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1.
1. Respiration of growing cultures of Acanthamoeba castellanii is inhibited less than 60% by azide (35 mM); the respiration of early-exponential-phase cultures differs from that of late-exponential-phase cultures in being stimulated by up to 120% by low concentrations (less than 1 mM) of this inhibitor. Azide (0.5 mM) plus 1 mM-salicylhydroxamic acid gives 80% inhibition of respiration in early- or late-exponential-phase cultures. 2. Lineweaver-Burk plots of 1/v against 1/[O2] for growing and stationary-phase cultures give values of less than 1 muM for the apparent Km for oxygen. 3. These values are not significantly altered when determined in the presence of 1 mM-salicylhydroxamic acid. 4. Higher values (greater than 7 muM) for apparent Km values for oxygen were obtained in the presence of azide, which gives non-linear Lineweaver-Burk plots. 5. Competitive inhibition of respiration by CO occurs with Ki 2.4 muM. 6. The results are discussed in terms of the presence of three terminal oxidases in this organism, namely two oxidases with high affinities for oxygen (cytochrome c oxidase of the main phosphorylating electron-transport chain and the salicylhydroxamic acid-sensitive oxidase) and a third oxidase with a low affinity for oxygen, sensitive to inhibition by cyanide but not by azide or salicylhydroxamic acid. The relative contributions to oxygen utilization by these oxidases change during the growth of a batch culture. 相似文献
2.
W N Poillon 《Journal of neurochemistry》1973,21(4):729-741
Abstract– Detergent-solubilized tyrosine hydroxylase from the caudate nucleus of the sheep was purified 3-fold by affinity chromatography on 3-iodotyrosine modified agarose. Supplementation of the standard assay with 1 mM Fe2+ resulted in only slight stimulation of the enzymic activity. The enzyme was, however, markedly inhibited by certain complexing agents specific for either Fe2+ or Fe3+. Double reciprocal plots of the kinetic data for a representative complexing agent, bathophenanthroline, showed the inhibition to be competitive with O2 (apparent Km 0.15 mM) and noncompetitive with either l -tyrosine or the synthetic tetrahydropterin cofactor DMPH4 (apparent Km's 0.12 and 0.29 mM, respectively). The combined inhibition and kinetics studies strongly suggest that brain tyrosine hydroxylase is an iron enzyme. Furthermore, the prosthetic iron very likely participates directly in catalytic function, presumably by binding molecular oxygen. The activity of ammonium sulphate-precipitated enzyme was found to be stimulated 2-fold by Fe2+, catalase or peroxidase, while untreated enzyme was markedly less affected by these agents. Since the only ostensible difference between the two preparations was the extensive aggregation present in the former case, the change in physical state evoked by ammonium sulphate precipitation appeared to be somehow related to this peculiar property of the enzyme. 相似文献
3.
L C Petersen 《Biochimica et biophysica acta》1977,460(2):299-307
1. The oxygen kinetics of purified beef heart cytochrome c oxidase were investigated. 2. The effect of addition of various fixed concentrations of the inhibitors CO, HN3, HCOOH, HCN and H2S on the double reciprocal plot of respiration rate against oxygen concentration was studied. 3. CO is strictly competitive, azide and formate are uncompetitive, and cyanide and sulfide are non-competitive inhibitors towards oxygen. 4. Binding constants for the various inhibitors from secondary plots of the oxygen kinetics at pH 7.4 are: CO: Ki = 0.32 micronM, azide: Ki = 33 micronM; formate: Ki = 15 mM; cyanide: Ki = 0.2 micronM and sulfide: Ki = 0.2 micronM. 5. The possible significance of these results in the elucidation of the reaction mechanism is discussed. 相似文献
4.
