Enzymatic Studies

Biosynthesis of Gangliosides. Since an abnormality of ganglioside catabolism in tumorigenic-virus-transformed cells was excluded, we 

TABLE II. Distribution of Gangliosides in AL/N and Swiss Mouse Cell Lines and in Polyoma- and SV40-Vims-Transformed Derivative Lines  

Cell type Growth in culture floia Gmi Gm2 Gm3 Total 

FIGURE 3. Scans of absorbance at 580 nm and radioactivity of thin-layer chromatograms of gangliosides extracted from NAL/N (control) and SVS AL/N cells grown in the presence of N-acetyl-[ H]mannanosamine in the tissue culture medium. Reproduced with permission from Brady and Mora, 1970. 

TABLE III. Uridine Diphosphate -Acetylgalactosamine Hematoside N Acetylgalactosaminyltransferase Activity in Normal and Transformed Mouse Cell Lines  

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Frimmer U, F Widdel (1989) Oxidation of ethanol by methanogenic bacteria. Growth experiments and enzymatic studies. Arch Microbiol 152 479-483. 

Gaafar H.A., Tantawy A., Melis A., Hennawy D.M. and Shehata H. (1998). The vomeronasal (Jacobson s) organ in adult humans frequency of occurrence and enzymatic study. Acta Otolaryngol 118, 409-412. 

Robin, J. P., Mercier, C., Charbormiere, R., and Guilbot, A. (1974). Lintnerized starches. Gel filtration and enzymatic studies of insoluble residues from prolonged acid treatment of potato starch. Cereal Chem. 51, 389 05. 

The metabolism of chloroform is well understood. Approximately 50% of an oral dose of 0.5 grams of chloroform was metabolized to carbon dioxide in humans (Fry et al. 1972). Metabolism was dose-dependent, decreasing with higher exposure. A first-pass effect was observed after oral exposure (Chiou 1975). Approximately 38% of the dose was converted in the liver, and < 17% was exhaled unchanged from the lungs before reaching the systemic circulation. On the basis of pharmacokinetic results obtained in rats and mice exposed to chloroform by inhalation, and of enzymatic studies in human tissues in vitro, in vivo metabolic rate constants (V, 3,C =15.7 mg/hour/kg, = 0.448 mg/L) were defined for humans (Corley et al. 1990). The metabolic activation of chloroform to its toxic intermediate, phosgene, was slower in humans than in rodents. 

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). 

Legionella pneumophila produces a siderophore named legiobactin, which shows no catecholate or hydroxamate reactions (206). Enzymatic studies suggest a citrate structure in agreement with the data obtained by mass spectrometry (molecular mass ca. 350 Da) and NMR (three carbonyl and ten aliphatic C atoms). It is not clear yet as to whether legiobactin is essential for the iron acquisition in the aqueous habitat of the bacterium or during lung infection (2, 65). 

The radioactive method used in enzymatic studies by Flodgaard and Brodersen (Fll) is based on work of Brodersen (B22) and Brodersen and Jacobsen (B24). Samples of a solution of bilirubin diglucuronoside... 

As reported below, recent enzymatic studies have provided insight into several of these problems in particular, many of the questions concerning the formation of / -glucogallin and its subsequent conversion to pentagalloylglucose have been answered by this technique. Other aspects outlined above remain rather obscure, but starting points for their eventual clarification will be discussed. 

In enzymatic studies with cell-free extracts from higher plants, however, no evidence has been found to date for galloyl-CoA s involvement in the biosynthesis of / -glucogallin, or its higher galloylated derivatives an eventual role in the formation of the chemically different meta-depside bonds of gallotannins has not been investigated. 

As documented in a review article (9), no experimental data was available to support hypothetical mechanisms for the biosynthesis of hydrolyzable tannins until recently. Enzymatic studies have now changed this unsatisfactory situation, at least as far as the formation of pentagalloylglucose is concerned. Future work will provide insight into those other challenges discussed in this contribution and that still require clarification. 

Mono-, di-, and trifluoro derivatives of fatty acids have been prepared in regio- and stereoselective manners, in order to evaluate the gauche effect induced by fluorine atoms on the conformation of hydrocarbon chains and for enzymatic studies. 

The pattern of urinary GAGs should be assessed in a case of a positive urinary MPS screening result or when clinical symptoms are suggestive of MPS despite normal GAG excretion. This also facilitates the choice of further enzymatic studies. In addition, approximately 5-6% of abnormal samples in the quantitative DMB method are false-positive results that can usually be identified as such by checking the distribution of GAG excretion [16]. 

