Activation and inhibition of mitochondrial adenosine triphosphatase by various anions and other agents

The activating or inhibiting actions of a variety of anion species and of oligomycin, aurovertin and Dio-9 on the ATPase of a sonic particle preparation of rat liver mitochondria have been characterized by measurements of the relevantV _max,K _i andK _m values.The normalV _max was increased by a factor near 7 by the anions: dichromate, chromate, pyrophosphate, orthophosphate, orthoarsenate and sulphate. The fully activating concentration varied from about 2 mM for dichromate to 150 mM for sulphate. The increase inV _max was accompanied by a time-dependent decrease in (K _i)ADP, but there was no change in (K _m)ATP. The increase inV _max by the activating anions was abolished by aurovertin; but in presence of oligomycin, the lowV _max was increased by the activating anions by the same factor as theV _max in absence of oligomycin.Certain anions, notably azide, decreasedV _max, but did not affect (K _i)ADP or (K _m)ATP. The decrease inV _max by azide and oligomycin were approximately additive. Even at high concentration, Dio-9 was without detectable effect on the ATPase, but it had a gramicidinlike effect on the intact mitochondria.The specificity of the ATPase for ATP relative to GTP was found to be attributable to the high value of (V _max)ATP compared with (V _max)GTP. The values of (K _m)ATP and (K _m)GTP were virtually the same.Some rationalization of these and other supporting observations is attempted in terms of present knowledge of the constitution of the ATPase complex.     

Stereological Studies of the Effects of α-MSH and cAMP on Melanosomes in Melanoma Cells

The effects of α-MSH and cAMP on melanosomes in Cloudman S91 melanoma cells were investigated by modern stereological techniques. Cells were cultured for 4 days in medium containing α-MSH or cAMP harvested at 24 hour intervals; some were frozen for melanin assay and the reminder embedded in Epon for light and electron microscopy. Cellular and melanosomal parameters were estimated by new stereological probes. We found that both stimulators induced increases in nuclear volume, cell volume, and the volume fractions and volumes of premelanosomes (V_Vpm,cell’V_pm) and mature melanosomes (V_Vmm,cell’V_mm) and the number of mature melanosomes (N_mm). Both stimulators also caused declines in the volume of individual mature melanosomes (_Vimm) the melanin content per mature melanosome unit volume and the melanin content per individual mature melanosome. The increases in the volume of individual premelanosomes and the number of premelanosomes were only induced by cAME The effect cAMP on some parameters occurred 24 hours prior to α-MSH and was more marked. The response of premelanosomes to the stimulators was more sensitive than mature melanosomes. These results suggest that both stimulators enchanced melanogenesis by increasing the V_Vpm,cell’V_Vmm,cell’V_pm, V_mm and N_mm. The melanogenic level did not depend on the _Vimm and melanin concentration in melanosomes. The maturation of premelanosomes was involved in melanogenesis induced by both stimulators, but, de novo synthesis and enlargement of premelanosomes were only stimulated by cAME It imply that exogenous cAMP may affect melanosomes, and hence melanogenesis in quantitatively or qualitatively different ways to α-MSH.     

Interacting effects of CO2 partial pressure and temperature on photosynthesis and calcification in a scleractinian coral

We show here that CO₂ partial pressure (pCO₂) and temperature significantly interact on coral physiology. The effects of increased pCO₂ and temperature on photosynthesis, respiration and calcification rates were investigated in the scleractinian coral Stylophora pistillata. Cuttings were exposed to temperatures of 25°C or 28°C and to pCO₂ values of ca. 460 or 760 μatm for 5 weeks. The contents of chlorophyll c₂ and protein remained constant throughout the experiment, while the chlorophyll a content was significantly affected by temperature, and was higher under the ‘high‐temperature–high‐pCO₂’ condition. The cell‐specific density was higher at ‘high pCO₂’ than at ‘normal pCO₂’ (1.7 vs. 1.4). The net photosynthesis normalized per unit protein was affected by both temperature and pCO₂, whereas respiration was not affected by the treatments. Calcification decreased by 50% when temperature and pCO₂ were both elevated. Calcification under normal temperature did not change in response to an increased pCO₂. This is not in agreement with numerous published papers that describe a negative relationship between marine calcification and CO₂. The confounding effect of temperature has the potential to explain a large portion of the variability of the relationship between calcification and pCO₂ reported in the literature, and warrants a re‐evaluation of the projected decrease of marine calcification by the year 2100.     

