共查询到20条相似文献,搜索用时 31 毫秒
1.
NADP malic enzyme (EC 1.1.1.40
[EC]
) from leaves of two C4 speciesof Cyperus (C. rotundus and C. brevifolius var leiolepis) exihibiteda low level of activity in an assay mixture that contained lowconcentrations of Cl. This low level of activity wasmarkedly enhanced by increases in the concentration of NaClup to 200 mM. Since the activity of NADP malic enzyme was inhibitedby Na2SO4 and stimulated by relatively high concentration ofTris-HCl (50100 mM, pH 78), the activation ofthe enzyme by NaCl appears to be due to Cl. Variationsin the concentration of Mg2+ affected the KA (the concentrationof activator giving half-maximal activation) for Cl,which decreased from 500 mM to 80 mM with increasing concentrationsof Mg2+ from 0.5 mM to 7 mM. The Km for Mg2+ was decreased from7.7 mM to 1.3 mM with increases in the concentration of NaClfrom zero to 200 mM, although the increase of Vmax was not remarkable.NADP malic enzyme from Cyperus, being similar to that from otherC4 species, was able to utilize Mn2+. The Km for Mn2+ was 5mM, a value similar to that for Mg2+. The addition of 91 mMNaCl markedly decreased the Km for Mn2+ to 20 +M. NADP malicenzyme from Setaria glauca, which contains rather less Clthan other C4 species, was inactivated by concentrations ofNaCl above 20 mM, although slight activation of the enzyme wasobserved at low concentrations of NaCl at pH7.6. (Received February 20, 1989; Accepted June 12, 1989) 相似文献
2.
Inokuchi Ritsuko; Itagaki Tadashi; Wiskich Joseph T.; Nakayama Katsumi; Okada Mitsumasa 《Plant & cell physiology》1997,38(3):327-335
NADP-glutamate dehydrogenase (EC 1.4.1.4
[EC]
; NADP-GDH) was purifiedto electrophoretic homogeneity from the multinuclear-unicellulargreen marine alga in Sipho-nales, Bryopsis maxima, and its propertieswere examined. Mr of the undenatured enzyme was 280 kDa, andthe enzyme is thought to be a hexamer of 46 kDa subunit protein.Optimum pHs for the reductive amination and oxidative deaminationwere 7.5 and 8.2-9.0 respectively. The enzyme displayed NADPH/NADH-specificactivities with a ratio of 18 :1. Apparent Km values for 2-oxoglutarate,ammonia, NADPH, glutamate and NADP+ were 3.0, 2.2, 0.03, 3.2and 0.01 mM respectively. The enzymochemical characteristicsof the GDH were studied and compared to those of other species.The B. maxima GDH was insensitive to 5 mM Ca2+ and to 1 mM EDTAin contrast to higher plant NAD-GDHs. Chemical modificationswith DTNB and pCMBS suggested that cysteine residues are essentialfor the enzymatic activity as in other species GDHs. The GDHwas not affected by 1 mM purine nucleotides, suggesting thatthe enzyme is not allosteric, in contrast to animal NAD(P)-GDHsand fungal NAD-GDHs. (Received August 12, 1996; Accepted January 7, 1997) 相似文献
3.
