to the calcium-unbound ATPase was studied by varying the pH (6.0-8.8) and temperature (0-20 degrees C) at a saturating concentration of 50-100 microM [Ca2+]. At pH 6.0 and 0 degrees C, calcium sites of the enzyme at a rate of t1/2 approximately 10 s. By increasing the pH from 6.0 to 8.8, about half of the total calcium sites were converted from a slow binding state to a rapid binding state (less than 2s). The maximum level was reached at about pH 7.4, and the midpoint of the conversion was observed at about pH 6.7. On the other hand, the slow binding reaction to the other sites was not significantly affected by the pH increase. At pH 7.0 and 20 degrees C, about 90% of the total calcium sites rapidly (less than 2s) bound calcium. The present results suggest that pH and temperature resolve the kinetics of two pools of calcium bound to the Ca2+-ATPase.     

Enzymatic Studies on the Conversion of Citric Acid Fermentation to Polyol Fermentation in Hydrocarbon-assimilating Yeast Candida zeylanoides

Production of polyols such as erythritol, d-mannitol and d-arabitol by citric acid-producing yeasts occurred only when the medium-pH was controlled at acidic pH, as described in the previous papers.

In order to elucidate the conversion mechanism of citric acid fermentation to polyol fermentation, the effect of pH on the activities of enzymes involved in polyol synthesis and tricarboxylic acid cycle was studied. Shifting down of the medium-pH from 5.5 to 3.5 led immediately to the change of intracellular pH, from 6.5~6.7 to 5.5~5.7. Such the change affected remarkably on the activities of intracellular enzymes. Citrate synthase was significantly depressed at pH 5.7, but isocitrate lyase and phosphoenolpyruvate carboxykinase were reversely stimulated at this pH.

In some yeast strains incapable of polyol production, the change of medium-pH reflected directly on intracellular pH, whereby almost all enzymes were inhibited.

From these results, the conversion of citric acid production to polyol production was explained by the change in the enzyme activities caused by lowering of intracellular pH.     

Optimization of dilution rate, pH and oxygen supply on optical purity of 2, 3-butanediol produced by Paenibacillus polymyxa in chemostat culture

The optimum dilution rate for 2, 3-butanediol (BDL) production by Paenibacillus polymyxa was 0.2 h1 and the optical purity of BDL remained above 98 % at all dilution rates. With decreasing culture pH, ethanol and BDL production increased, whereas the optical purity of BDL decreased to 94 % at pH 5.7. In the chemostat culture at pH 6.3 and a 0.1 h1 dilution rate, the optimum air supply for BDL production was 200 ml min–1 in which the O2 uptake rate was 6.7 mmol l–1 h–1. Under this condition, the optical purity of BDL decreased to 93 %. © Rapid Science Ltd. 1998     

Mechanisms of sorbate inhibition of Bacillus cereus T and Clostridium botulinum 62A spore germination.

The mechanism by which potassium sorbate inhibits Bacillus cereus T and Clostridium botulinum 62A spore germination was investigated. Spores of B. cereus T were germinated at 35 degrees C in 0.08 M sodium-potassium phosphate buffers (pH 5.7 and 6.7) containing various germinants (L-alanine, L-alpha-NH2-n-butyric acid, and inosine) and potassium sorbate. Spores of C. botulinum 62A were germinated in the same buffers but with 10 mM L-lactic acid, 20 mM sodium bicarbonate, L-alanine or L-cysteine, and potassium sorbate. Spore germination was monitored by optical density measurements at 600 nm and phase-contrast microscopy. Inhibition of B. cereus T spore germination was observed when 3,900 micrograms of potassium sorbate per ml was added at various time intervals during the first 2 min of spore exposure to the pH 5.7 germination medium. C. botulinum 62A spore germination was inhibited when 5,200 micrograms of potassium sorbate per ml was added during the first 30 min of spore exposure to the pH 5.7 medium. Potassium sorbate inhibition of germination was reversible for both B. cereus T and C. botulinum 62A spores. Potassium sorbate inhibition of B. cereus T spore germination induced by L-alanine and L-alpha-NH2-n-butyric acid was shown to be competitive in nature. Potassium sorbate was also a competitive inhibitor of L-alanine- and L-cysteine-induced germination of C. botulinum 62A spores.     

