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1.
A new L-amlnolactam-hydrolyzing enzyme was partially purifiedfrom cells of Cryptococcus laurentii which can grow on L-aminolactamas a carbon and nitrogen source. The enzyme required a bivalentmetal ion, such as Mn2+ or Mg2+, and its molecular weight wasroughly estimated to be 1.5?105 Some other properties were alsostudied. 1 Present address: Department of Biology, Faculty of Science,Osaka City University Sumiyoshi-ku, Osaka 558, Japan. (Received June 20, 1977; )  相似文献   

2.
Regulation by the active form of phytochrome (PFR) and the effectof Ca2+ and calmodulin was examined with glutamate dehydrogenase(GDH) of Zea mays. A brief irradiation (5 min) to 5 day oldplants with red light resulted in 5-6 fold increase in GDH activity.This effect was nullified when red light was followed immediatelyby far-red light. The photoreversibility showed that PFR regulatesGDH activity in vivo. To the enzyme extract obtained after EGTAtreatment, when Ca2+ was added in vitro, GDH was activated by6 fold. The maximum response by Ca2+ was obtained at 80 µM.Both PAR and Ca2+ effects were found to be age dependent. Theenzyme activity was inhibited by compound 48/80 in partiallypurified extracts and the effect was reversed by calmodulin.The purified GDH, however, was not activated directly by calmodulin;it required the presence of another protein factor which wasseparated by gel permeation column by HPLC. Neither anticalmodulindrugs nor addition of calmodulin had any effect on nitrate re-ductaseactivity. (Received July 13, 1988; Accepted October 31, 1988)  相似文献   

3.
The reaction of spinach RuBisCO activated with CO2 and Mg2+proceeded in two phases, an initial burst for a few minutesand the subsequent linear phase, in the presence of saturatingconcentrations of CO2, ribulose 1,5-bisphosphate (RuBP), andMg2+. The percentage of the activity in the linear phase tothat in the initial burst was 55% with RuBisCO prepared withpolyethylene glycol, and very close to the value with the enzymereleased immediately from isolated chloro-plasts. RuBisCO preparedwith ammonium sulfate had a much larger decrease of the activityin the linear phase. The Euglena enzyme had a linear courseof reaction with time for up to 20 minutes. The Km for CO2 of spinach RuBisCO activated beforehand was 20µM in the initial burst, and 28 µM in the linearphase. In the carboxylase reaction initiated with inactive enzyme,the activity was initially negligible, but in 5 minutes increasedto the level observed in the linear phase of the activated enzyme.The Km for CO2 in the linear phase of the pre-inactivated enzymewas 70 µM. The concentration of RuBP was the immediate cause of the two-phasiccourse of the carboxylase reaction of spinach RuBisCO. The curvatureof the time course was not observed below 35 µM RuBP.The enzyme required over 88 µM RuBP for the conventionaltwo-phasic course. Further increase of the concentration ofRuBP increased the extent of the curvature, but did not startthe curvature sooner after the start of the reaction. Even ifspinach RuBisCO was in the linear phase, dilution of RuBP orits consumption by the enzymatic reaction to less than 30 µMcaused the enzyme to show the resumed biphasic reaction courseafter addition of a high concentration of RuBP. 1This paper is the twenty-fourth in a series on PhotosyntheticCarbon Metabolism in Euglena gracilis. (Received September 19, 1988; Accepted November 25, 1988)  相似文献   

