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1.
The prime plasmalemma ATPase of the halophilic green alga Dunaliella bioculata has been solubilized by Triton X-100 from a plasmalemma-rich membrane fraction and purified by anion-exchange chromatography. Vanadate-sensitive ATPase activity was totally enriched about 230-fold to a specific activity of approx. 250 nkat·mg protein–1. The presence of Mg2+ or Mn2+ is essential for ATP hydrolysis by the enzyme. In addition to an equimolar requirement (11 Mg2+: ATP), there is further stimulation by Mg2+ (up to 20 mM) and by (100 mM) monovalent cations (K+ NH
4
+
>Rb+ -Na+ >Cs+ >Li+-choline+). Most anions have no or little effect. With a molecular mass of about 105 kDa for the single subunit, sensitivity to vanadate and N,N-dicyclohexylcarbodiimide (50% inhibition at about 1 M and 0.3 mM, respectively), strict ATP-specificity, and an acidic pH optimum, this enzyme shows the typical characteristics of the common type of H+-ATPase in the plasmalemma of higher plants and fungi. These results undermine the hypothesis of a wider distribution of a special (high salt) type of plasmalemma ATPase as found in the marine alga Acetabularia.Abbreviations BTP
1,3-bis[tris(hydroxymethyl)-methylamino]propane
- DCCD
N,N-dicyclohexylcarbodiimide
- DES
diethylstilbestrol
- Mega-9
nonanoyl-N-methyl-glucamide
- Mes
N-morpholinoethanesulfonic acid
- Mops
N-morpholinopropanesulfonic acid
- PAGE
polyacrylamide-gel electrophoresis
- PM
plasmalemma-enriched membrane fraction
- SDS
sodium dodecyl sulfate
This work was supported by the Deutsche Forschungsgemeinschaft; we thank Drs. M. Ikeda and D. Oesterhelt (MPI für Biochemie, Martinsried, FRG) for generous and valuable information about their work prior to publication. 相似文献
2.
3.
Summary Ultrastructural investigation of the flagellar root system ofAcetabularia gametes reveals one type of organization for both male and female gametes. There is a modified cruciate system with four microtubular bands X-2-X-2, with X=4. A prominent distal striated fiber and a small proximal striated fiber connect the flagellar bases. A striated root fiber type I underlies the microtubular root type II, and a short striated root fiber type I underlies the microtubular root type I (terminology ofMelkonian 1980 b). This specific root system has some details in common with theChlamydomonas type, and others with theUlvaphyceae and the siphonalean algaeDerbesia andBryopsis. This might indicate the phylogenetic relationships. 相似文献
4.
H. -U. Koop 《Protoplasma》1979,100(3-4):353-366
Summary The life cycle ofAcetabularia is described with special reference to nuclear divisions. Recent arguments, derived from the fields of cytology, genetics and systematics are in favour of the hypothesis, that meiosis occurs during the division of the primary nucleus. This hypothesis is summarized in a schematical representation of the whole life cycle. 相似文献
5.
Using monoclonal antibodies we have studied the interaction of tubulin with the plasma membrane of leaves of Nicotiana sylvestris (Speg. et Comes) and tobacco suspension-culture cells. The results show that isolated plasma membranes contain tightly bound -tubulins. Their association with the plasma membrane is resistent to non-ionic detergent and to low and high ionic strength. Only extraction with sodium dodecyl sulfate is capable of dissociating these cytoskeletal proteins. It is unlikely that this membrane-bound tubulin is present in its polymeric form because electron-microscopical analysis does not reveal the presence of filaments, whereas treatment of membranes with oryzalin (which has been shown to destabilize microtubules in vitro) does not remove the tubulins from isolated plasma membrane. When living cells are treated with oryzalin, the amount of membrane-associated tubulin is drastically reduced, which could mean that its presence is related to in-vivo microtubule dynamics.Abbreviations Mes
2 (N-morpholino) ethane sulfonic acid
- NP40
Nonidet P40 相似文献
6.
The ATPase activity present in plasmalemma-enriched preparations from maize coleoptiles shows an optimum at pH 6, a strong dependence on Mg2+, and is stimulated by K+ and other monovalent cations, both organic and inorganic. The activation of ATPase by K+ obeys Michaelis Menten kinetics, saturation being reached at 50 mM K+ concentration. K+, Mg2+-stimulated ATPase activity is strongly inhibited by N,N-dicyclohexylcarbodiimide and by diethylstilbestrol and, to a lesser extent, by octylguanidine.Abbreviations DCCD
N,N-dicyclohexylcarbodiimide
- DES
diethylstilbestrol
- DTE
dithioerythritol
- Ellmans r
5-5 dithiobis (2 nitrobenzoic) acid
- FC
fusicoccin
- NPA
naphthylphthalamic acid
- OG
octylguanidine
- PCMBS
p-chloromercuribenzensulphonate 相似文献
7.
