Different Effects of Tris and Histidine on a Membrane Bound NaK ATPase from Sugar Beet Roots

A membrane fraction of sugar beet roots prepared in the presence of dithiothreitol contains (Na⁺+ K⁺+ Mg²⁺) ATPase activity. This activity was studied in the presence of different concentrations of Tris and histidine. Tris was found to interact with the ATPase in the following way: (1) Tris at 50 mM increases, in the absence of Na and/or K, the activity in an uncompetitive way with respect to MgATP. (2) Concentrations of Tris > 50 mM cause inhibition in the absence of Na and K when the ratio between MgATP and Tris is relatively low. (3) Though Tris at 50 mM stimulates similarly to Na, it can not substitute for Na in the Na + K activation. (4) In the presence of Na and K, Tris acts as a competitive inhibitor. Histidine has little influence on the rate both in the presence and absence of Na and/or K.     

Lipid and Sterol Composition of Plasma Membranes Isolated from Stele and Cortex of Maize Roots

The phospholipid and sterol contents of plasma membranes isolatedfrom stele and cortex of maize roots were compared. The majorsterol present in both tissues was stigmasterol which was foundin significantly higher quantities in the cortex (27·4µg mg^–1 membrane protein) compared to the stele(17·4 µg mg^–1). Other sterols detected includedsitosterol, campesterol and small quantities of cholesterol.The major phospholipids found were phosphatidylethanolamine(PE) and phosphatidylcholine (PC), with PC more abundant inthe stele. The fatty acid composition indicated that the majorfatty acid in both stele and cortex was 16:0 (palmitic), withothers found in lesser amounts. Key words: Zea mays, cortex, phospholipids, plasma membrane, stele, sterols     

Lipid Composition of Plasma Membranes and Endomembranes Prepared from Roots of Barley (Hordeum vulgare L.) : Effects of Salt

Membrane fractions enriched in endoplasmic reticulum (ER), tonoplast and Golgi membranes (TG) and plasma membranes (PM) were prepared from barley (Hordeum vulgare L. cv CM 72) roots and the lipid compositions of the three fractions were analyzed and compared. Plants were grown in an aerated nutrient solution with or without 100 millimolar NaCl. Each membrane fraction had a characteristic lipid composition. The mole per cent of the individual phospholipids, glycolipids, and sterols in each fraction was not altered when roots were grown in 100 millimolar NaCl. The ER had the highest percentages of phosphatidylinositol and phosphatidylcholine of the three fractions (7 and 45 mole per cent, respectively, of the total lipid). The TG contained the highest percentage of glycosylceramide (13 mole per cent). The PM had the highest percentage of phosphatidylserine (3 mole per cent) and nearly equal percentages of phosphatidylethanolamine (15 mole per cent and phosphatidylcholine (18 mole per cent). The most abundant sterols in membranes prepared from barley roots were stigmasterol (10 mole per cent), sitosterol (50 mole per cent), and 24ζ-methylcholesterol (40 mole per cent of the total sterol). Salt-treated plants contained a slightly higher percentage of stigmasterol than controls. The percentage of stigmasterol increased with age and a simple cause and effect relationship between salt treatment and sterol composition was not observed.     

铝胁迫对小麦根系液泡膜ATP酶、焦磷酸酶活性和膜脂组成的效应

研究了铝胁迫下耐铝性不同的两个小麦品种根细胞液泡膜ＡＴＰ 酶、焦磷酸酶活性和膜脂的变化。与对照相比,经２０ 和１００μｍｏｌ／Ｌ的ＡｌＣｌ３ 处理后,耐铝品种Ａｌｔａｓ ６６ 的液泡膜Ｈ＋ＡＴＰ 酶和Ｃａ２＋ＡＴＰ 酶活性迅速下降; 铝敏感品种Ｓｃｏｕｔ ６６ 液泡膜Ｈ＋ＡＴＰ酶和Ｃａ２＋ＡＴＰ 酶活性则在２０ μｍｏｌ／Ｌ 时增加,１００ μｍｏｌ／Ｌ时下降。焦磷酸酶活性在Ａｌｔａｓ６６ 中下降,在Ｓｃｏｕｔ ６６ 中增加。与对照相比,在ＡｌＣｌ３ ２０ μｍｏｌ／Ｌ处理时,液泡膜磷脂含量增加,Ａｌｔａｓ ６６ 中的增加比Ｓｃｏｕｔ ６６ 更为明显;１００ μｍｏｌ／Ｌ 时,Ｓｃｏｕｔ ６６ 液泡膜磷脂含量迅速下降,而Ａｌｔａｓ ６６ 下降不显著。两品种小麦在铝处理后根液泡膜糖脂结合半乳糖含量均高于对照,而Ａｌｔａｓ ６６ 中的含量又高于Ｓｃｏｕｔ６６ 。铝处理后,两品种小麦根液泡膜的棕榈酸和油酸含量增加,亚麻酸含量下降, 不饱和指数也随之下降,其中Ｓｃｏｕｔ６６ 下降更为明显。表明Ａｌｔａｓ ６６ 根细胞液泡膜比Ｓｃｏｕｔ６６ 对铝胁迫有更强的适应能力。     

