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1.
Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Δ 9 position. We expressed two sunflower ( Helianthus annuus) oleate Δ 12 desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain ( trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Δ 9,12, the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15°C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 ( TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp + or Trp − strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30°C or 15°C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains. 相似文献
2.
Thermostability of the photosynthetic apparatus of abscisic acid (ABA)-treated seedlings of barley ( Hordeum vulgare) was studied by light-scattering and by fluorescence measurements of isolated chloroplasts. ABA treatment markedly decreased heat damage of the chloroplast ultrastructure; an exogenous ABA concentration of 10 −5 molar was most effective. Heat-induced increase of the 77 kilodalton fluorescence ratio F 740/F 685 was also smaller at this ABA concentration. The heat-induced increase of the initial chlorophyll fluorescence level (F o) was virtually eliminated in ABA-treated (10 −5 molar) chloroplasts up to 45°C and slightly increased at 50°C, relative to control chloroplasts where F o increased even at 35°C and reached its maximal value at 45°C. In control chloroplasts, F o increased with a 5-minute pretreatment temperature, an effect observed as low as 35°C. F o was maximal at 45°C. In contrast, chloroplasts treated with 10 −5 molar ABA did not exhibit a heat-induced increase in F o until 50°C. 相似文献
3.
Rotation of the γ subunit of the F 1-ATPase plays an essential role in energy transduction by F 1-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F 1-ATPase. In this study, we introduced a βY341W mutation as an optical probe for nucleotide binding to catalytic sites, and a βE190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric βY341W subunit and a mutant α3β3γ subcomplex containing the βY341W mutation with or without an additional βE190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/K d) of the nucleotides for the isolated β subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van’t Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated β subunit, NDPs and NTPs with the same base moiety exhibited similar Δ H0 and Δ G0 values at 25°C. The binding of nucleotides with different bases to the isolated β subunit resulted in different entropy changes. Interestingly, NDP binding to the α3β(Y341W) 3γ subcomplex had similar K d and Δ G0 values as binding to the isolated β(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy. 相似文献
4.
Through use of commercially available DnaK proteins and anti-DnaK monoclonal antibodies, a competitive enzyme-linked immunosorbent assay was developed to quantify this heat shock protein in Escherichia coli ATCC 25922 subjected to various heating regimens. For a given process lethality ( F7010 of 1, 3, and 5 min), the intracellular concentration of DnaK in E. coli varied with the heating temperature (50 or 55°C). In fact, the highest DnaK concentrations were found after treatments at the lower temperature (50°C) applied for a longer time. Residual DnaK after heating was found to be necessary for cell recovery, and additional DnaK was produced during the recovery process. Overall, higher intracellular concentrations of DnaK tended to enhance cell resistance to a subsequent lethal stress. Indeed, E. coli cells that had undergone a sublethal heat shock (105 min at 55°C, F7010 = 3 min) accompanied by a 12-h recovery (containing 76,786 ± 25,230 molecules/cell) resisted better than exponentially growing cells (38,500 ± 6,056 molecules/cell) when later heated to 60°C for 50 min ( F7010 = 5 min). Results reported here suggest that using stress protein to determine cell adaptation and survival, rather than cell counts alone, may lead to more efficient heat treatment. 相似文献
5.