Partially (6-fold) purified plasma membrane ATPase from an ethanol-sensitive yeast, Kloeckera apiculata, had an optimum pH of 6.0, an optimum temperature of 35°C, a K
m of 3.6 mm ATP and a V
max of 11 mol Pi/min.mg protein. SDS-PAGE of the semi-purified plasma membrane showed a major band of 106 kDa. No in vivo activation of the ATPase by glucose was observed. Although 4% (v/v) ethanol decreased the growth rate by 50% it did not affect the ATPase. Concentrations of ethanol 2% (v/v) did, however, inhibit the enzyme in vitro. The characteristics of the enzyme did not change during growth in the presence of ethanol. 相似文献
8.
A multitasking time-sharing computer system was implemented for studies of different circadian rhythms in individual cells
of the unicellular green alga,Acetabularia. This fully automatized system allows simultaneous data acquisition and analysis. Graphical presentation of untreated and
mathematically treated data is permanently available on three graphic displays and on a digital plotter. The sampling rate
for the data acquisition in each of the 60 channels connected to the system is 720/24 h. Provisions have been made to guarantee
uninterrupted data uptake for these long-term measurements by including an auto-restart module and by providing extremely
reliable software for the experimenter using menu techniques. 相似文献
9.
Summary The cytochemical localization of ATPase activity has been investigated in maize root cells using both lead and cerium-based capture methods. With both methods, staining at the plasma membrane was observed in all cells of the root, although the precipitate obtained with cerium was more uniform and granular than that with lead. Controls using no substrate or no magnesium, -glycerophosphate to replace ATP, vanadate or boiled tissue generally showed little or no staining. However, biochemical studies on purified plasma membrane fractions showed that ATPase activity was markedly inhibited by fixation, particularly by glutaraldehyde, and also by lead and cerium ions. Non-enzymic hydrolysis of ATP by cerium was greater than that by lead. The value and limitations of these procedures for the localization of plasma membrane H+-ATPase activity are summarized in relation to previous criticisms of these methods.Abbreviations DTT
dithiothreitol
- EDTA
ethylene diaminetetraacetic acid
- GP
B-glycerophosphate
- PCMBS
p-chloromercuribenzene sulphonic acid
- PMSF
phenylmethylsulphonyl fluoride 相似文献
10.
The high photosynthetic activity (O2 production and CO2 consumption) ofAcetabularia mediterranea Lamour. (=A. acetabulum (L.) Silva) characteristic of cells cultured in white light decreases slowly when cells are kept in continuous red light,
and is less than 20% of the original activity after three weeks. Subsequent blue irradiation restores the original activity
completely within 3–5 d. The polypeptide composition of the thylakoids from cells grown in either red or blue light and after
transfer from red to blue light was analyzed mainly with regards to photosystem II (PSII). The P700-containing reaction-centre
complex of photosystem I, CPI, showed only minor quantitative alterations as a consequence of the growth-light quality, which
correlated well with the activity of photosystem I under these conditions. In PSII, no drastic changes occurred in the quantity
of the reaction-centre components D1 (herbicide-binding polypeptide) and D2, as determined by immunoblots. Likewise, the proteins
associated with the water-splitting apparatus did not change detectably in thylakoids from red- or blue-light-treated cells
(the 16-kDa component could not be found inAcetabularia thylakoids). The level of the major light-harvesting complex was completely unaffected by the light quality. In contrast,
the quantities of the chlorophyll a-protein complexes of the core antenna, CP43 and CP47 (and probably CP29), changed, with
kinetics similar to those of total photosynthetic activity. We postulate that the function of the PSII antenna became increasingly
impaired in the absence of blue light (i.e. in red light), while blue light had a restoring effect. The peripheral antenna,
comprising the light-harvesting complexes, is probably functionally connected with the reaction-centre chlorophylls via the
core antenna chlorophyll-protein complexes (CP43, CP47 and probably CP29). A deficiency of these complexes would lead to uncoupling
of antenna and reaction centre in the majority of PSII complexes after long periods of red-light treatment. 相似文献
11.
Cryptococcus neoformans is a facultative intracellular pathogen, which can replicate in the acidic environment inside phagolysosomes. Deletion of the enzyme inositol-phosphosphingolipid-phospholipase-C (Isc1) makes C. neoformans hypersensitive to acidic pH likely by inhibiting the function of the proton pump, plasma membrane ATPase (Pma1). In this work, we examined the role of Isc1 on Pma1 transport and oligomerization. Our studies showed that Isc1 deletion did not affect Pma1 synthesis or transport, but significantly inhibited Pma1 oligomerization. Interestingly, Pma1 oligomerization could be restored by supplementing the medium with phytoceramide. These results offer insight into the mechanism of intracellular survival of C. neoformans. 相似文献
12.
