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1.
Spheroplasts from respiratory-deficient (rho) yeastswere transformed into rho+ cells by incubation with mitochondriaprepared from respiratory-sufficient (rho+) yeasts in the presenceof polyethylene glycol 4000 and CaCl2. Spheroplasts of buddingyeast rho strains were also transformed into rho+ cellsby treatment with mitochondria prepared from heterospecificbudding or fission yeast strains. All the transformed regeneratantsyielded rho+ yeast colonies which bore chromosomal genetic characteristicsof the spheroplasts used and cytoplasmic genetic characteristicsof the mitochondria used. These indicate that mitochondrialgenes or mitochondria themselves introduced by the incubationfunctioned normally in the rho cells regardless of thespecies difference of the recipient spheroplasts.
1A preliminary report appeared in ref. 17. (Received October 31, 1978; ) 相似文献
2.
We have devised conditions which produced isolated protoplastsof non-spherical shape and which, therefore, affected the mechanismsthat control the exchange of membrane material between the plasmamembrane and an intracellular membrane reservoir. Non-sphericalprotoplasts of Avena sativa were obtained if protoplasts weretreated with hypertonic shock in the presence of 1.0 mol m3LaCl3 at pH 8.3. This indicated that their ability to removeplasma membrane material via endocytotic vesiculation was suppressed.Non-spherical protoplasts were obtained under isotonic conditionsif protoplasts were incubated with 1.0 mol m-3 LaCl3 at pH 8.3and the proton carrier CCCP (12 mmol m3) was added. Thenon-spherical protoplasts had intact membranes as judged bystaining with fluorescein diacetate. The loss of the sphericalshape was reversible. On addition of EDTA protoplasts resphericulatedimmediately. Incubation in isotonic solution at pH 8.3 containingeither only 1.0 mol m3 LaCl3 or only CCCP did not influencethe protoplast shape. We conclude that the membrane hyperpolarizationinduced by CCCP at high pH acted to stimulate the incorporationof membrane material into the plasma membrane and, subsequently,produced nonspherical protoplasts if the removal of membranematerial was simultaneously suppressed. This demonstrates thatmembrane incorporation and removal are two largely independentprocesses. 相似文献
3.
Guard cell protoplasts were isolated enzymatically from theepidermis of Vicia faba L. and their photosynthetic activitieswere investigated. Time courses of light-induced changes inthe chlorophyll a fluorescence intensity of these protoplastsshowed essentially the same induction kinetics as found formesophyll protoplasts of Vicia. The transient change in thefluorescence intensity was affected by DCMU, an inhibitor ofphotosystem II; by phenylmercuric acetate, an inhibitor of ferredoxinand ferredoxin NADP reductase; and by methyl viologen, an acceptorof photosystem I. Low temperature (77 K) emission spectra ofthe protoplasts had peaks at 684 and 735 nm and a shoulder near695 nm. A high O2 uptake (175 µmol mg1 Chl hr1)was observed in guard cell protoplasts kept in darkness, whichwas inhibited by 2 mM KCN or NaN3 by about 60%. On illumination,this O2 uptake was partially or completely suppressed, but itssuppression was removed by DCMU, which indicates that oxygenwas evolved (150 µmol mg1 Chl hr1) photosynthetically.We concluded that both photosystems I and II function in guardcell chloroplasts and that these protoplasts have high respiratoryactivity. (Received January 30, 1982; Accepted May 15, 1982) 相似文献
4.
