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1.
Fatty acid synthesis from Na (1-14C) acetate in leucoplasts isolated from developing seeds of Brassica compestris was found to be maximum when leucoplasts were supplied with 0.8 mM acetate, 20 mM NaHCO3, 8 mM ATP, 8 mM MgCl2, 4 mM MnCl2, 0.6 mM CoA, 1 mM NADH, 1 mM NADPH and 0.2 M sorbitol and incubated at 30°C for 2 h. The rate of fatty acid synthesis was highest at pH 8.5 In presence of 0.4 M Bistris-propane buffer and linear for upto 4 h at 30°C with 80–110 μg plastid protein. Sorbitol was an essential requirement as it prevented the rupturing of leucoplasts by osmosis. ATP and divalent cations were almost absolute requirements, whereas nucleotides, CoA and bicarbonate improved the rate of fatty acid synthesis by two to ten folds. Mg2+ and NADH were the preferred cation and nucleotide, respectively. High concentration of dithiothreltol inhibited the incorporation of (14C) acetate Into fatty acids. The system developed as above could be used for in vitro studies. 相似文献
2.
Charles R. Hipkin Jaqueline A. Hermann-Smith Philip J. Syrett 《Biochimica et Biophysica Acta (BBA)/General Subjects》1985,838(2):191-196
The appearance of nitrate reductase activity in derepressed cultures of the Nit A mutant of Chlamydomonas reinhardtii required concomitant photosynthetic CO2 fixation and was inhibited when protein turnover was prevented. Provided leupeptin was included in the extraction buffer, a single species of nitrate reductase (molecular mass, m = 390 kDa) was extracted from Nit A cultures incubated in nitrate medium for 4 h. Cultures of the mutant incubated in nitrate-free medium contained a number of nitrate reductase species (m = 52–500 kDa). This evidence suggests that nitrate plays a role in the stabilisation of the structure of the mutant nitrate reductase. Only one species of nitrate reductase (m = 188 kDa) was extracted from wild type cultures grown with nitrate. 相似文献
3.
L. Li T. A. Freier P. A. Hartman J. W. Young D. C. Beitz 《Applied microbiology and biotechnology》1995,43(5):887-892
A resting-cell assay was established to evaluate the cholesterol reductase activity of Eubacterium coprostanoligenes ATCC 51222. Cell suspensions from cholesterol-free media rapidly reduced cholesterol to coprostanol. Optimal assay conditions
in a 1-ml reaction mixture were determined to be up to 1 h of incubation and up to 0.25 mg bacterial protein/assay with at
least 1 mM cholesterol as substrate. The cholesterol reductase activity in cells decreased as a function of storage time at
22°C, 4°C and −20°C. Filling the headspace of the reaction mixture with H2 increased the activity about 20%. Optimal cholesterol reductase activity occurred at pH 7.5 in sodium phosphate buffer. Pyruvate
and reducing agents in the buffer increased the activity. This study has validated assay conditions for determination of cholesterol
reductase activity in resting cells of E. coprostanoligenes.
Received: 2 August 1994/Received revision: 15 November 1994/Accepted: 8 December 1994 相似文献
4.
Hydrosulfite-reduced FMN served as an electron donor for nitratereductase purified from broad bean leaves. FMN was successfullyreplaced with BV. The flavine nucleotide nitrate reductase hadits pH optima at about 7.8 with phosphate buffer and at about7.4 with Tris-HCl buffer. The Km's for nitrate and FMN were3.7 ? 104 M and 3.7 ? 105 M, respectively. NADH2: nitrate reductase activity was completely inhibited by0.1 mM p-CMB, whereas FMNH2: nitrate reductase activity wasnot. Inhibited activity was restored by the addition of cysteine.A sulfhydryl enzyme is involved in the NADH2: nitrate reductasesystem but not in the FMNH2 : nitrate reductase system. NADH2and FMNH2 probably feed electrons into the electron transportchain at different sites. The nitrate reductase preparationhad an NADH2-specific diaphorase activity which was almost completelyinhibited by 0.1 mM p-CMB. The NADH2-specific diaphorase mayform the sulfhydryl enzyme which mediates electron transferbetween NADH2 and nitrate. (Received May 6, 1969; ) 相似文献
5.
