A specific uncoupler-binding protein in Tetrahymena pyriformis and Paracoccus denitrificans

The uncoupler of mitochondrial oxidative phosphorylation, 2-nitro-4-azido-carbonylcyanide phenylhydrazone (N₃CCP) which is capable of photoaffinity labeling has been used to examine the effect of uncouplers on the energy conserving membranes of Paracoccus denitrificans and Tetrahymena pyriformis. The N₃CCP uncouples respiration in P. denitrificans and T. pyriformis cells with $\text{U}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values of 1.05 μM and 0.24 μM, respectively. Binding studies show the presence of 0.65 ± 0.05 high affinity sites per cytochrome a with a K_d of 0.5 ± 0.1 μM in P. denitrificans membranes and 1.4 ± 0.2 sites per cytochrome a₂ with a K_d of 0.4 ± 0.1 μM in T. pyriformis membranes. Irradiation of [³H]-N₃CCP bound to the membranes leads to a covalent linking of the radioactive uncoupler to a peptide of 10–15 kdaltons as analyzed by SDS-polyacrylamide gel electrophoresis. It is concluded that these two microbial systems contain a specific high affinity uncoupler binding site very similar to that of mammalian mitochondria (Katre, N.V. and Wilson, D.F. (1978) Arch. Biochem. Biophys. 191, 647–656).     

Itaconic acid: An inhibitor of isocitrate lyase in Tetrahymena pyriformis in vitro and in vivo

The succinate analog itaconic acid was observed to be a competitive inhibitor of the glyoxylate cycle specific enzyme isocitrate lyase (EC 4.1.3.1) in cell-free extracts of Tetrahymena pyriformis. Itaconic acid also inhibited net in vivo glycogen synthesis from glyoxylate cycle-dependent precursors such as acetate but not from glyoxylate cycle-independent precursors such as fructose. The effect of itaconic acid on the incorporation of ¹⁴C into glycogen from various ¹⁴C-labeled precursors was also consistent with inhibition of isocitrate lyase by this compound. Another analog of succinate which shares a common metabolic fate with itaconic acid, mesaconic acid, had no effect on isocitrate lyase activity in vitro or on ¹⁴C-labeled precursor incorporation into glycogen in vivo. In addition, itaconic acid did not affect gluconeogenesis from lactate in isolated perfused rat livers, a system lacking the enzyme isocitrate lyase. These results are taken as evidence that itaconic acid is an inhibitor of glyoxylate cycle-dependent glyconeogenesis Tetrahymena pyriformis via specific competitive inhibition of isocitrate lyase activity.     

Purification and characterization of extracellular acid phosphatase of Tetrahymena pyriformis

An extracellular acid phosphatase secreted into the medium during growth of Tetrahymena pryiformis strain W was purified about 900-fold by (NH₄)₂SO₄ precipitation, gel filtration and ion exchange chromatography. The purified acid phosphatase was homogenous as judged by polycrylamide gel electrophoresis and was found to be a glycoprotein. Its carbohydrate content was about 10% of the total protein content. The native enzyme has a molecular weight of 120 000 as determined by gel filtration and 61 000 as determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis. The acid phosphatase thus appears to consist of two subunits of equal size. The amino acid analysis revealed a relatively high content of asparic acid, glutamic acid and leucine. The purified acid phosphatase from Tetrahymena had a rather broad substrate specificity; it hydrolyzed organic phosphates, nucleotide phosphates and hexose phosphates, but had no diesterase activity. The K_m values determined with p-nitrophenyl phosphate, adenosine 5′-phosphate and glucose 6-phosphate were 3.1·10^?4 M, 3.9·10^?4 M and 1.6·10^?3 M, respectively. The optima pH for hydrolysis of three substrates were similar (pH 4.6). Hg²⁺ and Fe³⁺ at 5 mM were inhibitory for the purified acid phosphatase, and fluoride, L-(+)-tartaric acid and molybdate also inhibited its cavity at low concentrations. The enzyme was competitively inhibited by NaF (K_i=5.6·10^?4 M) and by L-(+)-tartaric acid (K_i = 8.5·10^?5 M), while it was inhibited noncompetitively by molybdate K_i = 5.0·10^?6 M). The extracellular acid phosphatase purified from Tetrahymena was indistinguishable from the intracellular enzyme in optimum pH, K_m, thermal stability and inhibition by NaF.     