Characterization of the vacuolar-type H+-ATPase from guard cell protoplasts of Commelina 总被引:1,自引:0,他引:1
Willmer C.M.; Grammatikopoulos G.; Lascve G.; Vavasseur A. 《Journal of experimental botany》1995,46(4):383-389
Characteristics of the vacuolar-type (V-type) H+-ATPase fromguard cell protoplasts of Commelina communis L. were investigatedusing a linked enzyme assay and nitrate inhibition as a diagnosticindicator of the enzyme activity. ATPase activity was completelyinhibited by about 50 mol m3 nitrate and activity wasoptimal near pH 8.0. The temperature optimum for activity wasabout 37 C and an Arrhenius plot indicated changes in activationenergy for the ATPase at 15C and possibly at about 30 C. Theenzyme was stimulated by Cl while Ca2+ inhibited activity(l50 = 1.5 mol m3). The apparent Km (MgATP) was 0.62mol m3. Incubation of guard cell protoplasts for up to 5 h in 50 µMabscisic acid (ABA) or 25µM fusicoccin (FC) did not affectsubsequent ATPase activity. In vitro assays with FC or ABA alsodid not affect enzyme activity. Activity was not affected bylight or potassium ferricyanide, two factors which are knownto influence stomatal activity. Beticoline was a potent inhibitorof activity (l50 = 50 µM) while DCCD was less effective(l50 = 90µM). On chlorophyll, protein and protoplast bases, V-type ATPaseactivity was greater in guard cell protoplasts than mesophyllcell protoplasts by 66, 13.9 and 1.9, respectively. On atonoplast surface area basis the enzyme activity was 5.6 timeshigher in guard cell protoplasts than in mesophyll cell protoplasts Thus, although the characteristics of the V-type, H +-ATPaseof GCP are very similar to those found in other cell types,rates of activity and probably tonoplast enzyme density aremuch greater in guard cell protoplasts than mesophyll cell protoplastsof C. communis which corresponds with the large and rapid ionfluxes across the tonoplast associated with stomatal movements Key words: Guard cell protoplasts, stomata, V-type H +-ATPase 相似文献
5.
Abscisic Acid-Induced Membrane Potential Changes in Barley Aleurone Protoplasts: a Possible Relevance for the Regulation of rab Gene Expression 总被引:2,自引:0,他引:2
Heimovaara-Dijkstra Sjoukje; van Duijn Bert; Libbenga Kees R.; Heidekamp Freek; Wang Mei 《Plant & cell physiology》1994,35(5):743-750
The effect of ABA on the membrane potential of barley (Hordeumvulgare cv. Himalaya) aleurone protoplasts was studied by measuringthe distribution of the lipophilic cation tetraphenylphosphonium(TPP+). The resting membrane potential (Em) according to ourmeasurements with TPP+ is about 53 mV and is in agreementwith membrane potential values as measured with intracellularmicroelectrodes (about 55 mV). The TPP+-measurementscould demonstrate a clear dependence of the resting Em on theexternal pH (pHe). Stimulation of the protoplasts with ABA induced a transienthyperpolarization of the membrane to 62 mV as measuredwith TPP+. The hyperpolarization was ABA-concentration dependent. Inhibition of the H+-ATPases with the specific proton pump inhibitorsdiethylstilbestrol (DES) or Micanozole effectively preventedhyperpolarization. This indicates that the hyperpolarizationis consistent with the activation of plasma membrane H+-ATPases.The K+-inward rectifier inhibitor BaCl2 was able to prolongthe hyperpolarization. This result suggests that the hyperpolarizationcauses the opening of K+-channels. The ABA-induced proton-pump activation may be involved in ABA-inducedgene-expression, as DES was able to inhibit this gene expression.BaCl2 did only show a slight inhibitory effect on ABA-inducedgene-expression. (Received January 4, 1994; Accepted April 12, 1994) 相似文献
6.
