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1.
In extracts from Zea mays shoots, the presence of thiol compoundsin the extraction buffer was necessary to get an active 3 deoxy-D-arabinoheptulosonic acid 7-phosphate (DAHP) synthase. Its pH optimumfor activity was about 7.5. Of the different cations tested,only Mn++ was an activator. Enzyme stability was optimal inTris-HCl buffer, pH 7.5, that contained a reducing agent, Mn++and a polyol. Contrary to other reports, phosphoenolpyruvate(PEP) did not stabilize the preparation significantly. The synthaseexhibited high affinities for both erythrose-4-phosphate (Km:0.24 mM) and PEP (Km: 0.31 mM). Its specific activity was highestin young shoots. Corn DAHP synthase was inhibited in vitro by tryptophan. Moreover,the enzyme was retarded on a tryptophan agarose affinity column,but it was removed with the bulk of protein from the same supportwhen eluted with buffer containing tryptophan. Inhibition whichwas easily lost during storage at 4°C was pH dependent andincreased during development. Maximal inhibition, about 60%with 1 mM tryptophan, was observed in extracts from 8 day-oldshoots. Phenylalanine and tyrosine were not inhibitory, andno synergistic effects were observed when the aromatic aminoacids were tested in combination. Isoenzymes could not be demonstrated. (Received April 23, 1980; )  相似文献   

2.
NADP-specific isocitrate dehydrogenase from the soluble fractionof maturing castor bean endosperm was partially purified (approximately180-fold) and some of its enzymatic properties were studied.Mg++, Mn++, Cd++, Ba++, Co++, Zn++, and Sr++ were activatorsof the enzyme reaction at a concentration of 6.7x10 M. The optimumpH of this enzyme was about 8.5. The enzyme was stable in thenarrow range from pH 7.0 to pH 8.0. Km values for isocitrateand NADP at pH 8.5 were 3.5x10–6 M and 3.6x10–6M, respectively. Enzyme stability was not affected by NaCl concentrationand enzyme reaction was inhibited at 5x10–6 M PCMB (80%inhibition). It is suggested that the condensation product ofglyoxylate and oxalacetate also inhibits the reaction. NADP-IDHin the crude extract from maturing castor bean endosperm washeat-stable but the dialyzed enzyme preparation and the partiallypurified enzyme were labile against heat treatment at 57°C.When Mg++ was added to the partially purified enzyme in thepresence of isocitrate or NADP, the enzyme was stabilized againstheat treatment. Mn++, Ca++, Co++, Sr++ or Ba++ could be substitutedfor Mg++. Addition of only one of the factors, Mg++, isocitrateor NADP, had no effect on the heat stability. Moreover, a combinationof isocitrate and NADP did not establish stabilization. A divalentcation plays a central role, while adenine nucleotide, especiallyATP, may have an important part in stabilization. (Received August 14, 1972; )  相似文献   

3.
Enzymological basis for herbicidal action of glyphosate   总被引:8,自引:8,他引:0       下载免费PDF全文
The effects of 1 millimolar glyphosate (N-[phosphonomethyl]glycine) upon the activities of enzymes of aromatic amino acid biosynthesis, partially purified by ion-exchange chromatography from mung bean seedings (Vigna radiata [L.] Wilczek), were examined. Multiple isozyme species of shikimate dehydrogenase, chorismate mutase, and aromatic aminotransferase were separated, and these were all insensitive to inhibition by glyphosate. The activities of prephenate dehydrogenase and arogenate dehydrogenase were also not sensitive to inhibition. Two molecular species of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase were resolved, one stimulated several-fold by Mn2+ (DAHP synthase-Mn), and the other absolutely dependent upon the presence of Co2+ for activity (DAHP synthase-Co). Whereas DAHP synthase-Mn was invulnerable to glyphosate, greater than 95% inhibition of DAHP synthase-Co was found in the presence of glyphosate. Since Co2+ is a Vmax activator with respect to both substrates, glyphosate cannot act simply by Co2+ chelation because inhibition is competitive with respect to erythrose-4-phosphate. The accumulation of shikimate found in glyphosate-treated seedlings is consistent with in vivo inhibition of both 5-enolpyruvylshikimic acid 3-phosphate synthase and one of the two DAHP synthase isozymes. Aromatic amino acids, singly or in combination, only showed a trend towards reversal of growth inhibition in 7-day seedlings of mung bean. The possibilities are raised that glyphosate may act at multiple enzyme targets in a given organism or that different plants may vary in the identity of the prime enzyme target.  相似文献   

