High-yield extraction and purification of glutathione reductase from baker's yeast.

Glutathione reductase was extracted from toluene-treated baker's yeast cells by a two-stage buffer autolysis method. The yeast cells were treated with toluene for 1 h at 40 degrees C. After removal of the toluene, the cells were then allowed to autolysis in buffer for 72 h at 4 degrees C. The cells were collected and resuspended in buffer. A second stage autolysis was carried out for another 96 h at 4 degrees C. The enzyme was purified to 786-fold from the second stage cell autolysate by using two steps of affinity chromatography with triazine dyes (Yellow H-E4G and Yellow H-E6G) coupled to Sepharose CL-4B. By using this simplified method, 1.44 mg (165 units/mg) of glutathione reductase was obtained from 65 g (wet weight) of yeast cells, equivalent to 80% enzyme recovery.     

The heterogeneity of brewer's yeast old yellow enzyme

Heterogeneity of brewer's yeast old yellow enzyme (OYE) was found by anion-exchange high-performance liquid chromatography (HPLC) as well as by 13C-NMR spectroscopy of [4a-13C]FMN reconstituted into apo OYE. Though the OYE sample prepared according to the conventional procedure gave a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the OYE sample was found to consist of five species on anion-exchange HPLC. The 13C-NMR spectrum of the [4a-13C]FMN-reconstituted OYE gave multiple peaks corresponding to 4a-13C. This multiplicity indicates that this OYE preparation possesses heterogeneity in the environment surrounding FMN, i.e., the active site of OYE. The different species of OYE were separately obtained by preparative HPLC on an anion-exchange column. These species as well as the unresolved sample showed identical mobility on SDS-PAGE and similar but slightly different NADPH oxidase activities. This heterogeneity was shown not to have resulted from proteolytic modification during the conventional purification procedure, which includes autolysis of the yeast cells, since the enzyme extracted by mechanical destruction of the yeast cells in the presence of various protease inhibitors exhibited identical heterogeneity. The pure OYE forms obtained by preparative anion-exchange HPLC are homogeneous in the flavin environment as revealed by a single 13C-NMR signal for the [4a-13C]FMN-reconstituted species.     

Enzymatic Removal of Diacetyl from Beer : III. Enzyme Protection and Regeneration of Cofactor

Use of diacetyl reductase, a reduced nicotinamide adenine dinucleotide (NADH)-requiring enzyme, to eliminate diacetyl off-flavor in beer was studied. The crude enzyme was extracted from Aerobacter aerogenes and partially purified by ammonium sulfate precipitation or Sephadex chromatography. In the semipure state, the enzyme was inactivated by lyophilization; in a crude state, the lyophilized extract remained stable for at least 4 months at - 20 C. A 50% reduction in specific activity within 5 min was observed when crude diacetyl reductase was suspended (5 mg of protein/ml) in phosphate buffer at pH 5.5 or below; a similar inactivation rate was observed when the crude enzyme was dissolved in a 5% aqueous ethyl alcohol solution. Effective crude enzyme activity in beer at a natural pH of 4.1 required protection of the enzyme in 10% gelatin. Incorporation of yeast cells with the gel-protected enzyme provided regeneration of NADH. Combinations of yeast, enzyme, and gelatin were tested to obtain data analyzed by regression analysis to determine the optimal concentration of each component of the system required to reduce the level of diacetyl in spiked (0.5 ppm) beer to less than 0.12 ppm within 48 hr at 5 C. The protected enzyme system was also effective in removing diacetyl from orange juice (pH 3.8) and some distilled liquors.     

3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus).

3-Oxoacyl-[ACP] reductase (E.C. 1.1.1.100, alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.     