Millhouse, J. and Strother, S. 1987. Further characteristicsof salt-dependent bicarbonate use by the seagrass Zostera muelleri.J.exp. Bot. 38: 10551068. The contribution of HCO3to photosynthetic O2 evolutionin the seagrass Zostera muelleri Irmisch ex Aschers. increasedwith increasing salinity of the bathing seawater when the inorganiccarbon concentration was kept constant. K1/2 (seawater salts)for HCO3 -dependent photosynthesis was 66% of seawatersalinity. Both short- and long-term pretreatment at low salinitiesstimulated photosynthesis in full strength seawater. Twentyfour hours pre-incubation of seagrass plants in 3·0 molm3 NaHCO3 resulted in increased photosynthesis at allsalinities, apparently due to stimulation of HCO3 use(K1/2 (seawater salts) = 26%). Vmax (HCO3) was not affectedby low salinity pretreatment. The kinetics of HCO3 stimulationby the major seawater cations was investigated. Ca2+ was themost effective cation with the highest Vmax (HCO3) andwith K1/2(Ca2+) = 14 mol m3. Mg2+ was also very effectiveat less than 50 mol m3 but higher concentrations wereinhibitory. This inhibition cannot be accounted for solely byprecipitation of MgCO3. Na+ and K+ were both capable of stimulatingHCO3 use. Stimulation was in two distinct parts. Up to500 mol m3, both citrate and chloride salts gave similarresults (K1/2(Na+) 81 mol m3, Vmax(HCO3) 0·26µmol O2 mg1 chl min1), but use of citratesalts above 500 mol m2 caused a second stimulation ofHCO3 use (K1/2(Na+) 830 mol m3, Vmax(HCO3)0·68 µmol O2 mg1 chl min1). Vmax(HCO3)for the second-phase Na+ or K+ stimulation was of the same orderas for Ca2+-stimulated HCO3 use. To further characterizesalt-dependent HCO3 use, the sensitivity of photosynthesisto Tris and TES buffers was investigated. The effects of Trisappear to be due to the action of Tris+ causing stimulationof HCO3 -dependent photosynthesis in the absence of salt,but inhibition of HCO3 use in saline media. TES has noeffect on photosynthesis. External carbonic anhydrase, althoughimplicated in salt-dependent HCO3 use in Z. muelleri,could not be detected in whole leaves. Key words: Zostera muelleri, HCO3 use, salinity 相似文献
7.
Direct Transfer of Plasmid DNA from Intact Yeast Spheroplasts into Plant Protoplasts 总被引:1,自引:0,他引:1
Hatsuyama Yoshimichi; Sunaga Natsuo; Habu Yoshiki; Ishikawa Masayuki; Kawamoto Shinichi; Naito Satoshi; Ohno Takeshi 《Plant & cell physiology》1994,35(1):93-98
We developed a polyethylene glycol (PEG)-mediated direct DNAtransfer method from intact Saccharomyces cerevisiae spheroplastsinto Arabidopsis thaliana protoplasts. To monitor the DNA transferfrom yeast to plant cells, ß-glucuronidase (GUS) reportergene in which a plant intron was inserted was used as a reporter.This intron-GUS reporter gene on a 2µm-based plasmid vectorwas not expressed in yeast transformants, while it expressedGUS activity when the plasmid DNA was introduced into plantcells. When a mixture of 1 x 108 of S. cerevisiae spheroplastsharboring the plasmid and 2 x 106 of A. thaliana protoplastswas treated with PEG and high pH-high Ca2+ solution (0.4 M mannitol,50 mM CaCl2, 50 mM glycine-NaOH pH 10.5), GUS activity was detectedin the extract of the plant cells after a three-day culture.The GUS activity was higher than that of a reconstitution experimentin which the mixture of 1 x 108 of S. cerevisiae spheroplastswhich did not carry the reporter gene, 2 x 106 of A. thalianaprotoplasts and the same amount of the reporter plasmid DNAas that contained in 1 x 108 of S. cerevisiae spheroplasts,was treated with PEG and high pH-high Ca2+ solution. Moreover,the GUS gene expression was resistant to micrococcal nucleasetreatment before and during PEG treatment. From these results,we concluded that plasmid DNA can be directly transferred fromintact yeast spheroplasts to plant protoplasts by a nuclease-resistantprocess, possibly by the cell fusion.
2Deceased on September 15, 1992. 相似文献
8.
