Cell-free extracts

Biocatalysis Chemical reactions mediated by biological systems (microbial communities, whole organisms or cells, cell-free extracts, or purified enzymes aka catalytic proteins). 

An important tool for elucidating the steps in the pathway was the use of metabolie inhibitors. Adding an enzyme inhibitor to a cell-free extract caused an accumulation of intermediates in the pathway prior to the point of inhibition (Figure 18.12). Each inhibitor was specific for a particular site in the sequence of metabolic events. As the arsenal of inhibitors was expanded, the individual steps in metabolism were revealed. 

Herrera, A. A., Hastings, J. W., and Morin, J. G. (1974). Bioluminescence in cell free extracts of the scale worm Harmothoe (Annelida, Polynoidae). Biol. Bull. 147 480-481. 

Strehler, B. L., and Cormier, M. J. (1953). Factors affecting the luminescence of cell-free extracts of the luminous bacterium, Achromobacter fischeri. Arch. Biochem. Biophys. 47 16-33. 

Synthesis in a Cell-free Extract of Spinach Chloroplasts. 277... 

A wheat germ, cell-free, translation extract was fractionated into three concentrated parts using ammonium sulfate the 0 - 40 % saturated fraction, the 40 - 60 % saturated fraction, and the ribosome fraction. These fractions were tested for their ability to enhance the translational activity of the wheat germ, cell-free extract for dihydrofolate reductase. The fortified cell-free system supplemented with the 0 - 40 % ammonium sulfate fraction enhanced the efficiency of protein synthesis by 50 %. 

Cell-free translation system, used for the identification of cloned genes and gene expression, has been investigated extensively as a preparative production system of commercially interesting proteins after the development of continuous-flow cell-free translation system. Many efforts have been devoted to improve the productivity of cell-free system [1], but the relatively low productivity of cell-free translation system still limits its potential as an alternative to the protein production using recombinant cells. One approach to enhance the translational efficiency is to use a condensed cell-free translation extract. However, simple addition of a condensed extract to a continuous-flow cell-free system equipped with an ultrafiltration membrane can cause fouling. Therefore, it needs to be developed a selective condensation of cell-free extract for the improvement of translational efficiency without fouling problem. 

The catalytic activities of the fortified wheat germ cell-free systems supplemented with each fraction were investigated (Fig. 2). As shown in Fig. 2, only 0 - 40 % ammonium sulfate fraction showed an enhancement in DHFR protein synthesis. This enhancement of protein experimental results and the fact that the various eukaryotic initiation factors are contained in synthesis was also confirmed by SDS-PAGE and autoradiography (Fig. 3). From the above 0-40 % ammonium sulfate fraction [5, 6], it can be concluded that the amount of initiation factors in a conventionally prepared wheat germ cell-fi extract is deficient for the translation of DHFR with internal ribosome entry site. Therefore, it needs to supplement a wheat germ cell-free extract with the fraction containing the limited initiation factors for the efficient protein translation, and this fortified cell-free system can be easily made by simple... 

The occurrence of 4-hydroxybenzoate decarboxylase was also found in facultative anaerobic bacteria, E. cloacae P240, and the enzyme was purified and characterized. The activity of the cell-free extract of E. cloacae P240 was determined to be 13.7 ijumol min (mg protein) at 30°C, which was much higher than... 

The occurrence of 3,4-dihydroxybenzoate decarboxylase was also found widely in facultative anaerobes. Among them, Enterobacter cloacae P241 showed the highest activity of 3,4-hydroxybenzoate decarboxylase, and the activity of the cell-free extract of E. cloacae P241 was determined to be 0.629 p.mol min (mg protein) at 30°C, which was more than that of C. hydroxybenzoicum, 0.11 (xmol min mg protein)" at 25°C. The E. cloacae P241 enzyme has a molecular mass of 334 kDa and consists of six identical 50 kDa subunits. The value for 3,4-dihydroxybenzoate was 177 p.M. The enzyme is also characteristic of its narrow substrate specificity and does not act on 4-hydroxybenzoate and other benzoate derivatives. The properties of E. cloacae P241 3,4-hydroxybenzoate decarboxylase were similar to those of C. hydroxybenzoicum in optimum temperature and pH, oxygen sensitivity, and substrate specificity. 