Equilibria in the fibrinogen-fibrin conversion. IX. Effects of calcium ions on the reversible polymerization of fibrin monomer

An investigation was made of the role of calcium ions in the reversible stage of fibrin polymerization, using a direct and relatively simple approach. Purified fibrin monomer in solution (7.5 mg/ml) in 1.0 m NaBr (pH 5.3) was polymerized by raising the pH to 5.7–7.7 by the addition of aliquots of standard NaOH solution and the rate and total extent of proton release during polymerization were measured potentiometrically. In the presence of added CaCl₂ (10⁻⁵-10⁻²m) the rate of proton release was increased and the clotting time was decreased. The profile of equilibrium proton release vs pH of polymerization was also shifted, the maximum being increased and occurring at a lower pH. Sedimentation velocity studies in the intermediate pH range (5.7–6.0) showed that the altered profile of equilibrium proton release was due to a broadening of the pH range of polymerization, and that polymerization remained reversible in the presence of CaCl₂. At pH 5.3, where fibrin is essentially monomeric, addition of CaCl₂ resulted in the release of protons and small increases in sedimentation coefficient and reduced viscosity. Under the same conditions, a similar release of protons was observed from fibrinogen, but there was no effect on its sedimentation coefficient. It was concluded that the proton release at pH 5.3 was due mainly to binding of calcium ions to fibrinogen and fibrin monomer. The effect of CaCl₂ on the sedimentation coefficient of fibrin at pH 5.3 was found to decrease with decreasing protein concentration, indicating that it was the result of a small extent of polymerization, rather than a conformational change. Added MgCl₂ had no effect on fibrin monomer at pH 5.3 and no significant effect on the rate or extent of proton release during polymerization at higher pH, indicating that there are specific binding sites for calcium ions in fibrinogen and fibrin. The observed effects of bound calcium ions on reversible fibrin polymerization are explained most simply in electrostatic terms.     

Purification and characterization of acid beta-D-galactosidase from rabbit spleen

beta-D-Galactosidase has been purified to apparent homogeneity from rabbit spleen. The purification steps involved ammonium sulphate precipitation, DEAE-cellulose, concanavalin A-Sepharose, Sephadex G-200, and Sepharose 4B-(epsilon-aminocaproyl)-2-deoxy-beta-D-glucosylamine affinity chromatographies. In the DEAE-cellulose step, the beta-D-galactosidase was separated into two molecular forms, designated I and II, with similar pH optimum, Km, substrate specificity, and sensitivity to substrate analogues and other substances. Form I was purified 1,800-fold with a yield of about 2% of the total activity. This form is heat-labile, it has an acid optimal pH (4.0), an isoelectric point of 6.7 and a molecular weight of 75,000 daltons. Form II has an optimal pH of 3.6 and three different pI values (5.3, 5.7, and 6.7) whose relative proportions can be modified by treatment with neuraminidase. Form II appeared to be a multimeric form (IIA) of about 600,000 daltons at pH 4.0, which was reversibly dissociated to an oligomeric form (IIB) with an apparent molecular weight of 120,000 at neutral pH values. Both IIA and IIB were purified separately and showed an acid pH optimum and an heterogeneous pI (from 4.6 to 7.2). The dissociation of IIA into IIB can be generated spontaneously, but is increased by the presence of urea in the elution buffer, suggesting that both are aggregates of a common subunit.     

Evaluation of clastogenicity of formic acid, acetic acid and lactic acid on cultured mammalian cells

Using Chinese hamster ovary K1 cells, chromosomal aberration tests were carried out with formic acid, acetic acid and lactic acid, and the relationship between the pH of the medium and the clastogenic activity was examined. The medium used was Ham's F12 supplemented with 17 mM NaHCO3 and 10% fetal calf serum. All of these acids induced chromosomal aberrations at the initial pH of ca. 6.0 or below (about 10-14 mM of each acid) both with and without S9 mix. Exposure of cells to about pH 5.7 or below (about 12-16 mM of each acid) was found to be toxic. When the culture medium was first acidified with each of these acids and then neutralized to pH 6.4 or pH 7.2 with NaOH, no clastogenic activity was observed. Using F12 medium supplemented with 34 mM NaHCO3 as a buffer, no clastogenic activity was observed at doses up to 25 mM of these acids (initial pH 5.8-6.0). However, it was found that about 10% of the cells had aberrations at pH 5.7 or below (27.5-32.5 mM of each acid). Furthermore, when 30 mM HEPES was used as a buffer, chromosomal aberrations were not induced at doses up to 20 mM formic acid and acetic acid (initial pH 7.0-7.1), and at doses up to 30 mM lactic acid (initial pH 6.6). In the initial pH range of 6.4-6.7 (25-32.5 mM of each acid), chromosomal aberrations were observed. The above results show that these acids themselves are non-clastogenic, and the pseudo-positive reactions attributable to non-physiological pH could be eliminated by either neutralization of the treatment medium or enhancement of the buffering ability.     