Takuro Yamamoto Yasuo Nakata Chen Cao Yosuke Sugiyama Yoshihisa Asanuma Shuji Kanamaru Tomoko Matsuda 《Applied microbiology and biotechnology》2013,97(24):10413-10421
The gene encoding acetophenone reductase (APRD), a useful biocatalyst for producing optically pure alcohols, was cloned from the cDNA of Geotrichum candidum NBRC 4597. The gene contained an open reading frame that consisted of 1,029 nucleotides corresponding to 342 amino acid residues. The subunit molecular weight was calculated to be 36.7 kDa. The predicted amino acid sequence did not have significant similarity to those of the acetophenone reductase reported previously. The gene was inserted into the pET-21b(+) expression vector and expressed in Escherichia coli Rosetta?(DE3)pLysS by induction with 1 mM of isopropyl-β-d-thiogalactopyranoside. E. coli cell-free extract gave 21.9 U/mg APRD activity, which was 81 times that of the G. candidum cell-free extract. The enzyme was purified with a HisTrap FF crude column. The enzyme exhibited the highest activity at 60 °C, and optimum reducing and oxidizing activity were observed in a pH range around 7.0–8.0 and 8.5, respectively. The enzyme was most stable at 60 °C and pH?6.5–7.5. The Vmax and the apparent Km value of the reductase were 67.6 μmol/min per milligram of protein and 0.146 mM for acetophenone, respectively. From 4 % (v/v) 4-phenyl-2-butanone, (S)-4-phenyl-2-butanol was obtained with a yield >80 % and an enantiomeric excess >99 % in a 20 h reaction recycling NADH with 15 % (v/v) 2-propanol. 相似文献
6.
Maranville JW 《Plant physiology》1970,45(5):591-593
The addition of nickel (4 × 10−3m) to the extracting buffer enhances the nitrate reductase activity in preparations of young grain sorghum (Sorghum bicolor L. [Moench] leaf tissue by as much as 6-fold. Activities comparable to other plant species are obtained over an extraction pH range of 7 to 8 with tris buffer and reduced nicotinamide adenine dinucleotide as a cofactor for the reaction when the ratio of plant material to extraction medium is 1:20. The method also enhances nitrate reductase activity in sudangrass (Sorghum sudanense P. [Stapf]). 相似文献
7.
《Journal of Fermentation Technology》1987,65(4):489-491
Chemostat-grown cells of a methanol yeast, Candida boidini S2 AOU-1, were investigated for formaldehyde production. The productivity and catalytic stability were improved by preincubation of the cells at 37°C for 24 h in 0.1 M potassium phosphate buffer (pH 7.5) containing 50 mM NaN3. These cells produced 1150 mM formaldehyde after a 10-h reaction. 相似文献
8.
《Plant Physiology and Biochemistry》2002,40(5):401-405
Nitrite reductase (NiR; EC 1.7.7.1) from the eukaryotic microalga Monoraphidium braunii has been purified to electrophoretic homogeneity, resulting in a preparation with a specific activity of 3574 nkat mg–1 and a purification factor of 2553-fold. The enzyme is a single polypeptide chain with a molecular mass of 63 kDa, and absorption maxima at 690, 573, 385 and 280 nm. Kinetic data indicate Km values of 0.7 mM for nitrite, 10 μM for M. braunii ferredoxin (Fd) and 0.26 mM for methyl viologen. The enzyme showed an optimum pH of 7.5 in 100 mM Tris–HCl buffer and an optimum temperature of 40 °C. NiR activity was inhibited by the sulfhydryl reagent p-hydroxymercuribenzoate and the chelating reagent KCN. Immunological studies revealed the presence of common antigenic determinants, at the Fd-binding domain, in NiR and glutamate synthase (EC 1.4.7.1) from M. braunii. 相似文献
9.
《Journal of Fermentation and Bioengineering》1995,79(1):67-69
Citrate(si)-synthase (citrate oxaloacetate-lyasem EC 4.1.3.7) was purified as an electrophoretically homogeneous protein from an ammonia-oxidizing chemoautotrophic bacterium, Nitrosomonas sp. TK794. The molecular mass of the native enzyme was estimated to be about 287 kDa by gel filtration, whereas SDS-PAGE produced one band with Mr values of 44.7 kDa, suggesting that the enzyme is a hexamer consisting of identical subunits. The isoelectric point of the enzyme was 5.0. The pH and temperature optima for citrate synthase (CS) activity was about 7.5–8.0 and 40°C, respectively. The citrate synthase was stable over a pH range of 6.0–8.5 and up to 40°C. The apparent Km values for oxaloacetate and acetyl-CoA were about 11 μM and 247 μM, respectively. The activity of the citrate synthase was not inhibited by ATP, NADH or 2-oxoglutarate at 5mM, and was activated by potassium chloride at 0.1–100 mM. The N-terminal amino acid sequence of the enzyme protein was PPQDVATLSPGENKKTIELPILG. 相似文献
10.
