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1.
Phospholipid-Dependence of Plant UDP-Glucose Sterol beta-d-Glucosyl Transferase : IV. Reconstitution into Small Unilamellar Vesicles
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The phospholipid dependence of the UDP-glucose sterol glucosyl transferase (UDPG-SGTase) from maize coleoptiles was previously demonstrated using the partially purified and highly delipidated enzyme, in the presence of the detergent Triton X-100 (P Ullmann, P Bouvier-Navé, P Benveniste [1987] Plant Physiol 85: 51-55). We now report the reconstitution of the enzyme activity into unilamellar lipid vesicles. This was achieved by adding phospholipids, sterols and β-octylglucoside to the solubilized enzyme and passing the mixture through Sephadex G-50. The treatment led to almost complete removal of the detergents. The incorporation of UDPG-SGTase in the lipid vesicles was demonstrated by (a) coelution of the enzyme activity with the labeled lipid vesicles (average diameter: 260Å) on a Sephacryl S-1000 column and (b) flotation experiments on metrizamide density gradients. Release of dithiobis-(2-nitro-benzoic acid) (DTNB) from DTNB-preloaded vesicles was very slow, indicating good membrane integrity of the vesicles. Treatment of the intact vesicles with the nonpermeant reagent p-chloro-mercuribenzene sulfonate led to more than 95% inactivation of the total enzyme activity, i.e. the activity measured in the presence of Triton X-100 at permeabilizing concentration. This suggests an outward orientation for the active site of the enzyme. Finally, the enzyme was incorporated into vesicles of various phospholipid compositions and the kinetic parameters of the reactions were determined. Our results clearly show that the reconstituted UDPG-SGTase activity is stimulated to a large extent by negatively charged phospholipids. 相似文献
2.
Purification and Characterization of a Soluble Phosphatidylinositol 4-Kinase from Carrot Suspension Culture Cells 总被引:1,自引:0,他引:1
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Previously we reported the presence of a soluble phosphatidylinositol 4-kinase (PI 4-Kinase) in carrot (Daucus carota L.) suspension culture cells (C.M. Okpodu, W. Gross, W.F. Boss [1990] Plant Physiol 93: S-63). We have purified the enzyme over 1000-fold using Q-Sepharose ion exchange, hydroxylapatite, and G-100 gel filtration column chromatography. The Mr of the enzyme was estimated to be 83,000 by gel filtration. PI 4-kinase activity was recovered after renaturation of the 80-kD region of polyacrylamide gels, and an 80-kD peptide cross-reacted with antibodies to the yeast 55-kD membrane-associated PI 4-kinase on western blots. The isolated lipid kinase phosphorylated PI but not lysophosphatidylinositol or phosphatidylinositol monophosphate. Maximal PI kinase activity occurred when the substrate was added as Triton X-100/PI mixed micelles at pH 8. The enzyme required divalent cations. At low concentrations (1-5 mM), Mn2+ was more effective than Mg2+ in increasing enzyme activity; however, maximal activity occurred at 25 to 40 mM Mg2+. Calcium from 0.01 [mu]M to 1 mM had no effect on the enzyme activity. The Km of the enzyme for ATP was estimated to be between 400 and 463 [mu]M. The enzyme was inhibited by adenosine (100 [mu]M); however, ADP (up to 100 [mu]M) had no effect on the activity. The biochemical characteristics of the carrot soluble PI 4-kinase are compared with the previously reported PI 4-kinases from animals and yeast. 相似文献
3.
