Cell-free preparation

L-Phenylalanine can be synthesised from trims-cinnamic add (Figure A8.12) catalysed by a L-phenylalanine ammonia-lyase from Rhodococcus glutinis. The commercialisation of the process was limited by the low conversion (70%), low stability of the biocatalyst and die severe inhibition exerted by trims-cinnamic add. These problems were largely overcome by researchers at Genex. The process, commercialised for a short period by Gen ex, involves a cell-free preparation of phenylalanine-ammonia-lyase activity from Rhodotorula rubra. 

ChAT, first assayed in a cell-free preparation in 1943, has subsequently been purified and cloned from several sources [16]. The purification of ChAT has allowed... 

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). 

Horiguchi, M. and Rosenberg, H., Phosphonopyruvic acid a probable precursor of phosphonic acids in cell-free preparations of Tetrahymena, Biochim. Biophys. Acta, 404, 333, 1975. 

It obviously was difficult to establish details of the N2 fixation process with intact organisms, as the ammonia fixed was rapidly assimilated into other compounds. So a search was initiated in several laboratories for a cell-free preparation that would fix N2. 

We tried preparations from a variety of N2-fixing agents, but responses were variable. Sometimes we achieved rather good fixation with cell-free preparations, but the results were not consistent. For example, at a meeting in early 1958, we reported that cell-free preparations from Clostridium pasteurianum exposed under an atmo-... 

Carnahan et al. [19] in 1960 also reported N2 fixation with cell-free preparations from C. pasteurianum. They dried their cells in a rotary evaporator at about 40 °C and then extracted them to make their preparations. Their other innovation was to supply high levels of pyruvate to support fixation. The method was reproducible, and we promptly verified their results. [20] Fixation for 3 h in our tests with added pyruvate gave 1.334 atom% 1SN excess. Fixation with other substrates was less vigorous lactate supported fixation to give 0.028 and 0.033 atom% 1SN excess. 

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. 

Magee, W.E. and R.H. Burris. Oxidative activity and nitrogen fixation in cell-free preparations from Azotobacter vindandii. J. Bac-teriol. 71, 635-643 (1956). 

Schneider, K.C., C. Bradbeer, R.N. Singh, L.C. Wang, P.W. Wilson and R.H. Burris. Nitrogen fixation by cell-free preparations from microorganisms. Proc. Natl. Acad, Sci. USA 46, 726-733 (1960). 

McNary, J.H. and R.H. Burris. Energy requirements for nitrogen fixation by cell-free preparations from Clostridium pasteurianum. J. Bacterid. 84, 598-599 (1962). 

In vivo oxidation activity may not be expressed in vitro due to inclusion of excessive amounts of "inactive" tissue in the various cell-free preparations. The resulting tissue dilution artifact renders activity unmeasurable due to the sensitivity of the analytical procedures. This consideration warrants further experimental evaluation. 

TX-20 cell-free preparation (A), after and (B), before protease treatment. (-),... 

A range of symmetrical bicyclic /3-diketones can be converted to 2,3-disubstituted cycloalkanones in high yield with high diastereomeric and enantiomeric excess using a cell-free preparation of a retro-Claisenase enzyme, or /3-diketone hydrolase, the gene for which has been heterologously expressed in Escherichia coli. ... 

Procedure 4 Biotransformation of Fluorobenzene by Whole Cells of P. mendocina KRl Expressing T4MO in Tandem with a Cell-free Preparation of Tyrosinase from Mushroom... 

Heritage, A.D. and MacRae, I.C. Degradation of lindane by cell-free preparations of Clostridium sphenoides, Appl Environ. Microbiol, 34(2) 222-224, 1977a. 

The work by Scott and Lee 165) on the isolation of a crude enzyme system from a callus tissue culture of C. roseus was followed by studies of Zenk et al. on an enzyme preparation from a cell suspension system which produced indole alkaloids 166). The cell-free preparation was incubated with tryptamine and secologanin (34) in the presence of NADPH to afford ajmalicine (39), 19-epiajmalicine (92), and tetrahydroalstonine (55) in the ratio 1 2 0.5. No geissoschizine (35) was detected. In the absence of NADPH, an intermediate accumulated which could be reduced with a crude homogenate of C. roseus cells in the presence of NADPH to ajmalicine (39). Thus, the reaction for the formation of ajmalicine is critically dependent on the availability of a reduced pyridine nucleotide. 

The enzyme-catalyzed formation of anhydrovinblastine (8) from catharanthine (4) and vindoline (3) was first examined by Kutney and co-workers (170,219) using a cell-free preparation. [ao f- H]Catharanthine (4) and [acety/- C]vindoline (3) were incubated for 3-8 hr, both separately and jointly with a preparation from C. roseus, which led to the isolation of labeled anhydrovinblastine (8) and leurosine (11) incorporations were of the order of 0.54 and 0.36%, respectively. On this basis, anhydrovinblastine (8) was proposed as the key biosynthetic intermediate en route to vinblastine (1) and vincristine (2). 