Purification and some properties of phosphomannomutase from corms of Amorphophallus konjac C. Koch 总被引:1,自引:0,他引:1
Phosphomannomutase [Glazer et al.: Biochim. Biophys. Acta 33:522625 (1959)] was purified 1700-fold in a 39% yieldfrom cell-free extract of konjak (Amorphophallus konjac C. Koch)corms. The molecular weight of the enzyme as determined by gelfiltration was about 62,000. The enzyme required both Mg2+ and-D-glucose-l,6-bisphosphate for activity, although Mg2+ waspartially replaceable by either Co2+ or Ni2+. An apparent equilibriumconstant, Keq=(mannose-6-phosphate) (mannose-1-phosphate), wasdetermined to be 8.5. Activity was maximal at pH 6.5 to 7.0.Activation energy was 11.1 kcal/mole. The enzyme was the moststable at pH 7.5. The addition of substrate or cofactor markedlyincreased enzyme stability toward heat denaturation. The enzymewas more labile to heat than phosphoglucomutase from konjakcorms. Treatment with various metal ions in Tris buffer inhibited theenzyme. Cu2+ and Zn2+ were the most potent inhibitors amongthe metal ions tested, while Co2+ and Ni2+ were weak. When theenzyme was treated with metal ions in the presence of histidinebuffer, Cu2+ and Zn2+ showed no inhibitory effect on the enzyme,whereas Be2+ inhibited it to an extent similar to that in Trisbuffer. Plots of 1/v versus l/(mannose-l-phosphate) at different fixedconcentrations of glucose-1,6-bisphosphate and 1/v versus 1/(glucose-1,6-bisphosphate)at different fixed concentrations of mannose-1-phosphate wereseries of converging lines. Mannose-1-phosphate at high concentrationswas found to inhibit the enzyme competitively with respect toglucose-l,6-bisphosphate. Apparent Km and K1 values for mannose-1-phosphatewere calculated to be 0.2 mM and 1.2 mM, respectively. The Kmvalue for glucose-1,6-bisphosphate was 1.8 µM.
1This paper constitutes part 5 of a series of studies on konjakmannan biosynthesis. (Received May 24, 1976; ) 相似文献
4.
Enzymic mechanism of starch breakdown in germinating rice seeds V. Sucrose phosphate synthetase in the scutellum 总被引:1,自引:0,他引:1
Some enzymic Properties of a partially purified preparationof sucrose phosphate synthetase (E.C.2.4.1.14) from germinatingrice seed scutella were studied. Examination of the reactionkinetics revealed that the rate of synthesis of sucrose phosphatefollows the Michaelis-Menten equation at an optimum PH of 7.5,having Km of 25 mM for UDP-glucose, and of 4.9 mM for fructose6-phosphate. UDP inhibited the enzyme reaction competitively;K1 of 3.3 mM. Fe++ and Fe+++ activated the enzyme reaction about2-fold; Ka, 0.3 mM and 2.0 mM, respectively. Co++, Co(NH3)6+++,Mg++ and Mn++ also activated the enzyme reaction. At high concentrationK+ activated the enzyme reaction with the maximum activationof 24% at 400 mM. The molecular weight and S20,w value of theenzyme were determined as 4.5 ? 105 and 10.4S, respectively.
1Part IV of this series is Ref. (5).
2California Foundation for Biochemical Research Fellow (1973). (Received December 20, 1973; ) 相似文献
5.
Purification of NADP-Malic Enzyme and Phosphoenolpyruvate Carboxylase from Sugar Cane Leaves 总被引:1,自引:0,他引:1
A procedure is described for the purification of phosphoenolpyruvatecarboxylase (EC 4.1.1.31
[EC]
) and NADP-dependent malic enzyme (EC1.1.1.40
[EC]
) from sugar cane leaves. Each enzyme was purified tohomogeneity as judged by sodium dodecyl sulfate-polyacrylamidegel electro-phoresis, with about 30% yield. Phosphoenolpyruvatecarboxylase was purified 54-fold. A molecular weight of 400,000and a homotetrameric structure were determined for the nativeenzyme. The purified carboxylase had a specific activity of20.0 {diaeresis}mol (mg protein)1 min1, and wasactivated by glucose-6-phosphate and inhibited by L-malate.Km values at pH 8.0 for phosphoenolpyruvate and bicarbonatewere 0.25 and O.l0 mM, respectively. NADP-malic enzyme, 356-foldpurified, exhibited a specific activity of 71.2 {diaeresis}mol(mg protein)1 min1 and was characterized as ahomotetramer with native molecular weight of 250,000. Purifiedmalic enzyme showed an absolute specificity for NADP+ and requireda divalent metal ion for activity. Km values of 0.33 and 0.008mM for L-malate and NADP+, respectively, were determined. Thisenzyme was inhibited by several organic acids, including ketoand amino acids; while succinate and citrate increased the enzymeactivity when assayed with 10{diaeresis}M L-malate. The effectsshown by amino acids and by citrate were dependent on pH, beinghigher at pH 8.0 than at pH 7.0. (Received October 26, 1988; Accepted February 3, 1989) 相似文献
6.