4.
Inorganic pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase was detected in extracts of mung bean sprouts, the first such detection in C3 plants. The enzyme had an absolute requirement for a divalent metal (Mg++) as well as for D-fructose 6-phosphate and inorganic pyrophosphate. An examination of anomalous kinetics revealed that the enzyme was activated by a product of the reaction, D-fructose 1,6-bisphosphate; micromolar concentrations of this effector increased the activity of the enzyme about 20-fold. D-Glucose 1,6-bisphosphate at higher concentrations could substitute for D-fructose 1,6-bisphosphate as an activator, but not as a substrate in the reverse reaction. The enzyme was fully active under conditions wherein ATP: D-fructose-6-phosphate 1-phosphotransferase from the same source was inhibited >99% (e.g., in the presence of 10 μM phosphoenolpyruvate).  相似文献   

5.
An ATPase was extracted and purified from castor bean endospermmitochondria. The enzyme is stable at 60°C only in the presenceof ATP in the incubation medium. It is less stable at 0°Cthan at 30°C but is stabilized by ammonium sulfate or glycerol.Activity is dependent on the presence of Mg++, and in the presenceof Mg++ is enhanced by 2,4-dinitrophenol, but is not inhibitedby oligomycin. The enzyme hydrolyzes ITP in addition to ATP,but ITPase activity is hardly enhanced by 2,4-dinitrophenol.This preparation has many properties in common with the ATPase(coupling factor 1) from beef heart mitochondria. (Received November 8, 1969; )  相似文献   

6.
Mg++ -activated and -inhibited ATPases from mung bean hypocotyls   总被引:1,自引:0,他引:1  
Mg++-activated and inhibited ATPases were isolated from dark-grownmung bean hypocotyls. The enzymes hydrolyzed nucleoside tri-,di- and monophosphates and ß-glycerophosphate. Theeffect of Mg++ was most marked when ATP and other nucleosidetriphosphates were used as substrates. Mg++-activated ATPases: The activity of enzyme-I was localizedin the membranes and was not released by treatment with 0.1%deoxycholate. Enzyme-II was released and separated by CM-cellulosecolumn chromatography. Enzyme-V was separated from the solublefraction of the cell homogenate by DEAE-cellulose column chromatography.The rates of activivation by Mg++ of enzyme-II and enzyme-Vwere very small compared to that of enzyme-I. Mg++-inhibited ATPases: Enzyme-II and -IV were precipitatedwith 50–80% ammonium sulfate from the soluble fractionof the cell homogenate and were separated by successive columnchromatographies on Sepharose 6B and DEAE-cellulose. The activitiesof enzyme-III and -IV were inhibited by Mg++, when ATP, UTPand GTP were used as substrates. Enzyme-III was purified approximately38-fold, and was more remarkably inhibited by Mg++ than wasenzyme-IV. 1Present address: Institute for Plant Virus Research, 959 Aobacho,Chiba 280, Japan. (Received January 7, 1974; )  相似文献   

7.
The release of inorganic phosphate from ATP by mitochondriaisolated from endosperms of castor bean (Ricinus communis) wasstimulated by Mg++, but not by Ca++. EDTA, succinate, NADH2or oligomycin depressed the reaction. The depression by succinatewas removed by KCN, antimycin A or anoxia. DNP alone did notaffect activity but did stimulate the Pi release in the presenceof succinate under aerobic conditions. Enhanced Pi release inthe presence of succinate and DNP was cancelled by KCN, antimycinA, oligomycin or anoxia. On the basis of these results, themechanism of ATPase action in castor bean endosperm mitochondriais discussed. (Received January 27, 1969; )  相似文献   

8.
To understand the plant response to oxidative stresses, we studied the influence of magnesium (Mg++) deficiency on the formation of hydrogen peroxide (H2O2), malondialdehyde (MDA), and protease activity in kidney bean plants. The expression pattern of proteins under Mg++ deficiency also was examined via two-dimensional electrophoresis. The formation of H2O2 and MDA increased in the primary leaves of plants grown in a nutrient solution deficient in Mg++. Protease activity in Mg++-deficient plants was also higher than in those grown with sufficient Mg++. The expression pattern of the proteins showed that 25 new proteins were generated and 64 proteins disappeared under Mg++-deficient conditions. Therefore, a deficiency in Mg++ may cause oxidative stress and a change in protein expression. Some of these proteins may be related to the oxidative stress induced by Mg++ deficiency.  相似文献   