Interaction of L antibody with low potassium-type sheep red cells: Resolution of two separate functional antibodies

Summary Antibodies of two specificities in alloimmune sheep anti-L sera, anti-L _P and anti-L _l , were separated by a new technique and characterized. Absorption of anti-L serum with trypsinized LK (LL) sheep red cells left anti-L _P antibodies; the absorbed anti-L _l antibodies were then eluted. Anti-L _P was only weakly lytic in the presence of complement; it had no effect on passive K influx, but stimulated active K influx. The stimulation could be reversed by eluting the antibody in glycine buffer at low pH. Stimulatory activity in the eluted cells could be restored by resensitization with anti-L _P . Anti-L _l was more strongly lytic than anti-L _P in the presence of complement; it had no effect on active K influx, but inhibited passive K influx. Pig anti-ruminant IgG conjugated to hemocyanin was used to visualize by electron microscopy the number of L _P and L _l antigen sites onLL sheep red cells sensitized with anti-L _P and anti-L _l . The values obtained were 590 L _P sites/cell and 847 L _l sites/cell.     

松嫩平原苏打盐渍化旱田土壤表观电导率空间变异

在松嫩平原西部吉林省大安市乐胜乡,于2013年4月20日选择盐碱程度不均一的典型盐渍化旱田地块,面积为4.8 hm~2作为研究样地。利用EM38大地电导率仪测定结合田间定点采样,并通过经典统计和地统计相结合的方法研究了盐渍化旱田土壤表观电导率空间变异特征,分析了土壤表观电导率与土壤盐碱指标之间的关系。结果表明,盐渍化旱田土壤水平方向表观电导率(EC_h)经对数转换后具有强空间自相关,其变异特征主要是与地形地貌和水文状况等结构性因素有关。垂直方向表观电导率(EC_v)经对数转换后具有中等空间自相关性,其变异特征受结构性因素和随机因素共同作用。EC_h和EC_v半方差模拟的最优模型分别为球状模型和指数模型。Pearson分析表明土壤表观电导率(EC_h和EC_v)与土壤盐碱指标EC_(1∶5)、pH_(1∶5)、SAR、SC、Na~+、CO_3~(2-)、HCO_3~-呈正相关关系(P0.05),EC_h与土壤盐碱指标相关系数均大于EC_v。在实际应用中可以用EC_h来指示土壤的盐碱程度。回归分析表明土壤表观电导率(EC_h和EC_v)与土壤盐碱指标呈线性相关,且EC_h回归模型的决定系数均大于EC_v回归模型的决定系数,可用水平方向土壤表观电导率(EC_h)来计算土壤盐碱指标,进行土壤盐渍化的快速评估。     

Natural occurrence of Fusarium species and fumonisin-production by toxigenic strains isolated from poultry feeds in Argentina

Fusarium species and fumonisin production by toxigenic strains were investigated. During 1996–1998, 158 samples of poultry feeds were collected from a factory located in the department of Río Cuarto Córdoba province, Argentina. The most common species of Fusarium were F. moniliforme (60.7%) and F. nygamai (35.4%) followed by F. semitectum, F. subglutinans, F. proliferatum, F. dlamini, F. solani, F. oxysporum and F. napiforme. Fungal counts ranged from 1 × 10³ to 8 × 10⁵ CFU/g with mean values from 1.5 × 10³ to 2.3 × 10⁵ CFU/g. The highest counts were for F. dlamini, F. subglutinans, F. moniliforme and F. nygamai. Strains of F. moniliforme, F. nygamai, and F. proliferatum were screened for their potential to produce fumonisin B₁ (FB₁), fumonisin B₂ (FB₂) and fumonisin B₃ (FB₃) in corn grain. The samples were analysed using a modified high performance liquid chromatography method. The strains assayed, 43 strains, produced three fumonisins. There was a high degree of variability in the quantities of FB₁, FB₂, and FB₃ produced. The toxin produced in highest levels by the majority of the strains was FB₁. The range of concentration varied from 5.4 to 3,991, 1.01 to 189 and 0.4 to 765 ppm per gram of corn for FB₁, FB₂ and FB₃ respectively. The toxigenic pattern of strains was normal, although two strains of F. moniliforme produced exceptionally high concentrations of FB₃ and minor concentrations of FB₂ and FB₁. This is the first report from Argentina on Fusarium species in poultry feeds and fumonisin production by these strains.This revised version was published online in October 2005 with corrections to the Cover Date.     