Expanded bed affinity chromatography of dehydrogenases from bakers' yeast using dye-ligand perfluoropolymer supports

Malate dehydrogenase (MDH) and glucose 6-phosphate dehydrogenase (G6PDH) have been partially purified from preparations of homogenized yeast cells using Procion Yellow H-E3G and Procion Red H-E7B, respectively, immobilized on solid perfluoropolymer supports in an expanded bed. A series of pilot experiments were carried out in small packed beds using clarified homogenate to determine the optimal elution conditions for both MDH and G6PDH. Selective elution of MDH using NADH was effective but the yields obtained were dependent on the concentration of NADH used. Selective elution was found to be most effective when a low concentration of NaCl (0.1 M) was present. MDH could be recovered in 84% yield with a purification factor of 94 when this strategy was adopted. In the case of G6PDH, specific elution using NADP(+) was successful in purifying G6PDH 178-fold in 96% yield. The dynamic capacity of both affinity supports was estimated by frontal analysis, in an expanded bed with unclarified homogenate, and corresponded to 17 U MDH/mL of settled Procion Yellow H-E3G perfluoropolymer support and 7.7 U H6PDH/mL of settled Procion Red H-E7B perfluoropolymer support. Expanded bed affinity chromatography of MDH resulted in an eluted fraction containing 89% of the applied activity with a purification factor of 113. Expanded bed affinity chromatography of G6PDH resulted in an eluted fraction containing 84% of the applied activity with a purification factor of 172. With both enzymes, the overall recovery of enzyme activity was greater than 94%, showing that the expanded bed approach to purification was nondenaturing. (c) 1995 John Wiley & Sons, Inc.     

Immunological analysis of rat pancreatic prokallikrein activation

The present study shows that tissue kallikrein is present in rat pancreas as a proenzyme that can be converted by autolysis to a 38 000 Da active enzyme. The activation of pancreatic prokallikrein was examined by direct radioimmunoassay, enzymatic assays, active-site labeling with immunoprecipitation, and Western blot analyses. A monoclonal antibody (V1C3), which binds only active kallikrein, was used in a direct radioimmunoassay to monitor the appearance of the active enzyme. During a 22-h autolysis of pancreatic extract, a time-dependent increase in active kallikrein concentration paralleled the increase of kallikrein activities measured by both TosArgOMe esterase and kininogenase assays. The activation process was further analyzed by labeling the pancreatic extract with [14C]diisopropylphosphorofluoridate [( 14C]DFP) followed by immunoprecipitation with sheep anti-kallikrein antiserum. Pancreatic prokallikrein was not labeled by [14C]DFP; however, upon autolysis, a 38 000 Da active kallikrein can be labeled with [14C]DFP and increase in quantity with time. Western blot analysis, using a monoclonal antibody (V4D11) which recognizes both latent and active tissue kallikreins, identified a 39 000 Da pancreatic prokallikrein prior to autolysis and a 38 000 Da active kallikrein after 7 h of autolysis. The results indicate that the pancreatic prokallikrein exists as a 39 000 Da protein which may be converted to a 38 000 Da active kallikrein, indistinguishable from purified urinary, brain, spleen or submandibular gland kallikrein.     

Suitability of primary monolayer cultures of adult rat hepatocytes for studies of cholesterol and bile acid metabolism

Monolayer cultures of hepatocytes isolated from cholestyramine-fed rats and incubated in serum-free medium converted exogenous [4-14C]cholesterol into bile acids at a 3-fold greater rate than did cultures of hepatocytes prepared from untreated rats. Cholic acid and beta-muricholic acid identified and quantitated by gas-liquid chromatography and thin-layer chromatography were synthesized by cultured cells for at least 96 h following plating. The calculated synthesis rate of total bile acids by hepatocytes prepared from cholestyramine-fed animals was approximately 0.058 micrograms/mg protein/h. beta-Muricholic acid was synthesized at approximately a 3-fold greater rate than cholic acid in these cultures. Cultured hepatocytes rapidly converted the following intermediates of the bile acid pathway; 7 alpha-hydroxy[7 beta-3H]cholesterol, 7 alpha-hydroxy-4-[6 beta-3H] cholesten-3-one, and 5 beta-[7 beta-3H]cholestane-3 alpha, 7 alpha, 12 alpha-triol into bile acids. [24-14C]Chenodeoxycholic acid and [3H]ursodeoxycholic acid were rapidly biotransformed to beta-muricholic acid. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase activity measured in microsomes of cultured hepatocytes decreased during the initial 48 h following plating, but remained relatively constant for the next 72 h. In contrast, cholesterol 7 alpha-hydroxylase activity appeared to decrease during the first 48 h, followed by an increase over the next 48 h. Despite the apparent changes in enzyme activity in vitro, the rate of bile acid synthesis by whole cells during this time period remained constant. It is concluded that primary monolayer cultures of rat hepatocytes can serve as a useful model for studying the interrelationship between cholesterol and bile acid metabolism.     