MEMON ABDUL RAZAQUE; SIDDIQI M. YAEESH; CLASS ANTHONY D. M. 《Journal of experimental botany》1985,36(1):79-90
Barley (Hordeum vulgare L.) varieties differed in their raponseto [K+]0, in terms of their utilization efficiencies (UE = freshweight. concentration of [K+]11). At low [K+]0, Compana,an efficient-non-responder demonstrated superior utilizationof absorbed K+. On the other hand, at high [K+]0, Fergus (anefficient responder) and BT 334 (an inefficient responder) hadhigher UE values for K+ than Compana which performed poorlyat this [K+]0. Kinetic parameters for K+ activation of the enzyme pyruvatekinase from 12 barley varieties, representing a range of UEvalues, were determined. Varieties showed substantial differencesin their Vmax values (P<0·01). Compana, an efficientvariety, had the highest Vmax (31 µmol g1 freshwt. h1) which was about 50% higher than that of Mingo,an inefficient variety. By contrast, Km values for the enzymeswere not significantly different among varieties The mean valuesfor all varieties (3·9±0·15 mol m3K+) is far below the estimated cytoplasmic [K+] (100-200 molm3). It is, therefore, unlikely that differences in theutilization of K+ by these varieties can be explained on thebasis of differential requirements for (K+) activation of theseenzymes. Alternative possibilities for differences in the utilizationof K+ are discussed. Key words: K+ utilization efficiency, Pyruvate kinase, Barley varieties 相似文献
9.
Bouzinova EV Praetorius J Virkki LV Nielsen S Boron WF Aalkjaer C 《American journal of physiology. Cell physiology》2005,289(6):C1448-C1456
Several studies suggest the involvement of Na+ and HCO3 transport in the formation of cerebrospinal fluid. Two Na+-dependent HCO3 transporters were recently localized to the epithelial cells of the rat choroid plexus (NBCn1 and NCBE), and the mRNA for a third protein was also detected (NBCe2) (Praetorius J, Nejsum LN, and Nielsen S. Am J Physiol Cell Physiol 286: C601C610, 2004). Our goal was to immunolocalize the NBCe2 to the choroid plexus by immunohistochemistry and immunogold electronmicroscopy and to functionally characterize the bicarbonate transport in the isolated rat choroid plexus by measurements of intracellular pH (pHi) using a dual-excitation wavelength pH-sensitive dye (BCECF). Both antisera derived from COOH-terminal and NH2-terminal NBCe2 peptides localized NBCe2 to the brush-border membrane domain of choroid plexus epithelial cells. Steady-state pHi in choroidal cells increased from 7.03 ± 0.02 to 7.38 ± 0.02 (n = 41) after addition of CO2/HCO3 into the bath solution. This increase was Na+ dependent and inhibited by the Cl and HCO3 transport inhibitor DIDS (200 µM). This suggests the presence of Na+-dependent, partially DIDS-sensitive HCO3 uptake. The pHi recovery after acid loading revealed an initial Na+ and HCO3-dependent net base flux of 0.828 ± 0.116 mM/s (n = 8). The initial flux in the presence of CO2/HCO3 was unaffected by DIDS. Our data support the existence of both DIDS-sensitive and -insensitive Na+- and HCO3-dependent base loader uptake into the rat choroid plexus epithelial cells. This is consistent with the localization of the three base transporters NBCn1, Na+-driven Cl bicarbonate exchanger, and NBCe2 in this tissue. bicarbonate metabolism; BCECF; cerebrospinal fluid; acid/base transport; ammonium prepulse 相似文献
10.
The relationship between thef-ratio [NO3 uptake/(NO3+ NH4+) uptake] and ambient nitrate concentration was evaluatedfor eight data sets from coastal waters. The f-ratio increasedasymptotically with increase in nitrate concentration in mostdata sets. However, the rate at which f-ratio increased at lownitrate concentration (slope = m) and the maximum attained f-ratio(fmax) varied among regions; the initial slope varied most withvalues ranging in excess of an order of magnitude. The datawere analyzed in relation to environmental factors and methodologicalconsiderations known to influence the f-ratio. Ambient ammoniumconcentration was important in accounting for regional differencesin the f versus NO3 relationship. A further analysisof the data, relating f-ratio to the ratio of NO3/(NO3+ NH4+) concentrations yielded a much more regionally consistentand approximately linear relationship; slopes varied by lessthan a factor of two in the extreme cases. Inclusion of knownalternative (aside from NH4+) sources of reduced-N (e.g. urea)and correction for methodological/computational errors (isotopedilution) systematically reduce f-ratio estimates. Other factors,e.g. reduced-N uptake by microheterotrophs, may systematicallyincrease the f-ratio. 相似文献
11.
