共查询到20条相似文献,搜索用时 46 毫秒
1.
SYNOPSIS Two glutamate dehydrogenases, NADH-linked (EC 1.2.1.2) and NADPH-linked (EC 1.2.1.4.) were isolated from the epimastigote forms of Trypanosoma cruzi and purified. Both enzymes exist as hexamers. The molecular weights of the native NADH-and NADPH-linked glutamate dehydrogenases were estimated to be 360,000 and 265,000, respectively, and those of the subunits to be 58,000 and 43,000, respectively. The isoelectric point of the NADH-linked dehydrogenase is at pH 5.25 and that of the NADPH-linked enzyme at pH 5.1. The activities of both enzymes are regulated by product inhibition. In addition, purine nucleotides were shown to be potent inhibitors of the NADH-linked glutamate dehydrogenase. 相似文献
2.
Summary The induction of xylose reductase (XR) and xylitol dehydrogenase (XD) activities by D-xylose under different fermentation conditions was investigated in Neurospora crassa. The induction of NADPH-linked XR preceded NADH-linked XR and the ratio of NADH to NADPH-linked XR activity displayed variation from 0.02 to 0.2 suggesting the presence of two separate enzymes. Aerobic conditions were required by N. crassa for cell growth but not for ethanol production. Maximum ethanol of 0.3 g/g of D-xylose was produced when shifted to semiaerobic condition, where high NADH-linked XR and NAD-linked XD activities were observed. 相似文献
3.
Effects of 1-methyl-4-phenylpyridinium ion (MPP+) on the activities of NAD+- or NADP+-linked dehydrogenases in the TCA cycle were studied using mitochondria prepared from mouse brains. Activities of NAD+- and NADP+-linked isocitrate dehydrogenases, NADH- and NADPH-linked glutamate dehydrogenases, and malate dehydrogenase were little affected by 2 mM of MPP+. However, alpha-ketoglutarate dehydrogenase activity was significantly inhibited by MPP+. Kinetic analysis revealed a competitive type of inhibition. Inhibition of alpha-ketoglutarate dehydrogenase may be one of the important mechanisms of MPP+-induced inhibition of mitochondrial respiration, and of neuronal degeneration. 相似文献
4.
Summary Aerobic cultures harvested from the lag and early exponential growth phases fermented D-xylose poorly under anaerobic conditions whereas fermentation by late exponential and stationary phase cultures was rapid. These differences could be related to the ratios of NADH- to NADPH-linked xylose reductase (XR) and the levels of NADH-linked XR and NAD-linked xylitol dehydrogenase (XD) present. Under aerobic conditions, induction of NADPH-linked XR preceded NADH-linked XR which suggested the presence of two separate XR't's. Induction of XR and XD was more rapid under aerobic than anaerobic conditions. 相似文献
5.
NADH- and NADPH-linked aquacobalamin reductases occur in both mitochondrial and microsomal membranes of rat liver 总被引:1,自引:0,他引:1
F Watanabe Y Nakano S Maruno N Tachikake Y Tamura S Kitaoka 《Biochemical and biophysical research communications》1989,165(2):675-679
To elucidate the mechanism for the synthesis of the coenzyme forms of cobalamin in mammals, rat liver aquacobalamin reductase was partially characterized. Rat liver contained both NADH- and NADPH-linked aquacobalamin reductases. The NADH-linked enzyme was distributed in the mitochondria (approx. 40%) and microsomes (60%), identical to the distribution of the NADPH-linked enzyme. The two mitochondrial NADH- and NADPH-linked enzymes were located inside of the outer membrane. 相似文献
6.
