共查询到20条相似文献,搜索用时 31 毫秒
1.
Schmitz K 《Plant physiology》1979,63(6):1003-1009
Discs from mature regions of Macrocystis blades picked up significantly more [32P]phosphate from the ambient medium than similar discs from young meristematic regions, and this uptake was higher in light than in darkness. Double-labeling experiments with NaH14CO3 and [32P]phosphate, using intact fronds as well as cut frond segments, indicated that 32P was translocated from mature blades to sink regions at velocities of 25 to 45 centimeters per hour, velocities comparable to 14C translocation velocity in the same material. There was a slight delay in transport of 32P which may be due to a delay in loading or to a high metabolism of 32P in the transporting channels. Histoautoradiography of stipe segments in the translocation pathway indicated that transport of label occurred in the peripheral parts of medulla. An analysis of 32P-labeled compounds in the fed blade and in the sieve tube sap, collected from basal cut ends of stipes, indicated major differences in labeling patterns. In the blade, a high proportion of 32P was recovered as inorganic phosphate and relatively small amounts were found in hexose mono- and diphosphates, UDPG and ATP. In the sieve tube sap, however, only a small amount of 32P was present as inorganic phosphate, a large proportion was found in hexose mono- and diphosphates, and appreciable amounts were present in ATP and UDPG. 相似文献
2.
Acid-soluble phosphate esters were determined in extracts of plant material after a 24 h exposure of the roots of intact alfalfa plants to nutrient media labelled with32Pi. Similar phosphate ester patterns were found in 2-, 3-, 8-, and 9-week-old plants with the exception of Gra-P which could be detected only in shoot extracts. However, phosphate ester levels differed in the shoots and roots. Whereas Fru-P2, Glc-6-P, Fru-6-P, and adenine nucleotides were more abundant in the shoots, Grn-P and P-choline levels were higher in the roots. Certain differences in the levels of P-esters were also recorded between plants susceptible and resistant to bacterial wilt. 相似文献
3.
A new method has been developed for prelabeling tissue ATP pools with 32P inorganic phosphate (Pi) and for the subsequent isolation of [32P]cAMP and [32P]ATP. The new method of prelabeling eliminates the need to separate trace amounts of radioactive cAMP from radioactive breakdown products of adenine formed in tissues prelabeled with [3H]- or [14C]adenine. The effect of epinephrine to increase [32P]cAMP levels in rat ventral prostate tissue fragments has been studied in terms of increase in the ratio of [32P]cAMP/[32P]ATP in the absence and presence of various phosphodiesterase inhibitors. Tissue prelabeling with 32Pi labels GTP as well as ATP (and other nucleoside triphosphates); thus the method lends itself to the isolation of [32P]cGMP as well as [32P]cAMP from the same tissue sample. 相似文献
4.
We describe a method for studying the phosphorylation of the S6 ribosomal protein in intact cells. The procedure has the advantage of using few cells, little 32Pi, and by using an air-driven centrifuge, many samples can be processed in a short time. Metabolically labeling the ribosomes with [3H]uridine before the experiment provides a measure of ribosome yield. The amount of 32Pi incorporated into proteins other than S6, which cosediment with the ribosomes, increases by the same amount as the specific activity of [32P]ATP increases, when the cells are stimulated by prostaglandin F2α, insulin, epidermal, or fibroblast growth factor, or serum; whereas the 32Pi incorporated into S6 increases by a factor greater than the increase in the specific activity of [32P]ATP. We show that the phosphate on S6 turns over at least as rapidly as does the phosphate on ATP. This last observation allows us to use a procedure, which we have outlined for determining the absolute amount of phosphate added to S6 due to a stimulus. 相似文献
5.