High performance liquid chromatographic analysis of catecholamines in growing and non-growing Tetrahymena pyriformis

Tetrahymena pyriformis, strains NT-1 and W, harvested in logarithmic (growing) and stationary (non-growing) phases, were found by high-performance liquid chromatography to contain considerable quantities of dopamine. In addition, small amounts of epinephrine and norepinephrine were detected. Logarithmic-phase strain NT-1 cells contained 249±44 pg dopamine/10⁶ cells compared to 477±42 pg/10⁶ cells for logarithmic-phase strain W cells for logarithmic-phase strain W cells. The dopamine content of stationary-phase cells was approximately half the value of the logarithmic-phase cells. There was a significant amount of dopamine in the growth medium from stationary-phase cultures and, to a lesser extent, logarithmic-phase cells.     

Enzymes related to catecholamine biosynthesis in Tetrahymena pyriformis. Presence of GTP cyclohydrolase I.

We first identified GTP cyclohydrolase I activity (EC 3.5.4.16) in the ciliated protozoa, Tetrahymena pyriformis. The V_max value of the enzyme in the cellular extract of T. pyriformis was 255 pmol mg⁻¹ protein h⁻¹. Michaelis–Menten kinetics indicated a positive cooperative binding of GTP to the enzyme. The GTP concentration producing half-maximal velocity was 0.8 mM. By high-performance liquid chromatography (HPLC) with fluorescence detection, a major peak corresponding to -monapterin (2-amino-4-hydroxy-6-[(1′R,2′R)-1′,2′,3′-trihydroxypropyl]pteridine, -threo-neopterin) and minor peaks of -erythro-neopterin and -erythro-biopterin were found to be present in the cellular extract of Tetrahymena. Thus, it is strongly suggested that Tetrahymena converts GTP into unconjugated pteridine derivatives. In this study, dopamine was detected as the major catecholamine, while neither epinephrine nor norepinephrine was identified. Indeed, this protozoa was shown to possess the activity of a dopamine synthesizing enzyme, aromatic -amino acid decarboxylase. On the other hand, activities of tyrosine hydroxylase or tyrosinase which converts tyrosine into dopa, the substrate of aromatic -amino acid decarboxylase, could not be detected in this protozoa. Furthermore, neither dopamine β-hydroxylase activity nor phenylethanolamine N-methyltransferase activity could be identified by the HPLC methods.     

Inhibition of DNA synthesis does not influence the H1 histone synthesis in Tetrahymena pyriformis

In the protozoan Tetrahymena pyriformis the DNA synthesis is stopped immediately and completely after addition of one of the two DNA synthesis inhibitors methotrexate + uridine and hydroxyurea to a cell suspension. However, the present experiments show, that the accumulation of labeled H1 histone in the inhibited cells is almost totally unaffected for more than two-thirds of a cell cycle after addition of either inhibitor.     

Characterization of peroxisomes in Tetrahymena pyriformis

Peroxisomes from Tetrahymena pyriformis contained catalase, d-amino acid oxidase, cyanide-insensitive fatty acyl-CoA oxidizing system, carnitine acetyltransferase, isocitrate lyase, leucine:glyoxylate aminotransferase and phenylalanine:glyoxylate aminotransferase. These activities, except carnitine acetyltransferase, were found at the highest levels in the light mitochondrial fraction, whereas the highest activity of carnitine acetyltransferase was found in the micotchondrial fraction. Sucrose density gradient centrifugation showed that the density of peroxisomes was approx. 1.228 g/ml and that of mitochondria was approx. 1.213 g/ml. When the light mitochondrial fraction was treated with deoxycholate or by freeze-thawing, most of the activities of catalase and isocitrate lyase were solubilized, whereas about half of the original activity of aminotransferase remained in the pellet fraction. Addition of fatty acid and clofibrate increased the activities of the cyanide-insensitive fatty acyl-CoA oxidizing system and isocitrate lyase in the peroxisomes. The activity of catalase was slightly increased by glucose and clofibrate; leucine:glyoxylate aminotransferase activity was significantly increased by clofibrate treatment.     