Lipid Composition of Plasma Membranes Isolated from Lactating Bovine Mammary Gland

The light and heavy plasma membranes (PM) isolated from lactating bovine mammary glands contained 38~43% lipid of which 41~44% was phospholipid and 47~52% neutral lipid. The contents of phospholipid and neutral lipid were somewhat higher in the light PM than in the heavy PM. Cholesterol was contained 55 ~60% of neutral lipid and the ratio of cholesterol to phospholipid was 0.64 to 0.69. Phospholipid was composed of sphingomyelin (Sph) 29~38%, phosphatidylcholine (PC) 27~35%, phosphatidylethanolamine (PE) 16~20%, phosphatidylserine 10%, and phosphatidylinositol 6~7%. The content of Sph was higher in the heavy PM than in the light PM, while the values of PC and PE were opposite. The major fatty acids of lipid components were palmitic acid, stearic acid, and oleic acid and those of Sph were palmitic acid, stearic acid, C23:0 and 24:0. The fatty acid composition of individual lipid classes differed significantly from each other but were similar between the light and heavy PMs. Tetracosapentaenoic acid (C24:5) was the major fatty acid of the diacylglycerol fraction. The results indicated that the lipid composition, especially phospholipid components, of bovine mammary gland PMs was different from those of milk fat globule membranes which is derived from the PM of mammary secretory cells.     

渗透胁迫对小麦根质膜膜脂物理状态的影响

以两相法纯化的小麦(Triticum sativum L.)根质膜微囊为材料,研究了渗透胁迫下质膜物理状态的变化。结果表明,随着介质蔗糖浓度增加,质膜光散射值降低,二苯己三烯(DPH)荧光偏振值升高,MC540荧光强度增强,并且DPH长寿命组分的荧光寿命和平均寿命都缩短,暗示渗透胁迫使质膜微囊收缩变小,降低了质膜流动性和表面电荷密度,并且表明质膜的疏水性减弱。进一步实验发现,质膜内源色氨酸长寿命组分的寿命缩短,质膜H     

Sucrose Leakage from Isolated Parenchyma of Sugar Beet Roots

The kinetics of sugar efflux from slices of sugar beet rootswas investigated using washing solutions of different osmoticpressure and calcium concentration. The leakage of sucrose isstrongly reduced in solutions of high osmotic pressure (>0·8MPa) or high calcium concentration (10 mM). Turgor-dependentnecrosis of parenchyma cells (plasmoptysis) is the main causeof sucrose efflux from the tissue in hypotonic media with lowcalcium activity. This was shown by good correlation betweenthe percentage of leaked sucrose and the percentage of tissuewater, which was in the free space after the washing procedure.The kinetics of sugar leakage from beet root parenchyma is nobasis for the estimation of the sugar contents of the free spaceor the cytoplasm in situ.     

渗透胁迫对小麦根质膜膜脂物理状态的影响

以两相法纯化的小麦（TriticumsativumL．）根质膜微囊为材料,研究了渗透胁迫下质膜物理状态的变化。结果表明,随着介质蔗糖浓度增加,质膜光散射值降低,二苯己三烯（DPH）荧光偏振值升高,MC540荧光强度增强,并且DPH长寿命组分的荧光寿命和平均寿命都缩短,暗示渗透胁迫使质膜微囊收缩变小,降低了质膜流动性和表面电荷密度,并且表明质膜的疏水性减弱。进一步实验发现,质膜内源色氨酸长寿命组分的寿命缩短,质膜H －AT－Pase活力降低,暗示膜蛋白的构象和功能发生了改变。结果表明,质膜膜脂物理状态改变可能是植物感受渗透胁迫的原初响应。     