Cell-free extracts of a streptomycin-bleached strain of Euglena gracilis var. bacillaris have been examined for enzyme systems primarily responsible for the oxidation of reduced pyridine nucelotides. NADH lipoyl dehydrogenase, NADH and NADPH oxidase, NADH and NADPH diaphorase, and NADH and NADPH cytochrome c reductase have been demonstrated. The NADPH-linked enzymes had lower activity rates and were less sensitive to N-ethyl maleimide and p-hydroxymercuribenzoate than their NADH-linked counterparts. NADH cytochrome c reductase was the most sensitive to antimycin A. Michaelis-Menten constants (Km) determined were as follows: NADH diaphorase, 350 muM; NADPH oxidase 150 muM ; NADH lipoyl dehydrogenase, 0.35 muM. Enzyme activities after storage at -5 C indicate that the diaphorases are less labile than the other tested enzymes, and the differential activities of the NADH and NADPH linked enzymes suggest that functionally they may have different roles. 相似文献
7.
Purification and properties of glutamate synthase and glutamate dehydrogenase from Bacillus megaterium. 总被引:8,自引:0,他引:8
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Bacillus megaterium N.C.T.C. no. 10342 exhibits glutamate synthetase (EC 2.6.1.53) and glutamate dehydrogenase (EC 1.4.1.4) activities. Concentrations of glutamate synthase were high when the bacteria were grown on 3mM-NH4Cl and low when they were grown on 100mM-NH4Cl, whereas glutamate dehydrogenase concentrations were higher when the bacteria were grown on 100mM-NH4Cl than on 3mM-NH4Cl. Glutamate synthase and glutamate dehydrogenase were purified to homogeneity from B. megaterium grown in 10mM-glucose/10mM-NH4Cl. The purified enzymes had mol.wts. 840000 and 270000 for glutamate synthase and glutamate dehydrogenase respectively. The Km values for substrates with NADPH and coenzyme were (glutamate synthase activity shown first) 9 micron and 360 micron for 2-oxoglutarate, 7.1 micron and 8.7 micron for NADPH, and 0.2 mM for glutamine and 22 mM for NH4Cl, similar values to those of enzymes from Escherichia coli. Glutamate synthase contained NH3-dependent activity (different from authentic glutamate dehydrogenase), which was enhanced 4-fold during treatment at pH 4.6 NH3-dependent activity was generally about 2% of the glutamine-dependent activity. Amidination of glutamate synthase by the bi-functional cross-linking reagent dimethyl suberimidate inactivated glutamine-dependent glutamate synthase activity, but increased NH3-dependent activity. A cross-linked structure of mol.wt. approx 200000 was the main product formed. 相似文献
8.
Characterization of alanine and malate dehydrogenases from a marine psychrophile strain PA-43 总被引:2,自引:0,他引:2
Jane A. Irwin Haflidi M. Gudmundsson Viggo T. Marteinsson Gudmundur O. Hreggvidsson Anthony J. Lanzetti Gudni A. Alfredsson Paul C. Engel 《Extremophiles : life under extreme conditions》2001,5(3):199-211
Alanine dehydrogenase (AlaDH: EC 1.4.1.1), malate dehydrogenase (MDH: EC 1.1.1.37), and glutamate dehydrogenase (EC 1.4.1.2), all NAD+ dependent, were detected in extracts from a psychrophilic bacterium, strain PA-43, isolated from a sea urchin off the Icelandic coast. Characterization tests suggested that the strain had a close relationship to Vibrio, but sequencing of part of the 16S rDNA gene placed the bacterium among Shewanella species in a constructed phylogenetic tree. The bacterium had an optimum growth temperature of 16.5 degrees C, and maximum dehydrogenase expression was obtained in a rich medium supplemented with NaCl. Both AlaDH and MDH were purified to homogeneity. AlaDH is a hexamer, with an approximate relative molecular mass of 260,000, whereas MDH is dimeric, with an apparent relative molecular mass of approximately 70,000. Both enzymes were thermolabile, and the optimum temperatures for activity were shifted toward lower temperatures than those found in the same enzymes from mesophiles, 37 degrees C for MDH and approximately 47 degrees C for AlaDH. The pH optima for AlaDH in the forward and reverse reactions were 10.5 and 9, respectively, whereas those for MDH were 10-10.2 and 8.8, respectively. Partial amino acid sequences, comprising approximately 30% of the total sequences from each enzyme, were determined for N-terminal, tryptic, and chymotryptic fragments of the enzymes. The AlaDH showed the highest similarity to AlaDHs from the psychrotroph Shewanella Ac10 and the mesophile Vibrio proteolyticus, whereas MDH was most similar to the MDHs from the mesophiles Escherichia coli and Haemophilus influenzae, with lower identity to the psychrophilic malate dehydrogenases from Vibrio 5710 and Photobacterium SS9. 相似文献
9.