Poly(A) RNA metabolism during change of physiological state of Tetrahymena pyriformis cells

In the present work the metabolism of poly(A)+ RNA was investigated in cells of Tetrahymena pyriformis derived either from stationary cultures or from starved suspensions that were initiating growth. Under these circumstances the organisms derived from stationary cultures synthesize ribosomal and poly(A)+ RNA and form polysomes. In the presence of actinomycin D (actD) the observed expansion of the polysomal population is arrested. Pre-starved cells, on the other hand, start making polysomes in the virtual absence of ribosomal and poly(A)+ RNA synthesis soon after being transferred to peptone medium. In this case polysome formation is only partially sensitive to actD. These results have been interpreted as indicating that, in the beginning of growth, cells derived from stationary cultures are dependent on RNA synthesis for polysome formation, whereas pre-starved cells use pre-synthesized RNA for the same purpose.     

Measurement of membrane potential in polymorphonuclear leukocytes and its changes during surface stimulation

The membrane potential of guinea pig polymorphonuclear leukocytes has been assessed with two indirect probes, tetraphenylphosphonium (TPP⁺) and 3,3′-dipropylthiadicarbocyanine (diS-C₃-(5)). The change in TPP⁺ concentration in the medium was measured with a TPP⁺-selective electrode. By monitoring differences in accumulation of TPP⁺ in media containing low and high potassium concentrations, a resting potential of −58.3 mV was calculated. This potential is composed of a diffusion potential due to the gradient of potassium, established by the Na⁺, K⁺ pump, and an electrogenic potential. The chemotactic peptide fMet-Leu-Phe elicits a rapid efflux of accumulated TPP⁺ (indicative of depolarization) followed by its reaccumulation (indicative of repolarization). In contrast, stimulation with concanavalin A results in a rapid and sustained depolarization without a subsequent repolarization. The results obtained with TPP⁺ and diS-C₃-(5) were comparable. Such changes in membrane potential were observed in the absence of extracellular sodium, indicating that an inward movement of sodium is not responsible for the depolarization. Increasing potassium levels, which lead to membrane depolarization, had no effect on the oxidative metabolism in nonstimulated or in fMet-Leu-Phe-stimulated cells. Therefore, it seems unlikely that membrane depolarization per se is the immediate stimulus for the respiratory burst.     

Correlation between fluidity and fatty acid composition of phospholipid species in Tetrahymena pyriformis during temperature acclimation

The correlation between the fluidity of phospholipids and their fatty acid composition was studied by spin label technique and gas-liquid chromatography for three major phospholipid species in Tetrahymena pyriformis during temperature acclimation. The fluidity of 2-aminoethylphosphonolipid increased within the first 10 h of the cold-acclimation when the content of γ-linolenic acid in 2-aminoethylphosphonolipid was highest, and it then decreased up to 24 h. On the other hand, the fluidities of phosphatidylethanolamine and phosphatidylcholine showed a gradual decrease up to 24 h after the temperature shift, although γ-linolenic acid contents were highest at 10 h after the temperature shift. Thus the fluidity changes of these two phospholipids were interpreted as resulting from the altered content of other fatty acids in addition to γ-linolenic acid, since the γ-linolenic acid content was smaller than that of 2-aminoethylphosphonolipid. The results suggest that the content of γ-linolenic acid in 2-aminoethylphosphonolipid plays a role in regulating the thermal adaptation process.     