Understanding the factors that influence the distribution and abundance of marine diazotrophs is important in order to assess their role in the oceanic nitrogen cycle. Environmental DNA samples from four cruises to the North Atlantic Ocean, covering a sampling area of 0°N to 42°N and 67°W to 13°W, were analyzed for the presence and amount of seven nifH phylotypes using real-time quantitative PCR and TaqMan probes. The cyanobacterial phylotypes dominated in abundance (94% of all nifH copies detected) and were the most widely distributed. The filamentous cyanobacterial type, which included both Trichodesmium and Katagnymene, was the most abundant (51%), followed by group A, an uncultured unicellular cyanobacterium (33%), and gamma A, an uncultured gammaproteobacterium (6%). Group B, unicellular cyanobacterium Crocosphaera, and group C Cyanothece-like phylotypes were not often detected (6.9% and 2.3%, respectively), but where present, could reach high concentrations. Gamma P, another uncultured gammaproteobacterium, was seldom detected (0.5%). Water temperature appeared to influence the distribution of many nifH phylotypes. Very high (up to 1 × 10 6 copies liter −1) nifH concentrations of group A were detected in the eastern basin (25 to 17°N, 27 to 30°W), where the temperature ranged from 20 to 23°C. The highest concentrations of filamentous phylotypes were measured between 25 and 30°C. The uncultured cluster III phylotype was uncommon (0.4%) and was associated with mean water temperatures of 18°C. Diazotroph abundance was highest in regions where modeled average dust deposition was between 1 and 2 g/m 2/year. 相似文献
7.
Severe photoinactivation of catalase (EC 1.11.1.6) and a decline of variable fluorescence ( Fv), indicating photoinhibition of photosynthesis, were observed as rapid and specific symptoms in leaves exposed to a high heat-shock temperature of 40°C as well as in leaves exposed to low chilling temperatures in white light of only moderately high photosynthetic photon flux density of 520 μE m −2 s −1. Other parameters, such as peroxidase (EC 1.11.1.7), glycolate oxidase (EC 1.1.3.1), glutathione reductase (EC 1.6.4.2), or the chlorophyll content, were hardly affected under these conditions. At a compatible temperature of 22°C, the applied light intensity did not induce severe photoinactivations. In darkness, exposures to high or low temperatures did not affect catalase levels. Also, decline of Fv in light was not related to temperature sensitivity in darkness. The effective low-temperature ranges inducing photoinactivation of catalase differed significantly for chilling-tolerant and chilling-sensitive plants. In leaves of rye ( Secale cereale L.) and pea ( Pisum sativum L.), photoinactivation occurred only below 15°C, whereas inactivation occurred at 15°C in cucumber ( Cucumis sativus L.) and maize ( Zea mays L.). The behavior of Fv was similar, but the difference between chilling-sensitive and chilling-tolerant plants was less striking. Whereas the catalase polypeptide, although photoinactivated, was not cleaved at 0 to 4°C, the D1 protein of photosystem II was greatly degraded during the low-temperature treatment of rye leaves in light. Rye leaves did not exhibit symptoms of any major general photodamage, even when they were totally depleted of catalase after photoinactivation at 0 to 4°C, and catalase recovered rapidly at normal temperature. In cucumber leaves, the decline of catalase after exposures to bright light at 0 to 4°C was accompanied by bleaching of chlorophyll, and the recovery observed at 25°C was slow and required several days. Similar to the D1 protein of photosystem II, catalase differs greatly from other proteins by its inactivation and high turnover in light. Inasmuch as catalase and D1 protein levels depend on continuous repair synthesis, preferential and rapid declines are generally to be expected in light whenever translation is suppressed by stress actions, such as heat or chilling, and recovery will reflect the repair capacity of the plants. 相似文献
8.
1. Two species of double-helical RNA isolated from mycelium of Penicillium chrysogenum were titrated with acid at 25°C and 95°C (solvent 0.1 m-sodium phosphate buffer). At 25°C denaturation occurred at about pH3. At 95°C in the denatured form cytosine residues titrated as a simple monobasic acid of p K3.9 compared with p K2.5 for the native form at 25°C. 2. On thermal denaturation in neutral and acidic solutions one species of RNA (38% rG·rC) `melted' in three distinct stages, equivalent to a mixture of three species, namely one of about 25% rG·rC, another of about 33% rG·rC and a third of about 46% rG·rC: the relative proportions were 0.25:0.35:0.40. 3. On thermal denaturation in acidic solutions the increase in the fraction of ionized cytosine residues concomitant with the `melting' of rG·rC base pair also affects the spectrum especially at 280nm and serves to enhance the contribution of rG·rC base pairs at this wavelength. The increment in ε (P) at 280nm on `melting' an rG·rC base pair approaches 53501·mol −1·cm −1 depending on pH, compared with 33501·mol −1·cm −1 at pH7. In contrast ε (P) at 280nm is scarcely affected by `melting' rA·rU base pairs or by the protonization of adenine residues. 4. Changes in the spectrum of Escherichia coli rRNA on denaturation in acidic solutions were studied to yield the mole fractions of rA·rU and rG·rC base pairs `melting' at particular pH values. 相似文献
9.