4.
A vanadate-sensitive and nitrate-resistant ATPase was solubilizedwith Zwittergent 3–14 from a highly purified plasma membranefraction of mung bean hypocotyls and partially purified by glyceroldensity gradient centrifugation and phenyl-Sepharose columnchromatography. Either phosphatidylcholine or phosphatidylserinein addition to Mg2 + was required for the enzyme activity, whereasK+, phosphatidylethanolamine and lysophosphatidylcholine hadno effect on the activity. The purified enzyme preparation containedtwo major polypeptides with molecular masses of 67 and 55 kDaas analyzed by SDS-polyacrylamide gel electrophoresis. Whenthe plasma membrane fraction was incubated with [-32P]ATP, a45-70-kDa polypeptide(s) was labeled, and the label could berapidly chased with cold ATP. When the fraction was incubatedwith [14C]N,N'-dicyclohexylcarbodiimide, an inhibitor for theATPase, a 15-20-kDa polypeptide was labeled. We propose thatthe enzyme is a new type of higher plant plasma membrane ATP-aseand is composed of 67- and 55-kDa subunits and probably alsoa 15-20-kDa subunit. 1Present address: Takarazuka Institute, Sumitomo Chemical IndustriesLtd., Takatsukasa, Takarazuka, Hyogo 665, Japan (Received September 2, 1987; Accepted May 20, 1988)  相似文献   

5.
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   

6.
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 min–1, 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 min–1 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   

7.
Putrescine N-methyltransferase, a new enzyme catalyzing theformation of N-methylputrescine from putrescine and S-adenosyl-L-methioninewas found in roots of tobacco plants. The enzyme was purified30-fold from crude extracts of tobacco roots. NMethylputrescinewas identified as the reaction product by comparison with theauthentic compound. The enzyme had a pH optimum between pH 8and 9, and a molecular weight of about 60,000, as determinedby gel filtration. Km values for putrescine and 5-adenosyl-L-methioninewere 4.0 x 10–4 M and 1.1 x 10–4 M, respectively.Enzyme activity was inhibited by N-chloromercuribenzoate andAg+. No cofactors were required. Of the various substrates tested,only putrescine served as a methyl acceptor. The enzyme waslocalized exclusively in the roots and its activity was greadyenhanced by decapitation. The presence of putrescine N-methyltransferase in tobacco rootsstrongly suggests that N-methylputrescine participates as anintermediate in nicotine biosynthesis. (Received March 2, 1971; )  相似文献   

8.
A soluble, NADPH-dependent reductase catalyzing the reductionof (+)-dihydroquercetin to 3,4-cis-leucocyanidin (5,7,3',4'-tetrahydroxyflavan-3,4-cis-diol)was demonstrated in an enzyme preparation from a cell suspensionculture of Japanese cedar (Cryptomeria japonica D. Don). TheKm value for (+)-dihydroquercetin was 48µM. The enzyme,which was purified 26.2-fold, could also catalyze the reductionof (+)-dihydrokaempferol to 3,4-cis-leucopelargonidin (5,7,4'-trihydroxyflavan-3,4-cis-diol). The enzyme had a pH optimumof 7 and a molecular weight of 133,000. It was inhibited byCu2+ and iodoacetate, but not by p-chloromercuribenzoate. Duringthe growth stages of the cell suspension cultures, an increasein reductase activity proceeds an increase in procyanidin content,as might be expected. (Received November 25, 1987; Accepted April 11, 1988)  相似文献   

9.
The control of the activity of the matrix-located malic enzyme(EC 1.1.1.39 [EC] ) by Mn2+ was investigated in Percoll-purified mitochondriafrom potato (Solarium tuberosum) tuber. Malic enzyme activitywas tightly controlled by the amount of Mn2+ available in thematrix space and could be stimulated by the addition of exogenousMn2+. A net uptake of Mn2+ into the matrix space of energizedmitochondria was measured. The uptake of Mn2+ was mediated bythe active cation pump present in the mitochondria. The activityof this cation pump was shown to be dependent on the membranepotential sustained by the activity of the respiratory chain.The uptake of Mn2+ was totally abolished in the presence ofan uncoupler and strongly depressed in the presence of rutheniumred, a specific inhibitor of the Ca2+-pump which is presentin animal mitochondria. Thus, the effect of Mn2+ on matrix-locatedMn2+-dependent malic enzyme was strongly influenced by the presenceof an uncoupler or of ruthenium red. In addition, this effectwas reduced in the presence of Ca2+. The possible physiologicalsignificance of the presence of this cation pump is discussedin relation to the presence of a matrix-located, NAD+-dependentmalic enzyme in plant mitochondria. (Received November 21, 1988; Accepted March 6, 1989)  相似文献   