An Autolytic Process for Recovery of Antioxidant Activity Rich Carotenoprotein from Shrimp Heads

Studies were carried out to utilize in situ proteases of shrimp heads to recover carotenoproteins possessing antioxidant activity. Highest protease activity of the buffer extract was found at pH 8.0 (9.85 ± 0.61 units). The protease activity increased with temperature up to 50°C and reduced thereafter with highest activity being 19.32 ± 2.0 units. Thus, the autolysis of shrimp heads for recovery of carotenoprotein was carried out at pH 8.0 and at 50°C. Waste to buffer ratio had a significant (p < 0.05) effect on recovery of carotenoids in carotenoprotein filtrate with a maximum of 58.5 ± 6.4% recovery with a waste to buffer ratio of 1:2.5 (w:v). The carotenoid recovery increased significantly to 63.4% ± 3.6% at the end of a 4-h autolysis. The studies on combined effect of waste to buffer ratio and autolysis time indicated increase in protein recovery with increase in waste to buffer ratio but not with autolysis time. DPPH scavenging activity of the carotenoprotein isolate increased with autolysis time up to 100 min, and thereafter, reduced above 160 min of autolysis time. With increase in waste to buffer ratio, the scavenging activity increased, reaching more than 12.5 mg TBHQ equivalent/mg protein at waste to buffer ratio of 1:5. The optimum autolysis condition for obtaining antioxidant activity rich carotenoprotein from shrimp heads was found to be waste to buffer (pH 8.0) ratio of 1:5 and an autolysis time of 2 h at 50°C. The isolated carotenoprotein was found to have antioxidant activity with respect to singlet oxygen quenching, reducing power and metal chelating activity.     

Cell wall autolysis in log phase cells of Micrococcus lysodeikticus (luteus).

Log phase cells of Micrococcus lysodeikticus (luteus) IFO 3333 autolyzed when incubated at 37 C in 0.01 M sodium-phosphate buffer pH 7.5. The enzyme involved in the autolysis was recovered mainly in an aqueous phase from cytoplasmic membranes and cytoplasmic materials treated with n-butanol, and proved to be an N-acetylmuramyl-L-alanine amidase. The autolysis of log phase cells suspended in autolyzing buffer was depressed by the addition of trypsin to the buffer.     

A Convenient Synthesis of 2′-Deoxy-2-fluoroadenosine; a Potential Prodrug for Suicide Gene Therapy

Abstract

A convenient synthesis of 2′-deoxy-2-fluoro-adenosine (1) is described. Deaminative fluorination of 2-aminoadenosine (2) followed by silylation of the 3′, 5′-hydroxyl groups gave the corresponding 2-fluoroadenosine derivative 4 in good yield. Thiocarbonylation of 4 to thiocarbonylimidazolyl derivative 5a followed by treatment with an excess of tris(trimethylsilyl)silane (TTMSS) and tert-butyl peroxide in toluene at 80 [ddot]C was found to affect an efficient deoxygenation to the corresponding 2′-deoxy derivative 6. Desilylation of 6 by Et₄NF in CH₃CN afforded 1 in high yield.     

The Affinity Purification and Characterization of a Dehydrogenase from Aspergillus parasiticus Involved in Aflatoxin B1, Biosynthesis

Abstract

A two step scheme has been developed for the purification of a dehydrogenase from mycelia of 84 hours old Aspergillus parasiticus (1-11-105 Wh 1), which catalyzes the conversion of norsolorinic acid (NA) to averantin (AVN). The dehydrogenase was purified from cell-free extracts using reactive green 19-agarose and norsolorinic acid-agarose affinity chromatography. The latter affinity matrix was synthesised by attaching norsolorinic acid to ω-aminohexylagarose. The purified protein was shown to be homogenous on non-denaturing polyacrylamide gel electrophoresis. A final purification of 215-fold was achieved. Results of gel filtration chromatography indicated the approximate molecular mass of the native protein to be 140 000 daltons. The isoelectric point of the protein was about 5.5 as determined by chromatofocusing. The reaction catalyzed by the dehydrogenase was optimum at pH 8.5 and between 25[ddot] to 35[ddot]C. The Km of the enzyme for NA and NADPH was determined to be 3.45 μM and 103 μM respectively.     