Salt-induced pH changes in spinach chloroplast suspension. Changes in surface potential and surface pH of thylakoid membranes

A pH decrease in chloroplast suspension in media of low salt concentration was observed when a salt was added at pH values higher than 4.4, while at lower pH values a pH increase was observed. The salt-induced pH changes depended on the valence and concentration of cations of added salts at neutral pH values (higher than 4.4) and on those of anions at acidic pH values (lower than 4.4). The order of effectiveness was trivalent > divalent > monovalent. The pH value change by salt addition was affected by the presence of ionic detergents depending on the sign of their charges. These characteristics agreed with those expected from the Gouy-Chapman theory on diffuse electrical double layers. The results were interpreted in terms of the changes in surface potential, surface pH and the ionization of surface groups which result in the release (or binding) of H⁺ to (or from) the outer medium.The analysis of the data of KCl-induced pH change suggests that the change in the surface charge density of thylakoid membranes depends mainly on the ionization of carboxyl groups, which is determined by the surface pH. When the carboxyl groups are fully dissociated, the surface charge density reaches ?1.0 ± 0.1 · 10^?3 elementary charge/square Å.Dependence of the estimated surface potential on the bulk pH was similar to that of electrophoretic mobility of thylakoid membrane vesicles.     

Factors influencing the pattern of dopamine secretion in Tetrahymena pyriformis

Dopamine production and secretion by the unicellular eukaryote Tetrahymena pyriformis were examined through the use of high performance liquid chromatography (HPLC) with electrochemical detection and through labeling studies with radioactive precursors. Growing cultures maintained a steady state intracellular level of 1.6 ± 0.3 pmol dopamine/10⁶ cells while secreting dopamine into the medium at a rate of 0.2–0.3 pmol/10⁶ cells per min. Incorporation of [¹⁴C]tyrosine and l-[³H]dihydroxyphenylalanine (DOPA) into dopamine was most successful in a basal medium (1.3 mM Tris-HCl, 1 mM citric acid, and 1 mM Ca(OH)₂, (pH 6.5)). A rapid conversion of added l-[³H]DOPA into dopamine confirmed the dynamic pattern of dopamine synthesis and secretion first indicated by the quantitative chromatographic analyses. The intracellular concentration of dopamine dropped sharply after cells were resuspended in the basal medium at 10⁶ cells/ml, so that by approx. 1 h after resuspension, dopamine dropped below the level detectable by HPLC (0.15 pmol/10⁶ cells). Under these conditions, dopamine secretion continued at a high rate for some time, finally leading to a maximal extracellular concentration of 8.71 ± 1.73 pmol/ml by 1 h. At this concentration, the rate of secretion appears to match that of degradation. Pulse chase experiments confirmed the rapid 3urnover of intracellular dopamine. Approx. 90% of [³H]dopamine and l-[³H]DOPA disappeared from l-[³H]DOPA-prelabeled cells during a 5 min chase, with approx. 50% of this being recovered as [³H]dopamine in the cells' medium. Dopamine secretion could be increased by nearly 100-fold by adding high levels (15 nmol/ml) of l-DOPA to the medium. In contrast, NSD-1015, a potent inhibitor of dopamine synthesis, completely blocked dopamine production. 0.15 mM dibucaine and 0.02 mM reserpine reduced dopamine secretion by approx. 65% over a 25-min incubation, but 5 mM EGTA had no noticeable effect.     

Effect of surface potential on Rb+ uptake in yeast: The effect of pH

The apparent K_m of Rb⁺ uptake and the zeta potential of yeast cells are appreciably affected by changes in the pH, variation of the concentration of the buffer cation Tris⁺ and addition of Ca²⁺ to the suspending medium. Irrespective of the way in which the zeta potential is affected, a direct relationship between the apparent K_m of the Rb⁺ uptake and the zeta potential is observed. A reduction of 8 mV in the zeta potential is accompanied by a 20-fold increase in the apparent K_m, which illustrates that electrostatic effects in ion uptake cannot be ignored. Measured zeta potentials are, to a good approximation, linearly related to surface potentials evaluated from a kinetic analysis of the Rb⁺ uptake. This shows the practical use of the zeta potential as a measure of the surface potential in studies of electrostatic effects in ion uptake by yeast. It is concluded that Tris⁺ and the aikaline earth cations inhibit the Rb⁺ uptake in yeast exclusively via a reduction in the surface potential. Protons, in addition, exert a competitive inhibition.     