Clint, G. M. 1985. The investigation of stomatal ionic relationsusing guard cell protoplasts. 1. Methodology.J exp. Bot.36: 17261738. A study was made of the methodology for the production and useof guard cell protoplasts in ion transport studies, with particularemphasis placed on the effects of the composition of the externalmedium on protoplast survival and performance. Addition of externalKCl to media during the production of guard cell protoplastsfrom Commelina communis L. was found to improve viability andto increase K+ content and physiological competence of the isolatedprotoplasts. Addition of low levels (20 x 103 mol m3)CaCl2 increased protoplast yield and the maintenance of viabilityin long-term incubation. Ambiguities and uncertainties werefound in the application of methods commonly used for the assessmentof viability of isolated protoplasts. Poor yields (despite highpercentage recoveries) together with difficulties in the assessmentof viability were considered to pose major potential problemsin the use of guard cell protoplasts in ion transport studies. Key words: Guard cell protoplasts, ion transport, Commelina communis 相似文献
12.
K+ channel KVLQT1 located in the basolateral membrane of distal colonic epithelium is not essential for activating Cl- secretion 总被引:1,自引:0,他引:1
Liao T Wang L Halm ST Lu L Fyffe RE Halm DR 《American journal of physiology. Cell physiology》2005,289(3):C564-C575
The cellular mechanism for Cl and K+ secretion in the colonic epithelium requires K+ channels in the basolateral and apical membranes. Colonic mucosa from guinea pig and rat were fixed, sectioned, and then probed with antibodies to the K+ channel proteins KVLQT1 (Kcnq1) and minK-related peptide 2 (MiRP2, Kcne3). Immunofluorescence labeling for Kcnq1 was most prominent in the lateral membrane of crypt cells in rat colon. The guinea pig distal colon had distinct lateral membrane immunoreactivity for Kcnq1 in crypt and surface cells. In addition, Kcne3, an auxiliary subunit for Kcnq1, was detected in the lateral membrane of crypt and surface cells in guinea pig distal colon. Transepithelial short-circuit current (Isc) and transepithelial conductance (Gt) were measured for colonic mucosa during secretory activation by epinephrine (EPI), prostaglandin E2 (PGE2), and carbachol (CCh). HMR1556 (10 µM), an inhibitor of Kcnq1 channels (Gerlach U, Brendel J, Lang HJ, Paulus EF, Weidmann K, Brüggemann A, Busch A, Suessbrich H, Bleich M, and Greger R. J Med Chem 44: 38313837, 2001), partially (50%) inhibited Cl secretory Isc and Gt activated by PGE2 and CCh in rat colon with an IC50 of 55 nM, but in guinea pig distal colon Cl secretory Isc and Gt were unaltered. EPI-activated K+-secretory Isc and Gt also were essentially unaltered by HMR1556 in both rat and guinea pig colon. Although immunofluorescence labeling with a Kcnq1 antibody supported the basolateral membrane presence in colonic epithelium of the guinea pig as well as the rat, the Kcnq1 K+ channel is not an essential component for producing Cl secretion. Other K+ channels present in the basolateral membrane presumably must also contribute directly to the K+ conductance necessary for K+ exit during activation of Cl secretion in the colonic mucosa. HMR1556; K+ secretion; epinephrine; prostaglandin E2; cholinergic 相似文献
13.