A new model for relationship between irradiance and the rate of photosynthesis in <Emphasis Type="Italic">Oryza sativa</Emphasis>

The calculated maximum net photosynthetic rate (P _N) at saturation irradiance (I _m) of 1 314.13 μmol m⁻² s⁻¹ was 25.49 μmol(CO₂) m⁻² s⁻¹, and intrinsic quantum yield at zero irradiance was 0.103. The results fitted by nonrectangular hyperbolic model, rectangular hyperbolic method, binomial regression method, and the new model were compared. The maximum P _N values calculated by nonrectangular hyperbolic model and rectangular hyperbolic model were higher than the measured values, and the I _m calculated by nonrectangular hyperbolic model and rectangular hyperbolic model were less than measured values. Results fitted by new model showed that the response curve of P _N to I was nonlinear at low I for Oryza sativa, P _N increased nonlinearly with I below saturation value. Above this value, P _N decreased nonlinearly with I.     

Sugar transport in Coprinus cinereus

Two transport systems for glucose were detected: a high affinity system with a K_m of 27 μM, and a low affinity system with a K_m of 3.3 mM. The high affinity system transported glucose, 2-deoxy-d-glucose (K_m = 26 μM), 3-O-methylglucose (K_m = 19 μM), d-glucosamine (K_m = 652 μM), d-fructose (K_m = 2.3 mM) and l-sorbose (K_m = 2.2 mM). All sugars were accumulated against concentration gradients. The high affinity system was strongly or completely inhibited by N-ethylmaleimide, quercetin, 2,4-dinitrophenol and sodium azide. The system had a distinct pH optimum (7.4) and optimum temperature (45°C). The low affinity system transported glucose, 2-deoxy-d-glucose (K_m = 7.5 mM), and 3-O-methylglucose (K_m = 1.5 mM). Accumulation again occurred against a concentration gradient. The low affinity system was inhibited by N-ethylmaleimide, quercetin and 2,4-dinitrophenol, but not by sodium azide. The rate of uptake by the low affinity system was constant over a wide temperature range (30–50°C) and was not much affected by pH; but as the pH of the medium was altered from 4.5 to 8.9 a co-ordinated increase in affinity for 2-deoxy-d-glucose (from 52.1 mM to 0.3 mM) and decrease in maximum velocity (by a factor of five) occurred. Both uptake systems were present in sporelings germinated in media containing sodium acetate as sole carbon source. Only the low affinity system could initially be demonstrated in glucose-grown tissue, although the high affinity system was restored by starvation in glucose-free medium. The half-time for restoration of high affinity activity was 3.5 min and the process was unaffected by cycloheximide. Addition of glucose to an acetate-grown culture inactivated the high affinity system with a half-life of 5–7.5 s. Addition of cycloheximide to an acetate-grown culture caused decay of the high affinity system with a half-life of 80 min. Regulation is thus thought to depend on modulation of protein activity rather than synthesis, and the kinetics of glucose, 2-deoxy-d-glucose and 3-O-methylglucose uptake would be consistent with there being a single carrier showing negative co-operativity.     

Construction of a fusion flagellin complex and evaluation of the protective immunity of it in red snapper (Lutjanus sanguineus)