Radioenzymatic assay for reductive catalysis of N(5)N(10)-methylenetetrahydrofolate by methylenetetrahydrofolate reductase

Methylenetetrahydrofolate reductase catalyzes the reduction of N(5), N(10)-methylenetetrahydrofolate to N(5)-methyltetrahydrofolate. Because this substrate is unstable and dissociates spontaneously into formaldehyde and tetrahydrofolate, the customary method to assay the catalytic activity of this enzyme has been to measure the oxidation of [14C]N(5)-methyltetrahydrofolate to N(5), N(10)-methylenetetrahydrofolate and quantify the [14C]formaldehyde that dissociates from this product. This report describes a very sensitive radioenzymatic assay that measures directly the reductive catalysis of N(5),N(10)-methylenetetrahydrofolate. The radio-labeled substrate, [14C]N(5),N(10)-methylenetetrahydrofolate, is prepared by condensation of [C(14)]formaldehyde with tetrahydrofolate and the stability of this substrate is maintained for several months by storage at -80 degrees C in a pH 9.5 buffer. Partially purified methylenetetrahydrofolate reductase from rat liver, incubated with the radio-labeled substrate and the cofactors, NADPH and FAD at pH 7. 5, generates [14C]N(5)-methyltetrahydrofolate, which is stable and partitions into the aqueous phase after the assay is terminated with dimedone and toluene. A K(m) value of 8.2 microM was obtained under conditions of increasing substrate concentration to ensure saturation kinetics. This method is simple, very sensitive and measures directly the reduction of N(5), N(10)-methylenetetrahydrofolate to N(5)-methyltetrahydrofolate, which is the physiologic catalytic pathway for methylenetetrahydrofolate reductase.     

Postmortem Changes in Rat Brain: Studies on Membrane-Bound Enzymes and Receptors

The relationship between the stability of potential neurochemical markers and autolysis time was studied at 4 degrees C and 25 degrees C using postmortem brain samples from two rat strains. In general, qualitatively similar results were obtained with either N/Nih or Sprague-Dawley rats; however, quantitative differences were often observed, particularly in regard to benzodiazepine receptor changes. For every enzyme activity or binding property examined, no significant change was found when brains were kept at 4 degrees C for up to 72 h prior to freezing at -70 degrees C. Na,K-ATPase and low-affinity Ca-ATPase activities were also stable in brains kept at 25 degrees C for up to 72 h. Mg-ATPase activity was reduced in brains kept at 25 degrees C for 24 and 48 h. [3H]Guanidinoethylmercaptosuccinic acid [( 3H]GEMSA) binding to enkephalin convertase in the cytosol was not significantly changed in brains kept at 25 degrees C; however, a small increase was seen for [3H]GEMSA binding to the membrane fraction at 24, but not 48 and 72 h postmortem. [3H]Quinuclidinyl benzilate [( 3H]QNB) binding to muscarinic cholinergic receptors decreased in brains kept at 25 degrees C for 72 h. Opioid receptor binding also decreased in brains kept at 25 degrees C. Using [3H]2-D-alanine-5-D-leucine enkephalin to label delta opioid receptors, a statistically significant decrease in binding was observed as early as 6 h postmortem, and was completely abolished after 72 h at 25 degrees C. In contrast, [3H]naloxone binding was unchanged after 24 h at 25 degrees C, but was decreased after 48 and 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for acetyl coenzyme A and cinnamoyl coenzyme A in the anaerobic toluene mineralization pathway in Azoarcus tolulyticus Tol-4.