An amylase with a molecular mass of 55 kDa and an N-terminal sequence exhibiting similarity to enzyme from Bacteroides thetaitaomicron was isolated from fruiting bodies of the monkey head mushroom Hericium erinaceum. The purification scheme included extraction with distilled water, ion exchange chromatography on DEAE-cellulose and SP-sepharose, and gel filtration by FPLC on Superdex 75. The amylase of H. erinaceum was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and eluted with 0.2 M NaCl in the same buffer. The enzyme was subsequently adsorbed on SP-Sepharose in 10 mM ammonium acetate buffer (pH 4.5) and eluted with 0.3 M NaCl in the same buffer. This fraction was subsequently subjected to gel filtration on Superdex 75. The first peak eluted had a molecular mass of 55 kDa in SDS-PAGE. The amylase of H. erinaceum exhibited a pH optimum of 4.6 and a temperature optimum of 40°C. The enzyme activity was enhanced by Mn2+ and Fe3+ ions, but inhibited by Hg2+ ions. 相似文献
11.
Cell walls prepared from aged discs by extraction in 0·1 M acetate buffer, pH 4·8, possess ionically bound acid invertase which can be removed from the wall by incubation in 1 M sodium chloride in 0·1 M acetate buffer, pH 4·8, and more firmly attached enzyme which is not removed. Cell walls prepared in 0·195 M phosphate-0·003 M citrate-buffer, pH 8·0, do not possess ionically bound enzyme. Ionically bound invertase is density labelled when discs are aged in 90% deuterium oxide suggesting that at least part of the increase in activity observed during aging is due to de novo protein synthesis. 相似文献
12.
Abdullah B. Sipat 《Phytochemistry》1982,21(11):2613-2618
The activity of hydroxymethylglutaryl CoA reductase (NADPH) (EC 1.1.1.34) was studied in the latex of regularly tapped mature trees of Hevea brasiliensis. The reductase activity was found mainly (95% of the total activity) in the pellet fraction (40 000 g) of the centrifuged latex. The enzyme in this fraction had a specific requirement for NADPH as the cofactor and, while not obligatory for activity, was activated by dithiothreitol at the optimum concentration of 2 mM. The pH optimum was found to be 6.6–6.9 in 0.1 M phosphate buffer. Mevalonate and CoA (at 2 mM each) did not affect enzyme activity, while hydroxymethylglutarate (2 mM) was slightly inhibitory. p-Chloromercuribenzoate (1 mM) completely inhibited this enzyme. The reductase activity in the 40 000 g pellet was not easily solubilized either using Triton X-100 or by sonication. The apparent Km for the washed, membrane-bound enzyme (103 000 g pellet) was 56 μ M (RS-HMG-CoA). Magnesium-ATP (4 mM) inactivated the reductase but this effect was greatly diminished or was absent upon washing the 40 000 g pellet. 相似文献
13.
Inderjeet Singh Bakshi 《Biologia Plantarum》1983,25(4):259-265
Nitrate concentration required for maximal extractable level of nitrate reductase (NR) inWolffia varies with the conditions prior to the nitrate treatment. Maximal enzyme activity is obtained at 2 mM nitrate concentration
with asparagine grown plants and at 15 mM with nitrate grown. Both the level of enzyme activity and nitrate uptake by the
tissue are increased by irradiation. The radiant energy induced increase in enzyme activity is not due to photosynthetic activity
alone. An effect of radiant energy at the membrane level is sug gested. The extracted enzyme, which is labile, is protected
and activated by NADH at 0 °C. 相似文献
14.
Purification, structure and properties of the respiratory nitrate reductase of Klebsiella aerogenes.