Aims: The main aims of this study were to construct a bivalent subunit vaccine containing flagellin flaA gene and flagellin flaB gene from Vibrio alginolyticus strain HY9901 and to explore the potential application of the fusion protein FlaA‐(G₄S)₃‐FlaB as a vaccine candidate for red snapper (Lutjanus sanguineus). Methods and Results: Flagellin gene flaA and flaB of V. alginolyticus were linked by gene SOEing (gene splicing by overlap extension) technology. The expression of the fusion gene flaA‐(G₄S)₃‐flaB in Escherichia coli BL21(DE3) was confirmed by SDS‐PAGE. Western blot analysis showed that the fusion protein FlaA‐(G₄S)₃‐FlaB, which was purified by affinity chromatography on Ni‐NTA resin, had positive reaction with mouse anti‐FlaA serum and mouse anti‐FlaB serum, respectively. The immunoprotection of FlaA‐(G₄S)₃‐FlaB as a bivalent subunit vaccine was investigated in red snapper model by enzyme‐linked immunosorbent assay (ELISA) and challenge test. Red snapper vaccinated with FlaA‐(G₄S)₃‐FlaB produced specific antibodies and were highly resistant to infection by virulent V. alginolyticus. Conclusions: The fusion gene flaA‐(G₄S)₃‐flaB from V. alginolyticus strain HY9901 was cloned by gene SOEing and was expressed in E. coli. This fusion protein FlaA‐(G₄S)₃‐FlaB is a good protective antigen of V. alginolyticus and should be considered as an effective vaccine candidate against infection by V. alginolyticus in red snapper. Significance and Impact of the Study: Two flagellin genes, flaA and flaB, which are independent in structure and function, were first linked together by gene SOEing technology. The finding that red snapper did adequately respond to the fusion protein FlaA‐(G₄S)₃‐FlaB injection made it a promising candidate for vaccine treatment. To develop effective vaccine candidates against V. alginolyticus, more attention should be given to these immunogenic flagellins.     

Expression of a Soluble Functional Form of the Integrin α4β1 in Mammalian Cells

The integrin α₄β₁(VLA4) has been expressed as a soluble, active, heterodimeric immunoglobulin fusion protein. cDNAs encoding the extracellular domains of the human α₄ and β₁ subunits were fused to the genomic DNA encoding the human γ1 immunoglobulin Fc domain and functional integrin fusion protein was expressed as a secreted, soluble molecule from a range of mammalian cell lines. Specific mutations were introduced into the Fc region of the molecules to promote α₄β₁ heterodimer formation. The soluble α₄β₁ Fc fusion protein exhibited divalent cation dependent binding to VCAM-1, which was blocked by the appropriate function blocking antibodies. The apparent K_d for VCAM-1 binding were similar for both the soluble and native forms of α₄β₁. In addition, the integrin–Fc fusion was shown to stain cells expressing VCAM-1 on their surface by FACs analysis. This approach for expressing soluble α₄β₁ should be generally applicable to a range of integrins.     

Molecular mapping of the potato virus Y resistance gene Rysto in potato

Ry _sto is a dominant gene which confers resistance to potato virus Y (PVY) in potato. We have used bulked segregant analysis of an F₁ tetraploid potato population to identify three AFLP markers linked to and on either side of Ry _sto . The tomato homologue of one of these AFLP markers was assigned to linkage group XI by analysis of an F₂ mapping population of tomato, suggesting that Ry _sto is also on chromosome XI of the potato genome. This map position was confirmed by the demonstration that Ry _sto was linked to markers which had been previously mapped to chromosome XI of the potato genome. Four additional AFLP markers were identified that were closely linked to Ry _sto in a population of 360 segregating progeny of a potato cross between a resistant (Ry _sto ) and a susceptible parent. Two of these markers were on either side of Ry _sto , separated by only a single recombination event. The other two markers co-segregated with Ry _sto . Received: 29 July 1996 / Accepted: 30 August 1996     

Photosynthetic enhancement and conductance to water vapor of field-grown Solanum tuberosum (L.) in response to CO2 enrichment