气候变化对黄腹角雉潜在栖息地的影响

气候变化直接影响物种赖以生存的栖息地环境条件,进而影响物种的分布、数量和存活率。基于优化后最大熵(MaxEnt)模型预测气候变化下黄腹角雉(Tragopan caboti)过去、当前、未来时期的潜在栖息地格局。结果表明,降水量、温度、海拔是栖息地的主要影响因子。当前时期适宜栖息地面积较过去时期下降24.69%;未来2041—2060年间,共享社会经济路径(SSP)3-7.0与SSP5-8.5情景下黄腹角雉适宜栖息地面积较当前时期分别下降55.19%、58.10%。浙江、江西和福建是当前以及未来黄腹角雉核心适宜栖息地,适宜栖息地面积呈现下降的趋势,并往高纬度区域移动。     

气候变化情景下基于最大熵模型的青海云杉潜在分布格局模拟

青海云杉(Picea crassifolia)是我国青藏高原东北缘特有树种,在维系我国西北地区生态平衡、水土保持、水源涵养和生物多样性等方面发挥着重要作用。基于其分布范围内的69个地理分布样点,利用最大熵(Maxent)模型对现实气候条件下青海云杉的潜在分布及其分布的主导气候因子进行分析,同时结合3种大气环流模型模拟青海云杉在3种气候变化情景(温室气候排放量不同)下未来2050s和2080s潜在分布区的变化。结果表明:Maxent模型对青海云杉潜在分布区的预测具有极高的准确度,所有模型的平均受试者工作特征曲线下面积(AUC测试值)均高于0.99;Jackknife检验和气候因子响应曲线表明年最低降雨量是限制青海云杉分布的主导因子;当前青海云杉的潜在分布区主要集中于青海东部、甘肃东南部、宁夏大部分地区、西藏东部、四川西部山区以及陕西、新疆和内蒙古部分地区。在未来3种增温情景下,青海云杉在2050s和2080s的潜在分布总面积与当前相比变化不明显,但不同适生等级的潜在分布面积变化较大,其中,中度适生区和低度适生区受气候增温影响显著,中度增温下这些区域在2080s的面积明显增大,而高度适生区(核心分布)则在所有增温情景下均呈缩小趋势。同时,在未来3种增温情景下,青海云杉在2050s和2080s的潜在分布区有向北移动趋势,但其心分布区域(高度适生区)仍然以青海东部、甘肃北部为主,无明显变迁趋势。从气候因素角度考虑,本研究表明未来气候变化情景下,青海云杉依然在西部高山地区,特别是作为我国重要生态屏障的祁连山、贺兰山等山区具有重要的经济价值并将持续其生态服务功能。     

The electrochemical proton potential and the proton/electron ratio of the electron transport with fumarate in Wolinella succinogenes