Early isotope tracer experiments by David Shemin permitted the elucidation of the formation of the immediate precursor of the porphyrin needed for the cytochromes and for hemoglobin. These studies indicated that the glycine methylene carbon and nitrogen were incorporated along with both carbons of acetate. Subsequent enzymatic studies in both bacteria and animals revealed a condensation reaction between succinyl-CoA and glycine to yield 5-amino-levulinate and C02 (presumably by way of an enzyme-bound /3-keto acid, a-amino-/3-ketoadipate) (fig. 22.13). 

Wan C, Fiebig T, Schiemann O, Barton JK, Zewail AH (2000) Femtosecond direct observation of charge transfer between bases in DNA. Proc Nat Acad Sci USA 97 14052-14055 Wang J, Biedermann KA, Wolf CR, Brown JM (1993) Metabolism of the bioreductive cytotoxin SR 4233 by tumour cells enzymatic studies. Br J Cancer 67 321-325 Wang W-F, Schuchmann MN, Schuchmann H-P, KnolleW, von Sonntag J, von Sonntag C (1999) Radical cations in the OH-radical-induced oxidation of thiourea and tetramethylthiourea in aqueous solutions. J Am Chem Soc 121 238-245... 

Cell Culture. Human neuroblastoma IMR-32 (passaged through nude mice the cells were donated by Dr. Steven E. Brooks, Kingsbrook Jewish Medical Center, Brooklyn) and mouse neuroblastoma clones NIE-115, NS-20, and N-18) (donated by Dr. Shraga Makover, Hoffmann LaRoche, Inc., Nutley, New Jersey) were maintained in our laboratory as described previously (30,31). Confluent monolayers (6 to 8 x 10 cells per 250-ml Falcon plastic flask) were harvested for enzymatic studies with phosphate-buffered saline [7.0 mM potassium phosphate/0.14 M NaCl - buffer, pH 7.2 (Pi/NaCl)] containing 0.1% EDTA. 

Initial enzymatic studies show that 16a and 16b bind to CPA somewhat less strongly than the corresponding phosphonamidate ii (K3 of 11 = 1.2 pM). 

FIGURE 2.37 Chip design optimized for experiments using hydrodynamic flow control. The chip has the pair-well design suitable for dilution and enzymatic studies [295]. Reprinted with permission from Springer-Verlag. 

Saturated fatty acids are produced by the combination of an acetyl-CoA carboxylase and fatty acid synthetase (FAS). Although no direct enzymatic studies have been... 

Morse D. and Meighen E. A. (1987b) Pheromone biosynthesis enzymatic studies in lepidoptera. In Pheromone Biochemistry, eds G. D. Prestwich and G. J. Blomquist, pp. 121-158. Academic Press, Orlando, FL. 

The purity of the target protein can be expressed in terms of the percentage of total protein, although other types of contaminants can also be important. For enzymatic studies, 80-90% of purity is usually suitable, not taking into account the activity-interfering contaminants, which should be removed. For structural studies, the required purity should be equal to or higher than 95%. For therapeutic applications, any type of contaminants should be removed and purity higher than 99.9% is often required. 

Double reciprocal plots have been extensively used in enzymatic studies to quantify the Michaelis-Menten adjustable parameters Vmax and Km. Such plots, however, provide excellent correlations but poor accuracy in predicting Km and (Dowd and Riggs, 1965 Kinniburgh, 1986 Evangelou and Coale, 1987). Kinniburgh (1986) reported that the double reciprocal plot produces erroneous Michaelis-Menten adjust-... 

Another possibihty to gain the product encoded by a silent gene cluster can be achieved by a method called the in vitro reconstitution approach. A necessity is that the substrates of the pathway are predictable. The in vitro reconstitution of an entire biosynthetic pathway usually involves the separate overexpression of each gene, the purification of the resulting protein and the performance of enzymatic studies. Thus, the discovery of a fully elaborated metabolic product by this approach is likely to be very laborious. For example, after discovering a cryptic sesquiterpene synthase in the genome of S. coelicolor (64), the new epi-isozaene (8) could be produced successfully. This metabolite was shown to be an intermediate in the assembly line of the known Streptomyces sesquiterpene albaflavenone (9) (135). 

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