Summary The claim that osmium-containing deposits which lack lead are frequently and incorrectly interpreted as enzymatic reaction products in lead precipitation techniques for ATPase localization in plants is without foundation. Proper controls clearly demonstrate the enzymatic origin of membrane-located deposits and the presence of lead is confirmed by analytical electron microscopy. 相似文献
13.
Kinetic analysis of the Mg2+-dependence of tonoplast ATPase from suspension-cultured cells of sugarcane showed that the enzyme activity increased with increasing magnesium concentrations till 1–3 mM and then decreased consideably for higher concentrations. This kinetic could be explained by the assumption that MgATP2- is the substrate of ATPase: MgATP2- concentration increases with increasing concentration of magnesium till, at high concentrations of magnesium, Mg2ATP is formed. No evidence for a direct role of Mg2+ as activator or inhibitor was found. These data corroborate previous findings that MgATP2- is the sole substrate of the vacuolar ATPase of sugarcane (Thom and Komor 1984). High concentrations of ATP seemed to inhibit the ATPase. This result, however, could be traced back to interference of ATP with the Fiske-Subbarow method of phosphate determination. After adjustment of the test conditions, inhibition by ATP was no longer found. Reported data for ATPases of other plant materials, showing inhibition of enzyme activity with high magnesium or ATP concentrations, might be explicable in a similar way.Abbreviation Mes
2-(N-morpholino)ethane+Sulfonic acid 相似文献
14.
The calmodulin-stimulated ATPase of maize (Zea mays L.) coleoptiles has been purified by calcium-dependent binding to a calmodulin affinity column. In the presence of protease inhibitors (phenylmethylsulfonylfluoride and chymostatin) a polypeptide of relative molecular mass (Mr) 140000 (±10000) is obtained on sodium-dodecylsulphate polyacrylamide gels. This polypeptide is recognised specifically by an affinity-purified polyclonal antibody to mammalian calmodulin-stimulated calcium-pumping ATPases and is of similar Mr to the erythrocyte-membrane calcium pump (138000 Mr).Abbreviations EGTA
ethylene glycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid
- Mr
apparent molecular mass
- SDS
sodium dodecyl sulphate 相似文献
15.
In plants, the oxygen generated by photosynthesis can be excited to form reactive oxygen species (ROS) under excessive sunlight.
Excess ROS including singlet oxygen (1O2) inhibit the growth, development and photosynthesis of plants. To isolate ROS-resistant crop plants, we used paraquat (PQ),
a generator of O2
·− as a source of screening and mutagen, and obtained two PQ-resistant lines in Pisum sativum, namely R3-1 and R3-2. Both lines showed greater resistance to PQ than their wild type (WT) siblings with respect to germination, root growth, and
shoot growth. Biochemical analysis showed differences in these lines, in which ROS-scavenging enzymes undergo changes with
a distinguishable increase in Mn-SOD. We further observed that the cytosolic catalases (CATs) in leaves in both lines were
shifted in a native-PAGE analysis compared with that of the WT, indicating that the release of bound 1O2 was enhanced. Phenotypic analysis revealed distinguishable differences in leaf development, and in flowering time and position.
In addition, R3-1 and R3-2 showed shorter individual internode lengths, dwarf plant height, and stronger branching compared with the WT. These results
suggested that PQ-induced ROS-resistant Pisum have the potential pleiotropic effects on flowering time and stem branching, and that ROS including 1O2 plays not only important roles in plant growth and development as a signal transducer, but also appears as a strong inhibitor
for crop yield.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
16.
The auxin sensitivity of the plasma-membrane H+-ATPase from tobacco leaves (Nicotiana tabacum L. cv. Xanthi) depends on the physiological state of the plant (Santoni et al., 1990, Plant Sci. 68, 33–38). Results based on the study of auxin sensitivity according to culture conditions which accelerate or delay tobacco development demonstrate that the highest auxin sensitivity is always associated with the end of the period of induction to flowering. Auxin stimulation of H+-translocation activity corresponds to an increase of the apparent ATPase affinity for ATP. The plasma-membrane H+-ATPase content, measured with an enzyme-linked immunosorbent assay using a specific anti-H+-ATPase antibody, varies according to plant development, and was found to increase by 100% during floral induction. The specific molecular ATPase activity also changes according to plant development; more particularly, the decrease in molecular ATPase activity upto and during the floral-induction period parallels the increase of sensitivity to indole-3-acetic acid.Abbreviations ELISA
enzyme-linked immunosorbent assay
- PAGE
polyacrylamide gel electrophoresis
- SDS
sodium dodecyl sulfate
Authors are grateful to Mrs. Grosclaude (Lab. Virologie, INRA, Jouy-en-Josas, France) and Mrs. Boudon (Lab. Mycoplasmes, INRA, Dijon, France) for support and advice in the preparation of antibodies. This work was supported by grants No. 89/512/6 from the E.P.R of Bourgogne and No. 89 C 0662 from M.R.T. 相似文献
17.