An oxygen electrode was used to monitor the reaction between dopamine (DA, 1-20 mM) and oxygen at pH 7.4 and 37 degrees C, in both the presence and absence of ascorbate (10 mM). The selected concentrations approximate levels within DA neurons. Diethylenetriaminepentaacetic acid (DTPA, 0.1 mM) was used to suppress catalysis by trace metals in the reagents. Separate experiments with catalase showed that oxygen consumption could be equated with the formation of hydrogen peroxide. Depending upon the experimental conditions, ascorbate acted either as an antioxidant, suppressing oxygen consumption (H2O2 production) to 6-8% of the expected rate, or as a prooxidant, amplifying oxygen consumption by 640%. The antioxidant action is consistent with the scavenging of superoxide radicals by ascorbate. The prooxidant action is probably the result of redox cycling of a pre-melanin oxidation product derived from DA. Analyses conducted by high-performance liquid chromatography with electrochemical detection revealed formation of a product with a very low oxidation potential; the product was not 6-hydroxydopamine. These observations may be relevant to concepts of toxicity mediated by DA within neuronal systems. 相似文献
5.
The mannitol-2-dehydrogenase (MtDH) from Gibberella zeae was purified and the corresponding cDNA identified. Purification of MtDH was accomplished using a combination of ammonium sulfate fractionation, anion exchange and dye-ligand chromatography. Final purification was achieved following electroelution from a native gel. Molecular mass determination based on SDS-PAGE indicated that the denatured protein was 29 kDa. Native protein mass was determined to be 110 kDa using gel permeation chromatography, indicating a tetrameric form. The pH optima for mannitol oxidation and fructose reductase activities were 9.0, and 7.0, respectively. Activity with sorbitol as the substrate was 21% of activity with mannitol. Kinetic parameters were determined by direct-linear plots of enzyme activity vs. substrate concentrations. Fructose concentrations above 600 mM and NADPH concentrations above 0.3 mM caused substrate inhibition. Comparisons of predicted amino acid sequences of several fungal MtDHs indicated high conservation within the phyla. A possible role for MtDH in generation of turgor pressure for forcible ascospore discharge is discussed. 相似文献
6.
In the present study, carbonic anhydrase (CA) enzyme was purified from rainbow trout (RT) liver with a specific activity of 4318 EUxmg(-1) and a yield of 38% using Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. The overall purification was approximately 2260-fold. To check the purity and determine subunit molecular weight of enzyme, SDS-polyacrylamide gel electrophoresis was performed, which showed a single band and MW of approx. 29.4 kDa. The molecular weight of native enzyme was estimated to be approx. 31 kDa by Sephadex-G 200 gel filtration chromatography. Optimum and stable pH were determined as 9.0 in 1 M Tris-SO(4) buffer and 8.5 in 1 M Tris-SO(4) buffer at 4 degrees C, respectively. The optimum temperature, activation energy (E(a)), activation enthalpy ((DeltaH) and Q(10) from Arrhenius plot for the RT liver CA were 40 degrees C, 2.88 kcal/mol, 2.288 kcal/mol and 1.53, respectively. The purified enzyme had an apparent K(m) and V(max) of 0.66 mM and 0.126 micromol x min(-1) for 4-nitrophenylacetate, respectively. K(cat) of the CA was found to be 32.8 s(-1). The inhibitory effects of low concentrations of different metals (Co(II), Cu(II), Zn(II) and Ag(I)) on CA activity were determined using the esterase method under in vitro conditions. The obtained IC(50) values, 50% inhibition of in vitro enzyme activity, were 0.03 mM for cobalt, 30 mM for copper, 47.1 mM for zinc and 0.01 mM for silver. K(i) values for these substances were also calculated from Linewaever-Burk plots as 0.050 mM for cobalt, 1.950 mM for copper, 7.035 mM for zinc and 2.190 mM for silver respectively and determined that cobalt and zinc inhibit the enzyme a competitive manner and copper and silver inhibit the enzyme in an uncompetitive manner. 相似文献
7.