Guard cell protoplasts (GCP) were prepared from leaves of Commelinacommunis L. and phosphoenolpyruvate carboxylase (PEPc) activityrecorded after injection of the protoplasts directly into theassay medium. The GCP were lysed immediately by the presenceof Triton X-100 and a lowered osmotic concentration in the assaycuvette enabling PEPc activity to be measured with nascentenzyme. There was no light activation of the enzyme with KmPEP (about 3.7 mol m3) and Vmax being similar in light-ordark-treated protoplasts. Illumination of the GCP in the presenceof CO2-free air and KCI, a treatment which is known to swellGCP, did not change the kinetics. PEPc activity at saturating PEP was very sensitive to malateinhibition, 20 mmol m3 (the I50 value) inhibiting activityby about 50%. Inhibition was similar in light- or dark-treatedprotoplasts. Malate inhibition was, however, much less (I50= 500 mmol m3) if the enzyme source was a protoplastextract kept in the absence of glycerol. Inclusion of 20% glycerolin the extraction medium maintained the enzyme in the malate-sensitiveform as occurred in the in vivo assays. The high apparent KmPEP and the high sensitivity to malate inhibition of GCP PEPcare features unlike those observed with PEPc from leaf tissuesof C4 and CAM plants and from GCP extracts. PEPc activity increased slightly in the presence of KCI in theassay medium up to about 10 mol m3 and thereafter activityslowly declined as KCI concentrations increased further. Key words: Guard cell protoplasts, phosphoenolpyruvate carboxylase 相似文献
14.
Potassium-Ammonium Uptake Interactions in Tobacco Seedlings 总被引:6,自引:0,他引:6
SCHERER HEINRICH W.; MACKOWN CHARLES T.; LEGGETT J. EVERETT 《Journal of experimental botany》1984,35(7):1060-1070
Short-term (< 12 h) uptake experiments were conducted with67-week-old tobacco (Nicotiana tabacum L. cv. Ky 14)seedlings to determine absorption interactions between K+ andNH4+. At equal solution concentrations (0.5 mol m3) netK+ uptake was inhibited 3035% by NH4+ and NH4+ uptakewas decreased 924%. Removal of NH4+ resulted in completerecovery in K+ uptake rate, but NH4+ uptake rate did not recoverwhen K+ was removed. In both cases, inhibition of the uptakerate of one cation saturated as the concentration of the othercation was increased up to 0.5 mol m3. The relative effectof K+-NH4+ interactions was not altered when Cl- was replacedwith SO42, but the magnitudes of the uptake rates wereless in the absence of Cl-. The Vmax for NH4+ uptake was reducedfrom 128 to 105 µmol g1 dry wt. h1 in thepresence of 0.5 mol m3 K+ and the Km for NH4+ doubledfrom 12 to 27 mmol m3 in the presence of K+. The resultsof these K+-NH4+ experiments are interpreted as mixed-noncompetitiveinteractions. However, an enhanced efflux of K+ coupled to NH4+influx via an antiporter cannot be ruled out as contributingto the decrease in net K+ uptake. Key words: Nicotiana tabacum, K+, NH4+, Uptake interactions 相似文献
15.
The utilization of inorganic carbon and role of the coccolithswere investigated in intact cells and protoplasts of a marineunicellular calcareous alga, Emiliania huxleyi. Protoplastswith high photosynthetic activity were obtained by artificialdecalcification with 50 mM MES-NaOH (pH5.5). (1) The kineticsof the photosynthetic evolution of O2 at various concentrationsof externally added NaHCO3 were the same for intact cells andprotoplasts, indicating that the kinetic properties with respectto dissolved inorganic carbon (DIC) were not affected by thepresence or absence of the coccoliths on the cell surface. Double-reciprocalplots and plots of the concentration of substrate divided byvelocity (s/v) against the concentration of substrate (s) werebiphasic in the case of both intact cells and protoplasts. TheCO2-utilization reaction was, therefore, considered to involvetwo processes with different values of Km and Vmax. From thekinetic analyses, Km and Vmax [µmoles O2 (ml PCV)1h1] were deduced to be 92 µM and 76.3 for a "low-Km"reaction and 4.1 mM and 252 for a "high-Km" reaction, respectively.(2) In short-term (40-min) experiments, time courses of thetotal uptake of 14C-DIC and the incorporation of 14C into acid-stableproducts of photosynthesis and the internal pool of DIC, determinedas acid-labile compounds, under CO2-limiting conditions (80µM) were very similar for intact cells and protoplasts.However, incorporation of 14C into CaCO3 apparently occurredmore slowly in protoplasts than in intact cells. (3) In longterm (24-h) experiments, patterns of incorporation of 14C werealmost same for intact cells and protoplasts, with the exceptionthat the amount of 14C incorporated into CaCO3 was much smallerin the former than the latter. The production of Ca14CO3 increasedduring the course of 10 h after a 4-h lag. However, after 10h the level of Ca14CCO3 started to decrease. The decrease wasaccompanied by an increase in 14C in the products of photosynthesis,suggesting that CaCO3 was reutilized for the photosyntheticfixation of CO2 and, therefore, that the coccoliths functionas sites of storage of DIC. However, the internal level of DICremained at the same level even after the supply of externalDIC has been almost completely depleted. (Received July 25, 1995; Accepted December 11, 1995) 相似文献
16.