9.
Membrane-bound Mg++-activated ATPase was separated from thelower epidermis of tobacco leaves (Nicotiand tabacum L. SamsunNN) on stepwise sucrose density gradient centrifugation. Membrane-bound epidermal ATPase was localized in the interfaceof densities in sucrose of 1.12 to 1.16 in the sedimentary fractionbetween 1,500?g to 10,000?g from the homogenate of the lowerepidermis. The epidermal ATPase activity was activated by divalentcations (Mg++>Mn++Co++>Fe++>Zn++>Ca++) and furtherstimulated by KCl by ca. 20%. The pH optimum for Mg++-activationof the epidermal ATPase was ca. 6.0. The enzyme hydrolyzed ATPmore rapidly than other nucleoside triphosphates. The optimumtemperature for activation of the epidermal ATPase activitywas ca. 40?C. 50% of the epidermal ATPase activity was lostin 18 min at 55?C and in 2.5 days at 2.5?C. The apparent Kmvalue of the epidermal ATPase was 4.7?10–4 M and Vmaxwas 65.4 nmoles Pi/mg protein/min. The epidermal ATPase wasstrongly inhibited by N, N'-dicyclohexylcarbodiimide (DCCD)in vitro whereas oligomycin, carbonyl cyanide m-chlorophenylhydrazone(CCGP), indoleacetic acid (IAA) and abscisic acid (ABA) wereinsensitive to the epidermal ATPase activity. (Received May 23, 1978; )  相似文献   

10.
Isolated intact plant mitochondria, including those from turnipand mung bean, show low endogenous Mg2+-ATPase activity and,unlike mammalian mitochondria, lack significant uncoupler-stimulatedATPase activity. In contrast, the rates of respiration-drivenATP synthesis are comparable to those in mammalian mitochondria,suggesting the presence of an ATPase inhibitor. Disruption ofintact turnip mitochondria only results in limited increasesin ATPase activity, indicating that a permeability barrier toATP transport is not primarily responsible for the low endogenousactivity. The ATPase activity of turnip mitochondria and membraneparticles can be increased up to 50-fold when assayed underoptimum conditions. Time-dependent increases in activity inducedby ageing, exposure to salts and trypsin treatment, are allconsistent with an inhibitor protein being responsible for thelow endogenous activity and lack of uncoupler-stimulation. TheATPase activity of particles under optimum conditions and afterageing is sufficient to account for the rates of ATP synthesis.After activation, turnip mitochondrial ATPase activity is similarto the mammalian enzyme in inhibitor sensitivity, pH optimum,bivalent cation requirement, and sensitivity to ‘activatinganions’. In mung bean mitochondria, a permeability barrierto ATP is only partly responsible for the low endogenous ATPaseactivity, together with the inhibitory factor. On the basisof variation in the relative Ca2+ and Mg2+ -ATPase activitiesafter various treatments, a Ca2+-regulatory site which affectsATPase activity is proposed to exist in the F1ATPase complex. Key words: Plant mitochondrial ATPase, calcium/magnesium -ATPase, inhibitor+ nucleotide specificity, cation/anion effects  相似文献   

11.
Konjak phosphomannose isomerase was inactivated in a time-dependentprocess by metal binding agents, and the inactivated enzymewas instantaneously reactivated by adding such metal ions asZn++, Co++, Fe++, Mn++ and Cu++. However, neither Ca++ nor Mg++were effective for reactivation. Zn++, at a low concentration,brought about complete reactivation of the enzyme at pH 6–7. The EDTA-treated enzyme was more susceptible to heat denaturationwhen compared with the native enzyme, but the addition of variousmetal ions caused the recovery of the thermal stability of theEDTA-treated enzyme. The magnitude of the recovery dependedon the metal ion species and the concentrations. The most effectivemetal ion was Co++, which caused the recovery of thermal stabilityto a level higher than that of the native enzyme. Phosphomannoseisomerase was inhibited by pchloromercuribenzoate and HgCl2;the inhibition by p-chloromercuribenzoate being more pronouncedas incubation progressed. In contrast, the EDTA-treated enzymewas more readily inhibited by the mercurial ion than was thenative enzyme. Zn++, when added to the EDTA-treated enzyme,markedly restored its resistance to the mercurial-induced inhibition.The metal-substituted enzyme was also inhibited by EDTA in atime-dependent process. 1 This paper constitutes part 4 of studies on konjak mannanbiosynthesis. (Received March 3, 1975; )  相似文献   