The catalytic and regulatory properties of phosphoenolpyruvate(PEP) carboxylase (PEPC) are modulated remarkably by the increasein the level of bicarbonate in the assay medium. The activityof PEPC increased by two-fold as the concentration of bicarbonatewas raised from 0.05 to 10 mM. During this state, there wasonly marginal effect on Km for PEP, while the affinity of PEPCto Mg2+ increased by >2 fold. In contrast, the sensitivityof PEPC to malate decreased with increasing concentration ofHCO3. Similarly, the stimulation by glucose 6-phosphate(G-6-P) at optimal concentration (10 mM) of HCO3 wasmuch less than that at suboptimal concentration (0.05 mM). K1for malate increased by about 3 fold and Ka for G-6-P risedby fourfold as bicarbonate concentration was rised from 0.05to 10 mM. These results suggest that HCO3 desensitizesPEPC to both malate and G-6-P. Further, these changes were manifestedin both dark- as well as light-forms of the enzyme. Similarresults were obtained with PEPC in leaf extracts or in purifiedform. We therefore propose that bicarbonate-induced changesare independent of phospho-rylation and possibly through a significantchange in the conformation of the enzyme. This is the firstdetailed report indicating marked modulation of regulatory andcatalytic properties of PEPC by bicarbonate, one of its substrate. (Received April 14, 1998; Accepted September 22, 1998) 相似文献
7.
Structural and Kinetic Properties of NADP-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L. 总被引:1,自引:0,他引:1
Saitou Kazuyuki; Agata Waichi; Asakura Masae; Kubota Fumitake 《Plant & cell physiology》1992,33(5):595-600
NADP-malic enzyme (EC 1.1.1.40
[EC]
), which is involved in Crassulaceanacid metabolism (CAM), was purified to electrophoretic homogeneityfrom the leaves of the inducible CAM plant Mesembryanthemumcrystallinum. The NADP-malic enzyme, which was purified 1,146-fold,has a specific activity of 68.8 µmol (mg protein)1min1. The molecular weight of the subunits of the enzymewas 64 kDa. The native molecular weight of the enzyme was determinedby gel-filtration to be 390 kDa, indicating that the purifiedNADP-malic enzyme is a hexamer of identical subunits. The optimalpH for activity of the enzyme was around 7.2. Double-reciprocalplots of the enzymatic activity as a function of the concentrationof L-malate yielded straight lines both at pH 7.2 and at pH7.8 and did not reveal any evidence for cooperativity of bindingof L-malate. The Km value for L-malate was 0.35 mM. Hill plotsof the activity as a function of the concentration of NADP+indicated positive cooperativity in the binding of NADP+ tothe enzyme with a Hill coefficient (nH) of 2.0. An S0.5 value(the concentration giving half-maximal activity) of 9.9 µMfor NADP+ was obtained. Oxaloacetate inhibited the activityof the NADP-malic enzyme. Effects of succinate and NaHCO3 onthe activity of NADP-malic enzyme were small. (Received October 30, 1991; Accepted May 1, 1992) 相似文献
8.
A novel enzyme, UDP-D-galactose:flavonol 3-O-galactosyltransferase(F3GaT), catalyzing the transfer of D-galactose from UDP-D-galactoseto the 3 position of 5,7,4'-trihydroxyflavonol (kaempferol),was detected in and purified about 404-fold from seedlings ofVigna mungo by precipitation with ammonium sulfate, chromatographyon Sephadex G-100 and chromatofocusing. The enzyme was separatedby this procedure from a coexisting UDP-D-glucose:flavonol 3-O-glucosyltransferase(F3GT), which was simultaneously purified about 189-fold. F3GaTwas isolated as a soluble enzyme with pH optima of 8.0 in imidazole-HClbuffer and 7.5 in histidine-HCl buffer. F3GT had the same pHoptima. The Mr of both F3GaT and F3GT, which had isoelectricpoints of 5.1 and 6.1, respectively, was estimated by elutionfrom a column of Sephadex G-100 to be about 43,000. The activitiesof F3GaT and F3GT were stimulated by 14 mM 2-mercaptoethanoland strongly inhibited by 1 mM Cu2+, 1 mM Zn2+, and variousreagents that react with sulfhydryl groups. Among various possiblesubstrates for F3GaT that were tested, kaempferol, isorhamnetinand quercetin were the best. The Km values for kaempferol andUDP-D-galactose were determined to be 0.40 µM and 125µM, respectively. Similarly, F3GT had low Km values of0.69 µM for kaempferol and 1.67 mM for UDP-D-glucose.F3GaT and F3GT mediated the transfer of galactose and glucose,respectively, to the 3-hydroxyl groups exclusively of kaempferol,isorhamnetin and quercetin. Rhamnetin also functioned as a galactosylacceptor though less efficiently. (Received October 12, 1992; ) 相似文献
9.