The photosynthetic responses of potato [Solanum tuberosum (L.)] to CO₂ enrichment were studied in open-topped field chambers. Plants were raised in 2.4 m² plastic enclosures over three growing seasons from 1996 to 1998. Plots were continuously fertilized with 1, 1.5 and 2 times ambient daytime CO₂. These were the low (L), medium (M) and high (H) CO₂ treatments, respectively. Tuber dry matter yields were increased 9 and 40%, respectively, in the M and H treatments compared to the L CO₂ treatment. Net photosynthesis (P _n ) and conductance to water vapor (g _s) of upper canopy leaves were measured at 1 or 2-week intervals at the growth CO₂ partial pressure and then P _n of plants in the L treatment was determined at 70 Pa CO₂ (L₇₀). Leaflet P _n rates averaged over all measurement dates were 28, 49 and 84% greater, respectively, in the M, H and L₇₀ CO₂ treatments, compared to plants in the L treatment. Changes of P _n in response to the L, M and H CO₂ treatments were proportional to increases of internal CO₂ (C_i) and at low leaf-to-air vapor pressure deficits mid-day g _s was inversely related to growth CO₂. The ratio of P _n at H compared to L₇₀ was 0.81 when averaged over all measurement dates. Leaf soluble protein, Rubisco protein and chlorophyll (a + b) levels were unaffected by CO₂ treatment. Total Rubisco activity was decreased by CO₂ enrichment in 1998, but percent activation was similar in the L, M and H plots. Leaf starch was increased but sucrose, glucose and fructose were unaffected by CO₂ treatment. The above findings indicated that a down regulation of P _n in response to elevated CO₂ was consistently observed in field-grown potato. This was attributed to a decrease of total Rubisco activity that was potentially due to the presence of inhibitory compounds bound to the active site of the enzyme. The amount of photosynthetic acclimation observed here did not preclude a persistent enhancement of P _n under the elevated CO₂ growth conditions.     

A New Preparation Method of Coenzyme A with Microorganisms

The structure of aggregates formed by heating dilute BSA solution was analyzed with the fractal concept using light scattering methods. BSA was dissolved in HEPES buffer of pH 7.0 and acetate buffer of pH 5.1 to 0.1% and 0.001% solutions, respectively, and heated at 95°C, varying the heating time t_a. The fractal dimension D_f of the aggregate in the solution was evaluated from static light scattering experiments. The polydispersity exponent τ and the average hydrodynamic radius <R_h> of the aggregates were calculated from dynamic light scattering experiments using master curves obtained by Klein et al. The values of D_f and τ of heat-induced aggregates of BSA at pH 7.0 were about 2.1 and 1.5, respectively, the values of which agreed with those predicted by the reaction-limited cluster–cluster aggregation (RLCCA) model. On the other hand, D_f of heat-induced aggregates at pH 5.1 was about 1.8, which agreed with that predicted by the diffusion-limited cluster–cluster aggregation (DLCCA) model. The dependence of <R_h> for the sample of pH 7.0 on t_a was similar to that of the polystyrene colloids reported previously.     

Extraction, separation and labeling with 14C-glucose of HeLa cell polyglucose, RNA and DNA and comparison of the molecular weights and buoyant densities of polyglucose from poliovirus-infected and noninfected cultures

The aqueous phase of phenol extracts of HeLa cells contains polyglucose (CHO)_n, RNA, and DNA. These macromolecules were precipitated together and removed from 50% (v/v) ethanol solutions with a stirring rod. The viscous precipitate had the classical white appearance of DNA, but contained an average of 439, 670, and 220 μg (from 3 × 10⁷ cells) of (CHO)_n, RNA, and DNA, respectively. The (CHO)_n was separated from the RNA, either by CsCl density gradient centrifugation or by precipitating the RNA with trichloroacetic acid (TCA). Both methods of separation resulted in preparations of (CHO)_n with similar specific activities (radioactive counts/μg min). However, electron micrographs showed that the (CHO)_n separated by using TCA had a greater variation in particle size when compared with (CHO)_n separated by CsCl centrifugation. With the CsCl methods, the number-average molecular weights, as determined by electron microscope particle-counting, and the buoyant densities of (CHO)_n whose synthesis was stimulated by poliovirus infection and (CHO)_n from noninfected cultures, were found to be similar. When the (CHO)_n was extracted from HeLa cells with TCA, rather than phenol, the yield was 1.68-fold greater and its specific activity was an average of twice that of the (CHO)_n extracted with phenol. The time at which cells were pulse-labeled with ¹⁴C-glucose, after reducing the glucose in the culture medium to 0.01 of normal, was found to be important, in that the specific activity of the (CHO)_n increased 23.4-fold over a 4-hr period and the amount extracted decreased 8.2-fold. The increase in the specific activities of RNA and DNA was not as large as that of the (CHO)_n and the amounts extracted were not significantly changed. The sedimentation coefficients of RNA and (CHO)_n which were separated from each other with TCA were 6.4 and 116 S, respectively, whereas, without separation, two peaks were seen, with values of 25.4 and 31.4 S. Chloride ions reduce the sensitivity of the Burton test for DNA. However, the Burton reagent will detect (CHO)_n even in the presence of DNA if the assay mixture is heated. Chloride ions increase the sensitivity of the Burton reagent to detect melizitose and, at concentrations above l.5M, synthetic- polyglucose by increasing the absorption of the colored (CHO)_n reaction product(s).     

