Cell extracts

The phosphorodithioates DNA derivatives have been shown to bind specifically to complementary DNA or ENA sequences to form stable adducts. Because they are also highly resistant to degradation by cellular exonucleases, these derivatives can be useful both for appHcations in research and as therapeutic dmgs. Phosphorodithioate DNA has been shown to stimulate Rnase H activity in nuclear cell extracts and is a potent inhibitor of HIV type-1 reverse transcriptase (56). 

Qnadroni, M., et al., 1996. Analy.sis of global re.spon.ses by protein and peptide fingerprinting of protein.s i.solated by two-dimensional electrophore-.sis. Application to snlfate-starvation re.sponse of Escherichia coli. European Journal of Biochemistry 239 773-781. This paper de.scribes the n.se of tandem MS in the analysis of protein.s in cell extracts. 

The metabolite can be immobilized by covalendy conpling it to an insolnble matrix. snch as an agaro.se polymer. Cell extracts containing many individnal proteins may be pa.ssed dirongh die matrix. 

A practical application of SEE-CEST coupling is shown in Eigure 6.13, which displays the electropherogram obtained for a tomato sample contaminated with a pesticide, i.e. carbaryl. The sample was placed in the SEE cell, extracted with CO2... 

The major dasses of antibiotics are secondary metabolic products of micro-organisms. Many were discovered by empirically screening culture filtrates or cell extracts for antimicrobial activity. A range of techniques (examples are methods using, impregnated discs, porous cylinders, cut wells, see Figure 6.2) have been used to carry out such screening. 

Dabora, S.L. Sheetz, M.P. (1988). The microtubule-dependent formation ofatubulovesicular network with characteristics of the ER from cultured cell extracts. Cell 54, 27-35. 

Phosphorylation of HSF substantially enhances the transcriptional activity of HS gene expression which may be up to 100-fold of basal levels after HSFl binds to the promoter element. Heat shock will increase the C-terminal-domain-kinase activity in cell extracts, and this action may enhance the activity of RNA polymerase II that is bound to HS genes (Legagneux et al., 1990). Whether this kinase activity also affects HSFl phosphorylation is not known, but increased HS gene expression appears to occur as long as HSFl is bound to the promoter region. The CTD kinase complex contains multiple proteins, and it is quite possible that one or more of these proteins is also regulated by stress. 

Legagneux, V., Morange, M., Bensaude, O. (1990). Heat-shock and related stress enhance RNA polymerase II C-terminal-domain kinase activity in HeLa cell extracts. Eur. J. Biochemistry 193, 121-126. 

The easiest cells to grow are microbes that live independently in their natural environment. These include bacteria, yeasts, and molds. The hardest are the cells extracted from higher order plants and animals since they normally rely on complex interactions with other cells in the parent organism. Bacteria and yeasts are single-celled. Molds are multicelled but have relatively simple structures and nutritional requirements. 

CO oxidation occurs in two half-reactions (1) oxidation of CO to CO2 generating the two-electron reduced enzyme and (2) reoxidation of the enzyme by the electron acceptor. The ping-pong nature of this reaction was first proposed based on studies with cell extracts from C. thermoaceticum 54) and C. pasteurianum 155). 

Figure 4-4. Use of SDS-PAGE to observe successive purification of a recombinant protein. The gel was stained with Coomassie blue. Shown are protein standards (lane S) of the indicated mass, crude cell extract (E), high-speed supernatant liquid (H), and the DEAE-Sepharose fraction (D). The recombinant protein has a mass of about 45 kDa.

Recent evidence suggests that there is another possible mechanism of PIC formation and transcription regulation. First, large preassembled complexes of GTFs and pol II are found in cell extracts, and this complex can associate with a promoter in a single step. Second, the rate of transcription achieved when activators are added to limiting concentrations of pol II holoenzyme can be matched by increasing the concentration of the pol II holoenzyme in the absence of activators. Thus,... 