T Komabayashi M Tsuboi 《Nihon seirigaku zasshi. Journal of the Physiological Society of Japan》1976,38(11):447-456
This investigation was undertaken to clarify the mechanism of the stimulated--respiration caused by K+ or norepinephrine in brown adipose tissue. 1. The addition of 30 approximately 100 mM K+ stimulated remarkably oxygen uptake in brown adipose tissue, and similarly norepinephrine (0.1 or 1.0 mug/ml) caused a marked stimulation. 2. Even if Na+ in normal Ringer solution was replaced by Choline or Li+, oxygen uptake caused by K+ (30 mM) or norepinephrine (1.0 mug/ml) was unaffected. 3. K+ -induced oxygen uptake was not observed when a Ca2+ -deficient tissue was incubated in Ca2+ -free Ringer, while norepinephrine-induced oxygen uptake clearly observed. And the oxygen uptake of Ca2+ -deficient tissue due to K+ was recovered by the addition of 5 mM Ca2+. 4. Mn2+ (6 mM) or La3+ (10 mM) inhibited significantly oxygen uptake due to K+, but not oxygen uptake due to norepinephrine. 5. K+ -induced oxygen uptake was unaffected by 10(-4) or 10(-3)M ouabain, but norepinephrine-induced oxygen uptake was inhibited considerably by 10(-4)M ouabain. 6. The oxygen uptake due to K+ was unaffected by propranolol (33 muM), whereas that due to norepinephrine was significantly inhibited in the presence of propranolol. 7. In the tissue from reserpine-treated animal, the oxygen uptake caused by K+ was observed. According, from these positive results we are justified to suggest that K+ -induced oxygen uptake is dependent on the presence of Ca2+, and not always caused by catecholamines released secondarily from nerve terminal. 相似文献
8.
Purification and properties of an ethanolamine-serine base exchange enzyme of rat brain microsomes 总被引:4,自引:0,他引:4
The activity of an ethanolamine and serine base exchange enzyme of rat brain microsomes was copurified to near homogeneity. The purification sequence involved detergent solubilization, Sepharose 4B column chromatography, phenyl-Sepharose 4B column chromatography, glycerol gradient sedimentation, and agarose-polyacrylamide gel electrophoresis under non-denaturing conditions. The ratio of the ethanolamine and serine base exchange activities remained almost constant during purification, and both enzyme activities were enriched 25-fold over the initial microsomal suspension. The final enzyme preparation which contained both enzyme activities showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel, having an apparent molecular mass of about 100 kDa. Serine inhibited the ethanolamine incorporation by this preparation and ethanolamine inhibited the serine incorporation. The competitive nature of this inhibition was apparent from Lineweaver-Burk plots, suggesting that the enzyme catalyzes the incorporation of both ethanolamine and serine into their corresponding phospholipids. The Km and Ki values for ethanolamine were quite similar, being 0.02 and 0.025 mM, respectively. The Km and Ki values for serine were also quite similar being 0.11 and 0.12 mM, respectively. The pH optimum was the same at 7.0 with both substrates. The optimum Ca2+ concentration was 8 mM for serine incorporation. 相似文献
9.
Partial Purification and Characterization of an Inducible Indole-3-Acetyl-L-Aspartic Acid Hydrolase from Enterobacter agglomerans
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Indole-3-acetyl-amino acid conjugate hydrolases are believed to be important in the regulation of indole-3-acetic acid (IAA) metabolism in plants and therefore have potential uses for the alteration of plant IAA metabolism. To isolate bacterial strains exhibiting significant indole-3-acetyl-aspartate (IAA-Asp) hydrolase activity, a sewage sludge inoculation was cultured under conditions in which IAA-Asp served as the sole source of carbon and nitrogen. One isolate, Enterobacter agglomerans, showed hydrolase activity inducible by IAA-L-Asp or N-acetyl-L-Asp but not by IAA, (NH4)2SO4, urea, or indoleacetamide. Among a total of 17 IAA conjugates tested as potential substrates, the enzyme had an exclusively high substrate specificity for IAA-L-Asp. Substrate concentration curves and Lineweaver-Burk plots of the kinetic data showed a Michaelis constant value for IAA-L-Asp of 13.5 mM. The optimal pH for this enzyme was between 8.0 and 8.5. In extraction buffer containing 0.8 mM Mg2+ the hydrolase activity was inhibited to 80% by 1 mM dithiothreitol and to 60% by 1 mm CuSO4; the activity was increased by 40% with 1 mM MnSO4. However, in extraction buffer with no trace elements, the hydrolase activity was inhibited to 50% by either 1 mM dithiothreitol or 1% Triton X-100 (Sigma). These results suggest that disulfide bonding might be essential for enzyme activity. Purification of the hydrolase by hydroxyapatite and TSK-phenyl (HP-Genenchem, South San Francisco, CA) preparative high-performance liquid chromatography yielded a major 45-kD polypeptide as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 相似文献
10.