In the lipocalin family, the conserved interaction between the main α-helix and the β-strand H is an ideal model to study protein side chain dynamics. Site-directed tryptophan fluorescence (SDTF) has successfully elucidated tryptophan rotamers at positions along the main alpha helical segment of tear lipocalin (TL). The rotamers assigned by fluorescent lifetimes of Trp residues corroborate the restriction expected based on secondary structure. Steric conflict constrains Trp residues to two ( t, g
−) of three possible χ 1 ( t, g
−, g
+) canonical rotamers. In this study, investigation focused on the interplay between rotamers for a single amino acid position, Trp 130 on the α-helix and amino acids Val 113 and Leu 115 on the H strand, i.e. long range interactions. Trp130 was substituted for Phe by point mutation (F130W). Mutations at positions 113 and 115 with combinations of Gly, Ala, Phe residues alter the rotamer distribution of Trp130. Mutations, which do not distort local structure, retain two rotamers (two lifetimes) populated in varying proportions. Replacement of either long range partner with a small amino acid, V113A or L115A, eliminates the dominance of the t rotamer. However, a mutation that distorts local structure around Trp130 adds a third fluorescence lifetime component. The results indicate that the energetics of long-range interactions with Trp 130 further tune rotamer populations. Diminished interactions, evident in W130G113A115, result in about a 22% increase of α-helix content. The data support a hierarchic model of protein folding. Initially the secondary structure is formed by short-range interactions. TL has non-native α-helix intermediates at this stage. Then, the long-range interactions produce the native fold, in which TL shows α-helix to β-sheet transitions. The SDTF method is a valuable tool to assess long-range interaction energies through rotamer distribution as well as the characterization of low-populated rotameric states of functionally important excited protein states. 相似文献
10.
Alpha amylase family is generally defined as a group of enzymes that can hydrolyse and transglycosylase α-(1, 4) or α-(1, 6)
glycosidic bonds along with the preservation of anomeric configuration. For the comparative analysis of alpha amylase family,
nucleotide sequences of seven thermo stable organisms of Kingdom Archea i.e. Pyrococcus furiosus (100-105°C), Kingdom
Prokaryotes i.e. Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C), Bacillus amyloliquefaciens (72°C), Bacillus subtilis
(70°C) and Bacillus KSM K38 (55°C) and Eukaryotes i.e. Aspergillus oryzae (60°C) were selected from NCBI. Primary structure
composition analysis and Conserved sequence analysis were conducted through Bio Edit tools. Results from BioEdit shown only
three conserved regions of base pairs and least similarity in MSA of the above mentioned alpha amylases. In Mega 5.1 Phylogeny
of thermo stable alpha amylases of Kingdom Archea, Prokaryotes and Eukaryote was handled by Neighbor-Joining (NJ) algorithm.
Mega 5.1 phylogenetic results suggested that alpha amylases of thermo stable organisms i.e. Pyrococcus furiosus (100-105°C), Bacillus
licheniformis (90-95°C), Geobacillus stearothermophilus (75°C) and Bacillus amyloliquefaciens (72°C) are more distantly related as
compared to less thermo stable organisms. By keeping in mind the characteristics of most thermo stable alpha amylases novel and
improved features can be introduced in less thermo stable alpha amylases so that they become more thermo tolerant and
productive for industry. 相似文献
12.