D R Hodges H J Karavolas 《The Journal of steroid biochemistry and molecular biology》1992,41(1):79-84
Progesterone 5 alpha-reductase activity and 5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) enzymic activities (NADH-linked and NADPH-linked) were measured in anterior pituitaries (AP) from aged female rats during three stages of reproductive senescence (constant estrus: CE; repeated pseudopregnancies: PSP; and anestrus: AN). To assess ovarian influence on these enzymes during these stages of reproductive aging, we also determined enzyme levels from ovariectomized rats from each stage treated with estrogen or vehicle. Progesterone 5 alpha-reductase and NADH-linked 3 alpha-HSOR activities were 2-fold higher in pituitaries of CE rats as compared to those of PSP and AN rats. NADPH-linked 3 alpha-HSOR levels did not differ among the three stages. All three enzyme levels were elevated 2- to 5-fold as compared to the corresponding enzyme levels from young cycling rats. After ovariectomy (10 days), 5 alpha-reductase activity in PSP and AN rats was elevated 3- to 4-fold relative to mean levels in intact PSP and AN rats. Ovariectomy had no effect on 5 alpha-reductase levels in CE rats. Under similar conditions, young cycling rats exhibit a 10-12-fold increase. Treatment of ovariectomized PSP and AN rats for 3 days with estradiol benzoate (10 micrograms/day) restored 5 alpha-reductase levels. Ovariectomy had no effect on the NADPH-linked 3 alpha-HSOR levels in CE, PSP or AN animals which is similar to that observed with young rats. Ovariectomy also had no effect on the NADH-linked 3 alpha-HSOR levels except for the CE group. The ovariectomized CE rats exhibited reduced pituitary NADH-linked 3 alpha-HSOR levels (30%). In contrast, young rats exhibit elevated pituitary NADH-linked 3 alpha-HSOR levels after ovariectomy (4- to 5-fold). These changes suggest the possibility that altered processing of progesterone and its 5 alpha- and 3 alpha-reduced products may be one means by which the effectiveness of progesterone is reduced during aging. The results also suggest an altered ovarian role in the regulation of these enzymes. 相似文献
10.
Five different immobilized NAD+ derivatives were employed to compare the behavior of four amino acid dehydrogenases chromatographed using kinetic-based enzyme capture strategies (KBECS): S6-, N6-, N1-, 8'-azo-, and pyrophosphate-linked immobilized NAD+. The amino acid dehydrogenases were NAD+-dependent phenylalanine (EC 1.4.1.20), alanine (EC 1.4.1.1), and leucine (EC 1.4.1.9) dehydrogenases from various microbial species and NAD(P)+-dependent glutamate dehydrogenase from bovine liver (GDH; EC 1.4.1.3). KBECS for bovine heart L-lactate dehydrogenase (EC 1.1.1.27) and yeast alcohol dehydrogenase (EC 1.1.1.1) were also applied to assist in a preliminary assessment of the immobilized cofactor derivatives. Results confirm that the majority of the enzymes studied retained affinity for NAD+ immobilized through an N6 linkage, as opposed to an N1 linkage, replacement of the nitrogen with sulfur to produce an S6 linkage, or attachment of the cofactor through the C8 position or the pyrophosphate group of the cofactor. The one exception to this was the dual-cofactor-specific GDH from bovine liver, which showed no affinity for N6-linked NAD+ but was biospecifically adsorbed to S6-linked NAD+ derivatives in the presence of its soluble KBEC ligand. The molecular basis for this is discussed together with the implications for future development and application of KBECS. 相似文献
11.