10.
1-Aminocyclopropane-1-carboxylate (ACC) oxidase (ethylene-formingenzyme) was isolated from wounded mesocarp tissue of Cucurbitamaxima (winter squash) fruit, and its enzymatic properties wereinvestigated. The enzyme required Fe2+ and ascorbate for itsactivity as well as ACC and O2 as substrates. The in vitro enzymeactivity was enhanced by CO2. The apparent Km value for ACCwas 175 µM under atmospheric conditions. The enzyme activitywas inhibited by sulfhydryl inhibitors and divalent cationssuch as Co2+, Cu2+, and Zn2+. ACC oxidase activity was induced at a rapid rate by woundingin parallel with an increase in the rate of ethylene production.The exposure of excised discs of mesocarp to 2,5-norbornadiene(NBD),an inhibitor of ethylene action, strongly suppressed inductionof the enzyme, and the application of ethylene significantlyaccelerated the induction of the activity of ACC oxidase inthe wounded mesocarp tissue. These results suggests that endogenousethylene produced in response to wounding may function in promotingthe induction of ACC oxidase. (Received January 13, 1993; Accepted April 15, 1993)  相似文献   

11.
12.
A cell-wall fraction containing 10.0% dry weight of proteinwas isolated from tobacco (Nicotiana tabacum L.) XD-6 cellscultured in suspension. The fraction possessed high acid phosphataseactivity toward p-nitrophenyl phosphate, phenyl phosphate, inorganicpyrophosphate, adenosine diphosphate, nucleoside triphosphates,and bis- (p-nitrophenyl) phosphate; much less activity towardnaphthyl phosphate, ß-glycerophosphate, glucose-6-phosphate,fructose-6-phosphate, fructose-l,6-diphosphate, thiamine pyrophosphate;and no activity toward glucose-1-phosphate and inositol hexaphosphate.Metallic ions were not required for activation. The enzyme wasextracted from the wall with NaCl solution. The solubilizedenzyme resulted in a single peak on Sephadex G-150 chromatographywith activity toward p-nitrophenyl phosphate, pyrophosphate,and bis(p-nitrophenyl) phosphate. The activity of the solubilizedenzyme toward p-nitrophenyl phosphate was identical with thebound enzyme in its pH optimum, substrate specificity, ion inhibitionand Km value, but it was more sensitive to pH, substrate difference,inhibition, and heat treatment. 1Present address: Department of Biology, Japan Women's University,Mejiro-dai, Bunkyo-ku, Tokyo, Japan (Received September 13, 1972; )  相似文献   

13.
The thermal dependence of two of the reactions catalyzed bythe nitrate reductase from Chlorella vulgaris was determined.The activation energies for NADH:nitrate oxidoreductase (EC1.6.6.1 [EC] ) and NADH:Cytochrome c oxidoreductase (EC 1.6.99.3 [EC] )are 42.1 kJ?mol–1 and 21.5 kJ?mol–1, respectively.Since the thermal dependency of the two enzymes is different,ratios of the activities will vary with temperature. The importanceof both rigorous thermal control during nitrate reductase assaysas well as the need to specify the temperature at which theratio of activities for the enzyme are clearly established. 1Present Address: Cropping Systems Research Laboratory, USDA-ARS,Route 3, Box 215, Lubbock, TX 79401, U.S.A. (Received November 25, 1987; Accepted March 2, 1988)  相似文献   