Al-Induced, 51-Kilodalton,Membrane-Bound Proteins Are Associated with Resistance to Al in a Segregating Population of Wheat 总被引:4,自引:1,他引:3
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Incorporation of 35S into protein is reduced by exposure to Al in wheat (Triticum aestivum), but the effects are genotype-specific. Exposure to 10 to 75 [mu]M Al had little effect on 35S incorporation into total protein, nuclear and mitochondrial protein, microsomal protein, and cytosolic protein in the Al-resistant cultivar PT741. In contrast, 10 [mu]M Al reduced incorporation by 21 to 38% in the Al-sensitive cultivar Katepwa, with effects becoming more pronounced (31-62%) as concentrations of Al increased. We previously reported that a pair of 51-kD membrane-bound proteins accumulated in root tips of PT741 under conditions of Al stress. We now report that the 51-kD band is labeled with 35S after 24 h of exposure to 75 [mu]M Al. The specific induction of the 51-kD band in PT741 suggested a potential role of one or both of these proteins in mediating resistance to Al. Therefore, we analyzed their expression in single plants from an F2 population arising from a cross between the PT741 and Katepwa cultivars. Accumulation of 1,3-[beta]-glucans (callose) in root tips after 24 h of exposure to 100 [mu]M Al indicated that this population segregated for Al resistance in about a 3:1 ratio. A close correlation between resistance to Al (low callose content of root tips) and accumulation of the 51-kD band was observed, indicating that at least one of these proteins cosegregates with the Al-resistance phenotype. As a first step in identifying a possible function, we have demonstrated that the 51-kD band is most clearly associated with the tonoplast. Whereas Al has been reported to stimulate the activity of the tonoplast H+-ATPase and H+-PPase, antibodies raised against these proteins did not cross-react with the 51-kD band. Efforts are now under way to purify this protein from tonoplast-enriched fractions. 相似文献
4.
beta]-Glucan Synthesis in the Cotton Fiber (III. Identification of UDP-Glucose-Binding Subunits of [beta]-Glucan Synthases by Photoaffinity Labeling with [[beta]-32P]5[prime]-N3-UDP-Glucose 总被引:4,自引:3,他引:1
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Using differential product entrapment and photolabeling under specifying conditions, we identifIed a 37-kD polypeptide as the best candidate among the UDP-glucose-binding polypeptides for the catalytic subunit of cotton (Gossypium hirsutum) cellulose synthase. This polypeptide is enriched by entrapment under conditions favoring [beta]-1,4-glucan synthesis, and it is magnesium dependent and sensitive to unlabeled UDP-glucose. A 52-kD polypeptide was identified as the most likely candidate for the catalytic subunit of [beta]-1,3-glucan synthase because this polypeptide is the most abundant protein in the entrapment fraction obtained under conditions favoring [beta]-1,3-glucan synthesis, is coincident with [beta]-1,3-glucan synthase activity, and is calcium dependent. The possible involvement of other polypeptides in the synthesis of [beta]-1,3-glucan is discussed. 相似文献
5.
Purification and Characterization of Acetyl-Coenzyme A Carboxylase from Diclofop-Resistant and -Susceptible Lolium multiflorum 总被引:1,自引:0,他引:1
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Acetyl-coenzyme A carboxylase (ACCase) was purified >100-fold (specific activity 3.5 units mg-1) from leaf tissue of diclofopresistant and -susceptible biotypes of Lolium multiflorum. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified fractions from both biotypes contained a single 206-kD biotinylated polypeptide. The molecular mass of the native enzyme from both biotypes was approximately 520 kD. In some cases the native dimer from both biotypes dissociated during gel filtration to form a subunit of approximately 224 kD. The inclusion of 5% (w/v) polyethylene glycol 3350 (PEG) in the elution buffer prevented this dissociation. Steady-state substrate kinetics were analyzed in both the presence and absence of 5% PEG. For ACCase from both biotypes, addition of PEG increased the velocity 22% and decreased the apparent Km values for acetyl-coenzyme A (acetyl-CoA), but increased the Km values for bicarbonate and ATP. In the presence of PEG, the Km values for bicarbonate and ATP were approximately 35% higher for the enzyme from the susceptible biotype compared with the resistant enzyme. In the absence of PEG, no differences in apparent Km values were observed for the enzymes from the two biotypes. Inhibition constants (Ki app) were determined for CoA, malonyl-CoA, and diclofop. CoA was an S-hyperbolic (slope replots)-I-hyperbolic (intercept replots) noncompetitive inhibitor with respect to acetyl-CoA, with Ki app values of 711 and 795 [mu]M for enzymes from the resistant and susceptible biotypes, respectively. Malonyl-CoA competitively inhibited both enzymes (versus acetyl-CoA) with Ki app values of 140 and 104 [mu]M for ACCase from resistant and susceptible biotypes, respectively. Diclofop was a linear noncompetitive inhibitor of ACCase from the susceptible biotype and a nonlinear, or S-hyperbolic-I-hyperbolic, noncompetitive inhibitor of ACCase from the resistant biotype. For ACCase from the susceptible biotype the slope (Kis) and intercept (Kii) inhibition constants for diclofop versus acetyl-CoA were 0.08 and 0.44 [mu]M, respectively. ACCase from the resistant biotype had a Ki app value of 6.5 [mu]M. At a subsaturating acetyl-CoA concentration of 50 [mu]M, the Hill coefficients for diclofop binding were 0.61 and 1.2 for ACCase from the resistant and susceptible biotypes, respectively. The Hill coefficients for diclofop binding and the inhibitor replots suggest that the resistant form of ACCase exhibits negative cooperativity in binding diclofop. However, the possibility that the nonlinear inhibition of ACCase activity by diclofop in the enzyme fraction isolated from the resistant biotype is due to the presence of both resistant and susceptible forms of ACCase cannot be excluded. 相似文献
6.