1. The respiratory nitrate reductase of Klebsiella aerogenes was solubilized from the bacterial membranes by deoxycholate and purified further by means of gel chromatography in the presence of deoxycholate, and anion-exchange chromatography. 2. Dependent on the isolation procedure two different homogeneous forms of the enzyme, having different subunit compositions, can be obtained. These forms are designated nitrate reductase I and nitrate reductase II. Both enzyme preparations are isolated as tetramers having sedimentation constants (s20,w) of 22.1 S and 21.7 S for nitrate reductase I and II, respectively. The nitrate reductase I tetramer has a molecular weight of about 106. 3. In the presence of deoxycholate both enzyme preparations dissociate reversibly into their respective monomeric forms. The monomeric form of nitrate reductase I has a molecular weight of about 260 000 and a sedimentation constant of 9.8 S. For nitrate reductase II these values are 180 000 and 8.5 S, respectively. 4. Nitrate reductase I consists of three different subunits, having molecular weights of 117 000; 57 000 and 52 000, which are present in a 1:1:2 molar ratio, respectively. Nitrate reductase II contains only the subunits with a molecular weight of 117 000 and 57 000 in a equimolar ratio. 5. Treatment at pH 9.5 in the presence of deoxycholate and 0.05 M NaCl or ageing removes the 52 000 Mr subunit from nitrate reductase I. This smallest subunit, in contrast to the other subunits, is a basic protein. 6. The 52 000 Mr subunit has no catalytic function in the intramolecular electron transfer from reduced benzylviologen to nitrate. However, it appears to have a structural function since nitrate reductase II, which lacks this subunit, is much more labile than nitrate reductase I. Inactivation of nitrate reductase II can be prevented by the presence of deoxycholate. 7. The spectrum of the enzyme resembles that of iron-sulfur proteins. No cytochromes or contaminating enzyme activities are present in the purified enzyme. Only reduced benzylviologen was found to be capable of acting as an electron donor. 8. p-Chlormercuribenzoate enhances the enzymatic activity at concentrations of 0.1 mM and lower. At higher p-chlormercuribenzoate concentrations the enzymatic activity is inhibited non-competitively with either nitrate or benzylviologen as a substrate. The inhibition is not counteracted by cysteine. 相似文献
15.
Orawan Bunyatang Nion Chirapongsatonkul Nunta Churngchow 《Journal of plant biochemistry and biotechnology.》2013,22(2):185-192
Protease inhibitors (PIs) are one family of pathogenesis-related proteins (PR-proteins) that play essential roles in defense mechanisms against an attack by a pathogenic microorganism or insect. Cell suspension derived from a seed integument of rubber tree (Hevea brasiliensis) treated for 48 h with 20 μM copper sulphate, an abiotic elicitor, had an increased production of PIs. The intracellular PIs were detected in an extract of treated cells; however, much higher levels of PIs were found in the medium (extracellular). Using azocasein as substrate, these PIs possessed strong inhibitory activity against subtilisin A but not against trypsin, chymotrysin and papain. These extracellular PIs were purified by anion exchange chromatography, DEAE-Sepharose (CL-6B), eluted with 0.06 M NaCl in 20 mM Tris-HCl (pH 7.0). The active fractions were then subjected to native and SDS preparative gel electrophoresis, respectively. A single band of a purified PI with a molecular weight of 25 kDa was revealed after a tricine SDS-PAGE and stained with silver nitrate. The yield of this purified protein was 3.14 ng.g?1. The activity of the purified PI was stable up to 70 °C, and its activity was retained in the buffer pH values of 2–10. The biological activity of the obtained PI was investigated. It was found that the PI at 5 μg.mL?1 (0.2 μM) inhibited the mycelium growth of Phytophthora palmivora, a rubber tree pathogen. 相似文献
16.
《Biocatalysis and Biotransformation》2013,31(3):185-191
AbstractA novel reductase has been detected in cell-free extracts from growing/resting cultures of the fungus Aspergillus versicolor D-1, which specifically catalyzes NADPH-dependent reduction of the γ,δ-double bond of the lactone-conjugated unsaturated system in securinine to form 14,15-dihydrosecurinine. The localization of the reductase has been investigated using differential centrifugation techniques. It was found that the securinine reductase is a cytosolic enzyme. The reductase was highly inducible in growing/resting cultures when securinine was used as the substrate and inducer. Optimal incubation conditions for assay of the securinine reductase were determined by using the enzyme preparation from resting cultures of A. versicolor D-1. The optimum temperature and pH for the reductase activity were in the range of 20–24°C and 8.0–8.5 in 0.05 M Tris–HCl buffer, respectively. The thermal stability of the securinine reductase was poor. 相似文献
17.