NADP+-Dependent Sorbitol Dehydrogenase Found in Apple Leaves 总被引:1,自引:0,他引:1
An NADP+-dependent sorbitol dehydrogenase that catalyzes sorbitoland glucose was found in apple leaves. The partially purifiedenzyme had optimum activity at pH 9.6 and a Km value of 128mM for sorbitol. Among the polyols studied, this enzyme showedthe most activity for sorbitol.
1This paper is contribution A-173 of the Fruit Tree ResearchStation. (Received June 4, 1984; Accepted July 31, 1984) 相似文献
10.
Aldolase--An Important Enzyme in Controlling the Ribulose 1,5-bisphosphate Regeneration Rate in Photosynthesis 总被引:2,自引:0,他引:2
Iwaki Toshio; Wadano Akira; Yokota Akiho; Himeno Michio 《Plant & cell physiology》1991,32(7):1083-1091
This study was done to explore an enzymatic mechanism for thephotosynthetic carbon reduction cycle whereby the rate of synthesisof ribulose 1,5-bisphosphate (RuBP) could be changed while thelevels of intermediates other than 3-phosphoglycerate and RuBPwere kept constant. Chloroplast aldolase was purified to homogeneityfrom spinach leaves. When the enzyme was assayed in the directionof fructose 1,6-bisphosphate synthesis in the presence of theconcentrations of the substrates reported in vivo, the activitywas severely inhibited by physiological concentrations of RuBP.The aldolase reaction proceeded with a sequential mechanism.The Km for dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphatewere 0.45 mM and 40 µM, respectively. The activity wascompetitively inhibited by RuBP with respect to dihydroxyacetonephosphate. The KI was 0.78 mM. The maximum activity of aldolasein spinach leaves was calculated as 1,360µmol (mg Chl)1h1 An equation to express the reaction for the synthesisof fructose 1,6-bisphosphate by aldolase was constructed topredict the metabolic rate of this reaction in vivo. The calculationclearly showed that aldolase is an important enzyme in controllingthe rate of RuBP regeneration. (Received March 25, 1991; Accepted August 12, 1991) 相似文献
11.
Martinez-Reina G.; Matilla A.J.; Martin-Remesal C.; Gallardo M.; de Rueda P. Muoz 《Journal of experimental botany》1996,47(11):1771-1778
In the present work, certain biochemical characteristics ofthe enzyme 1-aminocyclopropane-1-carboxylate N-malonyltransferase(ACC N-MTase) which is responsible for the malonylation of 1-aminocyclopropane-1-carboxylate(ACC) in chickpea (Cicer arietinum) are described. Phosphatebuffer was the most appropriate buffer with regard to enzymestability and, therefore, ACC N-MTase was extracted, assayedand purified in the presence of this buffer. ACC N-MTase waspartially purified approximately 900-fold from embryonic axesof chick-pea seeds using ammonium sulphate precipitation, hydrophobicinteraction and molecular filtration chromatography. By gelfiltration chromatography on Superose-12, the molecular massof the enzyme was estimated to be 54 4 kDa. ACC N-MTase hadan optimal pH and temperature of 7.5 and 40C, respectively,as well as a Km for ACC and malonyl-CoA of 400 M and 90 M,respectively. D-Phenylalanine was a competitive inhibitor ofACC N-MTase with respect to ACC (Ki of 720 M), whereas co-enzymeA was a competitive product inhibitor with respect to malonyl-CoA(Ki of 300 M) and a non-competitive inhibitor with respectto ACC (Ki of 600 M). Under optimal assay conditions, ACC N-MTasewas strongly inhibited by (a)divalent [Zn2+>Mg2+>>Co2+>Co2+>(NH4)2+>Fe2+]and monovalent metal cations (Li+>Na+>K+), without activitybeing detected in the presence of Hg2+, and (b) PCMB or mersalicacid, suggesting that sulphydryl group(s) are involved at theactive site of the enzyme. Key words: ACC-N-malonyltransferase, Cicer arietinum, embryonic axes, ethylene, germination, seeds 相似文献
12.