Induction of UDP-Glucose:Salicylic Acid Glucosyltransferase Activity in Tobacco Mosaic Virus-Inoculated Tobacco (Nicotiana tabacum) Leaves 总被引:9,自引:4,他引:5
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Salicylic acid (SA) is a putative signal that activates plant resistance to pathogens. SA levels increase systemically following the hypersensitive response produced by tobacco mosaic virus (TMV) inoculation of tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaves. The SA increase in the inoculated leaf coincided with the appearance of a [beta]-glucosidase-hydrolyzable SA conjugate identified as [beta]-O-D-glucosylsalicylic acid (GSA). SA and GSA accumulation in the TMV-inoculated leaf paralleled the increase in the activity of a UDP-glucose:salicylic acid 3-O-glucosyltransferase (EC 2.4.1.35) ([beta]-GTase) capable of converting SA to GSA. Healthy tissues had constitutive [beta]-GTase activity of 0.076 milliunits g-1 fresh weight. This activity started to increase 48 h after TMV inoculation, reaching its maximum (6.7-fold induction over the basal levels) 72 h after TMV inoculation. No significant GSA or elevated [beta]-Gtase activity could be detected in the healthy leaf immediately above the TMV-inoculated leaf. The effect of TMV inoculation on the [beta]-GTase and GSA accumulation could be duplicated by infiltrating tobacco leaf discs with SA at the levels naturally produced in TMV-inoculated leaves (2.7-27.0 [mu]g g-1 fresh weight). Pretreatment of leaf discs with the protein synthesis inhibitor cycloheximide inhibited the induction of [beta]-GTase by SA and prevented the formation of GSA. Of 12 analogs of SA tested, only 2,6-dihydroxybenzoic acid induced [beta]-GTase activity. 相似文献
7.
Phosphoribosylpyrophosphate synthetase (PRS; EC 2.7.6.1) from Hevea brasiliensis Mull. Arg. latex was located in the cytosol. After purification, its apparent molecular weight under nondenaturing conditions was estimated at 200,000 [plus or minus] 10,000; a single band at 57,000 [plus or minus] 3,000 was detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme seemed to be a homotetramer. Its affinity constants were estimated at 200 [plus or minus] 30 [mu]M for adenosine triphosphate and 40 [plus or minus] 2 [mu]M for ribose-5-phosphate. The purified enzyme proved to be functional in a paraphysiological medium (cytosol deproteinized by ultrafiltration). Optimum pH was 7.5 in buffer and 6.5 in a paraphysiological medium. No PRS activity was detected in the absence of the Mg2+ ion. Of the numerous compounds tested, only Mn2+, phosphoribosylpyrophosphate and inorganic phosphate affected the enzymatic reaction. Mn2+ (inhibitor constant = 20 [mu]M) and phosphoribosylpyrophosphate (inhibitor constant = 30 [mu]M) were inhibitors. PRS responded allosterically (Hill's coefficient = 2.3) to ribose-5-phosphate in the presence of a physiological concentration of inorganic phosphate (10 mM). These results are set in the physiological context of laticifers. 相似文献
8.