Resting membrane potential and intracellular sodium and potassium concentrations were determined at 5 and 21°C in normal and veratridine-treated axons of the squid Doryteuthis plei. 300 μM veratridine produced an increase in the intracellular sodium concentration, which changed from 52 to 284 mM in 10 min of exposure at 21°C, and from 76 to 260 mM at 5°C. Under the same treatment the intracellular potassium concentration changed from 357 to 221 mM (21°C) and from 334 to 194 mM (5°C). All the changes could be prevented by adding 1 μM tetrodotoxin. Veratridine (30, 100 and 300 μM) increased the resting sodium permeability of the giant axon, and the effect was greater at 21°C. The affinity of the membrane for veratridine increases when the nerves are cooled, the three concentrations tested produce maximum activation of the sodium channels at 5°C. But only the higher two concentrations are saturating at 21°C. 相似文献
18.
Ravi Kant Bhatia Shashi Kant Bhatia Praveen Kumar Mehta Tek Chand Bhalla 《Journal of industrial microbiology & biotechnology》2013,40(1):21-27
The acyl transfer activity of the amidase of Alcaligenes sp. MTCC 10674 has been applied to the conversion of benzamide and hydroxylamine to benzohydroxamic acid. The unique features of the acyl transfer activity of this organism include its optimal activity at 50 °C and very high substrate (100 mM benzamide) and product (90 mM benzohydroxamic acid) tolerance among the hitherto reported enzymes. The bench scale production of benzohydroxamic acid was carried out in a fed-batch reaction (final volume 1 l) by adding 50 mM benzamide and 250 mM of hydroxylamine after every 20 min for 80 min in 0.1 M potassium phosphate buffer (pH 7.0) at 50 °C, using resting cells equal to 4.0 mg dcm/ml of reaction mixture. From 1 l of reaction mixture 33 g of benzohydroxamic acid was recovered with 24.6 g l?1 h?1 productivity. The acyl transfer activity of the amidase of Alcaligenes sp. MTCC 10674 and the process developed in the present study are of industrial significance for the enzyme-mediated production of benzohydroxamic acid. 相似文献
19.
The inactivation of nitrate reductase in the leaves and rootsof barley (Hordeum vulgare L. cv. Mazurka) during and afterextracting was investigated. At 0 °C in the absence of casein,25 per cent of total. i.e. maximal in vitro, nitratereductase activity was lost during the 2 min extraction process,followed by a slower loss of activity while the extract wasstored in ice. Activity was maintained by adding a minimum of1 per cent casein to the extraction medium containing 0·1M phosphate (pH 7·5), 1 mM EDTA and 1 mM dithiothreitol.Nitrate reductase was stable for several hours in these extracts,but declined in a first order manner in the absence of dithiothreitol.Casein also prevented the initial loss while making root extracts,but had less effect during storage. Using casein and thiols, nitrate reductase activity in light,(as product of maximal in vitro rates and wt g1) in leaveswas 98 per cent of the total activity in 31-day-old plants grownwith full nutrient in water culture and 60-day-old field-grownplants receiving no fertilizer. Field-grown plants, however,exhibited only 17 per cent of the activity of culture-grownplants. Nitrate reductase in leaves of barley plants grown in waterculture had a diurnal rhythm. During the first 3 h of the lightperiod, activity increased to 1·3 x the darkvalue. This was followed by a temporary decrease and then byanother increase to a maximum of 1·7 x the darkvalue, occurring about 8 h after illumination. Activity thendecreased during the rest of the light period and in darkness. Hordeum vulgare L., barley, nitrate reductase 相似文献
20.
The study of certain conditions for the extraction of seed proteins ofAllium cepa revealed that the best extractibility of proteins is obtained by the use of a buffered physiological solution at 20 °C in
comparison with TRIS-glycine buffer at 5 °C. Using potassium phosphate buffer with 0.01 M mercaptoethanol and 0.4 M NaCl,
an amount of proteins by up to 25 per cent higher passes into solution as compared with the physiological solution, but these
extracts are unsuitable for the electrophoretic separation in polyacrylamide gels. The defatting of the seed meal under low
temperature did not affect the qualitative composition of the protein complex studied, the addition of a 1 per cent soluble
starch to the polyacrylamide gel. improved its resolution. 相似文献