Comparisons of Plasma Membranes from Shoots and Roots of Winter Rye (Secale cereale L. cv. Puma): Polypeptide Composition, ATPase Activity and Specific Naphthylphthalamic Acid Binding Capacity

Polypeptide compositions, ATPase characteristics, and the N-1-naphthylphthalamicacid binding capacity of plasma membranes prepared from winterrye (Secale cereale L. cv. Puma) shoots and roots were examinedand compared. Some unique polypeptides were revealed in each plasma membraneby one- and two-dimensional slab gel electrophoresis. A differencewas also detected in glycopeptide compositions. The plasma membranesfrom both organs contained Mg²⁺-stimulated ATPase exhibitingslightly different properties in the divalent cation specificityand the kinetic constants. The ATPase activities from both organsshowed a similar optimum pH around 6.5, simple Michaelis-Mentensaturation with increasing ATP-Mg concentrations, and littleK⁺-stimulation at the optimum pH. Both ATPases were inhibitedby orthovanadate, however, the degree of inhibition was a littledifferent in each membrane sample. The specific N-1-naphthylphthalamicacid binding capacity in the shoot plasma membrane was 2.6-foldhigher than that in the root plasma membrane. These results suggest that polypeptide compositions of plasmamembranes vary corresponding with a difference in the physiologicalfunctions of plasma membranes between shoots and roots of winterrye. ¹ Contribution No. 2670 from the Institute of Low TemperatureScience. (Received May 17, 1984; Accepted October 9, 1984)     

An Investigation of Wound Healing in Sugar Beet Roots Using Light and Fluorescence Microscopy

Following impact, wound healing was investigated in roots ofsugar beet using fluorescence microscopy in conjunction witha conventional lignin test. Samples of sugar beet roots wereharvested at different stages of development and impacted inthe laboratory with a falling bolt delivering 1–4 Joules.A response in the form of deposition of brown material, presumedto be melanin, along the outer and inner walls of cells at thewound surface was observed within 3 d of impact. This materialeventually became granular in appearance. Formation of a ligno-suberizedboundary layer from cells present at the time of impact firstoccurred in 16-week-old roots 9 d after impact. Intensity ofcalcofluor fluorescence supported the findings made using lightmicroscopy. As wound healing progressed with time, the intensityof calcofluor fluorescence declined, demonstrating interferenceby wound healing products with calcofluor binding. Aggressiveharvest and subsequent storage dramatically reduced calcofluorfluorescence indicating that this dye may have potential valuein the assessment of tissue damage. Copyright 2001 Annals ofBotany Company Sugar beet, wound healing, impact damage, bruising, fluorescence, calcofluor, lignin     

Bolting Susceptibility of Sugar Beet (Beta vulgaris) in Relation to Contents of Sulfhydryls and Disulfides and to Protein Composition of Membranes

The aim of the investigation was to correlate the sensitivity to low temperature that leads to bolting of sugar beet with a property of seedlings or seeds, analyzable without cold treatment. Populations or inbred lines were investigated, the bolting percentages of which had been determined in field trials. Sulfhydryls (—SH) and disulfide sulphur (-S-) were analyzed by amperometric titration with silver nitrate. Disulfides were broken with sulphite, and proteins were unfolded with urea. Negative correlations were found between the bolting percentage and (-SH + -S-) per leaf fresh or dry weight unit and per leaf protein nitrogen, with urea at the titrations. Positive correlations were obtained between the bolting percentage and the ratio of (-SH + -S-) titratable in the absence of urea to that titratable in the presence of urea, when leaf homogenates or leaf or seed protein precipitates were examined. Centrifugation experiments showed that membrane proteins were responsible for the correlation. By polyacrylamide gel electrophoresis of the membrane proteins it was found that the proportion between some of these proteins was correlated with the bolting percentage. From these results and such from analyses of beet plants during and after cold treatment a hypothesis was constructed: Low bolting susceptibility, i.e., low sensitivity to low temperature, is caused by a high proportion of a hydrophobic membrane protein, rich in -SH or -S-. This protein interferes with the reactivity of the plants to gibberellins resulting from low temperatures or long days.     