Purification and Characterization of Phosphoribosylpyrophosphate Synthetase from Rubber Tree Latex
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Phosphoribosylpyrophosphate synthetase (PRS; EC 2.7.6.1) from Hevea brasiliensis Mull. Arg. latex was located in the cytosol. After purification, its apparent molecular weight under nondenaturing conditions was estimated at 200,000 [plus or minus] 10,000; a single band at 57,000 [plus or minus] 3,000 was detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme seemed to be a homotetramer. Its affinity constants were estimated at 200 [plus or minus] 30 [mu]M for adenosine triphosphate and 40 [plus or minus] 2 [mu]M for ribose-5-phosphate. The purified enzyme proved to be functional in a paraphysiological medium (cytosol deproteinized by ultrafiltration). Optimum pH was 7.5 in buffer and 6.5 in a paraphysiological medium. No PRS activity was detected in the absence of the Mg2+ ion. Of the numerous compounds tested, only Mn2+, phosphoribosylpyrophosphate, and inorganic phosphate affected the enzymatic reaction. Mn2+ (inhibitor constant = 20 [mu]M) and phosphoribosylpyrophosphate (inhibitor constant = 30 [mu]M) were inhibitors. PRS responded allosterically (Hill's coefficient = 2.3) to ribulose-5-phosphate in the presence of a physiological concentration of inorganic phosphate (10 mM). These results are set in the physiological context of laticifers. 相似文献
9.
A Plasma Membrane-Type Ca2+-ATPase of 120 Kilodaltons on the Endoplasmic Reticulum from Carrot (Daucus carota) Cells (Properties of the Phosphorylated Intermediate) 总被引:5,自引:4,他引:1
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Cytosolic Ca2+ levels are regulated in part by Ca2+-pumping ATPases that export Ca2+ from the cytoplasm; however, the types and properties of Ca2+ pumps in plants are not well understood. We have characterized the kinetic properties of a 120-kD phosphoenzyme (PE) intermediate formed during the reaction cycle of a Ca2+-ATPase from suspension-cultured carrot (Daucus carota) cells. Only one Ca2+-dependent phosphoprotein was formed when carrot membrane vesicles were incubated with [[gamma]-32P]ATP (W.L. Hsieh, W.S. Pierce, and H. Sze [1991] Plant Physiol 97: 1535-1544). Formation of this 120-kD phosphoprotein was inhibited by vanadate, enhanced by La3+, and decreased by hydroxylamine, confirming its identification as an intermediate of a phosphorylated-type Ca2+-translocating ATPase. The 120-kD Ca2+-ATPase was most abundant in endoplasmic reticulum-enriched fractions, in which the Ca2+-ATPase was estimated to be 0.1% of membrane protein. Direct quantitation of Ca2+-dependent phosphoprotein was used to examine the kinetics of PE formation. PE formation exhibited a Km for Ca2+ of 1 to 2 [mu]M and a Km for ATP of 67 nM. Relative affinities of substrates, determined by competition experiments, were 0.075 [mu]M for ATP, 1 [mu]M for ADP, 100 [mu]M for ITP, and 250 [mu]M for GTP. Thapsigargin and cyclopiazonic acid, specific inhibitors of animal sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, had no effect on PE formation; erythrosin B inhibited with 50% inhibition at <0.1 [mu]M. Calmodulin (1 [mu]M) stimulated PE formation by 25%. The results indicate that the carrot 120-kD Ca2+-ATPase is similar but not identical to animal plasma membrane-type Ca2+- ATPase and yet is located on endomembranes, such as the endoplasmic reticulum. This type of Ca2+ pump may reside on the cortical endoplasmic reticulum, which is thought to play a major role in anchoring the cytoskeleton and in facilitating secretion. 相似文献
10.
Nitrate Reductase from the Marine Diatom Skeletonema costatum (Biochemical and Immunological Characterization) 总被引:3,自引:1,他引:2
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Assimilatory nitrate reductase (NR) was purified from the marine diatom Skeletonema costatum (clone Skel) using Cibacron blue-Sepharose affinity chromatography. The single-step purification scheme yielded a 103-fold purification of specific activity with an overall recovery of 40.8%. Only NADH-dependent NR activity (form EC 1.6.6.1) was observed in this species. Kinetic analysis revealed that this form had apparent Michaelis constants of 3.6 [mu]M for NADH and 295 [mu]M for NO3- when purified from cells grown in NO3--enriched seawater. The S. costatum NR exhibits a pH optimum of 7.4, a temperature optimum of 14[deg]C, and enzyme activity not sensitive to Mg2+ inhibition. The strong temperature dependence of NR activity in S. costatum may contribute to the seasonal and latitudinal distributions and abundances of this bloom-forming species. Chromatographically isolated NR was further purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, yielding a single polypeptide with an apparent molecular mass of 110 kD. The 110-kD polypeptide was used to generate polyclonal antibodies. The antiserum recognized a single 110-kD polypeptide in western blots of total proteins from S. costatum, as well as the native enzyme. Western blot analysis also revealed an antigenic similarity of NR from two additional diatom species, whereas no cross-reactivity was observed with NR from other phytoplankton taxa, including prymnesiophytes, dinoflagellate, cyanobacterium, and green alga. This result suggests a structural diversity of NR in phytoplankton and identifies the potential for development of taxon-specific NR antisera for ecological studies. 相似文献
11.