Activation studies of the multiple forms of prochymosin (prorennin).

Activation of the four separate components of prochymosin (prorennin) at pH 5.0 demonstrated that each zymogen was the precursor to an electrophoretically distinct chymosin (rennin). When the increase in milk-clotting activity with time was analysed, the mechanism of activation of unfractionated prochymosin, individual prochymosin components, and a mixture of the prochymosin fractions at pH 5.0 was shown to follow essentially autocatalytic kinetics. The activation of prochymosin C was completed in 70 h, whereas the other three fractions each required more than 110 h for complete activation under the same conditions. Intact prochymosin, the mixture of four components and prochymosin C were activated at similar rates. Interaction of the individual fractions during activation is suggested to explain the increased rate of the activation for the mixture. Comparison of autocatalytic activation of unfractionated prochymosin purified chromatographically at pH 6.7 and 5.7 demonstrated an increased rate of reaction of the zymogen prepared at the lower pH value. The possibility that prochymosin became susceptible to activation during preparation at pH values slightly below 6.0, as a result of changes in the proportion of the components or a conformational change and exposure of the active site, is discussed.     

Catechol-o-methyltransferase in rat erythrocyte and three other tissues: comparison of biochemical properties after removal of inhibitory calcium

The biochemical characteristics of soluble catechol-O-methyltransferase (COMT) activity in rat erythrocytes were compared with the properties of the soluble enzyme in rat liver, heart, and brain. COMT was measured by a procedure that avoided artifacts of some other assay procedures including inhibition of the enzyme by endogenous calcium. After the removal of calcium from the reaction mixture the apparent Michaelis-Menten constants for the two cosubstrates of the COMT reaction, S-adenosyl-1-methionine (SAM) and 3,4-dihydroxybenzoic acid (DBA), were similar in tissue preparations of rat liver, brain, heart and blood. The apparent Km values for the four tissues ranged from 5.7 to 6.7 x 10(-6) M and from 0.9-1.4 x 10(-4) M for SAM and DBA, respectively. The optimal pH and the optimal concentration of magnesium for the assay of red blood cell COMT were also similar to those for the enzyme in the three other rat tissues. After the removal of endogenous calcium, COMT activity in all four tissues was inhibited by the addition of calcium, and the [CaCl2] necessary to inhibit the enzyme activity 50% was 3-5 x 10(-4) M in all cases. The relative activities of COMT in the rat heart, brain, erythrocyte, and liver when expressed per g tissue or per ml of packed red blood cells were 1 to 1.15 to 1.58 to 140, respectively.     

Effects of Membrane ATPase Inhibitors on Light-Induced Phase Shifting of the Circadian Clock in Neurospora crassa

Effects of several membrane ATPase inhibitors on light-induced phase shifting of the circadian conidiation rhythm in Neurospora crassa were examined using mycelial discs in liquid culture. Suppression of phase shifting by the inhibitors was strongly dependent on the pH of the liquid medium in which the discs were cultured during the time from light-dark transition (beginning of free-run) to light irradiation. When discs were cultured in pH 6.7 medium, azide, the inhibitors of plasma membrane ATPase (diethylstilbestrol and N, N′-dicyclohexylcarbodiimide), and ethanol completely suppressed the effect of light on the clock. In contrast, mycelial discs cultured in pH 5.7 medium were fully phase-shifted by light in the presence of the same and even higher concentrations of the chemicals. However, sensitivity to light of the discs cultured in relatively acidic medium was eight times higher than that of the discs cultured at neutral pH. Oligomycin and venturicidin, inhibitors of mitochondrial ATPase, did not suppress phase shifting by light at either pH.     

Proteolytic modification of staphylokinse.