John W. Runcie Raymond J. Ritchie Anthony W.D. Larkum 《Journal of applied phycology》2004,16(3):181-194
Uptake, assimilation and compartmentation of phosphate were studied in the opportunist green macroalgaUlva lactucaand the estuarine red algal epiphyteCatenella nipae. The Michaelis–Menten model was used to describe uptake rates of inorganic phosphate (Pi) at different concentrations. Maximum uptake rates (V
max) of P-starved material exceededV
maxof P-enriched material; this difference was greater forC. nipae. Uptake and allocation of phosphorus (P) to internal pools was measured using trichloroacetic acid (TCA) extracts and32P. Both species demonstrated similar assimilation paths: when P-enriched, most32P accumulated as free phosphate. When unenriched,32P was rapidly assimilated into the TCA-insoluble pool.C. nipaeconsistently assimilated more32P into this pool thanU. lactuca, indicatingC. nipaehas a greater P-storage capacity. In both species,32P release data showed two internal compartments with very different biological half-lives. The rapidly exchanging compartment had a short half-life of 2 to 12 min, while the slowly exchanging compartment had a much longer half-life of 12 days in P-starvedC. nipaeor 4 days in P-starvedU. lactuca. In both species, the slowly exchanging compartment accounted for more than 90% of total tissue.U. lactucaandC. nipaeresponded differently to high external Pi.U. lactucarapidly took up Pi, transferring this Piinto tissue phosphate and TCA-soluble P in a few hours (90% of total P).C. nipaetook up Piat lower rates and stored much of this P in less mobile TCA-insoluble forms. Long-term storage of refractory forms of P makesC. nipaea useful bioindicator of the prevailing conditions of Piavailability over at least the previous 7 days, whereas the P-status ofU.lactucamay reflect conditions over no more than the previous few hours or days.C. nipaeis a more useful bioindicator for P status of estuarine and marine waters thanU. lactuca. 相似文献
6.
Upon subcellular fractionation of (murine) Friend erythroleukaemic cells (FELCs), purified plasma membranes were identified by their high enrichment in specific marker enzymes and typical plasma membrane lipids. When FELCs were incubated for short periods with 32Pi before cell fractionation, the lipid-bound radioactivity was almost exclusively present in phosphatidylinositol-4-phosphate (DPI) and phosphatidylinositol-4,5-bisphosphate (TPI), and its distribution closely matched that of the plasma membrane markers. In addition, purified plasma membranes actively incorporated 32P from [γ-32P]ATP into polyphosphoinositides, and the specific activities of the involved kinases were again mostly enriched in the plasma membrane fraction. 相似文献
7.
Cholesterol ester-storage granules were isolated from luteinized rat ovary and rabbit ovarian interstitial tissue by centrifugal flotation and were investigated with regard to their structure and function. Cholesterol ester, protein, phospholipid and unesterified cholesterol accounted for the dry weight of granules from luteinized rat ovary. The protein and the phospholipid were resistant to removal by washing. Substrate specificities of nucleotide phosphatase and specific radioactivities of lipid-soluble P (determined after administration of [32P]Pi in vivo) were the same in granules and in a microsomal fraction from the same tissue. After administration of [32P]Pi in vivo, luteinizing hormone increased the specific radioactivity of lipid-soluble P in granules, mitochondria and the microsomal fraction. Since granules did not swell in hypo-osmotic media, whereas microsomal particles did, it is suggested that adherent phospholipid and protein in granule suspensions is unlikely to result from contamination with endoplasmic reticulum. Luteinizing hormone administered in vivo increased the phospholipid and unesterified cholesterol contents of isolated granules relative to their cholesterol ester content, and also tended to raise their protein content. This treatment decreased the ability of isolated granules to act as a substrate for cholesterol esterase in vitro and increased the activity of cholesterol esterase. Cycloheximide in vivo also raised the unesterified cholesterol/cholesterol ester ratio of isolated granules, and when administered with luteinizing hormone acted synergistically to bring about a further increase. These results are considered compatible with evidence obtained by microscopy which suggests that granules may be surrounded by a membrane, that they arise by pinching off from the endoplasmic reticulum, and that they shrink on trophic stimulation of the tissue. 相似文献
8.