Saito T Abe H Yamane H Sakurai A Murofushi N Takio K Takahashi N Kamiya Y 《Plant physiology》1995,109(4):1239-1245
ent-Kaurene synthase B (KSB) was purified 291-fold from a crude enzyme preparation from endosperm of pumpkin (Cucurbita maxima L.). Separation of ent-kaurene synthase A and KSB was achieved by hydrophobic interaction chromatography. The fractions containing KSB activity were further purified by diethylaminoethyl, phenyl, and hydroxyapatite column chromatography. Using sodium dodecyl phosphate-polyacrylamide gel electrophoresis, the purest enzyme preparation showed a major band at an apparent molecular mass of 81 kD. The amount of protein in this band was correlated with KSB activity after diethylaminoethyl and hydroxyapatite chromatography. The N terminus of the 81-kD protein was blocked. Therefore, the protein was partially digested with protease and the amino acid sequences of the resulting major peptide fragments were analyzed. A polyclonal antibody was raised against a synthetic peptide based on the longest peptide fragment combined with a keyhole limpet hemocyanin. The antibody recognized only the 81-kD denatured protein and not the native KSB. The properties of KSB were examined using the phenyl-purified enzyme preparation. The Km value for copalyl pyrophosphate was 0.35 [mu]M, and the optimal pH was 6.8 to 7.5. The KSB activity required divalent cations such as Mg2+, Mn2+, and Co2+, whereas Cu2+, Ca2+, and Ba2+ inhibited the activity. 相似文献
12.
Phosphatidate Kinase,a Novel Enzyme in Phospholipid Metabolism (Purification,Subcellular Localization,and Occurrence in the Plant Kingdom) 总被引:2,自引:1,他引:2
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Microsomal membranes from suspension-cultured Catharanthus roseus cells possess an enzymic activity that catalyzes the ATP-dependent phosphorylation of phosphatidic acid (PA) to form diacylglycerol pyrophosphate (H. Behrbohm, J.B. Wissing [1993] FEBS Lett 315: 95-99). This enzyme activity, PA kinase, was purified and characterized. Plasma membranes, obtained from C. roseus microsomes by aqueous two-phase partitioning, were extracted, and PA kinase was purified 3200-fold by applying different chromatographic steps that resulted in a specific activity of about 10 [mu]mol min-1 mg-1. Sodium dodecyl sulfate-gel electrophoresis of the fractions obtained from the final chromatographic step revealed a 39-kD protein that correlated with the enzyme activity; PA kinase activity could be eluted from this protein band. Subcellular localization, investigated with C. roseus cells, showed that the activity was confined to membrane fractions, and at least 80% was associated with plasma membranes. The data revealed the same distribution within the cellular membranes of PA kinase as reported for diacylglycerol kinase, which is a typical plasma membrane-located enzyme. Furthermore, PA kinase activity was detected in the calli of 16 different plant species and in the different organs of C. roseus plants and obviously occurs ubiquitously in the plant kingdom. 相似文献
13.
Uridine Diphosphate Glucose Metabolism and Callose Synthesis in Cultured Pollen Tubes of Nicotiana alata Link et Otto 总被引:3,自引:1,他引:2
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Membrane preparations from cultured pollen tubes of Nicotiana alata Link et Otto contain a Ca2+ -independent (1-3)-[beta]-D-glucan (callose) synthase activity that has a low affinity for UDP-glucose, even when activated by treatment with trypsin (H. Schlupmann, A. Basic, S.M. Read [1993] Planta 191: 470-481). Therefore, we investigated whether UDP-glucose was a likely substrate for callose synthesis in actively growing pollen tubes. Deposition of (1-3)-[beta]-glucan occurred at a constant rate, 1.4 to 1.7 nmol glucose min-1, in tubes from 1 mg of pollen from 3 h after germination; however, the rate of incorporation of radioactivity from exogenous [14C]-sucrose into wall polymers was not constant, but increased until at least 8 h after germination, probably due to decreasing use of internal reserves. UDP-glucose was a prominent ultraviolet-absorbing metabolite in pollen-tube extracts, with 1.6 nmol present in tubes from 1 mg of pollen, giving a calculated cytoplasmic concentration of approximately 3.5 mM. Radioactivity from [14C]-sucrose was rapidly incorporated into sugar monophosphates and UDP-glucose by the growing tubes, consistent with a turnover time for UDP-glucose of less than 1 min; the specific radioactivity of extracted UDP-[14C]glucose was equal to that calculated from the rate of incorporation of [14C]sucrose into wall glucans. Large amounts of less metabolically active neutral sugars were also present. The rate of synthesis of (1-3)-[beta]-glucan by nontrypsin-treated pollen-tube membrane preparations incubated with 3.5 mM UDP-glucose and a [beta]-glucoside activator was slightly greater than the rate of deposition of (1-3)-[beta]-glucan by intact pollen tubes. These data are used to assess the physiological significance of proteolytic activation of pollen-tube callose synthase. 相似文献
14.