In extension of a previous study with yeast glucose-6-P dehydrogenase (Kawaguchi, A., and Bloch, K. (1974) J. Biol. Chem. 249, 5793-5800), the structural changes accompanying the inhibition of glutamate dehydrogenase and several malate dehydrogenases by palmitoyl-CoA and by sodium dodecyl sulfate have been investigated. Palmitoyl-CoA converts liver glutamate dehydrogenase to enzymatically inactive dimeric subunits (Mr = 1.2 X 10(5)) and tightly binds to the dissociated enzyme. Removal of the inhibitor from the palmitoyl-CoA-dimer complex fails to regenerate enzyme activity. The Ki values for palmitoyl-CoA inhibition of malate dehydrogenases (oxalacetate reduction) are, for the enzyme from pig heart mitochondria, 1.8 muM, 500 muM from pig heart supernatant, and 10 muM from chicken heart supernatant. These inhibitions are readily reversible. Palmitoyl-CoA does not alter the quaternary structure of any of the malate dehydrogenases and binds only weakly to these enzymes. Mitochondrial malate dehydrogenase assayed in the direction malate to oxalacetate is much less sensitive to palmitoyl-CoA, with Ki values of 50 muM at pH 10 and greater than 50 muM at pH 7.4. While the differences in palmitoyl-CoA sensitivity in the forward and backward reactions catalyzed by mitochondrial dehydrogenase are unexplained, a physiological rationale for these differential effects is offered. Sodium dodecyl sulfate dissociates the various dehydrogenases to monomeric subunits in contrast to the more selective effects of palmitoyl-CoA. 相似文献
12.
It was possible to extract simultaneously several active enzymes involved in the carbohydrate or the amino acid metabolism from spruce needles [ Picea abies (L.) Karst.] when a) a 100 m M Na-Pi buffer of pH 7.5 containing 5% PVPP and 0.5% Triton X-100 was used and when b) the resulting crude extracts were freed from lowmolecular-weight compounds by gel-chromatography using the separation medium Fractogel TSK HW-40. Besides Triton X-100, Triton X-305, Myrij-52 and Brij-35 were tested, but 0.5% Triton X-100 brought about the most active enzyme extracts. In crude extracts prepared from spruce needles during the early summer a high increase in absorbance at 334 nm was observed when the co-substrate NADP+ was added, thus making reliable spectrophotometric assays impossible. The interfering low-molecular-weight substances could be eliminated by gel chromatography. As separation media Bio-Gel P-6 DG, Sephadex G-25 m, Trisacryl GF 05 and Fractogel TSK HW-40 (F) were tested, with Fractogel yielding the highest activities.
With the methods described in this paper the activities of the following enzymes were determined: glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), glucose-6-phosphate isomerase (EC 5.3.1.9), shikimate dehydrogenase (EC 1.1.1.25), NAD+ -malate dehydrogenase (EC 1.1.1.37), glutamate dehydrogenase (EC 1.4.1.2), aspartate aminotransferase (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2). The activities estimated for NAD+ -malate dehydrogenase and 6-phosphogluconate dehydrogenase are in the range of those published for the needle enzymes of white spruce and Scots pine, respectively. 相似文献
With the methods described in this paper the activities of the following enzymes were determined: glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), glucose-6-phosphate isomerase (EC 5.3.1.9), shikimate dehydrogenase (EC 1.1.1.25), NAD
13.
J Marchand J Torreilles M C Guerin B Descomps A C De Paulet M Gabriel D Larcher 《Biochimica et biophysica acta》1982,707(1):7-13
Carbonyl compounds such as alpha-ketoglutarate, pyruvate, oxaloacetate, butyraldehyde, acetaldehyde or acetone react with NAD or NADP to give adducts. Binding studies of adducts to dehydrogenases are performed by means of ultraviolet differential spectroscopy, circular dichroism and spectrofluorimetry. The dehydrogenases show a high degree of binding specificity toward the adducts which contain their specific oxidized substrate and their specific coenzyme. The high selectivity of the dehydrogenases for adducts is evidenced by binding studies of NAD(P)-pyruvate and NAD(P)-alpha-ketoglutarate adducts on glutamate dehydrogenase at pH 7.6 and 8.9. Evidence is presented showing that adducts bind to the active site of the enzymes. 相似文献
14.