A toluene-degrading denitrifier, Azoarcus tolulyticus Tol-4, was one of eight similar strains isolated from three petroleum-contaminated aquifer sediments. When the strain was grown anaerobically on toluene, 68% of the carbon from toluene was found as CO2 and 30% was found as biomass. Strain Tol-4 had a doubling time of 4.3 h, a Vmax of 50 micromol x min-1 x g of protein-1, and a cellular yield of 49.6 g x mol of toluene-1. Benzoate appeared to be an intermediate, since F-benzoates accumulated from F-toluenes and [14C]benzoate was produced from [14C]toluene in the presence of excess benzoate. Two metabolites, E-phenylitaconic acid (1 to 2%) and benzylsuccinic acid (<1%), accumulated from anaerobic toluene metabolism. These same products were also produced when cells were grown on hydrocinnamic acid and trans-cinnamic acid but were not produced from benzylalcohol, benzaldehyde, benzoate, p-cresol, or their hydroxylated analogs. The evidence supports an anaerobic toluene degradation pathway involving an initial acetyl coenzyme A (acetyl-CoA) attack in strain Tol-4, as proposed by Evans and coworkers (P. J. Evans, W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Young, Appl. Environ. Microbiol. 58:496-501, 1992) for another toluene-degrading denitrifier, strain T1. Our findings support a modification of the proposed pathway in which cinnamoyl-CoA follows the oxidation of hydrocinnamoyl-CoA, analogous to the presumed oxidation of benzylsuccinic acid to form E-phenylitaconic acid. Cinnamic acid was detected in Tol-4 cultures growing in the presence of toluene and [14C]acetate. We further propose a second acetyl-CoA addition to cinnamoyl-CoA as the source of benzylsuccinic acid and E-phenylitaconic acid. This pathway is supported by the finding that monofluoroacetate added to toluene-growing cultures resulted in a significant increase in production of benzylsuccinic acid and E-phenylitaconic acid and by the finding that [14C]benzylsuccinic acid was detected after incubation of cells with toluene, [14C]acetate, and cinnamic acid. Evidence for anaerobic toluene metabolism by methyl group oxidation was not found, since benzylsuccinic acid and E-phenylitaconic acid were not detected after incubation with benzylalcohol and benzaldehyde, nor were benzylalcohol and benzaldehyde detected even in 14C trapping experiments.     

Coordinate regulation of cholesterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A synthase but not 3-hydroxy-3-methylglutaryl coenzyme A reductase in C-6 glia

The effects on cholesterol biosynthesis of growth of cultured C-6 glial cells in serumfree medium ± supplementation with linoleic or linolenic acid were studied. Markedly higher activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) were observed in cells grown in linoleate- or linolenate-supplemented versus nonsupplemented medium. After 48 h HMG-CoA reductase activities were two-and four-fold higher in cells supplemented with 20 and 100 μm linoleate, respectively. The increase in activity became apparent after 24 h and was marked after 48 h. Rates of incorporation of [¹⁴C]acetate or ³H₂O into sterols did not reflect the changes in reductase activity. Thus, in cells supplemented with 50 μm linoleate for 24 and 48 h rates of incorporation of [¹⁴C]acetate were 75–80% lower than rates in nonsupplemented cells. This difference resulted because over the first 24 h of the experiment a fivefold increase in the rate of sterol synthesis occurred in the nonsupplemented cells, whereas essentially no change occurred in the linoleate-supplemented cells; little further change occurred between 24 and 48 h in the nonsupplemented and the linoleate-supplemented cells. That the difference in sterol synthesis under these experimental conditions could be mediated at the level of HMG-CoA synthase (EC 4.1.3.5) was suggested by two series of findings, i.e., first, similar quantitative and temporal changes in the activity of this enzyme, and, second, no change in the activity of acetoacetyl-CoA thiolase (EC 2.3.1.9) or the incorporation of [¹⁴C]mevalonate into sterols. Thus, the data suggest that HMG-CoA synthase, and not HMG-CoA reductase, may direct the rate of cholesterol biosynthesis under these conditions of serum-free growth ± supplementation with polyunsaturated fatty acid.     

Autolysis of disintegrated cells of the yeast saccharomyces cerevisiae

Optimum conditions for autolysis of disintegrated cells of S. cerevisiae are at 50–53°C and pH 5.5; the process is terminated after 6 h. In the presence of sodium chloride (3–5%) the autolysis is complete after 5 h. The yield of autolysis of disintegrated yeast cells is about 70% of autolytic product per yeast dry weight. The product obtained after centrifugation, filtration and drying has very good sensoric and physical properties.     