14.
Using permeabilized characean cells in which the ionic conditionsat the cytoplasmic side of the tonoplast are easily controlled,effects of Ca2+ ion on tonoplast potential were examined. Whenthe cell was treated with 1 µM Ca2+, the tonoplast potential(EM became positive in a complicated manner in Chara corallinawhile it simply became negative in Nitella axilliformis. Whenthe cell was treated with 9-antracenecarboxylic acid, a Cl-channelinhibitor, Em became more negative and the response of Em toCa2+ was significantly suppressed. It is suggested that Ca2+activates Cl-channel at a low concentration and inactivatesat a higher one in C. corallina while it simply inactivate Cl-channelin N. axilliformis. 1Present address: Biological Laboratory, The University of theAir, Wakaba 2-11, Wakaba, 260 Japan. (Received August 22, 1988; Accepted December 26, 1988)  相似文献   

15.
Blue light was specifically required for the induction of carbonicanhydrase (CA) activity in Chlamydomonas reinhardtii. The enhancingeffect of blue light (460 nm) was saturated at energy fluencerate as low as 0.6-0.8 W/m2. The wavelength dependency curvehad a peak at 460 nm with no effect at wavelengths above 510nm, thus showing the strong similarities to other blue lightresponses in microalgae. CA induction was strongly inhibitedby UV irradiation at 280 nm. Experiments with the flavin quencher,potassium iodide, suggested that flavin is somehow involvedin CA induction. 1On leave from the Institute of Biological Sciences, Collegeof Arts and Sciences, University of the Philippines at Los Banos,4031 College, Laguna, Philippines. (Received August 29, 1988; Accepted November 26, 1988)  相似文献   

16.
The effects of some amino acid analogues (o-, m- and p-fluorophenylalanineand azetidine-2-carboxylic acid) on uptake of42K and 45Ca intothe roots and transport to the shoots of whole wheat and mungbean seedlings were measured. The effect of each analogue oneither K+ or Ca2+ movement could be placed into one of fourcategories: (1) No effect on either ion uptake or transport;(2) No effect on ion uptake, but a reduction in transport; (3)Similar reductions in ion uptake and transport; (4) A relativelygreater reduction in ion transport than in uptake. At leasttwo independent sites of protein involvement in ion movementwere required to account for all four types of analogue effectobserved; one site of protein involvement was probably at theplasmalemma of root cortex cells and the second site, involvinga protein that turned over more quickly, was within the stele.Some evidence was found that Ca2+ transport is a passive process.Light did not stimulate uptake. Key words: Triticum aestivum, Vigna radiata, Two pump hypothesis  相似文献   

17.
Using butyl-TSK-gel chromatography, we purified NAD-malic enzyme(ME) (EC 1.1.1.39 [EC] ), which is involved in C4 photosynthesis,to electrophoretic homogeneity, from leaves of Amaran-thus tricolor.Molecular weights of the native and SDS-denatured enzyme fromA. tricolor were 490 kDa and 61 kDa, respectively. During assayof the enzyme there was a slow reaction transient in the formof a lag before a steady-state rate was reached. The durationof this lag was inversely proportional to the concentrationof each substrate and the activator, fructose- 1,6-bis-phosphate(FBP). The optimal pH of the reaction fell with decreasing concentrationsof either malate or FBP. High pH prolonged the lag in reaction. Double reciprocal plots of the enzymatic activity as a functionof the concentration of malate yielded straight lines and didnot show any cooperativity for binding of malate. The enzymefrom A. tricolor was not inhibited by either HCO3 orCO2. At different concentrations of malate, the nature of theactivating effect of FBP was compared among the purified enzymesfrom A. tricolor and the C4 monocots Eleusine coracana and Panicumdichotomiflorum. At low levels of malate, FBP markedly stimulatedthe enzyme from each species. In contrast, at saturating levelsof malate, the response of enzymes to increasing concentrationsof FBP was different and depended on the source of enzyme. The immunochemical properties of the enzymes from the threespecies were compared using an enzyme-linked immunoadsorbentassay with antisera raised against the purified enzymes fromthe three species. Different cross-reactivities were observedamong the enzymes from different sources. The N-terminal aminoacid sequences of NAD-MEs from the three species were determinedand some differences were found among the three enzymes. 2Permanent address; Tohoku National Agricultural ExperimentStation, Morioka, 020-01 Japan. 3Permanent address; National Grassland Research Institute, Nishinasuno,Tochigi, 329-27 Japan. (Received December 12, 1988; Accepted February 17, 1989)  相似文献   

