Effects of Aldehyde Fixation on Adenosine Triphosphatase and Peroxidase Activities in Maize Root Tips

The effects of aldehyde fixation of excised roots and subcellularfractions of Zea mays have been studied in relation to the preservationof ATP-ase and peroxidase activities. Total peroxidase was littleaffected by either formaldehyde or glutaraldehyde whereas ATP-aseshowed considerable loss of activity, particularly with glutaraldehyde.The activity remaining after fixation was dependent on boththe concentration of fixative and the pH of fixation. Subcellularfractions differed in their response to fixation with the cellwall fraction generally showing higher retention of activitythan the mitochondrial or microsomal fractions.     

Changes in the Ability of Photophosphorylation and Activities of Surface-Bound Adenosine Triphosphatase and Ribulose Diphosphate Carboxylase of Chloroplasts Isolated from the Barley Leaves Senescing in Darkness

Dark-induced aging of detached primary leaves of 11-day-old barley seedlings brings about a significant decline in the rates of ferricyanide [Fe(CN)₆]^3? reduction and photophosphorylations of isolated chloroplasts. Ferricyanide-supported noncyclic photophosphorylation is somewhat more susceptible to leaf aging than phenazine methosulfate (PMS)-supported cyclic phosphorylation. Non-latent membrane-bound adenosine triphosphatase (ATPase) and ribulosediphosphate carboxylase (RuDPCase) lose about half of their initial activities after 24 h, while during this period the electron transport and photophosphorylation activities are much less affected. Also, the loss of RuDPCase is almost complete, while chloroplasts still exhibit a significant level of [Fe(CN)₆]^3? reduction and photophosphorylations after 7 days of dark incubation. This would suggest that the enzymatic dark reactions are more sensitive to aging stress than the primary photochemical reactions of chloroplasts. Studies on the effect of divalent cations such as Mg²⁺ and Ca²⁺ on non-latent ATPase activity revealed that the dark stressed aging of detached leaves brings about a time dependent alteration in the response of this enzyme to Mg²⁺, but not to Ca²⁺. The former showed inhibitory as well as stimulatory response, whereas the latter always caused the usual stimulation. Addition of kinetin (50 μM) ensured retention of [Fe(CN)₆]^3? reduction, photophosphorylations and RuDPCase activity in chloroplasts during leaf aging, but it failed to preserve the initial loss in the activity of the non-activated membrane-bound ATPase.     

Sodium, Potassium, Chloride, and Betaine Concentrations in Isolated Vacuoles from Salt-Grown Atriplex gmelini Leaves

Vacuoles were isolated via protoplasts from the leaves of a halophyte Atriplex gmelini C.A.Mey., grown in culture solution supplemented with 250 millimolar NaCl. Lysis of the protoplasts was induced by lowering the medium osmolarity (1.2 to 1.0 molar sorbitol) and adding a detergent, a synthesized cholate derivative, 3-([3-cholamidopropyl] dimethylammonio)-1-propanesulfonate at a concentration of 0.5 millimolar and the resulting vacuoles were purified by successive dilution and floatation. Isolated vacuoles contained almost the same concentration of sodium (569 millimolar) and chloride (260 millimolar) as recorded in protoplasts (582 and 254 millimolar, respectively), suggesting that the vacuoles are the major sequestration site of NaCl in leaves of halophytes. Betaine concentration in the protoplasts was about 16 millimolar, while that in vacuoles was only about 0.24 millimolar, indicating that betaine is accumulated in the cytoplasm as a compatible solute.     

Comparison of Reductions in Adenosine Triphosphate Content, Plasma Membrane-associated Adenosine Triphosphatase Activity, and Potassium Absorption in Oat Roots by Diethylstilbestrol

The possibility was investigated that diethylstilbestrol (DES) inhibits potassium absorption in oat (Avena sativa L. cv. Goodfield) roots by inhibiting mitochondrial functions in addition to inhibiting the plasma membrane ATPase. DES at 10⁻⁶ molar stimulated the mitochondrial ATPase slightly, but higher concentrations had no effect. Oxidative phosphorylation by isolated mitochondria was inhibited 50% by 2.6 × 10⁻⁵ molar DES; concentrations of 10⁻⁴ molar or greater were completely inhibitory. After a lag of about 2 minutes, 10⁻⁴ molar DES produced a linear decrease in ATP content of excised roots. After 20 minutes, the ATP content of the tissue was about 50% of the control and remained at that level after 30 minutes in DES.     