Resin acids are tricyclic terpenoids occurring naturally in trees. We investigated the occurrence of resin acid-degrading bacteria on the Arctic tundra near the northern coast of Ellesmere Island (82°N, 62°W). According to most-probable-number assays, resin acid degraders were abundant (10 3 to 10 4 propagules/g of soil) in hydrocarbon-contaminated soils, but they were undetectable (<3 propagules/g of soil) in pristine soils from the nearby tundra. Plate counts indicated that the contaminated and the pristine soils had similar populations of heterotrophs (10 6 to 10 7 propagules/g of soil). Eleven resin acid-degrading bacteria belonging to four phylogenetically distinct groups were enriched and isolated from the contaminated soils, and representative isolates of each group were further characterized. Strains DhA-91, IpA-92, and IpA-93 are members of the genus Pseudomonas. Strain DhA-95 is a member of the genus Sphingomonas. All four strains are psychrotolerant, with growth temperature ranges of 4°C to 30°C (DhA-91 and DhA-95) or 4°C to 22°C (IpA-92 and IpA-93) and with optimum temperatures of 15 to 22°C. Strains DhA-91 and DhA-95 grew on the abietanes, dehydroabietic and abietic acids, but not on the pimaranes, isopimaric and pimaric acids. Strains IpA-92 and IpA-93 grew on the pimaranes but not the abietanes. All four strains grew on either aliphatic or aromatic hydrocarbons, which is unusual for described resin acid degraders. Eleven mesophilic resin acid degraders did not use hydrocarbons, with the exception of two Mycobacterium sp. strains that used aliphatic hydrocarbons. We conclude that hydrocarbon contamination in Arctic tundra soil indirectly selected for resin acid degraders, selecting for hydrocarbon degraders that coincidentally use resin acids. Psychrotolerant resin acid degraders are likely important in the global carbon cycle and may have applications in biotreatment of pulp and paper mill effluents. 相似文献
13.
The indole alkaloid gramine is toxic to animals and may play a defensive role in plants. Under certain conditions, shoots of barley cultivars such as `Arimar' and CI 12020 accumulate gramine ( N,N-dimethyl-3-aminomethylindole) and lesser amounts of its precursors 3-aminomethylindole (AMI) and N-methyl-3-aminomethylindole (MAMI); other cultivars such as `Proctor' do not. When grown at optimal temperatures (21°C/16°C, day/night), Arimar contained a high level of gramine in the first leaf (approximately 6 milligrams per gram dry weight), but progressively less accumulated in successive leaves so that the gramine level in the shoot as a whole fell sharply with age. In Arimar and CI 12020 plants transferred at the two- to three-leaf stage from 21°C/16°C to supra-optimal temperatures (≥30°C/25°C), there was massive gramine accumulation in leaves which developed at high temperature, so that gramine level in the whole shoot remained high (about 3-8 milligrams per gram dry weight). Proctor lacked both constitutive gramine accumulation in the first leaf and heat-induced gramine accumulation in later leaves. The following evidence indicates that this results from a lesion in the pathway of synthesis (tryptophan →→ AMI → MAMI → gramine) between tryptophan and AMI. (a) Proctor and Arimar leaves readily absorbed [14C]gramine, but neither cultivar degraded it extensively. (b) Arimar leaf tissue incorporated [14C]formate label into the N-methyl groups of gramine and MAMI, and converted [methylene-14C]tryptophan to AMI, MAMI, and gramine; Proctor leaf tissue did not, even when a trapping pool of unlabeled gramine was supplied. (c) Proctor converted [14C]MAMI to gramine as actively as Arimar. (d) Proctor incorporated [14C]formate label into gramine and MAMI when supplied with AMI; the ratio [14C]gramine/[14C]MAMI fell with leaf age, suggesting that the two N-methylations involve different enzymes. Inasmuch as Proctor leaf tissue did not methylate added tryptamine or tyramine, the N-methyltransferase(s) of gramine synthesis may be substrate specific. In sterile culture at optimal temperatures, 10 millimolar gramine did not affect autotrophic growth of Arimar or Proctor plantlets or heterotrophic growth of callus. At supra-optimal temperature, plantlet growth was reduced by gramine although callus growth was not. We speculate that gramine-accumulating cultivars may suffer autotoxic effects at high leaf temperatures. 相似文献
14.