There are three types of staphylokinase of different isoelectric points (6.7, 6.1 and 5.7). Staphylokinse of pI 6.7 was converted to that of pI 6.1 and then to that of pI 5.7 by the treatment with trypsin. Heterogeneity of staphylokinase might be the result of post-translational modification by proteolytic enzyme.     

Preparation and characterization of milk calcium salts by using casein phosphopeptide

Milk calcium salt solution was prepared by the following procedures using casein phosphopeptides (CPP). To CPP solution, 1 M citric acid, 1 M CaCl2 and 1 M K2HPO4 were added with stirring, while adjusting the pH to 6.7. The prepared solution was left for 12 hr at 25 degrees C and then used for subsequent experiments, or lyophilized. The concentrations of organic phosphate of CPP, calcium, inorganic phosphate, and citrate in the typical milk salt solution were 7, 30, 22, and 10 mM, respectively, which were close to those in bovine milk. The lyophilized sample was easily dissolved in water. No crystal structure of hydroxyapatite was shown in the lyophilized milk calcium salts by X-ray diffraction analysis, although the pattern of KCl crystal was observed. The X-ray diffraction pattern of commercial whey mineral, which was prepared by precipitation at alkaline pH from rennet whey, was similar to that of hydroxyapatite. It was confirmed by high-performance gel chromatographic analysis that the form of calcium phosphate in the milk calcium salts was similar to that of casein micelles.     

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.     

Redox potential of plastoquinone A in spinach chloroplasts.

The redox potential of plastoquinone A in spinach chloroplasts was determined. The midpoint potential of the quinone is about +80 mV at pH 7.0 with an n value of 2. The pH-dependence of the potential is -30 mV per pH between pH 4.0 and 5.7, and -60 mV per pH between pH 5.7 and 8.0. The change of the slope at pH 5.7 is interpreted as the protonation of the oxidized plastoquinone A.     

Calcium influx, arachidonic acid,and control of endothelial cell proliferation

Recent evidences suggest a role for arachidonic acid (AA) in the triggering of store-independent, ornon-capacitative, calcium entry in different cell types. Here, using patch clamp and fluorimetric single-cell calcium measurements, we provide evidence for AA-activated calcium influx in bovine aortic endothelial cells (BAEC). AA-activated calcium entry is independent from intracellular calcium stores depletion at low doses of the fatty acid (< 5 microM) and insensitive to a decrease of pH to 6.7. Single-channel analysis in inside-out configuration reveals the presence of a family of AA-activated calcium-permeable channels, with different conductances and reversal potentials. Treatment with AA or ETYA induces a proliferative effect, significantly affected by external EGTA application during the early period (up to 2h) of stimulation with the agonists. We conclude that low concentrations of arachidonic acid are able to evoke a store-independent calcium influx, exerting a mitogenic role in BAECs.     

Redox potential of plastoquinone A in spinach chloroplasts

The redox potential of plastoquinone A in spinach chloroplasts was determined. The midpoint potential of the quinone is about +80 mV at pH 7.0 with an n value of 2. The pH-dependence of the potential is ?30 mV per pH between pH 4.0 and 5.7, and ?60 mV per pH between pH 5.7 and 8.0. The change of the slope at pH 5.7 is interpreted as the protonation of the oxidized plastoquinone A.     

Comparison of the α-Amylase of Bacillus subtilis and Bacillus amyloliquefaciens

The alpha-amylase (alpha-1,4-glucan 4-glucanohydrolase, EC 3.2.1.1) of Bacillus subtilis strain W23 is less negatively-charged than the alpha-amylase of B. amyloliquefaciens strain F, as determined by electrophoretic mobility in polyacrylamide gel at pH 8.6. The alpha-amylase of strain W23 is immunologically unrelated to the alpha-amylase of strain F, as judged by lack of cross-reaction in Ouchterlony immunodiffusion studies. The pH range of maximal activity for the enzyme of strain W23 was 5.7 to 6.7, with a maximum at 6.3. The pH range of activity for the alpha-amylase of strain F was 5.5 to 6.5, with a maximum at 5.9. No significant difference was found in the effect of temperature on the activity of the alpha-amylase of strain W23 and strain F. alpha-Amylase production by strain W23 occurs throughout the 7-hr growth period, whereas enzyme production by strain F does not begin until the culture enters the stationary phase of growth. The total amounts of enzyme produced by strains W23 and F after 7 hr of growth were 0.3 and 25.5 units/ml, respectively.