Beef-heart mitochondrial F1F0-ATP synthase contained six molecules of bound inorganic phosphate (Pi). This phosphate exchanged completely with exogenous 32Pi when the enzyme was exposed to 30% (v/v) dimethyl sulfoxide (DMSO) and then returned to a DMSO-free buffer (Beharry and Bragg 2001). Only two molecules were replaced by 32Pi when the enzyme was not pretreated with DMSO. These two molecules of 32Pi were not displaced from the enzyme by the treatment with 1 mM ATP. Similarly, two molecules of bound 32Pi remained on the DMSO-pretreated enzyme following addition of ATP, that is, four molecules of 32Pi were displaced by ATP. The ATP-resistant 32Pi was removed from the enzyme by pyrophosphate. It is proposed that these molecules of 32Pi are bound at an unfilled adenine nucleotide-binding noncatalytic site on the enzyme. Brief exposure of the enzyme loaded with two molecules of 32Pi to DMSO, followed by removal of the DMSO, resulted in the loss of the bound 32Pi and in the formation of two molecules of bound ATP from exogenous ADP. A third catalytic site on the enzyme was occupied by ATP, which could undergo a Pi ATP exchange reaction with bound Pi The presence of two catalytic sites containing bound Pi is consistent with the X-ray crystallographic structure of F1 (Bianchet, et al., 1998). Thus, five of the six molecules of bound Pi were accounted for. Three molecules of bound Pi were at catalytic sites and participated in ATP synthesis or Pi ATP exchange. Two other molecules of bound Pi were present at a noncatalytic adenine nucleotide-binding site. The location and role of the remaining molecule of bound Pi remains to be established. We were unable to demonstrate, using chemical modification of sulfhydryl groups by iodoacetic acid, any gross difference in the conformation of F1F0 in DMSO-containing compared with DMSO-free buffers. 相似文献
9.
A suspension culture of Catharanthus roseus has been used to study the permeability of cell membranes after treatment with various concentrations of a permeabilizing agent (DMSO). The uptake and release (after permeabilization) of inorganic phosphate (Pi) by cells have been investigated by 32P radiotracer and non-invasive phosphorus-31 NMR experiments. These studies have demonstrated that measurements of the Pi-efflux from plant cells provide a reliable measure of the permeability of the tonoplast.Abbreviations DMSO
dimethylsulfoxide
- NMR
nuclear magnetic resonance
- Pi
inorgainic phosphate 相似文献
10.
1. Slices of mouse brain grey matter were incubated with [32P]phosphate and [1-14C]acetate. Doubly labelled phospholipids were extracted from subcellular fractions prepared from the slices in a mixture of metabolic inhibitors, under conditions where there was negligible change in radioactive labelling during the preparation. Two tissue fractions were studied in detail; one contained a high proportion of mitochondria and the other was mainly microsomal. 2. In all tissue fractions the highest incorporations of both [32P]phosphate and [1-14C]acetate occurred into phosphatidylcholine. 3. After incubation for 1hr., the 32P/14C ratios for phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid in the mitochondrial fraction were similar to those in the microsomal fraction. 4. The 32P/14C ratios were similar in phosphatidylcholine and phosphatidylethanolamine and much lower than those in phosphatidic acid and phosphatidylinositol. 相似文献
11.
The effects of starvation and subsequent addition of phosphate-containing medium on the phosphate metabolic intermediates were studied by 31P-NMR spectroscope of perchloric acid extracts and intact cells of Heterosigma akashiwo (Hada) Hada. When orthophosphate in the medium was completely depleted the medium was enriched with orthophosphate (4.5 μM). In the phosphate starved condition, the P cell quota was 76 fmol·cell−1 and the major components of phosphate intermediates were phosphodiester, sugar phosphate and orthophosphate (Pi). After addition of Pi, rapid uptake of Pi was observed and the P cell quota increased to 108 fmol·cell−1 in 2 h, 134 fmol·cell−1 in 5 h and 222 fmol·cell−1 in 1 day after addition of phosphate. The 31P-NMR spectrum indicated that a major portion of P was stored as polyphosphate, in which the average chain length of polyphosphate increased from 10 to 20 phosphate residues in one day after addition of Pi. 相似文献
12.