Sugar-Induced Increase of Calcium-Dependent Protein Kinases Associated with the Plasma Membrane in Leaf Tissues of Tobacco 总被引:9,自引:3,他引:6
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The sugar-inducible expression of genes for sporamin and [beta]-amylase in leaf explants of sweet potato (Ipomoea batatas) and that of a [beta]-glucuronidase-fusion gene, with the promoter of the gene for [beta]-amylase in leaves of tobacco (Nicotiana tabacum), requires Ca2+ signaling (M. Ohto, K. Hayashi, M. Isobe, K. Nakamura [1995] Plant J 7: 297-307), and it was inhibited by staurosporin and K252a, inhibitors of protein kinases. Autophosphorylation activities of several potential protein kinases in leaves of tobacco were significantly higher in younger leaves than in mature leaves. However, the autophosphorylation activities of these proteins in mature leaves, especially those of the major autophosphorylatable proteins with apparent molecular masses of 56 and 54 kD, increased upon treatment of leaf discs with a 0.3 M solution of sucrose, glucose, or fructose, did not increase with sorbitol or mannitol treatments, and the increase by sucrose was inhibited by cycloheximide. Autophosphorylation of the 56- and 54-kD protein in vitro was dependent on Ca2+ and inhibited by staurosporine, K-252a, and by W-7. These results suggest that they belong to the family of calcium-dependent protein kinases. They were concentrated in the plasma membrane fraction and were released from membrane vesicles by high salt or with sodium carbonate. The possible functions of these sugar-inducible calcium-dependent protein kinases associated with the plasma membrane are discussed. 相似文献
15.
Dependence of the Extent and Direction of Average Stomatal Response in Zea mays L. and Phaseolus vulgaris L. on the Frequency of Fluctuations in Environmental Stimuli 总被引:1,自引:1,他引:0
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Three-day-old seedlings of an Al-sensitive (Neepawa) and an Al-resistant (PT741) cultivar of Triticum aestivum were subjected to Al concentrations ranging from 0 to 100 [mu]M for 72 h. At 25 [mu]M Al, growth of roots was inhibited by 57% in the Al-sensitive cultivar, whereas root growth in the Al-resistant cultivar was unaffected. A concentration of 100 [mu]M Al was required to inhibit root growth of the Al-resistant cultivar by 50% and resulted in almost total inhibition of root growth in the sensitive cultivar. Cytoplasmic and microsomal membrane fractions were isolated from root tips (first 5 mm) and the adjacent 2-cm region of roots of both cultivars. When root cytoplasmic proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, no changes in polypeptide patterns were observed in response to Al stress. Analysis of microsomal membrane proteins revealed a band with an apparent molecular mass of 51 kD, which showed significant accumulation in the resistant cultivar following Al exposure. Two-dimensional gel analysis revealed that this band comprises two polypeptides, each of which is induced by exposure to Al. The response of the 51-kD band to a variety of experimental conditions was characterized to determine whether its pattern of accumulation was consistent with a possible role in Al resistance. Accumulation was significantly greater in root tips when compared to the rest of the root. When seedlings were subjected to Al concentrations ranging from 0 to 150 [mu]M, the proteins were evident at 25 [mu]M and were fully accumulated at 100 [mu]M. Time-course studies from 0 to 96 h indicated that full accumulation of the 51-kD band occurred within 24 h of initiation of Al stress. With subsequent removal of stress, the polypeptides gradually disappeared and were no longer visible after 72 h. When protein synthesis was inhibited by cycloheximide, the 51-kD band disappeared even when seedlings were maintained in Al-containing media. Other metals, including Cu, Zn, and Mn, failed to induce this band, and Cd and Ni resulted in its partial accumulation. These results indicate that synthesis of the 51-kD microsomal membrane proteins is specifically induced and maintained during Al stress in the Al-resistant cultivar, PT741. 相似文献
16.