Wang HX Ng TB Ooi VE 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2000,125(2):247-253
Cultured mycelia of the edible mushroom Tricholoma lobayense were extracted with cold saline. Proteins were precipitated from the extract by addition of (NH4)2SO4. The precipitate was dissolved and dialyzed before ion exchange chromatography on DEAE-cellulose. Ability to inhibit translation in a rabbit reticulocyte lysate was located in the unadsorbed fraction which was then subjected to affinity chromatography on Affi-gel Blue gel. The strongest activity was again retained by the unadsorbed fraction. Ion exchange chromatography on CM-cellulose resulted in fractionation of this fraction into an unadsorbed and two adsorbed peaks. Cell-free translation inhibitory activity was concentrated in the fraction eluted with 100 mM NaCl in 10 mM NH4OAc (pH 5.4). The translation-inhibitory protein possessed a molecular weight of 30 kDa as estimated by gel filtration using a fast protein liquid chromatography system and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 相似文献
11.
Binding of Clostridium botulinum neurotoxin to gangliosides 总被引:3,自引:0,他引:3
The binding characteristics of Clostridium botulinum neurotoxins of types B, C1, and F to gangliosides was studied by thin layer chromatography plate and microtiter plate methods at low (10 mM NaCl in 10 mM Tris-HCl buffer, pH 7.2) or high (150 mM NaCl in 10 mM Tris-HCl buffer, pH 7.2) ionic strengths and at 0 or 37 degrees C. The three types of toxins bound exclusively to three kinds of gangliosides, GD1a, GD1b, and GT1b, in both the thin layer chromatography plate and the microtiter plate methods. Type C1 toxin bound to the three gangliosides under all the conditions, while type B and F toxins bound only at low ionic strength and 37 degrees C. At low ionic strength, the binding kinetics for the three toxins was monophasic in Scatchard plots, and the association constants obtained in the microtiter plate system were 2-4 X 10(8) M-1. In contrast, the binding kinetics of type C1 toxin in high ionic strength was biphasic in the Scatchard plot, and two association constants were obtained in the microtiter plate system. The heavy chain facilitated the binding of the toxin to the gangliosides. These results indicate that different types of botulinum toxins bind to the gangliosides under different optimal conditions and that gangliosides may not be the common receptor for all types of botulinum toxins. The gangliosides may bind to type C1 toxin together with other potential receptor(s) on synaptosomal membranes. 相似文献
12.
Microbial L-phenylalanine ammonia-lyase. Purification, subunit structure and kinetic properties of the enzyme from Rhizoctonia solani. 总被引:4,自引:0,他引:4
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1. Phenylalanine ammonia-lyase (EC 4.3.1.5) was purified to homogeneity from the acetone-dried powders of the mycelial felts of the plant pathogenic fungus Rhizoctonia solani. 2. A useful modification in protamine sulphate treatment to get substantial purification of the enzyme in a single-step is described. 3. The purified enzyme shows bisubstrate activity towards L-phenylalanine and L-tyrosine. 4. It is sensitive to carbonyl reagents and the inhibition is not reversed by gel filtration. 5. The molecular weight of the enzyme as determined by Sephadex G-200 chromatography and sucrose-density-gradient centrifugation is around 330000. 6. The enzyme is made up of two pairs of unidentical subunits, with a molecular weight of 70000 (alpha) and 90000 (beta) respectively. 7. Studies on initial velocity versus substrate concentration have shown significant deviations from Michaelis-Menten kinetics. 8. The double-reciprocal plots are biphasic (concave downwards) and Hofstee plots show a curvilinear pattern. 9. The apparent Km value increases from 0.18 mM to as high as 5.0 mM with the increase in the concentration of the substrate and during this process the Vmax, increases by 2-2.5-fold. 10. The value of Hill coefficient is 0.5. 11. Steady-state rates of phenylalanine ammonia-lyase reaction in the presence of inhibitors like D-phenylalanine, cinnamic, p-coumaric, caffeic, dihydrocaffeic and phenylpyruvic acid have shown that only one molecule of each type of inhibitor binds to a molecule of the enzyme. These observations suggest the involvement of negative homotropic interactions in phenylalanine ammonia-lyase. 12. The enzyme could not be desensitized by treatment with HgCl2, p-chloromercuribenzoic acid or by repeated freezing and thawing. 相似文献
13.