18.
GUPTA  S. C. 《Annals of botany》1956,20(1):179-190
The incorporation of sodium chloride in a synthetic medium stimulatedthe pectolytic activity of cultures of Pythium de Baryanum.The Cl ion appeared to be mainly responsible for thiseffect; on the other hand, presence of the Ca++ ion depressedenzymic activity. Glucose, fructose, and mannose were about equally suitable forgrowth and enzyme production. Sucrose, if used as sole carbohydratesource, gave good mycelial growth but poor enzyme production,but if a small proportion was replaced by glucose, enzyme productionwas as good as on glucose itself. Galactose gave very poor growthand negligible enzyme production. For optimum production of pectolytic enzyme, glucose (or fructoseor mannose) requires to be autoclaved in a somewhat alkalinemedium—very conveniently with the K2HPO4 or K3PO4 of thenutrient medium. A yellow to brown coloration (due to caramelization)is produced in the process, but the stimulating factor is notbound up with the colouring substance. The same stimulatingeffect on enzyme production was obtained by adding to the nutrientmedium a small quantity of glucose which had been dry heatedat 150° C. for 20 minutes. Chromatographic analysis suggestedthat the stimulating substance was probably glyceraldehyde,though it is not excluded that other breakdown products of sugarsmay also play a part.  相似文献   

19.
The urea-degrading enzyme of Cyclotella cryptica was testedin crude cell-free extracts for effects from chemical reagentsknown to distinguish between urease and ATP:urea amidolyase.Inhibition of the enzyme by hydroxyurea and its indifferenceto added ATP, Mg2+ or K+ avidin or biotin clearly characterizedthe enzyme as urease (EC 3.5.1.5 [EC] ). The Cyclotella urease wasunaffected by thiourea addition, as was also the growth of thediatom in the presence of this substrate analogue. Indirectevidence was obtained from growth studies of the diatom andcorresponding urease production showing that the enzyme: (i)contains Ni2+ tightly bound to an apoprotein; (ii) is producedconstitutively even from growth on nitrate and does not requireextracellular urea for its synthesis, although quantitativelythe activity is greatest from growth on urea. It is concludedthat Cyclotella urease is a Ni2+ constitutive enzyme similarin many respects to those previously reported from Phaeodactylumtricornutwn and Tetraselmis maculata.  相似文献   

20.
A thiol proteinase was purified 6,400-fold from leaves of Hordeumdistichum L. by a sequence of ammonium sulfate fractionation,gel filtration, ion exchange chromatography, hydrophobic chromatographyand chromatofocusing. This enzyme also had nitrate reductase(NR)-inactivating activity, which was associated with proteolyticactivity in almost constant proportions during the purificationprocedures. Its molecular weight was estimated as 74,000 bygel filtration, and it had an isoelectric point of 4.05 andan apparent Km of 0.83 mg ml–1 for azocasein. The respectiveoptimum pH for proteolytic and NR-inactivating activities were6.0 and 7.0. On electrophoresis, the proteinase gave a majorband that coincided with both activities; it also produced afaint band associated with no activity. Our purified thiol proteinase inactivated FMNH2-NR and MVH-NRas well as NADH-NR, but it had only a slight effect on NADHcytochrome c reductase activity. This enzyme also inactivatedglutamine synthetase. (Received September 16, 1983; Accepted January 26, 1984)  相似文献   

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