Dihydroxybenzoate decarboxylase activity of these bacteria was induced specifically by 2,6-dihydroxybenzoate. The enzyme activity in a cell-free extract of A. tumefaciens 1AM 12048 was stable during storage at 4°C for 7 days in potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol. Different from 4-hydroxybenzoate decarboxylase and 3,4-dihydroxybenzoate decarboxylase, 2,6-dihydroxybenzoate decarboxylase was much less labile and barely... 

Recently, the distribution of 2,3-dihydroxybenzoate decarboxylase has been found in a variety of fungal strains (unpubhshed data), and the carboxylation activity for catechol is confirmed by the reaction using resting cells (or cell-free extract) in the presence of 3M KHCO3. The detailed comparative studies of enzyme structures and catalytic properties between 2,3-dihydroxybenzoate decarboxylase and 3,4-dihyroxybenzoate decarboxylase might explain how the decarboxylases catalyze the regioselective carboxylation of catechol. 

Suzuki T (1978) Enzymatic methylation of pentachlorophenol and its related compounds by cell-free extracts of Mycobacterium sp isolated from soil. J Pesticide Sci 3 441-443. 

Cardini G, P Jurtshuk (1968) Cytochrome P-450 involvement in the oxidation of -octane hy cell-free extracts of Corynebacterium sp. strain 7E1C. J Biol Chem 243 6070-6072. 

D Ari L, WA Barker (1985) p-cresol formation by cell-free extracts of Clostridium difficile. Arch Microbiol 143 311-312. 

Ohisa N, M Yamaguchi, N Kurihara (1980) Lindane degradation by cell-free extracts of Clostridium rectum. Arch Microbiol 125 221-225. 

Ince JE, CJ Knowles (1986) Ethylene formation by cell-free extracts of Escherichia coli. Arch Microbiol 146 151-158. 

Nebreda, A. R., and Hunt, T. (1993). The c-mos proto-oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell-free extracts of Xenopus oocytes and eggs. EMBO J. 12 1979-1986. 

In summary, we showed that DNA damage can elicit a variety of responses in Xenopus early embryos. While y-irradiation induces apoptosis in the embryos, DSB-containing DNA prevents initiation of DNA replication in cell-free extracts derived from eggs. 

Gautier Yes. One would argue that everything required for apoptosis is present in the egg. If you dissociate the egg and make cell-free extract from the one cell stage embryo, you can in certain conditions have a system to follow apoptosis. 

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... 

The activities of NHases from Rhodococcus sp. Adpl2 and Gordonia sp. BR-1 strains have been partially characterized [25]. In reactions that catalyze the hydration of a-hydroxynitriles such as lactonitrile or glycolonitrile, the substrate can dissociate to produce HCN and the corresponding aldehydes. HCN can inhibit and/or inactivate NHase, and it was determined that these two enzymes remain active in the presence of cyanide ion at concentrations up to 20 him. The dependence of the NHase activity of cell-free extracts of Rhodococcus rhodochrous J1 and Gordonia sp. BR-1 on cyanide ion concentration is illustrated in Figure 8.1, demonstrating the improved cyanide stability of BR-1 NHase relative to that of Jl. 

Figure 8.1 Dependence of the NHase activity of cell-free extracts of Rhodococcus rhodochrous J1 (a) and Gordonia sp. BR-1 ( ) on cyanide ion concentration...

The regioselectivity of a Rhodococcus rhodochrous nitrilase has been demonstrated for the conversion of 5-fluoro-l,3-dicyanobenzene to 5-fluoro-3-cyano-benzoic acid [62]. The nitrilase was expressed in an Escherichia coli transformant, and a cell-free extract was employed as catalyst (0.14wt% cell-free extract) in 0.1m sodium phosphate buffer (pH 7.2) at 25 °C containing 0.18 m 5-fluoro-l,3-dicyanobenzene. After 72 h, the conversion was >98% and the reaction was stopped by addition of phosphoric acid (pH 2.4) to yield 5-fluoro-3-cyano-benzoic acid as a crystalline product (97% isolated yield). 

Oldfield s work confirmed the complete pathway and identified all the intermediates via an exhaustive experimental scheme, which included whole cell assays, cell-free extracts of IGTS8 as well as extracts from clones containing individual genes expressed in Escherichia coli. The need for NADH was clearly demonstrated in cell-free assays by amendment of NADH. The experimental evidence for involvement of FMN in the pathway was demonstrated [53,66,67],... 

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