A Pfohl-Leszkowicz G Keith G Dirheimer 《Comptes rendus des séances de la Société de biologie et de ses filiales》1985,179(5):679-687
Beef brain DNA-(cytosine-5-)-methyltransferase was partially purified by chromatography on Ultrogel AcA34 and Dyematrex Blue A. The purification was of 360 times and the recovery of 75%. The pH optimum of the reaction is 7.6 NaCl inhibits double stranded DNA methylation, but stimulates single stranded DNA methylation up to 50 mM, before inhibiting. EDTA (1 mM) and MgCl2 (4 mM) stimulate DNA methylation. Polyamines inhibit the reaction. 相似文献
14.
Inaba Y Tokishita S Hamada-Sato N Kobayashi T Imada C Yamagata H Watanabe E 《Biosensors & bioelectronics》2004,20(4):833-840
Agmatine (Agm) is an indicator of squid freshness. The Agm sensor was developed using flow injection analysis (FIA) that consisted of the putrescine oxidase (PuOx) reactor, the agmatinase (AUH)-PuOx reactor and two oxygen electrodes. In the proposed sensor, the first step is that coexisting cadaverine (Cad) and putrescine (Put) are removed by passing through the PuOx reactor and the initial decomposition is determined by the amount of oxygen consumed, simultaneously. The second step is that the amount of Agm is determined by the amount of oxygen consumed in the AUH-PuOx reactor. The optimum conditions for the use of the Agm sensor were as follows: 50 mM HEPES containing MnSO4 at a final concentration of 5 mM, pH 8.0, flow rate of 0.6 mL min(-1) and injection volume of 50 microL. A single assay could be completed in approximately 3 min. A linear relationship was obtained between the output and the Agm concentration in the range of 0.01-1 mM Agm with a correlation coefficient of 0.999. The detection limit was 0.005 mM. The relative standard deviations (RSDs) were 3.14 and 1.19% (n = 20) for 0.1 and 0.3 mM Agm, respectively. The extracts of squid were injected into the proposed sensor and the results were compared with those obtained using the conventional high-performance liquid chromatography (HPLC) method. A correlation was observed between the results obtained by the proposed sensor and those obtained by the conventional method. The determination of squid freshness is one of the good uses of the proposed Agm sensor. 相似文献
15.
A novel ribonuclease with antiproliferative activity from fresh fruiting bodies of the edible mushroom Hypsizigus marmoreus 总被引:1,自引:0,他引:1
An 18-kDa ribonuclease (RNase) with a novel N-terminal sequence was purified from fresh fruiting bodies of the mushroom Hypsizigus marmoreus. The purification protocol comprised ion exchange chromatography on DEAE cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose and Q-Sepharose and gel filtration by fast protein liquid chromatography on Superdex 75. The starting buffer was 10 mM Tris-HCl buffer (pH 7.2), 10 mM Tris-HCl buffer (pH 7.2), 10 mM NH(4)OAc buffer (pH 5), 10 mM NH(4)HCO(3) buffer (pH 9.4) and 200 mM NH(4)HCO(3) (pH 8.5), respectively. Absorbed proteins were desorbed using NaCl added to the starting buffer. A 42-fold purification of the enzyme was achieved. The RNase was unadsorbed on DEAE cellulose, Affi-gel blue gel and CM-cellulose but adsorbed on Q-Sepharose. It exhibited maximal RNase activity at pH 5 and 70 degrees C. Some RNase activity was detectable at 100 degrees C. It demonstrated the highest ribonucleolytic activity (196 U/mg) toward poly C, the next highest activity (126 U/mg) toward poly A, and much weaker activity toward poly U (48 U/mg) and poly G (41 U/mg). The RNase inhibited [(3)H-methyl]-thymidine uptake by leukemia L1210 cells with an IC(50) of 60 microM. 相似文献
16.