Excitatory effect of ATP on rat area postrema neurons

ATP-induced inward currents and increases in the cytosolic Ca²⁺ concentration ([Ca]_in) were investigated in neurons acutely dissociated from rat area postrema using whole-cell patch-clamp recordings and fura-2 microfluorometry, respectively. The ATP-induced current (I _ATP) and [Ca]_in increases were mimicked by 2-methylthio-ATP and ATP-γS, and were inhibited by P2X receptor (P2XR) antagonists. The current–voltage relationship of the I _ATP exhibited a strong inward rectification, and the amplitude of the I _ATP was concentration-dependent. The I _ATP was markedly reduced in the absence of external Na⁺, and the addition of Ca²⁺ to Na⁺-free saline increased the I _ATP. ATP did not increase [Ca]_in in the absence of external Ca²⁺, and Ca²⁺ channel antagonists partially inhibited the ATP-induced [Ca]_in increase, indicating that ATP increases [Ca]_in by Ca²⁺ influx through both P2XR channels and voltage-dependent Ca²⁺ channels. There was a negative interaction between P2XR- and nicotinic ACh receptor (nAChR)-channels, which depended on the amplitude and direction of current flow through either channel. Current occlusion was observed at V _hs between −70 and −10 mV when the I _ATP and ACh-induced current (I _ACh) were inward, but no occlusion was observed when these currents were outward at a V _h of +40 mV. The I _ATP was not inhibited by co-application of ACh when the I _ACh was markedly decreased either by removal of permeant cations, by setting V _h close to the equilibrium potential of I _ACh, or by the addition of d-tubocurarine or serotonin. These results suggest that the inhibitory interaction is attributable to inward current flow of cations through the activated P2XR- and nAChR-channels.     

Axial‐to‐radial water permeability of leaf major veins: a possible determinant of the impact of vein embolism on leaf hydraulics?

The leaf hydraulic conductance (K_L) was measured in Prunus laurocerasus L. and Juglans regia L. in which previous measurements had revealed different impacts of dehydration on K_L. Leaves of P. laurocerasus lost 8% of their K_L at water potentials (Ψ_L) of ?2.0 MPa. Leaflets of J. regia showed K_L losses of 40% at Ψ_L = ?1.0 MPa. When major veins were blocked using cyanoacrylate to simulate their embolism, the K_L of P. laurocerasus was reduced by 57% but that of J. regia leaflets was reduced by 80%. Such differences were hypothesized to be due to different axial‐to‐radial permeabilites of major veins. Infiltration of leaves with Phoxine B revealed that P. laurocerasus major veins were largely leaky in the radial direction whereas those of J. regia leaflets showed prevailing axial water transport. Differences between species in terms of axial‐to‐radial water permeability were confirmed by measurements of changes of hydraulic resistance along the midrib. The two hydraulic models are discussed in terms of leaf vulnerability to embolism and plant adaptation to dry habitats.     

Different Pathways are Involved in the Enhancement of Photosynthetic Rate by Sodium Bisulfite and Benzyladenine, a Case Study with Strawberry (Fragaria×Ananassa Duch) Plants

In order to understand the pathway involved in the chemical enhancement of photosynthetic rate, sodium bisulfite (NaHSO₃) and benzyladenine (BA), a growth regulator, were applied to strawberry plants. The influence of these compounds on gas exchange and millisecond delayed light emission (ms-DLE) was investigated using 2-month-old plants. Results showed the net photosynthetic rate (A) in leaves was promoted by both NaHSO₃ and BA. Stomatal conductance (g) and transpiration rate (E) were significantly increased only by BA, while intercellular CO₂ concentration (C_i) was significantly decreased by NaHSO₃. The enhancement of A by NaHSO₃ and BA was only a short-term effect, lasting approximately 5 days for NaHSO₃ and 30 h for BA. Plants treated with NaHSO₃, BA or NaHSO₃ + BA, showed no significant fluctuations in carboxylation efficiency (CE), photorespiration (R_P) or dark respiration (R_D). These results suggest that the influences of NaHSO₃ and BA on gas exchange particularly A, could be via different mechanisms: the enhancement of A by the application of low concentrations of NaHSO₃ appears to be associated with increased cyclic electron flow, while BA enhancement of A is at least partially due to increased g and/or E.     