12.
ATPase in isolated glycerinated flagella of Chlamydomonas snowiaeis shown to be inhibited by high ATP concentrations, by DNPat pH 6.0 and by salyrgan. The enzyme is activated by Mg++ andthe activation is reversed by Ca++. A number of other factorswhich can affect the phototactic behaviour of the cells do notaffect ATPase activity, indicating that the response of directionalmotility is under separate control in the intact cell. 1 This work is based on part of an M. Sc. thesis of I.C.-K.  相似文献   

13.
Summary The activity of 3-dehydroshikimate dehydratase was detected in an extract prepared from cells of mung bean (Vigna mungo) that had been cultured in the presence of shikimate while such activity was not detectable in an extract prepared from cells cultured without shikimate. The enzyme was partially purified and characterized. The maximum activity of the enzyme was observed at pH 7.4. The activity was inhibited to a small extent by EDTA and sulfhydryl inhibitors. The partially purified enzyme was sensitive to thermal denaturation but was stabilized by Mg2+ ions. These results suggest that 3-dehydroshikimate dehydratase might be induced in mung bean cultured cells in the presence of shikimic acid.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - DHS 3-dehydroshikimic acid - PCA protocatechuic acid - QA quinic acid - SA shikimic acid - SORase shikimate - NAEP oxidoreductase  相似文献   

14.
Dehydroquinate synthase from Phaseolus mungo seedlings was purified120-fold by DE-23, hydroxylapatite and Sephadex G-100 columnchromatography. The final preparation was free of dehydroquinatehydro-lyase and NAD(P)H2 oxidase. The dehydroquinate synthaserequired Co2+ and NAD as cofactors. Co2+ could be replaced byCu2+ at 0.1 mM, but Cu2+ at higher levels was inhibitory. Noneof the other metal ions tested activated the enzyme. Some activitywas observed in the absence of added Co2+ and this activitywas inhibited by EDTA but not by diethyldithiocarbamate, NaN3or NaCN. Heavy metal ions, such as Ag+ and Hg2+, and p-chloromercuribenzoatestrongly inhibited the enzyme activity. Of the pyridine nucleotidestested only NAD was required for the maximum activity of theenzyme. In the absence of NAD, the enzyme retained 30 to 40%of the activity obtained with added NAD. The apparent Km valuefor DAHP at pH 7.4 was about 23 µM. The enzyme activityappeared to be maximum at about pH 8.5. However, the characteristicsof the enzyme were studied at pH 7.4, because of the labilityof the enzyme under alkaline conditions. An Arrhenius plot ofthe enzyme reaction showed a break at about 21?C, and belowthis critical temperature the activation energy increased. (Received March 4, 1977; )  相似文献   

15.
ACC synthase, isolated from mung bean hypocotyl segments treatedwith IAA and BA, was inactivated by its substrate, SAM, duringits catalytic action. The reaction products, ACC and MTA, hadno effect on ACC synthase activity. The half-life of the enzymewas 12 min with an initial concentration of 150µM SAM,but this was extended to 23.5 min when the SAM concentrationwas reduced to 40 µM, near to the endogenous concentrationof SAM in mung bean hypocotyl tissue. Addition of AVG, a competitiveinhibitor of ACC synthase, to the reaction mixture containing40 µM SAM, prevented ACC synthase inactivation and increasedthe half-life about 2-fold. We suggest that ACC synthase inactivationis caused by SAM acting as an enzyme-activated irreversibleinactivator (kcat-type inactivator), besides being the substratefor the enzyme. This SAM-dependent inactivation of ACC synthasemay explain the rapid inactivation of the enzyme in intact mungbean hypocotyl segments previously found by Yoshii and Imaseki(1982). (Received October 15, 1985; Accepted December 6, 1985)  相似文献   