1. Diaminopimelate decarboxylase from a soluble extract of Escherichia coli A.T.C.C. 9637 was purified 200-fold by precipitation of nucleic acids, fractionation with acetone and then with ammonium sulphate, adsorption on calcium phosphate gel and chromatography on DEAE-cellulose or DEAE-Sephadex. 2. The purified enzyme showed only one component in the ultracentrifuge, with a sedimentation coefficient of 5·4 s. One major peak and three much smaller peaks were observed on electrophoresis of the enzyme at pH8·9. 3. The mol.wt. of the enzyme was approx. 200000. The catalytic constant was 2000mol. of meso-diaminopimelic acid decomposed/min./mol. of enzyme, at 37°. The relative rates of decarboxylation at 25°, 37° and 45° were 0·17:1·0:1·6. At 37° the Michaelis constant was 1·7m m and the optimum pH was 6·7–6·8. 4. There was an excess of acidic amino acids over basic amino acids in the enzyme, which was bound only on basic cellulose derivatives at pH6·8. 5. The enzyme had an absolute requirement for pyridoxal phosphate as a cofactor; no other derivative of pyridoxine had activity. A thiol compound (of which 2,3-dimercaptopropan-1-ol was the most effective) was also needed as an activator. 6. In the presence of 2,3-dimercaptopropan-1-ol (1m m), heavy-metal ions (Cu 2+, Hg 2+) did not inhibit the enzyme, but there was inhibition by several amino acids with analogous structures to diaminopimelate, generally at high concentrations relative to the substrate. Penicillamine was inhibitory at relatively low concentrations; its action was prevented by pyridoxal phosphate. 相似文献
15.
α-Amylase produced by Bacillus licheniformis CUMC305 was purified 212-fold with a 42% yield through a series of four steps. The purified enzyme was homogeneous as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and discontinuous gel electrophoresis. The purified enzyme showed maximal activity at 90°C and pH 9.0, and 91% of this activity remained at 100°C. The enzyme retained 91, 79, and 71% maximal activity after 3 h of treatment at 60°C, 3 h at 70°C, and 90 min at 80°C, respectively, in the absence of substrate. On the contrary, in the presence of substrate (soluble starch), the α-amylase enzyme was fully stable after a 4-h incubation at 100°C. The enzyme showed 100% stability in the pH range 7 to 9; 95% stability at pH 10; and 84, 74, 68, and 50% stability at pH values of 6, 5, 4, and 3, respectively, after 18 h of treatment. The activation energy for this enzyme was calculated as 5.1 × 10 5 J/mol. The molecular weight was estimated to be 28,000 by sodium dodecyl sulfate-gel electrophoresis. The relative rates of hydrolysis of soluble starch, amylose, amylopectin, and glycogen were 1.27, 1.8, 1.94, and 2.28 mg/ml, respectively. Vmax values for hydrolysis of these substrates were calculated as 0.738, 1.08, 0.8, and 0.5 mg of maltose/ml per min, respectively. Of the cations, Na +, Ca 2+, and Mg 2+, showed stimulatory effect, whereas Hg 2+, Cu 2+, Ni 2+, Zn 2+, Ag +, Fe 2+, Co 2+, Cd 2+, Al 3+, and Mn 2+ were inhibitory. Of the anions, azide, F −, SO 32−, SO 43−, S 2O 32−, MoO 42−, and Wo 42− showed an excitant effect. p-Chloromercuribenzoic acid and sodium iodoacetate were inhibitory, whereas cysteine, reduced glutathione, thiourea, β-mercaptoethanol, and sodium glycerophosphate afforded protection to enzyme activity. α-Amylase was fairly resistant to EDTA treatment at 30°C, but heating at 90°C in presence of EDTA resulted in the complete loss of enzyme activity, which could be recovered partially by the addition of Cu 2+ and Fe 2+ but not by the addition of Ca 2+ or any other divalent ions. 相似文献
16.