In apple fruit, active ATP-dependent microsomal Ca2$ uptakeand respiration-dependent mitochondrial Ca2$ uptake were observed. The mitochondrial Ca2$ uptake was depressed by the calmodulinantagonists chlorpromazine hydrochloride (CPZ) and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamidehydrochloride (W-7). The Ca2$-ATPase from apple mitochondriawas also inhibited by CPZ or W-7. The apparent Km value forCa2$ in mitochondrial Ca2$ uptake (Km=0.35 mM) was similar tothat of mitochondrial Ca2$-ATPase (Km=0.32 mM). The inhibitoryeffect of W-7 on the activity of the mitochondrial Ca2$ uptakewas closely correlated with the inhibition by W-7 of mitochondrialCa2$-ATPase (r=0.996). These findings indicate that the mitochondrialuptake of Ca2$ in apple fruit depends on the calmodulin-mediatedactivation of Ca2$-ATPase. The microsomal Ca2$ uptake was depressed by CPZ, suggestingthat the microsomal Ca2$ uptake may also be modulated by calmodulin.
1 Contribution No. C-72, Fruit Tree Research Station. (Received June 7, 1982; Accepted October 19, 1982) 相似文献
13.
A novel O-glucosyltransferase (I4'GT) which catalyzes the transferof D-glucose from UDP-D-glucose to position 4' of prunetin (4',5-dihydroxyl-7-methoxyisoflavone)was isolated from the leaves of Prunus ? yedoensis Matsum. andpurified 66-fold by precipitation with ammonium sulfate andchromatography on DEAE-cellulose. UDP-glucose:flavonol 3-O-glucosyltransferase(F3GT) was also isolated and purified 50-fold in the same manner.The molecular weights of both I4'GT and F3GT were estimatedby elution from a column of Sephadex G-100 to be about 51,000Da. The pH optima for I4'GT and F3GT activities were 8.0 and7.5, respectively. The specificities of I4'GT and F3GT for thesugar donor were quite strict, and only UDP-glucose could serveas glucosyl donor, both ADP-D-glucose and GDP-D-glucose beingineffective. The apparent Km values for UDP-glucose and prunetinwere 10.0µM and 1.20µM, respectively, for I4'GT.The Km values for UDP-glucose and quercetin were 9.8 µMand 1.21 µM, respectively, for F3GT. The activities ofboth I4'GT and F3GT were stimulated by 1 mM Mg*+ and stronglyinhibited by 1 mM Cu2+, 1 mM Zn2+ and various reagents thatreact with sulfhydryl groups. (Received May 16, 1990; Accepted September 3, 1990) 相似文献
14.