Isolation and Characterization of a Protein Associated with Carotene Globules in the Alga Dunaliella bardawil
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The halotolerant alga Dunaliella bardawil accumulates very large amounts of [beta]-carotene when exposed to high light intensity. The accumulated [beta]-carotene is concentrated in small, oily globules within the chloroplast and has been suggested to protect the alga against photodamage by high irradiation (A. Ben-Amotz, A. Katz, M. Avron [1982] J Phycol 18:529-537;A. Ben-Amotz, M. Avron [1983] Plant Physiol 72: 593-597; A. Ben-Amotz, A. Shaish, M. Avron [1989] Plant Physiol 91: 1040-1043). A 38-kD protein was identified and purified from [beta]-carotene globules and was designated carotene globule protein (Cgp). Induction of Cgp occurs in parallel with [beta]-carotene accumulation in D. bardawil grown under different inductive conditions. Cgp is overproduced in a constitutive mutant strain that overproduces [beta]-carotene and is not detected in Dunaliella salina, a species that does not accumulate [beta]-carotene. Cgp production was not suppressed by norflurazon, an inhibitor of [beta]-carotene synthesis that leads to accumulation of the carotenoid precursor phytoene. Immunogold-labeling analysis by electron microscopy demonstrates that the protein is localized at the periphery of the globules. Proteolytic cleavage by trypsin enhances the coalescence and destruction of the globules, in parallel with Cgp disappearance. It is suggested that the function of Cgp is to stabilize the structure of the globules within the chloroplast. 相似文献
17.
Autophagy in Tobacco Suspension-Cultured Cells in Response to Sucrose Starvation 总被引:16,自引:2,他引:14
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The response of tobacco (Nicotiana tabacum) suspension-cultured cells (BY-2) to nutrient starvation was investigated. When the cells that were grown in Murashige-Skoog medium containing 3% (w/v) sucrose were transferred to the same medium without sucrose, 30 to 45% of the intracellular proteins were degraded in 2 d. An analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that proteins were degraded nonselectively. With the same treatment, protease activity in the cell, which was measured at pH 5.0 using fluorescein thiocarbamoyl-casein as a substrate, increased 3- to 7-fold after 1 d. When the cysteine protease inhibitor (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methyl-butane (10 [mu]M) was present in the starvation medium, both the protein degradation and the increase in the protease activity were effectively inhibited. Light microscopy analysis showed that many small spherical bodies accumulated in the perinuclear region of the cytosol 8 h after the start of the inhibitor treatment. These bodies were shown to be membrane-bound vesicles of 1 to 6 [mu]m in diameter that contained several particles. Quinacrine stained these vesicles and the central vacuole; thus, both organelles are acidic compartments. Cytochemical enzyme analysis using 1-naphthylphosphate and [beta]-glycerophosphate as substrates showed that these vesicles contained an acid phosphatase(s). We suggest that these vesicles contribute to cellular protein degradation stimulated under sucrose starvation conditions. 相似文献
18.
Uridine diphosphoglucose pyrophosphorylase activity and differentiation in the cellular slime mold Physarum polycephaluno 总被引:3,自引:1,他引:2
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G D Kuehn 《Journal of bacteriology》1974,120(3):1151-1157
The specific activity of uridine 5'-triphosphate:alpha-d-glucose 1-phosphate uridyltransferase (EC 2.7.7.9) (also called uridine 5'-diphosphate [UDP]-glucose pyrophosphorylase) has been found to increase up to eightfold during spherule formation by the slime mold Physarum polycephalum. The enzyme accumulates during the first 8 to 9 h after initiation of spherule formation, declines to basal levels found in vegetative microplasmodia by 15 h, and is undetectable in completed spherules. Specific activities for UDP-glucose pyrophosphorylase in vegetative microplasmodia range from 15 to 30 nmol of UDP-glucose formed per min per mg of protein, whereas accumulated levels during spherule formation can attain a specific activity as high as 125 nmol of UDP-glucose formed per min per mg of protein. The scheduling and extent of accumulation are critically dependent on an early log-phase age of microplasmodia originally induced to form spherules. Spherule induction by 0.2 M or 0.5 M mannitol delays this schedule in a variable and unpredictable manner. Spherule-forming microplasmodia which have accumulated high levels of UDP-glucose pyrophosphorylase spontaneously excrete the enzyme when transferred to salts medium containing 0.2 M or 0.5 M mannitol. The excreted enzyme is subsequently destroyed or inactivated. Studies with preferential inhibitors of macromolecular synthesis indicate that accumulation of UDP-glucose pyrophosphorylase requires concomitant protein synthesis and prior ribonucleic acid synthesis. 相似文献
19.