Purification and characterization of bisulfite reductase (desulfofuscidin) from Desulfovibrio thermophilus and its complexes with exogenous ligands

A dissimilatory bisulfite reductase has been purified from a thermophilic sulfate-reducing bacterium Desulfovibrio thermophilus (DSM 1276) and studied by EPR and optical spectroscopic techniques. The visible spectrum of the purified bisulfite reductase exhibits absorption maxima at 578.5, 392.5 and 281 nm with a weak band around 700 nm. Photoreduction of the native enzyme causes a decrease in absorption at 578.5 nm and a concomitant increase in absorption at 607 nm. When reduced, the enzyme reacts with cyanide, sulfite, sulfide and carbon monoxide to give stable complexes. The EPR spectrum of the native D. thermophilus bisulfite reductase shows the presence of a high-spin ferric signal with g values at 7.26, 4.78 and 1.92. Upon photoreduction the high-spin ferric heme signal disappeared and a typical 'g = 1.94' signal of [4Fe-4S] type cluster appeared. Chemical analyses show that the enzyme contains four sirohemes and eight [4Fe-4S] centers per mol of protein. The molecular mass determined by gel filtration was found to be 175 kDa. On SDS-gel electrophoresis the enzyme presents a main band of 44 to 48 kDa. These results suggest that the bisulfite reductase contains probably one siroheme and two [4Fe-4S] centers per monomer. The dissimilatory bisulfite reductase from D. thermophilus presents some homologous properties with desulfofuscidin, the bisulfite reductase isolated from Thermodesulfobacterium commune (Hatchikian, E.C. and Zeikus, J.G. (1983) J. Bacteriol. 153, 1211-1220).     

溴化1-己基-3-甲基咪唑对斜生栅藻谷胱甘肽及其代谢酶的影响

研究了浓度为0、1、5、10、15、20 mg/L的新兴离子液体溴化1-己基-3-甲基咪唑([C6mim]Br)在24h、48h、72h和96h对斜生栅藻还原型谷胱甘肽（GSH）及其代谢酶-谷胱甘肽过氧化物酶（GPX）、谷胱甘肽转硫酶（GST）和谷胱甘肽还原酶（GR）的影响。结果表明：GSH含量在24h、48h和72h时,在最低处理浓度下不变,其他处理浓度下随胁迫浓度增加而降低,96h时则与对照无差异或较小;GPX和GST的活性在72h之前明显升高（最高浓度组的GST活性有波动）,96h时均降低至对照水平;GR活性在24h时,[C6mim]Br=1 mg/L时升高,之后降低,在48h增高至对照水平,72h时,[C6mim]Br≥10 mg/L的处理组高于对照水平,96h时,除最低处理组外,均降至对照水平以下。GR是GSH系统中的限速酶,GST则是该系统中活性和灵敏性最高的酶,可作为[C6mim]Br胁迫时的敏感的生物标志物。1 mg/L的[C6mim]Br可引起藻细胞的氧化胁迫,具有环境毒性。     

Pathways of glucose metabolism in Candida 107, a lipid-accumulating yeast.

Phosphofructokinase was not detected in extracts of Candida 107 prepared in a variety of ways but was highly active in cells treated with toluene. Disruption of these cells destroyed activity of phosphofructokinase indicating that the enzyme is extremely labile. As patterns of labelling from [I-14C]glucose and [6-14C]glucose showed that 60% of glucose was metabolized via the pentose cycle, augmentation of this cycle is necessary to account for the high molar growth yields of this yeast. Phosphoketolases, reacting with xylulose 5-phosphate and fructose 6-phosphate, were found but the extent to which they contribute to glucose metabolism was not assessed.     

Induzione Sperimentale Della Maschio-Sterilità in Triticum

Abstract

Experimental induction of male sterility in Triticum. — Prolonged artificial illumination (12-24 h. every 24 hours) and high temperature (20-25[ddot]C.) induce in « Denti de cani » plants (Meletti, 1955) the transformation of the stamens, which tend to take morphological characteristics of female organs (Meletti, 1960). The prolonged illumination induces, also at temperatures inferior to 20[ddot]C., a remarkable increase of the percentage of « Denti de cani » flowers which have reduced anthers without pollen grains.