The activity of plasma membrane H+-ATPase was measured withmembrane fragments of guard-cell protoplasts isolated from Viciafaba L. ATP hydrolytic activity was slightly inhibited by oligomycinand ammonium molybdate, and markedly inhibited by NO3and vanadate. In the presence of oligomycin, ammonium molybdateand NO3, the ATP-hydrolyzing activity was strongly inhibitedby vanadate. It was also inhibited by diethylstilbestrol (DES),p-chloromercuribenzoic acid (PCMB) and Ca2+, but slightly stimulatedby carbonyl cyanide m-chlorophenylhydrazone (CCCP). The acitivityhad higher specificity for ATP as a substrate than other phosphoricesters such as ADP, AMP, GTP and p-nitrophenylphosphate; theKm was 0.5 mM for ATP. The activity required Mg2+ but was notaffected by K+, and it was maximal around pH 6.8. When guard-cellprotoplasts were used instead of membrane fragments, the ATPaseactivity reached up to 800µmol Pi.(mg Chl)1.h1in the presence of lysolecithin. These results indicate thatthe guard cell has a high plasma membrane H+-ATPase activity. (Received December 23, 1986; Accepted April 28, 1987) 相似文献
17.
H+ translocation driven by NO3, NO2 and N2O reductionswith endogenous substrates in cells of Rhodopseudomonas sphaeroidesforma sp. denitrificans was investigated by the oxidant pulsemethod. Upon injection of nitrogenous oxides to anaerobic cellsin darkness, an alkaline transient in the external medium wasobserved, followed by acidification. The alkaline transientwas enhanced by carbonyl cyanide m-chlorophenylhydrazone. When a viologen dye was used as an electron donor in the presenceof 1 mM Af-ethylmaleimide and 0.1 mM 2-n-heptyl-4-hydroxyquinoline-N-oxideto preclude respiration-linked H+ extrusion, addition of KNO3,KNO2 and N2O caused only a rapid alkalinization. The H+ consumptionstoichiometries, H+/2e ratios for NO3 reductionto NO2, NO2 reduction to 1/2 N2O and N2O reductionto N2 were 1.90, 3.18 and 2.04, respectively.These values agreed well with the fact that all reductions ofnitrogenous oxides in denitrification occur on the periplasmicside of the cytoplasmic membrane. When corrected for H+ consumption in the periplasm, the H+ extrusionstoichiometries, H+/2e ratios with endogenous substratesin the presence of K+/valinomycin for NO3 reduction toNO2, NO2 reduction to 1/2 N2O and N2O reductionto N2 were 4.05, 4.95 and 6.01, respectively. (Received August 4, 1982; Accepted January 13, 1983) 相似文献
18.
Effect of Inorganic Phosphate on the Biosynthesis of Purine and Pyrimidine Nucleotides in Suspension-Cultured Cells of Catharanthus roseus 总被引:1,自引:0,他引:1
The levels of purine and pyrimidine nucleotides in suspensioncultures of Catharanthus roseus were determined 24 h after stationary-phasecells were transferred to fresh complete (+Pi)or phosphate-deficient (Pi) Murashige-Skoogmedium. The levels of ATP, GTP, UTP and CTP were from approx.3 to 5-fold greater in the cells grown in +Pimedium than in the cells grown in Pi medium.The levels of almost all other nucleotides were slightly higherin the cells in +Pi medium. The rates of de novoand salvage biosynthesis of purine and pyrimidine nucleotideswere estimated from the rates of incorporation of radioactivityfrom [14C]formate, [214C]glycine, NaH14CO3, [614C]orotate,[814C]adenine, [814C]adenosine, [214C]uraciland [214C]uridine. The results indicated that the activityof both the de novo and the salvage pathway was higher in thecells in +Pi medium than in the cells in Pimedium. The rate of degradation estimated from the rate of releaseof 14CO2 from labelled purines and pyrimidines indicated thatdegradation of uridine was significantly reduced in the cellsin +Pi medium, but no significant difference wasfound in the degradation of adenine, adenosine and uracil. Thepossible role of Pi in the control of the biosynthesis of nucleotidesand in the degradation of uridine is discussed. Catharanthus roseus, Madagascar periwinkle, suspension culture, inorganic phosphate, nucleotides, purines, pyrimidines, biosynthesis, degradation 相似文献
19.