More recently, Kutney and co-workers (220) have investigated whether the same dihydropyridinium intermediate 109 is involved in the enzymatic conversion of catharanthine (4) and vindoline (3) to anhydrovinblastine (8) as is involved in the chemical conversion. Use of a cell-free preparation from a 5-day culture of the AC3 cell line gave 18% of the bisindole alkaloids leurosine (11), Catharine (10), vinamidine (25), and hydroxy-vinamidine (110), with 10 predominating. When the incubations were carried out for only 5-10 min, the dihydropyridinium intermediate was detected followed by conversion to the other bisindole alkaloids, with FAD and MnClj required as cofactors. Clearly a multienzyme complex is present in the supernatant, but further purification led to substantial loss of enzymatic activity. The chemical formation of anhydrovinblastine (3) is carried out with catharanthine A-oxide (107), but when this compound was used in the enzyme preparation described, no condensation with vindoline (3) occurred to give bisindole alkaloids. This has led Kutney and co-workers to suggest (220) that the A-oxide 108 is not an intermediate in the biosynthetic pathway, but rather that a 7-hydroperoxyindolenine... 

The conversion of anhydrovinblastine (8) to vinblastine (1) has been examined by several different groups, using intact plants, cell suspension systems, and cell-free preparations. From the studies discussed above it was clear that 3, 4 -anhydrovinblastine (8) was probably the initially formed intermediate in the condensation of vindoline (3) and catharanthine (4) prior to vinblastine (1). Kutney and co-workers have reported (225,226) on the biotransformation of 3, 4 -anhydrovinblastine (8) using cell suspension cultures of the 916 cell line from C. roseus a line which did not produce the normal spectrum of indole alkaloids. After 24 hr the major alkaloid products were leurosine (11) and Catharine (10) in 31 and 9% yields, respectively, with about 40% of the starting alkaloid consumed. 

When 3, 4 -[ao - H]anhydrovinblastine (8) was incubated with a cell-free preparation at pH 6.3 for 50 hr, leurosine (11) and Catharine (10) were labeled to the extent of 8.15 and 15.15%, respectively, and vinblastine (1) was labeled to 1.84% (776,227). Approximately the same level of incorporation was obtained by Scott s group, using 3, 4 -[21 - H]anhydrovinblas-tine (8) and isolating vinblastine (1) from cell-free extracts of C. roseus (228). Scott s failure (87) to observe incorporation of the same precursor into vinblastine (1) in whole plants was explained by the established (82) instability of anhydrovinblastine (8). 

The question of the possible artifactual nature of leurosine (11) was examined by Kutney and co-workers (229) using a cell-free preparation at pH 6.3. After a 3-hr incubation, a 22% yield of leurosine (11) was formed from anhydrovinblastine (8), and when anhydrovinblastine (8) was incubated with horseradish peroxidase in the presence of HjOj, leurosine (11) was formed in 65% yield. 

The extremely low yield of vincristine (2) from intact plants has made pursuit of its biosynthesis a very challenging problem, which at this point in time remains unsolved. Kutney et al. have used both anhydrovinblastine (8) (227) and catharanthine N-oxide (107) (233) as precursors to vincristine (2) in a cell-free preparation, but incorporation levels were extremely low. Therefore, the question of whether vinblastine (1) is an in vivo, as well as an in vitro, precursor remains to be answered. Several possibilities exist for the overall oxidation of vinblastine (1) to vincristine (2), including a direct oxidation of the A-methyl group or oxidative loss of the N-methyl group followed by N-formylation. 

These observations suggest that the oxidative reactions occurring on toxaphene are similar in nature to the system described for camphor degradation (M). It is unfortunate that the above experiments could only be conducted with washed intact cells as cell-free preparations in all cases did not exhibit metabolic capabilities. The reason for this phenomenon has not been found, but to our knowledge no other research group has been able to demonstrate pesticide degrading activities of such oxygenase systems in cell-free preparations. 

Hao, D. Y. and Yeoman, M. M. 1996. Nicotine N-demethylase in cell-free preparations from tobacco cell cultures. Phytochemistry, 42(2) 325-329. 

ZO031 Surh, Y. J., and S. S. Lee. Enzymatic reduction of [6]-gingerol, a major pungent principle of ginger, in the cell-free preparation of rat liver. Life Sci 1994 54(19) PL 321-326. 

Kinetic studies of the incorporation of the " C-labelled precursors mevalonic acid, isopentenyl pyrophosphate, and phytoene into C40 carotenes by Halobac-terium cutirubrum cell-free preparations" produced results consistent with the pathways outlined in Schemes 2 and 3. Only the trans-isomers seemed to be involved. A mutant strain, " PGl, of the green alga Scenedesmus obliquus accumulates phytoene (135), phytofluene (136), and -carotene (137) in place of the... 

Enzyme Systems. Carotenoid biosynthesis by crude cell-free preparations from Halobacterium cutirubrum 0-carotene), Phycomyces blakesleeanus mutants (/8-carotene), and a Neurospora crassa mutant (phytoene) has been demonstrated. Detailed studies of carotenogenic enzymes from tomato fruit... 

As already indicated, control points in the regulation of the rate of glyeosylation of proteins may be (a) the availability of Dol-P (Section 11,1,a), (b) the D-glucosylation of the lipid-linked oligosaccharide, or its transfer to protein, or both (Section 1,2,a), and, as already discussed, (c) the metabolic fate of GDP-Man. Furthermore, GDP-Man inhibits fonnation of Glc-P-Dol in cell-free preparations from liver,157 and activates formation of GlcNAe-PP-Dol in cell-free preparations from tissues of chick embryo.156... 

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