Use of Antibody to Membrane Adenosine Triphosphatase in the Study of Bacterial Relationships

An antiserum to Ca(2+)-activated adenosine triphosphatase from membranes of Micrococcus lysodeikticus cross-reacted in agar gels with membrane adenosine triphosphatases from other pigmented micrococci and related species. Species of Micrococcus and Sarcina showed different levels of inhibition of adenosine triphosphatase activities in heterologous reactions with antiserum. Inter- and intraspecific relationships based on the inhibition reaction were compared with an independent parameter, namely the quantitative and qualitative composition of the bacterial membrane phospholipids and fatty acids. The guanine plus cytosine contents in the deoxyribonucleic acid of the species studied correlated well with the serological cross-reactivity of adenosine triphosphatases from their membranes. The types of cross-bridges found in the peptidoglycans of these cocci were also compared with the other properties. The results suggest that an antiserum specific for a major membrane protein may be a reliable and most useful adjunct in studying bacterial serotaxonomy.     

Lipids from Sugar Beet in Relation to the Preparation and Properties of (Sodium + Potassium)-Activated Adenosine Triphosphatases

Field grown leaves of sugar beet contained 0.89% of their fresh weight as chloroform : methanol 1 :2 extractable material, whereas climate chamber grown material contained 0.34, 0.15, and 0.16% in leaves, stalks, and roots respectively. A striking feature was the high proportion of sulfolipid: 7% of the total extractable of the field grown leaves, 19.5, 28.0, and 37.0% of the total extractable of respectively leaves, stalks, and roots from the climate chamber grown material. Among the fatty acids, all chain lengths from C₁₂ to C₂₈ were found, except only C₁₇ and C₁₉—Exceptionally high contents of fatty acids with a chain length of C₂₆ or C₂₈ were noted in some cases. The 2500–20,000 g fraction of root homogenates contained 19% of the total root lipids. Almost all of the phosphatidyl choline and about half of the phosphatidyl ethanolamine, but only 5% of the sulfolipid followed the fraction. A fractionation of conjugate lipid types was evident, with a loss of 18/2 and 18/3 conjugates, and with an increase in the proportions of 16/0 and, possibly, of the long-chain (around C₂₆) conjugates. The unspecific ATPase activity of the 2500–20,000 g fraction was rendered specific for (Na⁺+ K⁺) stimulation by treatment with 0.1% deoxycholate for 1 hour. This induced a more than 2-fold swelling of the preparation. About half of its total lipids were lost. Again, this loss was a fractional one, so that the phosphatidyl choline lost its long-chain (about C₂₆) fatty acid conjugate while the short to medium length chain conjugates remained; whereas the reverse was the case with the sulfolipid. The ATPase activity of the 2500–20,000 g fraction was destroyed by a 24 hour treatment with deoxycholate. As compared with the 1 hour treatment, the preparation lost about 20% both of its volume and of its chloroform : methanol extractable material. The quantitatively dominating loss was found in the (pigment + neutral fat) fraction. The monogalactosyl diglyceride, the phosphatidyl inositol, and a strongly acidic unknown fraction survived the deoxycholate treatments comparatively well. In the sulfolipid the fractionating effect of the prolonged deoxycholate treatment expressed itself as a loss mainly from the long-chain (about C₂₆) fatty acid conjugate. The (Na⁺+ K⁺) stimulation of the ATPase function of the particulate preparation is thus correlated with the balance between the long-chain (about C₂₆) fatty acid conjugates of zwitterionic phosphatidyl choline and anionic sulfolipid. This is of theoretical interest, since it indicates that the specific lipid composition under appropriate conditions may influence the charge and conformation of a lipoprotein complex, thereby determining its functional capacities.     

Photosynthetic and Stomatal Responses of the Grey Mangrove, Avicennia marina, to Transient Salinity Conditions

Measurements of gas exchange characteristics were made on intact, attached leaves of hydroponically grown seedlings of Avicennia marina (Forstk.) Vierh. var australasica (Walp.) Moldenke as the NaCl concentration of the culture solution was varied by step changes of 50 millimolar NaCl every 2nd day from 50 to 500 to 50 millimolar NaCl. The CO₂ assimilation rate, stomatal conductance, intercellular CO₂ concentration, and evaporation rate decreased at salinities above 250 millimolar NaCl and recovered substantially upon return to the original salinity.

The assimilation rate was measured as a function of the intercellular CO₂ concentration [A(c_i) curve]. The lower linear portion of this curve was insensitive to variation in salinity, whereas the upper nonlinear portion declined with increasing salinity, indicating a reduction in the capacity for CO₂ assimilation which recovered upon return to the original salinity. Stomatal conductance changed such that the intercellular CO₂ concentration measured under normal atmospheric conditions occurred in the transition between the lower, linear and upper nonlinear portions of the A(c_i) curve. Thus, stomatal conductance and photosynthetic capacity together co-limited the assimilation rate. The changes in gas exchange characteristics were such that water loss was minimal relative to carbon gain.