Soluble proteins extracted from leaves of Chenopodium albumcatalyzed the conversion of pheophorbide a to a precursor ofpyropheophorbide a, putatively identified as C-132-carboxyl-pyropheophorbidea. The precursor was then decarboxylated non-enzymatically toyield pyropheophorbide a. Soluble proteins and pheophorbidea, as the substrate, were required for the formation of theprecursor, and boiled proteins were enzymatically inactive.The maximum rate of conversion of pheophorbide a to the precursoroccurred at pH 7.5. The Km for pheophorbide a was 12.5 µMat pH 7.0. Both pheophorbide b and bacteriopheophorbide a couldserve as substrates, but protopheophorbide a could not. Formationof methanol was detected during the enzymatic reaction, an indicationthat the enzyme is an esterase. Among seven alcohol analogstested, only methanol inhibited the enzymatic activity uncompetitively,with a K1 of 71.6 mM. Mass-spectrometric (MS) analysis of theprecursor yield a peak at m/z 579 that indicated the releaseof a methyl group from pheophorbide a. It appears thereforethat the enzyme catalyzes the demethylation of the carbomethoxygroup at C-132 of pheophorbide a by hydrolysis to yield methanoland the precursor, C-132-carboxyl-pyropheophorbide a, whichis converted to pyropheophorbide a by spontaneous decarboxylation.We have tentatively designated the enzyme "pheophorbidase".The presence of the enzyme was dependent on plant species andit was expressed constitutively.
1Present address: Faculty of Science, Shizuoka University, Ohya,Shizuoka, 422 Japan 相似文献
15.
Dependence of Na+-K+ pump current-voltage relationship on intracellular Na+, K+, and Cs+ in rabbit cardiac myocytes 总被引:2,自引:0,他引:2
Hansen PS Buhagiar KA Kong BY Clarke RJ Gray DF Rasmussen HH 《American journal of physiology. Cell physiology》2002,283(5):C1511-C1521
To examine effects of cytosolicNa+, K+, and Cs+ on the voltagedependence of the Na+-K+ pump, we measuredNa+-K+ pump current (Ip)of ventricular myocytes voltage-clamped at potentials(Vm) from 100 to +60 mV. Superfusates weredesigned to eliminate voltage dependence at extracellular pump sites.The cytosolic compartment of myocytes was perfused with patch pipette solutions with a Na+ concentration ([Na]pip)of 80 mM and a K+ concentration from 0 to 80 mM or withsolutions containing Na+ in concentrations from 0.1 to 100 mM and K+ in a concentration of either 0 or 80 mM. When[Na]pip was 80 mM, K+ in pipette solutionshad a voltage-dependent inhibitory effect on Ipand induced a negative slope of theIp-Vm relationship. Cs+ in pipette solutions had an effect onIp qualitatively similar to that ofK+. Increases in Ip with increasesin [Na]pip were voltage dependent. The dielectriccoefficient derived from[Na]pip-Ip relationships at thedifferent test potentials was 0.15 when pipette solutions included 80 mM K+ and 0.06 when pipette solutions were K+ free. 相似文献
16.
Regulation of K+ Influx in Barley: Evidence for a Direct Control of Influx by K+ Concentration of Root Cells 总被引:1,自引:0,他引:1
Siddiqi, M. Y. and Glass, A. D. M. 1987. Regulation of K+ influxin barley: Evidence for a direct control of influx by K+ concentrationof root cells.J. exp. Bot. 38: 935947. The kinetics of K+ (86Rb+) influx into intact roots of barley(Hordeum vulgare L. cv. Fergus) seedlings having different combinationsof root and shoot [K+], different growth rates and differentroot:shoot weight ratios were studied. K+ influx was stronglycorrelated with root [K+]; shoot [K+], growth rates, and root:shoot ratios appeared to have little effect on K+ influx. Adetailed study showed that both Vmax and Km for K+ influx wereaffected by root [K+] but not by shoot [K+]. We have suggestedthat factors such as growth rates and root: shoot ratio mayaffect K+ influx indirectly primarily via their influence onroot factors such as root [K+]. We have reiterated that othertypes of kinetic control, e.g. increased or decreased synthesisof carrier systems, may operate in addition todirect (allosteric?) control of K+ influx by root [K+]. Thenegative feedback signal from root [K+] appeared to be the primeeffector in the regulation of K+ influx. Key words: Barley, K+ influx 相似文献
17.