Internal oxygen levels decrease during the growing season and with increasing stem height 总被引:8,自引:0,他引:8
L. Eklund 《Trees - Structure and Function》2000,14(4):177-180
The oxygen levels in the sapwood of Norway spruce [Picea abies Karst. (L.)] trees were measured by GC-MS (gas chromatography – mass spectrometry) as stem growth progressed during the growing
season. The measurements were made on 30-year-old, 13–15 m high, trees growing on three plots which were irrigated, left undisturbed
or subjected to drought. The oxygen measurements were done at 1.5, 4 and 8 m above the ground. The internal oxygen levels
dropped to about 5% of the level in air in irrigated trees, 1–3% in control trees and to below 1% in trees subject to drought
between mid-July and mid-August. In the trees subjected to drought there where no differences in internal oxygen concentrations
between the positions on the tree during the growing season except for a faster increase back to ambient concentration occurring
in late August in the 1.5 m position. In trees growing on the two other plots the internal oxygen concentration was higher
near the ground than at 4 or 8 m above ground during July and August. At all other dates there were no differences between
the positions. The results indicate that there is an oxygen gradient with levels decreasing acropetally, giving indirect support
to the hypothesis that oxygen is supplied to the growing stem in the transpiration stream.
Received: 26 January 1999 / Accepted: 1 June 1999 相似文献
17.
In this study, the in vitro effects of some sulfonamide derivatives, which are carbonic anhydrase inhibitors, on the enzymes activities of glucose-6-phosphate dehydrogenase, 6-phospho gluconate dehydrogenase and glutathione reductase were investigated. For this purpose, these three enzymes were purified from human erythrocytes. Purification procedure composed of four steps; preparation of the hemolysate, ammonium sulfate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and gel filtration chromatography on Sephadex G-200. 5-(3alpha-Hydroxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (1), 5-(3alpha,12alpha-Dihydroxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (2), 5-(3alpha,7alpha,12alpha-Trihydroxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (3), 5-(3alpha,Acetoxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (4), 5-(3alpha,7alpha,12alpha-Triacetoxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (5), 5-(3,7,12-Trioxo-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (6), acetazolamide, and dorzolamide were tested in this experiment. Compounds 3, 5, and dorzolamide showed inhibitory effects on the activity of 6-phosphogluconate dehydrogenase, and I(50) values and K(i) constants were calculated as 0.0601 mM, 0.00253 mM, and 1.41 mM and 0.0878 +/- 0.0274 mM, 0.0042 +/- 0.0009 mM, and 3.1446 +/- 0.2081 mM, respectively. Glutathione reductase was also inhibited by 1 and 2. I(50) values and K(i) constants were 0.0471 mM and 0.0723 +/- 0.0388 mM for 1 and 0.0045 mM and 0.0061 +/- 0.0014 mM, for 2. If these sulfonamide derivatives are proposed as drugs, some of which are being used in glaucoma treatment such as acetazolamide and dorzolamide, these results should be taken into consideration concerning via these enzymes. 相似文献
18.