Stimulation of the incorporation of 32Pi and myo-(2-3H)inositol into the phosphoinositides in polymorphonuclear leukocytes during phagocytosis 总被引:4,自引:0,他引:4
The effect of phagocytosis on the incorporation of 32Pi and myo-[2-3H]inositol into the phosphoinositides (phosphatidylinositol, diphosphoinositide, and triphosphoinositide) by polymorphonuclear leukocytes from guinea pig peritoneal exudates has been studied. The results show that phagocytosis enhanced the incorporation of 32Pi and myo-[2-3H]inositol into all three inositides in polymorphonuclear leukocytes. Pulse-chase experiments revealed that phagocytosis did not stimulate the loss of the label from the inositides. The findings indicate that the increased radioactivity of the phosphoinositides in polymorphonuclear leukocytes during phagocytosis is due to a greater rate of synthesis of these phospholipids at the time of labeling, rather than due to an increase in the rate of their turnover. 相似文献
13.
Lewis D. Dove 《Planta》1969,86(1):1-9
Summary Root systems from plants grown in nutrient solution were exposed to air and either transferred to fresh nutrient solution containing 32P-labeled phosphate or placed in a psychrometer to determine their water potential. The amount of 32P absorbed by maize and soybean roots in the hour following their exposure to air was proportional to their water potential at the time they were transferred. Some cells, probably located in the stele, were more resistant to moisture stress than others. Absorption of 32P by all cells was severely inhibited by water potentials below-12 to-15 bars. Nearly normal amounts of the radioisotope and total phosphate were absorbed within 72 hr following root exposure of 4 of 5 species of detopped plants; some phosphorus was lost to the nutrient solution. Uptake of 32P by passive processes was increased slightly by exposure of roots of intact maize plants to air, but the increase did not compensate for the substantial reduction in actively-absorbed 32P. 相似文献
14.
J.W. Herzig J.W. Peterson J.C. Rüegg R.J. Solaro 《Biochimica et Biophysica Acta (BBA)/General Subjects》1981,672(2):191-196
Tension development, immediate stiffness and ATPase of chemically skinned myocardial strips were measured in solutions with varying concentrations of phosphate (Pi) or vanadate (predominantly H2VO4? at pH 7) ion. Vanadate and Pi decreased stiffness in proportion to tension. The results show that, like Pi, vanadate accelerates the turnover rate of cross-bridges, but is effective at about 1/500 the concentration required for the Pi effect. Both Pi and vanadate increased the energy cost of isometric tension maintenance (that is, the ratio of ATPase to tension) and increased the velocity of delayed tension development following quick stretch of the chemically skinned myocardial strips. The results also show that changes in the rate of rise of delayed tension during stretch activation probably reflect changes in the kinetics of the biochemical cycle of the cross-bridges. 相似文献
15.
Inorganic phosphate labelled with 32P was applied to giant axons excised from squid (Loligo pealeii) by addition of 32Pi to the bathing solution, by injection into the axon, or by addition to axoplasm which had been separated from the sheath. The preparations were kept at 10 to 25° for various times up to 4 hr. When 32Pi was supplied by way of the bathing solution, axoplasm and sheath were usually separated at the end of incubation before extraction of the lipids. Lipids were extracted with chloroform-methanol and resolved by paper chromatography. The lipids which became labelled appeared to be the same in sheath and axoplasm. They were identified by cochromatography with known lipids and by chromatography of products formed from them by mild alkaline hydrolysis. They included phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, and probably somelysophosphatidylethanolamine. Some labelled components remained unidentified. Phosphatidylcholine was apparently present, but did not become significantly labelled either in sheath or in axoplasm, or in a squid's stellate ganglion. There was no evidence that separation from the sheath impaired the capacity of the axoplasm for lipid synthesis. 相似文献
16.