     

High Inorganic Triphosphatase Activities in Bacteria and Mammalian Cells: Identification of the Enzymes Involved

Background

We recently characterized a specific inorganic triphosphatase (PPPase) from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities.

Methodology/Principal Findings

Recombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPP_i) is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPP_i but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility.

Conclusions and General Significance

We show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPP_i in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPP_i, which could be cytotoxic because of its high affinity for Ca²⁺, thereby interfering with Ca²⁺ signaling.     

Ion Uptake in Higher Plants and KCl Stimulation of Plasmalemma Adenosine Triphosphatase: Comparison of Models

Criteria for the acceptance or rejection of the dual, the cooperative, and the multiphasic model of ion uptake are given and are used to evaluate the models on the basis of previously published analyses. In addition, mathematical representations of the models are fitted to concentration-dependence data for various ions and plant tissues by a general curve fitting program. The calculated parameters are evaluated for biological relevance, and the fit is compared by statistical analysis. The kinetics of ion uptake in higher plants are found to be consistent with the concept of multiphasic uptake mechanisms, while the dual and the cooperative model must be rejected. KCl stimulation of plasmalemma-bound ATPases is also shown to obey multiphasic kinetics, thus strengthening the correlation between ion uptake and membrane-bound ATPases.     

Histochemical Observations on the Distribution of Adenosine Triphosphatase in Phloem and Motor Organs in Higher Plants

In a previous communication (Kuo 1964), it was shown by histochemical means that the phloem of cucurbit, as compared with the ground parenchyma, is particularly rich in adenosine triphosphatase (ATPase). A concurrent research by Yen et al. (1965) has succeeded in actually extracting from the vascular bundle of tobacco a crude protein fraction which not only possesses high ATPase activity, but also exhibits viscosity changes on addition of ATP or AMP. Such properties have been known to be characteristic of the contractile proteins constituting the muscle in animals. Although little is known about the actual role played in food transport by the living tissues in phloem, evidence has been accumulating that the active participation of the protoplasm in the phloem is necessary to facilitate sap movement. All these results have tempted us to think that higher plants, just like animals, also derive the energy required for performing mechanical work directly from ATP through the mediation of the contractile protein which is the ATPase itself. Accordingly, the present investigation is engaged in further histochemical observations to locate more precisely ATPase in the sieve element and to map out the distribution of the enzyme in various motor organs of higher plants, with the purpose in mind to see whether the map of enzyme distribution in the tissue has anything to do with its peculiar way of movement. 1. Presence of ATPase in the slime body of the functioning sieve element. In the functioning sieve element, high ATPase activity is displayed at the slime body and the connecting strands traversing the pores of the sieve plate (Plate Ⅰ, figs. 2, 3 & 4). However, as the pores of the sieve plate are partially blocked by the callose, the enzyme reaction becomes lessened. If the plate is completely covered by the callose pad, none could be detected. Since the slime body has been claimed by many workers to be pro- teinaceous in nature (cf. Esau 1950), there should be no surprise that it also possesses enzyme activity and contractile action. This seems to indicate that the slime body and the connecting strands are not mere hindrance to the sap flow but may actively participate in propelling the translocation stream. The fact that the intense ATPase reaction is given in the companion cell (Plate I, figs. 1 & 3) is in conformity with the common notion that the cell is actively involved in the normal functioning of phloem. 2. Enzyme distribution in various motor organs of different sensitivity. The ATPase content in the various motor organs (pulvinus, stamen, tendril, etc.) of different sensitivity has been compared histochemically, ranging from. 1) the highly sensitive organs; e.g. the pulvinus of Mimosa pudica (Plate Ⅱ, fig. 5) and of Oxalis piota (Plate Ⅱ, fig. 6), the stamen of Berberis Watsonae (Plate Ⅲ, fig. 10), the young tendril of Cucurbita maxima Duchesne and Vitis vinifera (Plate Ⅲ, figs. 11 & 12); 2) the moderately sensitive organs; e.g. the pulvinus of Sesbania cannabina (Plate Ⅱ, fig. 8); 3) the feebly sensitive or insensitive organs, e.g. the pulvinus of Robinia pseudoacacia (Plate Ⅱ, fig. 7), the stamen of Brassica campestris var. oleifera (Plate Ⅲ, fig. 9). From the results, it can be seen that the higher the sensitivity of the organ, the greater its enzyme activity. In addition to the morphological assymetry which is typical of plant motor organs, there also exists a physiological gradient, as evidenced by the uneven distribution of ATPase, usually being more intense on the more irritable side. The claim made recently by Aimi (1963) and others that the sudden collapse of the pulvinus of Mimosa on stimulation is not a direct result of the loss of turgidity as is generally assumed, but an immediate manifestation of the active vacuolar contraction in the motor cells is supported by the high ATPase content in the motor cells in our investigation.     

