Enzymes pulse-chase experiments

Direct observation of the E p/t dNTP complex was obtained using pulse-chase experiments. In such experiments, incorporation of labeled nucleotide to an E p/t complex is either quenched by the addition of HC1 or allowed to proceed after the addition of a large excess of cold unlabeled dNTP (the chase step) followed by acid quench. In the HC1 quench experiments, the acid quenches all the enzyme-bound species. On the other hand, when the reaction is chased with cold dNTP, each of the enzyme-bound species is allowed to partition both in the forward and reverse directions. The amount of partitioning in the forward direction is observed as an excess of labeled product, compared with the acid quench experiment, while the dNTP that partitions in the reverse direction is diluted and remains unobservable. As an excess was observed and because the binding of dNTP to the E p/t complex is rapid, the observed flux or excess is mainly due to the E p/t dNTP complex. 

As shown in Figure 15, two pathways are of course susceptible to lead to the ureido ring, the carhamoylation taking place at N-7 or N-8. After trapping experiments, which were not entirely conclusive, a NMR study established that carhamoylation takes place at N-7. When [7- N]DAPA and C02 were incubated with an excess of enzyme, only the N-7 carbamate was observed, and pulse-chase experiments showed that this carbamate was kinetically competent. ... 

Successful isolation of these specialized ER structures, now called the MAM, indicated that they are selectively enriched in a subset of lipid synthetic enzymes, especially PS synthase (J.E. Vance, 1990). In mammalian cells, pulse-chase experiments coupled with subcellular fractionation have now established that the PS destined for the mitochondria must transit through the MAM and some step of this process requires ATP (Y. Shiao, 1995). A MAM structure has also been identified and isolated from yeast cells (G. Daum, 1997). 

However, pulse-chase experiments in vitro, or the addition to extracts of inhibitors of proteolytic enzymes, have failed to provide any indication that cleavage or correct processing of large polypeptides occurred. However, one consistent finding is that uninfected cell extracts translate EMC virus ENA to produce a major polypeptide approximately 20,000 daltons larger than is produced by translation of the same ENA in extracts from infected cells (5, 12). The smaller product from infected cells is the same size as the... 

Pulse-chase/pulse-quench experiments with KF ° indicated accumulation of the nucleotide bound enzyme species, which would not be possible if the forward reaction was much faster than the rate of conformational closing. To explain this observation, the authors proposed the presence of a kinetic road block - a slow step after the phosphodiester bond formation. However, the results of the pulse-chase/ pulse-quench experiments can also be explained by designating chemistry as the slow step, meaning that the chemical step itself plays the role of the road block. The conclusion that chemistry is a fast step in the KF reaction pathway was made based on the observation of a small thio-efifect magnitude,which, as elaborated in the following section, should not be used as a solid evidence of the chemical step being nonrate limiting. 

Thiolactomycin (TLM) selectively Inhibits type 11 fatty acid synthases from E. coli and higher plants, but does not type 1 fatty acid synthases from Tungi and nKunmals. In this paper, the effect of TLM on fatty acid synthesis is confirmed in enzyme and cell levels. Pulse- and chase-experiments with plant tissues in the presence of TLM accounts for the controlled synthesis of lipids in higher plants. Finally the modulated lipid composition of plant tissues by continuous administration of TLM is examined in relation to the chloroplast structure and the photosynthetic activity of the tissues. 

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