Arabidopsis thaliana lines have been isolated that are insensitive to the fungal toxin fusicoccin (FC). Initial screening was done by selecting for plants that either grew well on high concentrations of FC or did not respond to FC by increases in H+-extrusion. All selected plants were tested, in several additional rounds of screening, for binding to microsomal proteins of a 3H-labeled radioligand of fusicoccin. A novel assay allowing for the direct selection of individual plants exhibiting reduced binding of FC was developed and used as screening procedure. Independent variant lines (43) with stably expressed, reduced binding of FC were isolated and subjected to a detailed characterization of their binding sites. The lines could be subdivided into several distinct classes with respect to these characteristics. In class-I lines, the data indicate a partial conversion of high-affinity binding sites to a low-affinity state. In class-II lines, the affinity of the binding site to FC is strongly reduced while the number of sites, as well as several other biochemical parameters, is completely unchanged, suggesting a specific alteration in the properties of the fusicoccin-binding protein. In class-III lines, the ligand-binding protein complex, while retaining its high affinity, is destabilized at supraoptimal concentrations of FC (such as those used for screening). In wild-type plants, only the high-affinity binding site was detected. Combined, these data prove that the high-affinity sites represent the plant's FC receptor.Abbreviations Ao
binding site concentration
- FC
fusicoccin
- FCBP
fusicoccin-binding protein
- FCol
9-nor-8-hydroxyfusicoccin
- KD
dissociation constant of the FCBP-radioligand complex
We are grateful to Iris Sandorf and Gudrun Henrichs for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany and by Fonds der Chemischen Industrie (literature provision). 相似文献
18.
Jan Ahlers 《生物化学与生物物理学报:生物膜》1981,649(3):550-556
The reaction of plasma membrane ATPase from yeast with Mg2+ and Mg · ATP was studied in a temperature range of 10 – 30°C. The random mechanism of activation by Mg2+ and the pseudocompetitive inhibition at higher concentrations was not altered when the temperature was varied, nor were the kinetic constants representing substrate binding. However, at low temperature, the affinity of the enzyme for Mg2+ is greatly reduced. The Arrhenius plot of log V vs. 1/T shows straight lines with an inflection point at 24°C, which disappears in the presence of detergent. Calorimetric studies of the plasma membranes show a transition point at the same temperature. From these findings we suppose that Mg2+ is bound at a regulatory site of the ATPase, which is influenced by the surrounding phospholipids. 相似文献
19.
Separation and purification of the tonoplast ATPase and pyrophosphatase from plants with constitutive and inducible Crassulacean acid metabolism 总被引:7,自引:0,他引:7
Tonoplast vesicles were isolated from Kalanchoe daigremontiana Hamet et Pierrer de la Bâthie and Mesembryanthemum crystallinum L., exhibiting constitutive and inducible crassulacean acid metabolism (CAM), respectively. Membrane-bound proteins were detergent-solubilized with 2% of Triton X-100. During CAM induction in M. crystallinum, ATPase activity increases four-fold, whereas pyrophosphatase activity decreases somewhat. With all plants, ATPase and pyrophosphatase could be separated by size-exclusion chromatography (SEC, Sephacryl S 400), and the ATPase was further purified by diethylaminoethyl-ion-exchange chromatography. Sodium-dodecyl-sulfate electrophoresis of the SEC fractions from K. daigremontiana containing maximum ATPase activity separates several protein bands, indicating subunits of 72, 56, 48, 42, 28, and 16 kDa. Purified ATPase from M. crystallinum in the C3 and CAM states shows a somewhat different protein pattern. With M. crystallinum, an increase in ATP-hydrolysis and changes in the subunit composition of the native enzyme indicate that the change from the C3 to the CAM state is accompanied by de-novo synthesis and by structural changes of the tonoplast ATPase.Abbreviations CAM
Crassulacean acid metabolism
- DTT
dithiothreitol
- kDa
kilodalton
- PAGE
polyacrylamide gel electrophoresis
- PPiase
pyrophosphatase
- SEC
size exclusion chromatography
- SDS
sodium dodecyl sulfate
- Tris
2-amino-2-(hydroxymethyl)-1,3-propanediol 相似文献