跖肌肌球蛋白腺苷三磷酸酶与琥珀酸脱氢酶染色的相关性探讨

研究大鼠、家兔和人跖肌的肌纤维型构成比例与分布。应用肌球蛋白腺苷三磷酸酶(ATP酶)和琥珀酸脱氢酶(SDH酶)染色法,观察比较各型肌纤维的组织化学特性。结果表明：肌球蛋白ATP酶染色,大鼠、家兔和人跖肌的肌纤维可分成I型、ⅡA型、ⅡB型肌纤维,横切面呈多边形或椭圆形,分别约占25％、35％和40％;琥珀酸脱氢酶染色,肌纤维呈蔚蓝色,以镶嵌交叉排列,可分为中染S0、深染FOG和浅染FG三型,分别约占25％、30％和45％,种系间无显著性差异。ATP酶活性反应肌纤维收缩的生理特性,而SDH活性反应肌纤维的代谢特征,两种分型的方法有所差异。     

Seasonal Changes of Polyamine Concentrations and Arginine Decarboxylase Activities in Leaves of 4 Ecotyes of Reeds

The seasonal changes of polyamine concentrations and arginine decarboxylase (ADC, EC 4.1.1. 1. 9)activities were investigated in the leaves of 4 ecotypes of reeds (Phragamites comrnunis Trinius)distributed over Hexi Corridor of Gansu province. The leaves of all ecotypes of reeds contained the same kind of polyamines and showed the same trend of decrement in total amuonts of potyamines with change of seasons. From May to September, the reeds which grow in arid and saline habitat maintained higher level of spermidine (Spd)and spermine (Spm)with no accumulation of putrescine (Put), resulting in low ratios of Put to other polyamine (Spd and Spm), whereas opposite results were observed in swamp reeds. These results indicate that the adaption of reeds to drought and salt stresses may correlate with Put synthesis via ADC pathway and the quick transformation of Put into Spd and Spm.     

Concentrations of Sodium, Potassium, Magnesium, and Iron in the Serum of Dairy Cows with Subclinical Ketosis

Serum concentrations of sodium, potassium, magnesium, and iron were measured in dairy cows with subclinical ketosis. Compared with healthy cows, the subclinically ketotic cows had significantly higher levels of non-esterified fatty acids and β-hydroxybutirate in serum and significantly lower levels of blood glucose (p?相似文献   

Membrane Mg-(Ca)-Activated Adenosine Triphosphatase of Escherichia coli: Characterization in the Membrane-Bound and Solubilized States

The membrane-associated Mg(2+)-activated and Ca(2+)-activated adenosine 5'-triphosphatase (EC 3.6.1.3; ATPase) activities of Escherichia coli were further characterized. The degree of inhibition of membrane-bound Mg(2+)-(Ca(2+))-ATPase by a series of anions (i.e., sodium salts of nitrate, iodide, chloride, and acetate) was found to correlate with the relative chaotropic, or solubilizing, effectiveness of these anions. The enzyme was solubilized from washed membrane ghosts by treatment with 0.04% sodium lauryl sulfate at pH 9.0 and 37 C. Solubilized Mg(2+)-(Ca(2+))-ATPase exhibited an initial increase in activity, followed by fairly rapid inactivation, both ATPase activities being particularly cold-labile. The combined stabilizing effects of lauryl mercaptan (1-dodecanethiol), 0.01 m tris(hydroxymethyl)amino-methane-hydrochloride buffer (pH 9.0), 0.2 mm MgCl(2), and ambient temperature facilitated partial purification of the enzyme, the molecular weight of which was estimated to be approximately 100,000 by the gel filtration technique. In general, the membrane-associated Mg(2+)-(Ca(2+))-ATPase of E. coli resembles both mitochondrial membrane ATPase and the well-characterized membrane ATPases of Bacillus megaterium and Microcococcus lysodeikticus. It is of particular interest that N,N'-dicyclohexylcarbodiimide (DCCD), a known inhibitor of mitochondrial ATPase, of mitochondrial oxidative phosphorylation, and of the membrane-bound Mg(2+)-ATPase of Streptococcus faecalis was found to inhibit both the membrane-bound and the solubilized forms of E. coli Mg(2+)-(Ca(2+))-ATPase. The sensitivity of the membrane-associated Mg(2+)-(Ca(2+))-ATPase of E. coli to both anions and cations, its allotopic behavior, and its susceptibility to inhibition by DCCD favor the idea that this enzyme plays a key, probably polyfunctional, role in such biological activities of the membrane as oxidative phosphorylation and ion transport.