Soluble chlorophyllase (chlorophyll-chlorophyllido-hydrolase,EC 3.1.1.14
[EC]
) was purified 650-fold from tea leaf sprouts byammonium sulfate fractionation and gel filtration through SephadexG-200 and Sepharose CL-6B. The purified enzyme showed two bandson polyacrylamide gel electrophoresis and the specific activitywas 2.6 µmol chlorophyll a hydrolyzed min1 mg1of protein. The molecular weights determined by Sepharose CL-6Bwere 910,000 and 350,000, indicating high molecular aggregates.The subunit molecular weight estimated by sodium lauryl sulfate-polyacrylamidegel electrophoresis was 38,000. The isoelectric point was 3.9.The optimum pH was 5.5 in acetate buffer and the Km value forchlorophyll a was 10 µM. This enzyme did not require athiol compound nor metal ion such as Mg2+. (Received January 26, 1981; Accepted April 3, 1981) 相似文献
18.
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
19.
Cell suspension cultures of Corydalis sempervirens have provenideal for the study of fusicoccin action [Schulz et al. (1990)Planta 183: 83] and express the fusicoccin-binding protein aswell as a plasma membrane H+-ATPase which is activated by thefungal toxin. Microsomal vesicles prepared from these cellsaccumulate Ca2+ in the presence of Mg-ATP. The protonophorecar-bonylcyanide m-chlorophenylhydrazone did not inhibit theMg-ATP dependent Ca2+-transport into the vesicles. This processis thus due to the activity of at least one primary active,ATP-driven, Ca2+-pump. The enzyme was characterized in detail.It has a pH optimum of 7.2, an apparent Km of 0.3 mu (ATP),12pm (Ca2+), accepts ATP>ITP GTP>CTP UTP, and is strongly(Ki, app 0.75 µmM) inhibited by erythrosine B but lessso (Ki, app 95 µM) by or-thovanadate. These characteristicsare typical for the plasma membrane Ca2+-ATPase characterizedfrom differentiated tissues [Graf and Weiler (1990) Physiol.Plant. 75: 634]. Fusicoccin activates the erythrosine-sensitiveCa2+-pump by lowering its Km for ATP, when added to living cellsprior to tissue homogenization. Thus, fusicoccin appears toactivate at least two ion-translocating ATPases in one and thesame tissue, suggesting that the toxin's mechanism of actionis complex and not restricted to activation of the H+-ATPase.FC has no effect when administered to microsomes. The microsomalenzyme was solubilized and reconstituted into asolec-tin liposomesin functional form. The reconstituted, erythrosine sensitiveCa2+-ATPase was insensitive to fusicoccin. Thus, componentsessential for toxin action are either lost or inactivated duringsubcellular fractionation. It is likely that FC action requiressoluble components. (Received April 22, 1991; Accepted July 24, 1991) 相似文献
20.
Three isoforms (Types 1, 2 and 3) of phosphoenolpyruvate (PEP)carboxylase in young leaves of the Crassulacean acid metabolism(CAM) plant Kalanchoe daigremontiana were separated by DEAE-cellulosecolumn chromatography and preparative polyacrylamide-agarosegel electrophoresis, and their enzymatic properties were characterized. All three isoforms had similar molecular weights of about 234,000.At pH 8.0 Type 1 showed a high affinity to PEP, (Km=0.08 mM),whereas Type 3 showed a low affinity (Km=1.0mM). Km values forMgCl2 were 0.26 HIM in Types 1 and 3 and 0.5 nut in Type 2.All three types exhibited the same pH optimum at 8.0, but Type1 showed relatively low activity below pH 6.0, whereas Type3 showed high activity. Type 3 was more acid stable than theother forms. In the presence of glucose-6-phosphate, the Kmvalues of Types 1, 2 and 3 for PEP lowered to 0.027, 0.037 and0.044 mu at pH 8.0, respectively. Inhibition of activity byorganic acids such as malate and pyruvate was pronounced inType 3. Type 2 exhibited properties intermediate to Types 1and 3 with regard to pH curve, affinity to PEP and its effectof various metabolites. The physiological significance of PEPcarboxylase isoforms in CAM plants is discussed on the basisof these findings.
1Present address: Agricultural Chemicals Research Lab., SankyoCo., Ltd., Yasu-cho, Yasugun, Shiga 520-23, Japan. (Received November 30, 1983; Accepted March 24, 1984) 相似文献