Cell-free extracts, experiments

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). 

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. 

These experiments make it clear that removing competition with release factors leads to more efficient incorporation of the desired amino acid. Unfortunately, the technology to incorporate nonstandard nucleotides into mRNAs coding for full-length proteins is not yet available. Alternatives that have been tested include using (i) a 4-nucleotide codon-anticodon pair, dubbed frame-shift suppression (Sect. 6.1), (ii) a rare codon, and (iii) cell-free extracts from organisms that are either deficient in a release factor (Sect. 5.1) or unable to translate one or more codons (Sect. 6.2). 

Incubation of geissoschizine (35) with a cell-free extract from C. roseus 210) in the presence of NADPH caused the accumulation of an isomer of isositsrikine whose structure was established chemically to be the (167 ) isomer 58. None of the 16-epi isomer 95 was detected in the cell-free incubations or in feeding experiments with intact plants. Additionally, Stdck-igt has reviewed enzymatic studies on the formation of strictosidine (33) and cathenamine (76) (277), and Zenk has provided a very elegant summary of the enzymatic synthesis of ajmalicine (39) (272). 

An effort was made to investigate the involvement of cell-free extracts of a bacterial mixed-function oxidase ystem in pesticide degradation. In this experiment, the degradation of C-mexacarbate was examined using the 10,000 g supernatant of lysozyme-treated B. megaterium cells in 0.03 M phosphate buffer at pH 7.0, Incubation was... 

It was interesting that the cell-free extract had the capacity to support the biosynthesis all the way to FAc 1, an end product of one of the fluorometabolite pathways. This observation indicates that all of the enzymes and cofactors required to support FAc biosynthesis were present and active in the cell-free extract, even though the integrity of the cells had been destroyed. This experiment showed that organic fluoride production was achievable in vitro from the S. cattleya protein extract. Subsequent purification of the fluorinase (5 -fluoro-5 -deoxyadenosine synthase), using standard purification protocols revealed that the true substrate for the enzyme was SAM 8 and not ATP 7 [8]. It transpired that ATP 7 and L-methionine (L-Met) were converted to SAM 8 in the crude cell-free extract and that the resultant SAM 8 was then processed by the fluorinase with the release of L-Met. Thus, a catalytic cycle where L-Met was regenerated to drive these two reactions had been inadvertently established (Scheme 1). The fluorinase catalyses the conversion of SAM 8 and fluoride ion to make 5 -FDA 5 as shown in Scheme 1 [8]. 

In early experiments it was found that sodium pyruvate was required for fixation of N2 in cell-free extracts, and that large amounts of C02 and H2 accumulated. Investigation showed that cleavage of pyruvate supplies cells with two important products ... 

In the presence of its natural substrates, propionyl-CoA, methylmalonyl-CoA, and NADPH, DEBS 1-TE was initially shown to catalyze the formation of lactone in a cell-free system [36]. Concomitant work on a similar bimodular system called DEBS 1+TE (Fig. 9c) [37] in a cell-free extract and with partially purified protein, demonstrated that it too was competent for biosynthesis of the triketide lactone [33], These experiments set the stage for more rigorous investigation of mechanistic aspects of erythromycin biosynthesis. 

The stereochemistry of the reaction catalyzed by the lysine 2,3-aminomutase in C. subterminah SB4 was elucidated in detail by Aberhardt and Gould [32]. Incubation experiments with cell-free extracts of C. subterminah SB4 and (2RS)-[3-13C,2-lsN]lysine and NMR spectroscopy of the isolated /Mysine as the di-N-phthaloyl ethyl ester derivative revealed that the amino group migrates in an intramolecular reaction to position 3S in /Mysine 25. 

N, in a cell-free extract of Cephalosporium acremonium.154 Only the naturally occurring tripeptide with the LLD-configuration was utilized for biosynthesis, from which it is clear that L-valine is built into the tripeptide with inversion of configuration and that later formation of penicillin N involves an epimerization in the a-aminoadipyl moiety. Although in these experiments only the valine fragment of the precursor was labelled, intact incorporation is indicated by the failure of L-cysteinyl-D-valine or 6-aminopenicillanic acid (160) to act as penicillin precursors, and by the observation that the formation of labelled valine could not be detected in the feeding experiment with (159). 

The sequence of the various steps between uroporphyrinogen III and cobyrinic acid has been the subject of much recent work. A major advance in this area was the observation that sirohydrochlorin (77), the iron-free prosthetic group of the enzyme siroheme, could be modified to accommodate its role as a biosynthetic intermediate. Subsequently a dimethyl isobacteriochlorin (factor II) isolated from P. shermanii was shown to be identical with sirohydrochlorin from E. coli sulphite reductase. The complete stereostructure of (77) was elucidated by a series of biosynthetic experiments using [4- C]- and [5- C]-ALA and independently by more classical structural arguments (B-79MI10401, B-79MI10402>. Sirohydrochlorin labelled biosynthetically from [4- C]ALA and [ CHsJmethionine is incorporated into cobyrinic acid by cell-free extracts of F. shermanii without loss or migration of label. 

The specific and proximate precursor of the mCyN unit in ansamycin polyketides is 3-amino-5-hydroxybenzoic acid 59 (AHBA) [94]. The biosynthesis of AHBA has recently been described by Floss and co-workers from the initial branch point of the shikimic acid pathway prior to 3-deoxy-D-flra/jzno-heptulo-sonic acid 7-phosphate (DAHP) [95]. The pathway shown in Scheme 25 was delineated by feedings of the proposed AHBA precursors, in labelled forms, to cell-free extracts of both the rifamycin B producer A. mediterranei S699 and the ansatrienin A producer S. collinus Tul892. In these experiments each of the compounds 61-64 was converted into AHBA with generally increasing efficiency. Most importantly the shikimate pathway compound DAHP cannot replace phosphoenolpyruvate 61 and erythrose 4-phosphate 60, or aminoDAHP 62 as the precursor of AHBA 59. 

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