Tamplin et. al. (54) observed that V. cholerae and A. hydrophila cell extracts contained substances with TTX-like biological activity in tissue culture assay, counteracting the lethal effect of veratridine on ouabain-treated mouse neuroblastoma cells. Concentrations of TTX-like activity ranged from 5 to 100 ng/L of culture when compared to standard TTX. The same bacterial extracts also displaced radiolabelled STX from rat brain membrane sodium channel receptors and inhibited the compound action potential of frog sciatic nerve. However, the same extracts did not show TTX-like blocking events of sodium current when applied to rat sarcolemmal sodium channels in planar lipid bilayers. 

Figure 2.17 Application of the reverse DEPT pulse sequence to monitor C-labeled glucose by mouse liver-cell extract. (A) Normal FT spectrum. (B) Reverse DEPT spectrum showing the a- and )3-anomeric proton resonances. (C) Two different CH2 proton resonances, a and b, appear after 1.5 h of metabolism. (D) Edited H spectrum confirming that the CH2 resonances arise from metabolic products. (Reprinted from J. Magn. Resonance 56, Brooks et al., 521, copyright 1984, Academic Press.)...

Applying the reverse DEPT pulse sequence to monitor C-labeled glucose by mouse liver-cell extract is shown in Fig. 2.17. The a- and /3-anomeric proton resonances are shown in the starting material these are transformed to CH.2 proton resonances in the metabolite. 

Phenol is an important intermediate in the anaerobic degradation of many complex and simple aromatic compounds. Tschech and Fuchs proposed that the carboxylation of phenol to 4-hydroxybenzoate is the first step in the degradation of phenol under denitrifying conditions. However, 4-hydroxybenzoate is not detected in the cultures or cell extracts of the denitrifying Pseudomonas species in the presence of CO2 and phenol, but it is detected if phenol is replaced by phenolphosphate. In contrast, 4-hydroxybenzoate is readily detected as an intermediate of phenol degradation in the iron-reducing bacterium GS-15, and 4-hydroxybenzoate may prove to be a common intermediate in the anaerobic transformation. Thus, in anaerobic degradation of phenolic compounds, it has been postulated that carboxylation reactions may play important roles. 

When a cell extract prepared from A. nicotianae FI1612 cells was stored without the addition of sulfhydryl-protecting reagents, 80% of the initial activity was lost after storage at 4°C for 4 days. The enzyme activity was stabilized... 

When cell-wall fragments are incubated in molar NaCl, ionically bound proteins are released into the incubation medium. All investigated crude cell extracts deesterified Citrus pectin (Table 2) but the deesterification rates were clearly higher when the enzymes were still bound to the cell walls, indicating a major loss of activity during the solubilization process. 

Only the R(+) enantiomer of the herbicide 2-(2-methyl-4-chlorophenoxy)propionic acid was degraded (Tett et al. 1994), although cell extracts of Sphingomonas herbicidovorans grown with the R(-) or S -) enantiomer, respectively, transformed selectively the R -) or S(-) substrates to 2-methyl-4-chlorophenol (Nickel et al. 1997). 

Hydroxylation of n-octane by cell extracts of Gordonia (Corynebacterium) sp. strain 7E1C (Cardini and Jurtshuk 1968), and of some strains of Acinetobacter calcoaceticus induced with -hexadecane (Asperger et al. 1981). 

The hydroxylation of cyclohexane by a strain of Xanthobacterium sp. (Trickett et al. 1990). In cell extracts, a range of other substrates was oxidized including cyclopentane, pinane, and toluene (Warburton et al. 1990). 

Hydroxylation of progesterone and closely related compounds at the 15-(3 position by cell extracts of Bacillus megaterium (Berg et al. 1976). 

Neilson AH, C Lindgren, P-A Hynning, M Remberger (1988) Methylation of halogenated phenols and thiophenols by cell extracts of Gram-positive and Gram-negative bacteria. Appl Environ Microbiol 54 524-530. 

Allen JR, SA Ensign (1996) Carboxylation of epoxides to 3-keto acids in cell extracts of Xanthobacter strain Py2. J Bacterial 178 1469-1472. 

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