The electrochemical proton potential across the cytoplasmic membrane ( ) as well as the H⁺ / e ratio, which were brought about by the electron transport of Wolinella succinogenes, was measured with the aim of understanding the mechanism of electron-transport-coupled phosphorylation in this anaerobic bacterium. (1) Inverted vesicles derived from the bacterial membrane were found to take up protons from the external medium on initiation of fumarate reduction by H₂. Proton uptake was dependent on the presence of K⁺ within the vesicles, was enhanced by the presence of valinomycin and DCCD and high internal buffer concentration, and was abolished by protonophores. The maximum H⁺ / e ratio slightly exceeded 1. (2) The vesicles accumulated thiocyanate in the steady state of fumarate reduction by H₂. The concentration ratio (internal / external) was close to 1000 at an external thiocyanate concentration below 10 μM. Under the same conditions the uptake of methylamine was negligible. Thiocyanate uptake was abolished by the presence of a protonophore. (3) Cells of W. succinogenes accumulated tetraphenylphosphonium cation (TPP) in the steady state of fumarate reduction with H₂ or formate. Under the same conditions the uptake of benzoic acid was negligible. From the amount of TPP taken up by the bacteria, the free internal concentration of TPP was evaluated according to the procedure of Zaritsky et al. (Zaritsky, A., Kihara, M. and MacNab, R.M. (1981) J. Membrane Biol. 63, 215–231). The concentration ratio (internal / external) was 700 in the absence and close to 1 in the presence of a protonophore or in the absence of external Na⁺. (4) The experimental results are consistent with the view that the energy transduction from electron transport to phosphorylation is done by means of the across the bacterial membrane.     

气候变化条件下苦参在我国潜在分布区的预测分析

为了解气候变化情景下苦参在中国的潜在分布区变化,探讨生物气候因子与苦参适宜分布格局的关系.该文通过收集苦参的地理分布点并结合19项生态因子,运用最大熵模型(MaxEnt)和地理信息系统(ArcGIS)对末次盛冰期、当前气候、未来气候三种气候情景下苦参在我国适生区的分布格局进行模拟,并分析影响苦参生长的主导生态因子.结果...     

气候变化情景下大花杓兰在中国的适生区预测

大花杓兰(Cypripedium macranthos)隶属兰科杓兰属,是国家二级重点保护野生植物,与大多数杓兰属植物分布在我国西南山区不同,主要分布于我国的华北、东北和台湾等地区。多年来,过渡采挖等导致了大花杓兰种群数量和个体数目急剧下降。鉴于大花杓兰特殊的分布格局和濒危现状,选择过去、当前和未来8个气候情景,利用MaxEnt物种分布模型结合38个环境变量及来源于数据库和最新实地调查的80个分布位点进行建模,分析了影响大花杓兰分布的关键环境变量,预测了其在当前、过去和未来气候情景下的适生区及其分布中心和迁移趋势。结果表明：当前情景下,大花杓兰适生区主要分布在我国东北和华北地区。影响其分布的5个关键环境变量分别是：UV-B最强月份均值(UV-B3,贡献率：54.0%)、森林覆盖率(FOR,贡献率：14.3%)、降水量季节性变化(BIO15,贡献率：7.4%)、温度季节性变动系数(BIO4,贡献率：6.8%)和草/灌木/林地(GRS,贡献率：4.6%)。其中,紫外辐射相关变量是首次被运用在杓兰属植物的适生区分布预测中,并被证实对大花杓兰的分布具有重要影响。过去3个气候情景下大花杓兰总适生...     

Fluorimetric determination of the resting potential changes associated with the chemotactic response in Paramecium

1. (1) Evidence is presented which indicates that the carbocyanine dye (3,3′ dipropyl thiadicarbocyanine) can be used as a spectroscopic probe for monitoring the resting potential across the plasma membrane of the ciliated protozoan Paramecium.

2. (2) The dye at low concentrations ( 1 μM) does not affect either the viability or the motility of the cells, nor does it induce a chemotactic response.

3. (3) The fluorescence of the dye bound to the cells alters as the potential across the membrane is changed by increasing the external cation concentration.

4. (4) The absorbance of the bound dye also changes in response to an alteration of the membrane potential.

5. (5) The membrane potential changes as measured by the fluorescence method have been correlated with the measurements of the potential estimated by microelectrode methods.

6. (6) Both cations which induce a negative chemotactic response in Paramecium (K⁺, Na⁺, Ba²⁺) and several non-toxic cations bring about a rapid depolarization of the plasma membrane. The significance of these rapid changes in relation to the swimming behaviour of the ciliate is discussed.