Nicholas J. Hoogenraad 《Archives of biochemistry and biophysics》1974,161(1):76-82
The reaction mechanism of aspartate transcarbamylase from mouse spleen has been determined, using steady-state kinetics, isotope-exchange experiments, inhibition studies with a transition-state analog, and product-inhibition studies. Intersecting reciprocal plots obtained when one substrate was varied against different concentrations of the second substrate indicate that the mechanism is sequential. The transition-state analog, N-(phosphonacetyl)-l-aspartate, was a powerful inhibitor of aspartate transcarbamylase, with an inhibition constant (Ki) of 2.6 × 10?8m at 37 °C and pH 7.4 in 0.05 m Na HEPES buffer. PALA gave competitive inhibition with carbamyl phosphate and noncompetitive inhibition with l-aspartate, indicating that carbamyl phosphate must bind before aspartate for catalysis to occur. A ping-pong mechanism in which carbamyl phosphate binds first was excluded by isotope-exchange experiments, since [32P]inorganic phosphate was not incorporated into carbamyl phosphate in the absence of aspartate. Product-inhibition studies showed that only inorganic phosphate and carbamyl phosphate gave a competitive pattern; all other combinations of substrate and product gave noncompetitive inhibition patterns when incubations were carried out at subsaturating concentrations of the second substrate. These inhibition patterns showed that carbamyl phosphate binds first, aspartate binds second, carbamyl aspartate dissociates first, and phosphate dissociates second. 相似文献
17.
Summary Resting cells ofStaphylococcus aureus displayed a phosphate (Pi) exchange that was induced by growth with glucose 6-phosphate (G6P) orsn-glycerol 3-phosphate (G3P). Pi-loaded membrane vesicles from these cells accumulated32Pi, 2-deoxyglucose 6-phosphate (2DG6P) or G3P by an electroneutral exchange that required no external source of energy. On the other hand, when vesicles were loaded with morpholinopropane sulfonic acid (MOPS), only transport of32Pi (andl-histidine) was observed, and in that case transport depended on addition of an oxidizable substrate (dl-lactate). In such MOPS-loaded vesicles, accumulation of the organic phosphates, 2DG6P and G3P, could not be observed until vesicles were preincubated with both Pi anddl-lactate to establish an internal pool of Pi. Thistrans effect demonstrates that movement of 2DG6P or G3P is based on an antiport (exchange) with internal Pi.Reconstitution of membrane protein allowed a quantitative analysis of Pi-linked exchange. Pi-loaded proteoliposomes and membrane vesicles had comparable activities for the homologous32PiPi exchange (K
i's of 2.2 and 1.4mm;V
max's of 180 and 83 nmol Pi/min per mg protein), indicating that the exchange reaction was recovered intact in the artificial system. Other work showed that heterologous exchange from either G6P- or G3P-grown cells had a preference for 2DG6P (K
i=27 m) over G3P (K
i=1.3mm) and Pi (K
i=2.2mm), suggesting that the same antiporter was induced in both cases. We conclude that32PiPi exchange exhibited by resting cells reflects operation of an antiporter with high specificity for sugar 6-phosphate. In this respect, Pi-linked antiport inS. aureus resembles other examples in a newly described family of bacterial transporters that use anion exchange as the molecular basis of solute transport. 相似文献
18.
Phosphorylation of ribosomal proteins induced by auxins in maize embryonic tissues 总被引:4,自引:2,他引:2
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The effect of auxin on ribosomal protein phosphorylation of germinating maize (Zea mays) tissues was investigated. Two-dimensional gel electrophoresis and autoradiography of [32P] ribosomal protein patterns for natural and synthetic auxin-treated tissues were performed. Both the rate of 32P incorporation and the electrophoretic patterns were dependent on 32P pulse length, suggesting that active protein phosphorylation-dephosphorylation occurred in small and large subunit proteins, in control as well as in auxin-treated tissues. The effect of ribosomal protein phosphorylation on in vitro translation was tested. Measurements of poly(U) translation rates as a function of ribosome concentration provided apparent Km values significantly different for auxin-treated and nontreated tissues. These findings suggest that auxin might exert some kind of translational control by regulating the phosphorylated status of ribosomal proteins. 相似文献
19.