Thermothrix thiopara did not appear to be stressed at high temperature (72°C). Both the actual and theoretical yields were higher than those of analogous mesophilic sulfur bacteria, and the specific growth rate (μ max) was more rapid than that of most autotrophs. The specific growth rate (0.58 h −1), specific maintenance rate (0.11 h −1), actual molar growth yield at μ max ( Ymax = 16 g mol −1), and theoretical molar growth yield ( YG = 24 g mol −1) were all higher for T. thiopara (72°C) than for mesophilic (25 to 30°C) Thiobacillus spp. The growth efficiencies for T. thiopara at 70 and 75°C (0.84 and 0.78) were significantly higher than at 65°C (0.47). Corresponding specific maintenance rates were highest at 65°C (0.41 h −1) and lowest at 70 and 75°C (0.11 and 0.15 h −1, respectively). Growth efficiencies of metabolically similar mesophiles were generally higher than for T. thiopara. However, the actual yields at μ max were higher for T. thiopara because its theoretical yield was higher. Thus, at 70°C, T. thiopara was capable of deriving more metabolically useful energy from thiosulfate than were mesophilic sulfur bacteria at 25 and 30°C. The low growth efficiency of T. thiopara reflected higher maintenance expenditures. T. thiopara had higher maintenance rates than Thiobacillus ferroxidans or Thiobacillus denitrificans, but also attained higher molar growth yields. It is concluded that sulfur metabolism may be more efficient overall at extremely high temperatures due to increased theoretical yields despite increased maintenance requirements. 相似文献
17.
Nuclear magnetic resonance (NMR) was used to determine Na + complexing in muscle and liver (at 23°C) from bullfrogs ( Rana catesbeiana) and to study the influence of temperature on Na + complexing in muscle from leopard frogs ( Rana pipiens). The Na + complexed in muscle and liver was found to be 36.6 ± 4.6% and 66.1 ± 3.5% respectively. A temperature decrease from +34°C to -2°C results in a 20% decrease in the mobility of the free Na + in the fresh muscle. This 20% decrease in mobility results in about 50% of the free Na + at 34°C being complexed at the lower temperature. 相似文献
18.
The effects of low-temperature acclimation and oxygen stress on tocopheron production were examined in the unicellular phytoflagellate Euglena gracilis Z. Cells were cultured photoheterotrophically at 27.5 ± 1°C with 5% carbon dioxide-95% air and 740 microeinsteins m −2 s −1 (photosynthetically active radiation) and served as controls. Low-temperature acclimation (12.5 ± 1°C) and high-oxygen stress (5% carbon dioxide-95% oxygen) were individually examined in the mass culturing of the algae. Chromatographic analyses demonstrated a six-to sevenfold enhancement of α-tocopherol production in temperature-stressed cells, along with a concomitant decline in the levels of α-tocotrienol and the absence of other tocopherol homologs. Oxygen-stressed cultures demonstrated the presence of high levels of α-tocopherylquinone; α-tocopheron and its homologs and precursors were absent or declined markedly. These findings are discussed in terms of the feasibility of microbial production of natural tocopherols. In addition, these results lend themselves to speculation regarding the biological role(s) of tocopherols as antioxidants and free radical scavengers in reducing photo-induced oxidative damage or lipid peroxidation toxicities or both in photosynthetically active E. gracilis Z. 相似文献
19.