Barry J. Shelp Craig S. Walton Wayne A. Snedden Lucie G. Tuin Ivan J. Oresnik David B. Layzell 《Physiologia plantarum》1995,94(2):219-228
In the present study we investigated the proposal that the γ-aminobutyrate (Gaba) shunt in developing soybean (Glycine max [L.] Merr.) seeds is associated with hypoxia. The ontogeny and pH profile of enzymes associated with glutamate metabolism (glutamate decarboxylase [EC 4.1.1.15]. Gaba transaminase [EC 2.6.1.19], succinic semialdehyde dehydrogenase [EC 1.2.1.16], glutamate dehydrogenase [EC 1.4.1.2], glutamate:oxaloacetate transaminase [EC 2.6.1.1], glutamate:pyruvate transaminase [EC 2.6.1.2] and 2-oxoglutarate dehydrogenase complex [EC 1.2.4.2]) and hypoxia (alcohol dehydrogenase [ADH, EC 1.1.1.1] and pyruvate decarboxylase [PDC, EC 4.1.1.1]) were determined in cotyledons, nucellus and seed-coat tissues. Gaba-shunt enzymes were ubiquitous in the developing seed. Activities of enzymes catalyzing glutamate-C entry into the Krebs cycle via 2-oxoglutarate were generally greater than those of Gaba-shunt enzymes. In cotyledons, the activity of ADH increased throughout seed development (up to 72 days after anthesis [DAA]), whereas PDC was static during early development, then increased. In contrast, the activities of ADH and PDC in maternal tissues (nucellus and seed coat) were initially high, then declined dramatically after 37 DAA. The adenylate energy charge (AEC) = ([ATP]+0.5 [ADP])/ ([ATP] + [ADP] + [AMP]) of soybean seeds from fruits (37 DAA) frozen in situ was low (0.67±0.01) compared to the AEC of adjacent pod tissue (0.82 ± 0.04) and cotyledons exposed to air (0.84 ± 0.01). A 60-min time-course study showed that the rate of [U-14C]-glutamate catabolism by an intact excised cotyledon at 37 DAA was markedly lower at 8 and 0% O2 than at 21%; the pool size of [14C]-Gaba was unaffected. The data indicated that: (1) Gaba-shunt activity is not a response to limited glutamate deamination/transamination: (2) the soybean seed is hypoxic; and (3) the relative partitioning of glutamate-C through glutamate decarboxylase is increased by hypoxia. 相似文献
15.
Glutamate dehydrogenase (EC 1.4.1.2-4) from Peptostreptococcus asaccharolyticus has a strong preference for NADH over NADPH as a coenzyme, over 1000-fold in terms of kcat/Km values. Sequence alignments across the wider family of NAD(P)-dependent dehydrogenases might suggest that this preference is mainly due to a negatively charged glutamate at position 243 (E243) in the adenine ribose-binding pocket. We have examined the possibility of altering coenzyme specificity of the Peptostreptococcus enzyme, and, more specifically, the role of residue 243 and neighbouring residues in coenzyme binding, by introducing a range of point mutations. Glutamate dehydrogenases are unusual among dehydrogenases in that NADPH-specific forms usually have aspartate at this position. However, replacement of E243 with aspartate led to only a nine-fold relaxation of the strong discrimination against NADPH. By contrast, replacement with a more positively charged lysine or arginine, as found in NADPH-dependent members of other dehydrogenase families, allows a more than 1000-fold shift toward NADPH, resulting in enzymes equally efficient with NADH or NADPH. Smaller shifts in the same direction were also observed in enzymes where a neighboring tryptophan, W244, was replaced by a smaller alanine (approximately six-fold) or Asp245 was changed to lysine (32-fold). Coenzyme binding studies confirm that the mutations result in the expected major changes in relative affinities for NADH and NADPH, and pH studies indicate that improved affinity for the extra phosphate of NADPH is the predominant reason for the increased catalytic efficiency with this coenzyme. The marked difference between the results of replacing E243 with aspartate and with positive residues implies that the mode of NADPH binding in naturally occurring NADPH-dependent glutamate dehydrogenases differs from that adopted in E243K or E243D and in other dehydrogenases. 相似文献
16.
