Cell with -leucine incubation

Fig. 12. The separation of globin chains prepared from cells incubated with leucine- H by chromatography on a column of CM-cellulose. Solid line represents the optical density at 280 nm, and the dotted line, the radioactivity. (A) A normal subject the P chain is eluted in tubes 20-30 and the a chain in tubes 42-52. The a p ratio of radioactivity is 1.0. (B) A patient with /3-thalassemia major the y chain is found in tubes 10-20, the p chain in tubes 22-32, and tiie a chain in tubes 38-50. The a. p ratio of radioactivity is greater than 60. (C) A patient with Hb-S-/3-thalassemia the normal p chain is found in tubes 25-33, the p chain in tubes 35-42, and the a chain in tubes 46-55. The a -P ratio of radioactivity is 2.9. From Marks and Bank (M7) with permission of the authors and publisher.

Besides the synthetic inhibitors, a variety of natural compounds is known to inhibit the CP. One of these natural inhibitors, lactacystin, was discovered by its ability to induce neurite outgrowth in a murine neuroblastoma cell line. Incubation of cells in the presence of radioactive lactacystin leads to the labelling of the yS5 subunit (Fenteany et al. 1995) and to irreversible inhibition of the CP. As shown by X-ray analysis, the inhibitor is covalently attached to subunit fS5 by an ester bond with the N-terminal ThrlO (Groll et al. 1997) (see Figure 10.7A). The subunit selectivity of lactacystin can be attributed to its dimethyl group, which mimics a valine or a leucine side chain and closely interacts with Met45 in the hydrophobic SI pocket of subunit j85. 

Overton and coworkers discovered a leucine 2,3-aminomutase in plant tissue cultures of Andrographis paniculata that converts (S)-leucine in (R)-f-leucine [43] (Scheme 1.6.10). The enzyme activity was investigated in cell free extracts by incubation with (S)-[U-14C]leucine and by measuring the radioactivity of the methyl ester camphanamide derivatives of the reaction mixtures by radio-GC. The stereochemistry of the /i-am i no acid was determined by radio-GC comparison of the enzyme reaction product as methyl ester camphanamide derivative with an authentic sample. The enzyme is not dependent on cobalamin, because addition of intrinsic factor does not induce its inhibition. 

After incubation, the cells are plated with 9 ml of soft top agar onto minimal selection plates (the agar and plates both containing 1 M sorbitol, 8% glucose, and amino acid supplement lacking leucine). 

Incubate the reticulocytes with [3H]leucine, which will be incorporated into proteins. Prepare electron microscope autoradiographs and count silver grains per cell and the number of polysomes. The latter appear as rosettes of five ribosomes in these cells. A statistical comparison between the number of polysomes and the amount of protein synthesized during the incubation time (proportional to the number of silver grains) indicates whether there are nonactive polysomes. In fact, many of the polysomes are inactive i.e., they are switched off (see Chap. 17 for the control of protein synthesis). 

Table II DNA-binding studies on furazolidone Pig hepatocytes were incubated with 50 nM C-furazolidone for 16 hr (starting 26 hr after isolation), or C-leucine for 40 hr (starting 4 hr after isolation). Cells were homogenized, the chromatin collected by centrifugation and DNA purified by phenolic extraction...

Fig. 5. Rate of degradation of poly(ADP-iibose) synthetase in HeLa cells. HeLa 3S cells (2.5 X 10 in leucine-deprived Dulbecco s modified Eagle s medium (DIFCO Laboratories) containing 5% dialyzed fetal bovine serum were metabolically labeled with 50 pCi pHJleucine (120 Q/mmol) in a 5% COj incubator for 1 hr at 37°C. After the addition of a 5,000 times excess amount of cold leucine (0.5 mM), cells were either harvested immediately or incubated further for chase. The radiolabeled cells were disrupted and immunoprecipitated using al20K (O) or a54K (9) as described in Fig. 3. Quantification of the radiolabeled poly(ADP-iibose) synthetase was carried out by densitometric trace of fluorogram using a chromatoscanner (Shimadzu CD-900).

Other experiments were based n the "Ca -pulse technique here the incubation of the culture with Ca proceeded for only 10 min and the concentration of amino acid could be substantially reduced. The amino acid mixture was replaced by a single amino acid (L-leucine), which did not affect either growth or the activity of the proteinase and which suppressed partially the enzyme synthesis. A decrease of leucine concentration in the medium and the rate of its incorporation into proteins were determined simultaneously. The results (Figure 3B) indicate that the saturation concentrations of leucine for both the repression of the proteinase and for the incorporation into proteins were approximately the same. It seems therefore that the rate of the entry of the amino acid into the cell and/or... 

Sevag and Ishii (1958) have set about to identify the fluorescent compounds accumulated in a sulfathiazole-inhibited PABA-less mutant of E. coli incubated with PABA. Of three fluorescent compounds isolated, one resembled OF, another was assumed to be a reduced pteroic acid derivative, and the third resembled pteroic acid. Cell extracts incubated with PABA formed a butanol-soluble yellow pigment. On hydrolysis, the pigment yielded PABA, glutamic acid, alanine, and leucine the pigment was inactive for S. faecalis and the E. coli mutant. A yellow pigment absorbing at... 

Fia. 7. Time curve of the incorporation of leucine-C into the RNA and protein of the ribonucleoprotein particles and the soluble fraction in intact ascites cells. The tumor cells (about 10 gm of packed cells) were incubated at 25° in 50 ml of their own ascitic fluid fortifled with glucose and Tris buffer, and contiuning 3 jimoles with 1.06 X 10 cpm of L-leucine. The NaCl-insoluble and -soluble fractions were prepared from the supernatant of a 16,000 g centrifugation. [Taken from Hoagland et al. (186).]... 

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