Purification of cytochrome a 1 c 1 from Nitrobacter agilis and characterization of nitrite oxidation system of the bacterium

Cytochrome a ₁ c ₁ was highly purified from Nitrobacter agilis. The cytochrome contained heme a and heme c of equimolar amount, and its reduced form showed absorption peaks at 587, 550, 521, 434 and 416 nm. Molecular weight per heme a of the cytochrome was estimated to be approx. 100,000–130,000 from the amino acid composition. A similar value was obtained by determining the protein content per heme a. The cytochrome molecule was composed of three subunits with molecular weights of 55,000, 29,000 and 19,000, respectively. The 29 kd subunit had heme c.Hemes a and c of cytochrome a ₁ c ₁ were reduced on addition of nitrite, and the reduced cytochrome was hardly autoxidizable. Exogenously added horse heart cytochrome c was reduced by nitrite in the presence of cytochrome a ₁ c ₁; K _m values of cytochrome a ₁ c ₁ for nitrite and N. agilis cytochrome c were 0.5 mM and and 6 M, respectively. V _max was 1.7 mol ferricytochrome c reduced/min·mol of cytochrome a ₁ c ₁ The pH optimum of the reaction was about 8. The nitrite-cytochrome c reduction catalyzed by cytochrome a ₁ c ₁ was 61% and 88% inhibited by 44M azide and cyanide, respectively. In the presence of 4.4 mM nitrate, the reaction was 89% inhibited. The nitrite-cytochrome c reduction catalysed by cytochrome a ₁ c ₁ was 2.5-fold stimulated by 4.5 mM manganous chloride. An activating factor which was present in the crude enzyme preparation stimulated the reaction by 2.8-fold, and presence of both the factor and manganous ion activated the reaction by 7-fold.Cytochrome a ₁ c ₁ showed also cytochrome c-nitrate reductase activity. The pH optimum of the reaction was about 6. The nitrate reductase activity was also stimulated by manganous ions and the activating factor.     

Growth inhibition of hybridoma cells by ammonium ion: correlation with effects on intracellular pH

The effect of NH₄Cl addition on intracellular pH (pH _i ) was determined by flow cytometric measurements of the fluorescence of a pH-sensitive dye. The effects of NH₄Cl on growth were determined for batch growth of cells in flasks in an incubator. The addition of NH₄Cl caused a cytoplasmic acidification. A new lower steady-state value of pH _i was attained within 20–40 min of NH₄Cl addition. A correlation was found between the effects of NH₄Cl on growth and on pH _i : whereas 3 mM NH₄Cl had little effect on growth and on pH _i , 10 mM NH₄Cl caused a substantial growth inhibition and a pH _i decrease of 0.2–0.3 units. The effects of NH₄Cl on growth and on pH _i were found to be independent of the external pH value (pH _e over the range 6.8 to 7.6, except that 10 mM NH₄Cl was more toxic at pH _e 7.6. The addition of NH₄Cl caused an increase in the average cell volume at pH _e 7.6, but had no effect on the average cell volume at pH _e 's 6.8 and 7.2. For comparison, the effects of pH _e alone on growth and on pH _i were determined. There was little difference in cell growth at pH _e 's 6.8, 7.2 and 7.6. At pH _e 6.6, there was a substantial growth inhibition. Some measurements of the effects of pH _e on pH _i were made, although the steady-state value of pH _i as a function of pH _e was not determined due to limitations in the pH _i -measuring technique. These measurements showed that pH _i remained constant from pH _e 7.6 to 6.8, but fell by 0.2 units at pH _e 6.6, in agreement with the growth results.