Purification and properties of D-mannitol-1-phosphate dehydrogenase and D-glucitol-6-phosphate dehydrogenase from Escherichia coli. 总被引:8,自引:6,他引:2
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
D-Mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) and D-glucitol-6-phosphate dehydrogenase (EC 1.1.1.140) were purified to apparent homogeneity in good yields from Escherichia coli. The amino acid compositions, N-terminal amino acid sequences, sensitivities to chemical reagents, and catalytic properties of the two enzymes were determined. Both enzymes showed absolute specificities for their substrates. The subunit molecular weights of mannitol-1-phosphate and glucitol-6-phosphate dehydrogenases were 40,000 and 26,000, respectively; the apparent molecular weights of the native proteins, determined by gel filtration, were 40,000 and 117,000, respectively. It is therefore concluded that whereas mannitol-1-phosphate dehydrogenase is a monomer, glucitol-6-phosphate dehydrogenase is probably a tetramer. These two proteins differed in several fundamental respects. 相似文献
17.
Microsomal vesicle were centrifuged through sucrose density gradients containing deoxycholate. With 0.15% detergent electron transport enzymes and phosphatases could be separated. Increasing the deoxycholate concentration to 0.19% resulted in separation of the microsomal material into five bands containing (in order from the top of the gradient) adenosine monophosphatase, inosine diphosphatase and some glucose-6-phosphatase (band 1); NADH-linked (band 2) and NADPH-linked (band 3) electron transport enzymes; and glucose-6-phosphatase (bands 4 and 5). It appears that enzymes are arranged in specialized patchers in the microsomal membrane. 相似文献
18.
Coenzymic activities of the following NADP derivatives were investigated: 2'-O-(2-carboxyethyl)phosphono-NAD (I), N6-(2-carboxyethyl)-NADP (II), 2'-O-(2-carboxyethyl)phosphono-N6-(2-carboxyethyl)-NAD (III), 2'-O-[N-(2-aminoethyl)carbamoylethyl]phosphono-NAD (IV), N6-[N-(2-aminoethyl)carbamoylethyl]-NADP (Va), 2',3'-cyclic NADP, and 3'-NADP. Derivatives I and IV show the effects of modification at the 2'-phosphate group, and derivatives II and Va show those at the 6-amino group of NADP. As for enzymes, alcohol, isocitrate, 6-phosphogluconate, glucose, glucose-6-phosphate, and glutamate dehydrogenases were used. These enzymes were grouped on the basis of the ratio of the activities for NAD and NADP into NADP-specific enzymes (ratio less than 0.01), NAD(P)-specific enzymes (0.01 less than ratio less than 100), and NAD-specific enzymes (ratio greater than 100). For NADP-specific enzymes, modifications at the 2'-phosphate group of NADP caused great loss of cofactor activity. The relative cofactor activities (NADP = 100%) of derivatives I and IV for these enzymes were 0.5-20 and 0.01-0.5%, respectively. On the other hand, NAD(P)-specific enzymes showed several types of responses to the NADP derivatives. The relative cofactor activities of I and IV for Leuconostoc mesenteroides and Bacillus stearothermophilus glucose-6-phosphate dehydrogenases and beef liver glutamate dehydrogenase were 60-200%; whereas, for B. megaterium glucose dehydrogenase and L. mesenteroides alcohol dehydrogenase, the values were 0.8-8%. For NAD-specific enzymes, these values were 20-50%. The relative cofactor activities of 2',3'-cyclic NADP and 3'-NADP were very low (less than 0.2%) except for beef liver glutamate dehydrogenase, B. stearothermophilus glucose-6-phosphate dehydrogenase, and horse liver alcohol dehydrogenase. Kinetic studies showed that the losses of the cofactor activity of NADP by these modifications were mainly due to the increase of the Km value. The mechanisms of coenzyme specificity of dehydrogenases are discussed. Unlike the 2'-phosphate group, the 6-amino group is common to NAD and NADP, and the effects of modification at the 6-amino group were independent of the coenzyme specificity of enzymes used for the assay. Derivatives II and Va had high relative cofactor activities (65-130%) for most of the enzymes except for isocitrate and glucose dehydrogenases (less than 1%) and L. mesenteroides alcohol dehydrogenase (20-60%). The cofactor activity of derivative III was generally lower than those of I and II. 相似文献
19.