1. Superovulated rat ovary slices from rats treated with 20μg. of luteininzing hormone/100g. body wt. 2hr. before death and from control animals have been incubated in vitro. Output of Δ 4-3-oxo steroids (0·2μmole/g. wet wt./hr. in control tissue) was linear for 4hr., and was increased by approx. 70% in slices from luteinizing hormone-treated rats. Rate of oxygen consumption (90·0±4·6μmoles/g. wet wt./hr.) was linear for 3hr. and unaltered by luteinizing hormone treatment or addition of glucose (1mg./ml.) to the medium. 2. In slices from control animals, steady-state rate of glucose uptake was 78·0±2·9μg. atoms of carbon/g. wet wt./hr.; steady-state rates of lactate output, pyruvate output and incorporation of [U- 14C]-glucose carbon atoms into carbon dioxide and total lipid extract were 60·7±0·9, 2·4±0·1, 18·0±1·1 and 0·7±0·1μg. atom of carbon/g. wet wt./hr. and accounted for 104·5±1·9% of the glucose uptake. In slices from luteinizing hormone-treated rats, glucose uptake and outputs of lactate, pyruvate and [ 14C]carbon dioxide were increased by approx. 25%, and 108·4±3·2% of the glucose uptake could be accounted for. 3. The total lipid extract was separated by thin-layer chromatography and saponification. Of the 14C incorporated into this fraction during incubation with [U- 14C]glucose 97% was found in the fractions containing glyceride glycerol and less than 3% in the fractions containing sterols, steroids or fatty acids. Appreciable quantities of 14C were incorporated into these lipid fractions from [1- 14C]acetate. 4. From a consideration of the tissue glycogen content, the specific activities of [ 14C]lactate and glucose 6-phosphate (C-1) derived from [1- 14C]-, [6- 14C]- and [U- 14C]-glucose, and the ratio of [ 14C]carbon dioxide yields from [1- 14C]glucose and [6- 14C]glucose, it was concluded that there was no appreciable glycogenolysis or flow through the pentose phosphate cycle. 5. In ovary slices from both control and luteinizing hormone-treated animals, glucose in vitro raised the incorporation rate of 14C from [1- 14C]acetate into sterols and steroids. Luteinizing hormone in vivo stimulated the incorporation rate in vitro but only in the presence of glucose. 6. In slices incubated in medium containing [ 3H]water, [ 14C]sorbitol and glucose (1mg./ml.), the total water space (865±7·1μl./g.) and the extracellular water space (581±22μl./g.) were unchanged by luteinizing hormone treatment in vivo but the glucose space was raised from 540±23·6μl./g. to 639±31·3μl./g. 7. Luteinizing hormone treatment was found to lower the tissue concentration of the hexose monophosphates and to increase the total activity of hexokinase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and possibly of phosphofructokinase. 8. The kinetic properties of a partially purified preparation of phosphofructokinase were found to be qualitatively similar to those from other mammalian tissues. 9. The results are discussed with reference to both the role of glucose metabolism in steroidogenesis and the mechanism by which luteinizing hormone increases the rate of glucose uptake. 相似文献
20.
The recent development of mutant-selective inhibitors for the oncogenic KRASG12C allele has generated considerable excitement. These inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRAS G12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain. Here, we show by NMR and an independent biochemical assay that the pK a of the C12 thiol is depressed (pK a ∼7.6), consistent with susceptibility to chemical ligation. Using a validated fluorescent KRAS Y137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRAS G12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that at low temperatures, ARS-853 addition elicited both a rapid first phase of fluorescence change (attributed to binding, K d = 36.0 ± 0.7 μM) and a second, slower pH-dependent phase, taken to represent covalent ligation. Consistent with the lower pK a of the C12 thiol, we found that reversible and irreversible oxidation of KRAS G12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRAS G12C Cys12 to a sulfinate altered RAS conformation and dynamics to be more similar to KRAS G12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRAS G12C drug discovery efforts, and identify the occurrence of G12C oxidation with currently unknown biological ramifications. 相似文献
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