16.
Mg++-activated (A) and -inhibited (B) ATPases were isolatedfrom dark-grown mung bean hypocotyls and the effect of in vivoand in vitro IAA-treatment on these ATPases was observed. The activity of the A-ATPase, which was localized in the plasmamembrane-rich fractions, was enhanced by exogenously added l0–10M IAA (ca. 35%). The stimulatory effect of in vitro IAA-treatmentwas particularly found in the A ATPase isolated from sectionstreated with 10–5 M IAA for 60 min (ca. 280%). The activity of the B-ATPase, which was localized in the solublefraction of the cell homogenate, was markedly increased by invivo treatment with 10–7 M IAA for 60 min (ca. 360%).The stimulatory effect of IAA-treatment was observed even ifprotein synthesis was inhibited by cycloheximide. These results suggest that the enhancement of the activitiesof the A- and B-ATPases caused by in vivo and in vitro IAA-treatmentsoccur as a result of the activation of already present enzymes,rather than of the synthesis of these enzymes. 1Present address: Institute for Plant Virus Research, 959 Aobacho,Chiba 280, Japan. (Received March 27, 1974; )  相似文献   

17.
Cucumber (Cucumis sativus L.) seedlings were grafted onto cucumber-(CG) or figleaf gourd- (FG, Cucurbita ficifolia Bouché)seedlings in order to determine the effect of solution temperature(12, 22, and 32°C) on the mineral composition of xylem sapand the plasma membrane K+-Mg++-ATPase activities of the roots.Low solution temperature (12°C) lowered the concentrationof NO3 and H2PO4 in xylem sap of CG plants butnot of FG plants. Concentrations of K+, Ca++ and Mg++ in xylemsap were less affected than anions by solution temperature.The plasma membrane of FG plants grown in 12°C solutiontemperature showed the highest K+- Mg++-ATPase activity at allATP concentrations up to 3 mM and at low reaction temperatureup to 12°C, indicating resistance of figleaf gourd to lowroot temperature. (Received December 27, 1994; Accepted March 10, 1995)  相似文献   

18.
The water-soluble compounds synthesized by the weed, Pluchea lanceolata, and released by it into the soil significantly reduced seed germination, number of nodes, internode length, shoot and root lengths, nodule number and weight, and Chl a and b and Chl a/b ratio of asparagus bean plants. The pattern of accumulation of nutrients in shoot and root of asparagus bean was also affected. In contrast, the net photosynthetic rate and stomatal conductance of fully expanded leaves were higher in plants grown with treated soil. The concentrations of Mg++, Zn++, and PO43- were higher and K+ was lower in shoots of plants grown with treated soil as compared to those grown with the control soil. Also, roots of plants grown with treated soil showed greater accumulation of Mg++ and NO3-. Shoot/root ratio of nutrients in plants grown with control soil were higher for Zn++, Na+, Ca++, and NO3-, whereas plants grown with treated soil had higher ratios for PO43-. These results provide evidence for allelopathic interference by P. lanceolata to the growth of asparagus bean.  相似文献   

19.
20.
An enzyme, which catalyzes the formation of dihydrofolate fromdihydropteroic acid and L-glutamic acid, was found in pea seedlings.The enzyme was purified approximately 25-fold from the crudeextracts of pea seedlings, and its some properties were investigated.Optimum pH for the enzyme activity was found to be 8.8. Pteroicand tetrahydropteroic acids were not active as substrate. Theenzymatic reaction required as cofactors ATP, divalent (Mg2+or Mn2+) and univalent (K+, NH4+ or Rb+) cations. The productwas characterized as dihydrofolic acid by bioautography. MICHAELIS constants for L-glutamic acid, ATP, dihydropteroicacid and Mg2+ were 7.0x10–4, 9.0x10–5, 3.5x10–6and 1.2x10–3 M, respectively. The MICHAELIS constant forMn2+ was 3.0x10–4. The enzyme was inhibited by PCMB orsilver nitrate and, to some extent, by L-aspartic acid. Inhibitionby PCMB was completely reversed by addition of 2-mercaptoethanol.Enzyme activity was distributed widely among plants. The importanceof magnesium and potassium ions for enzyme catalysis is discussed. 1For the previous paper, Part V, see Reference (30). (Received March 28, 1970; )  相似文献   

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