Inhibition of ATPase Activity in Pea Plasma Membranes by Fungal Suppressors from Mycosphaerella pinodes and Their Peptide Moieties

The effects of two suppressors of the defense reactions of hostplants, which had been purified from the pea pathogen Mycosphaerellapinodes, as well as the effects of peptide moieties, on theATPase activity in pea plasma membranes were examined in vitro.One of the suppressors, Supprescin B, inhibited the ATPase activityin a non-competitive manner, but the other suppressor, SupprescinA, did not. Supprescin A was observed to reduce the inhibitoryeffect of Supprescin B. A tripeptide, Ser-Ser-Gly, and a hexapeptide,Ser-Ser-Gly-Asp-Glu-Thr, which were the respective peptide moietiesof Supprescin A and B, inhibited the ATPase activity in a competitivemanner. Supprescin B and fragments of the hexapeptide, suchas Asp-Glu-Thr and Gly- Asp-Glu, inhibited not only the ATPaseactivity but also the acid phosphatase activity of plasma membranesin vitro. These results indicate that the acidic amino-acidresidues of the "Asp-Glu" moiety seem to act as inhibitors ofthe phosphatase activity. Thus, the peptide moiety of SupprescinB consists of at least two functional elements. (Received October 23, 1992; Accepted January 18, 1993)     

Mechanism of Action of Pseudomonas syringae Phytotoxin, Syringomycin : Stimulation of Red Beet Plasma Membrane ATPase Activity

Syringomycin, a peptide toxin produced by the phytopathogen Pseudomonas syringae pv syringae preferentially stimulated (2-fold) the vanadate-sensitive ATPase activity associated with the plasma membrane of red beet storage tissue. The toxin had a very slight effect on the tonoplast ATPase and had no detectable effect on the mitochondrial ATPase. Optimal stimulation was achieved with 10 to 50 micrograms of syringomycin per 25 micrograms of membrane protein. Treatment of membranes with 0.1% (weight/volume) deoxycholate eliminated the activation effect, and enzyme solubilized with Zwittergent 3-14 was not affected by syringomycin. ATPase activity was activated to the same extent at KCl concentrations ranging from 0 to 50 millimolar. Valinomycin, nigericin, carbonylcyanide p-trifluoromethoxyphenylhydrazone, and gramicidin did not increase the plasma membrane ATPase activity. However, these ionophores did not hinder the ability of syringomycin to stimulate the activity. We suggest that syringomycin does not increase ATPase activity by altering membrane ion gradients nor directly interacting with the enzyme, but possibly through regulatory effectors or covalent modification of the enzyme.     

Observations on Fertilization in Sugar Beet

The whole process of double fertilization in sugar beet has been observed, the main results are as follows: About 2 hours after pollination, the pollen grains germinate, the sperms in the pollen tube are long-oval. 15 hours after pollination, the pollen tube destroys a synergid and releases two sperms on one side or at the chalazal end of the egg cell. The sperms are spherical each having a cytoplasmic sheath. 17 hours after pollination, one sperm enters the egg cell, and the sperm nucleus fuses with the egg nucleus rapidly. 21 hours after pollination, the zygote is formed. In the meantime, the primary endosperm nucleus has divided into two free endosperm nuclei. 25 hours after pollination, the zygote begins to divide, forming a two-celled proembryo. The dormancy stage of the zygote is about 4 hours. In the meantime the endosperm is at the stage of four free nuclei. 17 hours after pollination, the sperm nucleus comes into contact and fuses with the secondary nucleus. The sperm nucleus fuses with the secondary nucleus, faster than the sperm with the egg. he first division of the primary endosperm nucleus is earlier than that of the zygote, it takes place about 20 hours after pollination, the dormancy stage of the primary endosperm is about 2 hours. The endosperm is free nuclear. The fertilization of sugar beet belongs to premitotic type of syngamy. From the stage of zygote to the two-celled proembryo, it can be seen that addition- al sperms enter the embryo sac, but polyspermy has not been observed yet.     

Controlling and Measuring Local Composition and Properties in Lipid Bilayer Membranes

Local composition, structure, morphology, and phase are interrelated in lipid bilayer membranes. This gives us the opportunity to control one or more of these properties by manipulating others. We investigate theserelationships with combinations of simultaneous two-color widefield fluorescence imaging, three-dimensional rendering of vesicle domains, andmanipulation of the vesicle morphology via optical trapping and micropipetteaspiration. We describe methods to modulate, to measure, and to probe thelocal structure of model membranes through control of membrane curvature inliposomes.