The effects of starvation and subsequent addition of phosphate-containing medium on the phosphate metabolic intermediates were studied by 31P-NMR spectroscopy of perchloric acid extracts and intact cells of Heterosigma akashiwo (Hada) hada. When orthophosphate in the medium was completely depleted the medium was enriched with orthophosphate (4.5 μM). In the phosphate starved condition, the P cell quota was 76 fmol-cell−1 and the major components of phosphate intermediates were phosphodiester, sugar phosphate and orthophosphate (Pi) After addition of Pi' rapid uptake of Pi was observed and the P cell quota increased to 108 fmol. cell−1 in 2 h, 134 fmol. cell−1 in 5 h and 222 fmol. cell−1 in 1 day after addition of phosphate. The 31P-NMR spectrum indicated that a major portion of P was stored as polyphosphate, in which the average chain length of polyphosphate increased from 10 to 20 phosphate residues in one day after addition of Pi- 相似文献
20.
Metabolism of 3- and 4-phosphorylated phosphatidylinositols in stomatal guard cells of Commelina communis L. 总被引:2,自引:0,他引:2
Paroo N. Parmar Charles A. Brearley 《The Plant journal : for cell and molecular biology》1995,8(3):425-433
Within the plant kingdom the stomatal guard cell is presented as a model system of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-mediated signal transduction. Despite this it is only recently that the phosphoinositide components of animal signal transduction pathways have been identified in stomatal guard cells. Interestingly, stomatal guard cells contain both 3- and 4-phosphorylated phosphatidylinositols though their relative contributions to signalling remain undefined. An appraisal of the routes of synthesis and rates of turnover of these phosphatidylinositols would appear timely as the in vivo biosynthesis of these components is a much neglected facet of the phosphoinositide-mediated signalling paradigm as purported to apply to plants. A non-equilibrium [32P]Pi labelling strategy and enzymic and chemical dissection of labelled phosphatidylinositols have been used to address not only the route of synthesis but also the rates of turnover of phosphatidylinositols in stomatal guard cells of Commelina communis L. The specific activity of the ATP pool of isolated guard cells was found to increase over a 4 h period when labelled from [32P]Pi. In separate experiments, isolated guard cells were labelled over a 40–240 min period, their lipids extracted, deacylated and resolved by HPLC. Glycerophosphoinositol phosphate (GroPInsP) and glycerophosphoinositol bisphosphate (GroPInsP2) peaks were desalted and enzymically cleaved with alkaline phosphatase and human erythrocyte ghosts, respectively. The monoester phosphate in phosphatidylinositol 4-monophosphate (PtdIns4P) accounted for 90–97% of the [32P]Pi label while the 4- and 5-monoester phosphates of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] accounted for typically 39% and 61% respectively. Therefore, the evidence is consistent with synthesis of PtdIns(4,5)P2 by successive 4- and 5-phosphorylation of phosphatidylinositol (PtdIns). This study therefore represents the first report of the pathway of the synthesis of 4- and 5-phosphorylated phosphatidylinositols in a single defined hormone-responsive plant cell type. The monoester phosphate in phosphatidylinositol 3-monophosphate (PtdIns3P) accounted for 83–95% of the 32P label. It was not possible, however, to determine the route of synthesis of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2] owing to the rapid attainment of equilibrium between the 3- and 4-monoester phosphates of PtdIns(3,4)P2, each containing approximately 50% of the label at just 40 min of labelling. Turnover of PtdIns3P was quicker than that of PtdIns4P. Similarly, turnover of PtdIns(3,4)P2 was quicker than that of PtdIns(4,5)P2, and in mass terms PtdIns(3,4)P2 appeared to predominate over PtdIns(4,5)P2. By analogy with animal systems, in which signalling molecules such as PtdIns(4,5)P2 show considerable basal turnover, the evidence presented is consistent with signalling roles for PtdIns3P and PtdIns(3,4)P2 in addition to those previously indicated for PtdIns(4,5)P2 in stomatal guard cells. 相似文献