Peroxygenase metabolism of N-acetylbenzidine by prostaglandin H synthase. Formation of an N-hydroxylamine. 总被引:1,自引:0,他引:1
T V Zenser V M Lakshmi F F Hsu B B Davis 《The Journal of biological chemistry》1999,274(21):14850-14856
Synthesis of prostaglandin H2 by prostaglandin H synthase (PHS) results in a two-electron oxidation of the enzyme. An active reduced enzyme is regenerated by reducing cofactors, which become oxidized. This report examines the mechanism by which PHS from ram seminal vesicle microsomes catalyzes the oxidation of the reducing cofactor N-acetylbenzidine (ABZ). During the conversion of 0.06 mM ABZ to its final end product, 4'-nitro-4-acetylaminobiphenyl, a new metabolite was observed when 1 mM ascorbic acid was present. Similar results were observed whether 0.2 mM arachidonic acid or 0.5 mM H2O2 was used as the substrate. This metabolite co-eluted with synthetic N'-hydroxy-N-acetylbenzidine (N'HA), but not with N-hydroxy-N-acetylbenzidine. The new metabolite was identified as N'HA by electrospray ionization/MS/MS. N'HA represented as much as 10% of the total radioactivity recovered by high pressure liquid chromatography. When N'HA was substituted for ABZ, PHS metabolized N'HA to 4'-nitro-4-acetylaminobiphenyl. Inhibitor studies demonstrated that metabolism was due to PHS, not cytochrome P-450. The lack of effect of 5,5-dimethyl-1-pyrroline N-oxide, mannitol, and superoxide dismutase suggests the lack of involvement of one-electron transfer reactions and suggests that hydroxyl radicals and superoxide are not sources of oxygen or oxidants. Oxygen uptake studies did not demonstrate a requirement for molecular oxygen. When [18O]H2O2 was used as the substrate, 18O enrichment was observed for 4'-nitro-4-acetylaminobiphenyl, but not for N'HA. A 97% enrichment was observed for one atom of 18O, and a 17 +/- 7% enrichment was observed for two 18O atoms. The rapid exchange of 18O-N'HA with water was suggested to explain the lack of enrichment of N'HA and the low enrichment of two 18O atoms into 4'-nitro-4-acetylaminobiphenyl. Results demonstrate a peroxygenase oxidation of ABZ and N'HA by PHS and suggest a stepwise oxidation of ABZ to N'-hydroxy, 4'-nitroso, and 4'-nitro products. 相似文献
19.
The soluble Ca2+-ATPase from Escherichia coli had a distinctive behavior with respect to inhibition by Na+ measured at 36 degrees C and 19 degrees C. At the first temperature the Hill plots are linear and show a slope of 1.1 while at 19 degrees C the plots are biphasic, with slopes of 1.8 and 0.8 before and after the break, respectively. The break occurs at about 50 nM NaCl. Gel chromatography was performed in jacketed Sepharose 4B columns kept at 2 temperatures in the presence of different concentrations of NaCl. It was found that the Stokes' radius of the enzyme was dependent on the temperature and on the salt concentration. Equilibrium sucrose gradients run at 19 degrees C showed that the sedimentation constant of the enzyme remained constant irrespective of the NaCl concentration used. It is concluded that a "folding" of the enzyme takes place in the presence of NaCl, the process being complete at about 50 mM NaCl at 19 degrees C and at about 20 mM at 36 degrees C. The results are in excellent agreement with the kinetic data: the "folded" or "compact" configuration would show no cooperative response towards Na+ while the "expanded" conformer would present strong cooperativity. This is also in agreement with the results obtained with the enzyme embedded in the membrane: when the membrane is fluid a high n value (Hill coefficient) is found; when the membrane is more rigid the value of n falls. A model explaining all our results is proposed and discussed. 相似文献
20.
The plastidic class I and cytosolic class II aldolases of Euglena gracilis have been purified to apparent homogeneity. In autotrophically grown cells, up to 81% of the total activity is due to class I activity, whereas in heterotrophically grown cells, it is only 7%. The class I aldolase has been purified to a specific activity of 20 units/mg protein by anion-exchange chromatography, affinity chromatography, and gel filtration. The native enzyme (molecular mass 160 kD) consisted of four identical subunits of 40 kD. The class II aldolase was purified to a specific activity of 21 units/mg by (NH4)2SO4 fractionation, anion-exchange chromatography, chromatography on hydroxylapatite, and gel filtration. The native enzyme (molecular mass 80 kD) consisted of two identical subunits of 38 kD. The Km (fructose-1,6-bisphosphate) values were 12 [mu]M for the class I enzyme and 175 [mu]M for the class II enzyme. The class II aldolase was inhibited by 1 mM ethylenediaminetetraacetate (EDTA), 0.8 mM cysteine, 0.5 mM Zn2+, or 0.5 mM Cu2+. Na+, K+, Rb+, and NH4+ (but not Li+ or Cs+) enhanced the activity up to 7-fold. After inactivation by EDTA, the activity could be partially restored by Mn2+, Cu2+, or Co2+. A subclassification of class II aldolases is proposed based on (a) activation/inhibition by Cys and (b) activation or not by divalent ions. 相似文献