Casein Kinase II-Type Protein Kinase from Pea Cytoplasm and Its Inactivation by Alkaline Phosphatase in Vitro 总被引:4,自引:0,他引:4
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A casein kinase II-type protein kinase has been purified from the cytosolic fraction of etiolated pea (Pisum sativum L.) plumules to about 90% purity as judged from Coomassie blue stained sodium dodecyl sulfate-polyacrylamide gels. This kinase has a tetrameric [alpha][alpha]'[beta]2 structure with a native molecular mass of 150 kD, and subunit molecular masses of 41 and 40 kD for the two catalytic subunits ([alpha] and [alpha]') and 35 kD for the putative regulatory subunit ([beta]).Casein and phosvitin can be used as artificial substrates for this kinase. Both serine and threonine residues were phosphorylated when mixed casein, [beta]-casein, or phosvitin were used as the substrate, whereas only serine was phosphorylated if [alpha]-casein or histone III-S was the substrate. The kinase activity was stimulated 130% by 0.5 mM spermine (the concentration required for 50% of maximal enzyme activity [A50] = 0.1 mM) and 80% by 2.5 mM spermidine (A50 = 0.4 mM), whereas putrescine and cadaverine had no effect. The kinase was very sensitive to inhibition by heparin (concentration for 50% inhibition [I50] = 0.025 [mu]g/mL). In contrast to most other casein kinase II-type protein kinases, this preparation was inhibited by K+ and Na+, with I50 values of 75 and 65 mM, respectively. Pretreatment of the purified kinase preparation in vitro with alkaline phosphatase caused a 5-fold decrease in its activity. Additionally, this kinase also lost its activity when its [beta] subunit was autophosphorylated in the absence of substrate. These results suggest that the activity of this casein kinase II protein kinase may be regulated by the phosphorylation state of two different sites in its multimeric structure. 相似文献
20.
Partial Purification and Characterization of Hydroxycinnamoyl-Coenzyme A:Tyramine Hydroxycinnamoyltransferase from Cell Suspension Cultures of Solanum tuberosum
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A pathogen elicitor-inducible soluble acyltransferase (tyramine hydroxycinnamoyltransferase [THT], EC 2.3.1), which catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-coenzyme A (CoA) esters to tyramine in the formation of N-hydroxycinnamoyltyramine, was partially purified with a 380-fold enrichment and a 6% recovery from cell-suspension cultures of potato (Solanum tuberosum L. cv Datura). The enzyme showed specific activities of 33 mkat (kg protein)-1 (formation of feruloyltyramine). The apparent native Mr was found to be approximately 49,000. Highest activity was at pH 6.8 in K-phosphate. The isoelectric point of the enzyme was approximately pH5.2. The apparent energy of activation was calculated to be 96 kJ mol-1. The enzyme activity was stimulated more than 5-fold by 10 mM Ca2+ or Mg2+. The apparent Km values were 36 [mu]M for feruloyl-CoA and 85 and 140 [mu]M for cinnamoyl- and 4-coumaroyl-CoA, respectively. The Km value for tyramine in the presence of feruloyl-CoA was 22 [mu]M. In the presence of 4-coumaroyl-CoA, however, the Km for tyramine increased to about 230 [mu]M. The mode of action was an iso-ordered bi bi mechanism in which A, B, P, and Q equal hydroxycinnamoyl-CoA, tyramine, N-hydroxycinnamoyltyramine, and CoA, respectively. Thus, the reaction occurred in a ternary complex of the enzyme and substrates. The equilibrium constant of the reaction was determined to be 1.3 x 104. This gave a [delta]G[deg][prime] eq value of -23.5 kJ mol-1. 相似文献