L-Leucine Transport in Isolated Protoplasts of Vinca Suspension Culture: Characterization of Uptake 总被引:1,自引:0,他引:1
The uptake of L-leucine into Vinca protoplasts was studied undervarious conditions. The uptake was highly pH-dependent, withthe optimal pH between 3.0 and 4.0. The uptake was also energydependent, since azide, 2,4-dinitrophenol (DNP), carbonyl cyanidem-chlorophenyl hydrazone (CCCP), and iodoacetate inhibited theuptake. Oligomycin, N,N'-dicycIohexyI carbodiimide (DCCD) andvanadate, but not ouabain, inhibited the uptake, suggestingthat ATPase for H+ electrogenic extrusion was necessary to theuptake of L-leucine. The uptake showed stereospecificity, butwas partially inhibited by other L-amino acids. A kinetic studyof the uptake showed that the uptake was multiphasic with threesaturable phases and one unsaturable phase which occurred atconcentrations of L-leucine over 1 mM. The Km values of thethree affinity sites were 1.4 x 103 M, 1.3 x 104M, 4.3 x 105 M; the maximum velocity values were 3.3x 108, 4.5 x 109, 1.8 x 109 mol/10 min/4x 106 cells. (Received April 18, 1981; Accepted August 25, 1981) 相似文献
20.
Proton Transport and pH Control in Sinapis alba Root Hairs: A Study carried out with Double-Barrelled pH Micro-electrodes 总被引:3,自引:0,他引:3
Continuous measurements of cytoplasmic pH (pHc) in Sinapis roothairs have been carried out with double-barrelled pH-micro-electrodesin order to gain information on translocation of protons acrossthe plasmalemma and cytoplasmic pH control. (i) The cytoplasmicpH of Sinapis (733 ? 012, standard conditions)changes no more than 0.1 pHc, per pHo-unit, regardless of whethercyanide is present or not. (ii) Weak acids rapidly acidify pHcand hyperpolarize, while weak bases alkalize pHc and depolarizethe cells, (iii) 1.0 mol M,3 NaCN acidifies the cytoplasm by0.4 to 0.7 pH-units, but alkalizes the vacuole. (iv) 20 mmolm3 CCCP has no significant effect on pHc, if added atpH 9.6 or 7.2, but acidifies pHc by 1.3 units at pH 4.3. Inthe presence of CCCP, cyanide acidifies the cytoplasm, (v) Chloridetransiently acidifies pHc, while K+, Na+, and have no significant effects, (vi) Cytoplasmic buffer capacityforms a bell-shaped curve versus pHc with an optimum of about50 mol m3 H+pHc-unit. The modes of proton re-entry and the effects of active and passiveproton transport on cellular pH control are critically discussed.It is suggested that the proton leak, consisting of H+-cotransport(e.g. H+/Cl) rather than H+-uniport, is no threat topHc. The proton export pump, although itself reacting to changesin pHc, influences pHc only to a minor extent. It is concludedthat buffer capacity and membrane transport play moderate rolesin pHc control in Sinapis, while the interlocked H+-producingand -consuming reactions of cellular metabolism are the mainregulating factors. This makes pH control in Sinapis quite differentfrom bacterial and animal cells. Key words: Cytoplasmic pH, double-barrelled pH micro-electrode, pH control, proton transport, Sinapis 相似文献