Cell-free experiments

Cell-free Experiments. Cell-free extracts were prepared from fermentation samples (12 mL) by centrifugation (2700g for 10 minutes) of the clarified supernatant. Tannin (15 mg) and enriched anthocyanin (400 xL, adjusted to pH... 

EXPERIMENTAL FIGURE 16-4 Cell-free experiments demonstrate that translocation of secretory proteins into microsomes is coupled to translation. Treatment of microsomes with EDTA, which chelates Mg ions, strips them of associated ribosomes, allowing isolation of ribosome-free microsomes, which are equivalent to ER membranes (see Figure... 

In one of the early experiments designed to elucidate the genetic code, Marshall Nirenberg of the U.S. National Institutes of Health (Nobel Prize in physiology or medicine, 1968) prepared a synthetic mRNA in which all the bases were uracil. He added this poly(U) to a cell-free system containing all the necessary materials for protein biosynthesis. A polymer of a single amino acid was obtained. What amino acid was polymerized ... 

Experiments in cell-free in vitro assays have shown that formation of a PNA triplex invasion complex with a homopurine motif on the template strand results in the steric hindrance of RNA polymerases, leading to complete arrest of further... 

Experiments in 500 ml Erlenmeyer flasks and Fernbach flasks contained 200 ml and 1 L of EPl and EP2 medium respectively. Inocuia added to these cultures was 2 ml of spore suspension (5.0 optical density at 540 nm) for each 100 ml EP medium. All cultures were grown at 37°C in a shaking incubator (New Brunswik Sci. Co., USA), at 200 rpm. Then 10 ml of sample were withdrawn each 24 h during fermentation and immediately filtered through Millipore membranes of 0.45 pm pore size these cell-free filtrates were used for enzymatic assays and extracellular protein determinations by the Lowry method (14). Experiments in the 14 L fermentor (Microgen Fermentor New Brunswik Sci. Co., USA) were carried with lOL of fermentation medium EP2 and inoculum added was IL of mycelium grown 24 h in... 

Subsequently, similar experiments were done with viral nucleic acids. The pure viral nucleic acid, when added to cells, led to the synthesis of complete virus particles the protein coat was not required. This process is called transfection. More recently, DNA has been used in cell-free extracts to program the synthesis of RNA that functions as the template for the synthesis of proteins characteristic of the DNA... 

Further experiments focused therefore on [RuCl(en)(r 6-tha)]+ (12) and [RuCl(rj6-p-cym)(en)]+ (22), which represent the two different classes, and their conformational distortion of short oligonucleotide duplexes. Chemical probes demonstrated that the induced distortion extended over at least seven base pairs for [RuCl(rj6-p-cym)(en)]+ (22), whereas the distortion was less extensive for [RuCl(en)(rj6-tha)]+ (12). Isothermal titration calorimetry also showed that the thermodynamic destabilization of the duplex was more pronounced for [RuCl(r 6-p-cym)(en)]+ (22) (89). DNA polymerization was markedly more strongly inhibited by the monofunctional Ru(II) adducts than by monofunctional Pt(II) compounds. The lack of recognition of the DNA monofunctional adducts by HMGB1, an interaction that shields cisplatin-DNA adducts from repair, points to a different mechanism of antitumor activity for the ruthenium-arenes. DNA repair activity by a repair-proficient HeLa cell-free extract (CFE) showed a considerably lower level of damage-induced DNA repair synthesis (about six times) for [RuCl(en)(rj6-tha)] + compared to cisplatin. This enhanced persistence of the adduct is consistent with the higher cytotoxicity of this compound (89). 

H, receptors in brain slices can also stimulate glycogen metabolism [5] and can positively modulate receptor-linked stimulation of cAMP synthesis. The activation of brain cAMP synthesis by histamine is a well studied phenomenon that reveals a positive interaction between histamine receptors [35]. When studied in cell-free preparations, this response shows characteristics of H2, but not H receptors. When similar experiments are performed in brain slices, however, both receptors appear to participate in the response. Subsequent work showed that H receptors do not directly stimulate adenylyl cyclase but enhance the H2 stimulation, probably through the effects of calcium and PKC activation on sensitive adenylyl cyclase iso forms (see Ch. 21). 

Reaction (52) occurs at the gradient interface of the bolus addition until local Hb(02) concentrations have been reduced, at which point additional NO reduces the iron(III) to iron(II) which can further react with free NO to form Hb(NO). The validity of this mechanism was verified by the observation that addition of CN- ion, which binds irreversibly to metHb to form metHb(CN), significantly attenuated the formation of Hb(NO) in both cell-free Hb and RBC. Mathematical models used to simulate bolus addition of NO to cell-free Hb and RBC were compatible with the experimental results (147). In the above experiments, SNO-Hb was a minor reaction product and was formed even in the presence of 10 mM CN, suggesting that RSNO formation does not occur as a result of (hydrolyzed) NO+ formation during metHb reduction. However, formation of SNO-Hb was not detectable when NO was added as a bolus injection to RBC or through thermal decomposition of DEA/NO in cell free Hb (DEA/NO = 2-(A/ A/ diethylamino)diazenolate). SNO-Hb was observed... 

When 1-methyl-2-nitrosoimidazole became available190 it was found that addition of excess GSH to solutions of l-methyl-2-nitrosoimidazole led to a rapid loss of the characteristic absorbance at 360 mn within a few seconds. Preliminary experiments suggested that formation of GSSG and the hydroxylamine was followed by formation of stable thio ethers. It should be noted, however, that detection of free hydroxylamine was unsuccessful57. In cell-free systems l-methyl-2-nitrosoimidazole reacted with excess GSH to form adducts in a 1 3 stoichiometric reaction191. 

In their 1960 paper, Carnahan et al. reported that ATP was inhibitory to nitrogenase activity in their cell-free preparations. Hence, when McNary and Burris [24] reported that ATP was needed to support fixation, the report was met with a good deal of skepticism. But experiments in a number of other laboratories verified the absolute need for ATP. Not only is ATP needed, it is needed in substantial amounts. Under ideal conditions 16 ATP are required per N2 reduced to 2 NH3. Under normal conditions in nature the requirement probably is in the 20 to 30 ATP per N2 range. N2 reduction is energy demanding whether it is accomplished chemically in the Haber process or enzymatically by the nitrogenase system. 

A. bronchisepticus was cultivated aerobically at 30 °C for 72 h in an inorganic medium (vide supra) in 1 liter of water (pH 7.2) containing 1 % of polypeptone and 0.5 % of phenylmalonic acid. The enzyme was formed intracellularly and induced only in the presence of phenylmalonic acid. All the procedures for the purification of the enzyme were performed below 5 °C. Potassium phosphate buffer of pH 7.0 with 0.1 mM EDTA and 5 mM of 2-mercaptoethanol was used thoughout the experiments. The enzyme activity was assayed by formation of pheylacetic acid from phenylmalonic acid. The summary of the purification procedure is shown in Table 2. The specific activity of the enzyme increased by 300-fold to 377 U/mg protein with a 15% yield from cell-free extract [9]. One unit was defined as the amount of enzyme which catalyzes the formation of 1 mmol of phenylacetic acid from phenylmalonic acid per min. 

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