Stabilization of Enzymes against Thermal Stress and Freeze-Drying by Mannosylglycerate 总被引:5,自引:1,他引:4
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A. Ramos N. Raven R. J. Sharp S. Bartolucci M. Rossi R. Cannio J. Lebbink J. Van Der Oost W. M. De Vos H. Santos 《Applied microbiology》1997,63(10):4020-4025
2-O-(beta)-Mannosylglycerate, a solute that accumulates in some (hyper)thermophilic organisms, was purified from Pyrococcus furiosus cells, and its effect on enzyme stabilization in vitro was assessed. Enzymes from hyperthermophilic, thermophilic, and mesophilic sources were examined. The thermostabilities of alcohol dehydrogenases from P. furiosus and Bacillus stearothermophilus and of glutamate dehydrogenases from Thermotoga maritima and Clostridium difficile were improved to a significant extent when enzyme solutions were incubated at supraoptimal temperatures in the presence of 2-O-(beta)-mannosylglycerate, but no effect on the thermostability of glutamate dehydrogenase from P. furiosus was detected. On the other hand, there was a remarkable effect on the thermal stabilities of rabbit muscle lactate dehydrogenase, baker's yeast alcohol dehydrogenase, and bovine liver glutamate dehydrogenase, which were used as model systems to evaluate stabilization of enzymes of mesophilic origin. For all of the enzymes examined and at the highest temperatures tested, 2-O-(beta)-mannosylglycerate was a better thermoprotectant than trehalose. The stabilizing effect exerted by 2-O-(beta)-mannosylglycerate on enzymes suggests a role for this compound as a protein thermostabilizer under physiological conditions. 2-O-(beta)-Mannosylglycerate was also effective in the protection of enzymes against stress imposed by freeze-drying, with its protecting effect being similar to or better than that exerted by trehalose. The data show 2-O-(beta)-mannosylglycerate to be a potential enzyme stabilizer in biotechnological applications. 相似文献
20.
Evidence for the identity of glutathione-dependent formaldehyde dehydrogenase and class III alcohol dehydrogenase 总被引:17,自引:0,他引:17
Formaldehyde dehydrogenase (EC 1.2.1.1) is a widely occurring enzyme which catalyzes the oxidation of S-hydroxymethylglutathione, formed from formaldehyde and glutathione, into S-formyglutathione in the presence of NAD. We determined the amino acid sequences for 5 tryptic peptides (containing altogether 57 amino acids) from electrophoretically homogeneous rat liver formaldehyde dehydrogenase and found that they all were exactly homologous to the sequence of rat liver class III alcohol dehydrogenase (ADH-2). Formaldehyde dehydrogenase was found to be able at high pH values to catalyze the NAD-dependent oxidation of long-chain aliphatic alcohols like n-octanol and 12-hydroxydodecanoate but ethanol was used only at very high substrate concentrations and pyrazole was not inhibitory. The amino acid sequence homology and identical structural and kinetic properties indicate that formaldehyde dehydrogenase and the mammalian class III alcohol dehydrogenases are identical enzymes. 相似文献