Pulse-chase experiments with

Fig. 3,—Pulse-chase Experiment with Acetobacter xylinum.10 Incorporation of D-[14C]glucose (3,300 c.p.m. per nmol) into the water- and alkali-soluble fractions, and its subsequent transfer from these fractions into cellulose. In the pulse, cells were incubated in 3 mM D-[14C]glucose at 0° in buffer at pH 6.0 at the time of the chase, cells were diluted in cold buffer, centrifuged, and re-incubated at 30° in buffer either containing 40 mM unlabeled D-glucose (-) or lacking D-glucose (—). ...

In experiments performed with B. megaterium, Rothman (1977) used chemical modification with TNBS, under conditions where the probe did not enter the cell, to distinguish between PE molecules located on the outer and inner sides of the cell membrane. This technique was coupled with pulse-chase experiments with [ P]inorganic phosphate and [ HJglycerol and demonstrated that newly synthesized PE is initially found on the cytoplasmic surface of the cell membrane and is rapidly translocated to the outer leaflet of the membrane with a tyj of 3 min at 37°C. Although the translocation is rapid, it does not occur coincident with synthesis, but rather, with a significant delay after the molecule is synthesized. In addition, the translocation can continue in the absence of PE synthesis. These findings indicate that lipid synthesis and translocation are two distinct events. 

Transport of newly synthesized PC from the ER to the mitochondria. Using conventional subcellular fractionation techniques, the transport of nascent PC to the mitochondria of baby hamster kidney cells was examined by pulse-chase experiments with a [ H]choline precursor (M.P. Yaffe, 1983). These experiments show that the newly made PC pool equilibrates between the outer mitochondrial membrane and the ER in approximately 5 min (Fig. 8). Similar studies performed in yeast (G. Daum, 1986) also revealed that the PC pool rapidly equilibrates between the ER and the mitochondria. Addition of metabolic poisons did not eliminate the PC radioequilibration in yeast. Studies with isolated mitochondria demonstrate that PC loaded into the outer mitochondrial membrane can be transported to the inner membrane in an energy-independent manner (M. Lampl, 1994). Consistent with this finding is the observation that PC rapidly moves across the membrane of vesicles derived from mitochondrial outer membranes prepared ifom either mammalian cells or yeast (D. Dolis, 1996 ... 

Transport of cholesterol to and from the plasma membrane. Following its synthesis at the ER, cholesterol is transported throughout the cell and becomes enriched in the plasma membrane [7]. The transport of newly synthesized cholesterol to the plasma membrane has been examined in tissue culture cells using pulse-chase experiments with either the rapid plasma membrane isolation procedure (M. Kaplan, 1985), caveolae isolation (A. Uittenbogaard,... 

As far as the cytokinin donor function is concerned there are indications that tRNA cytokinins may contribute to the pool of free cytokinins. Based on pulse-chase experiments with labelled cytokinin precursors it was estimated that 40-50% of the free cytokinins in plant cells may be of tRNA origin [66,67]. However, there are serious limitations to tRNA as a possible source of free cytokinins ... 

Maturation of ConA precursor. Pulse-chase experiments with radioactive amino acids strongly support a mechanism of transpeptidation, rather than proteolytic cleavage followed by ligation. The only other known precedent for such a mechanism is the last step of peptidoglycan synthesis in bacteria (see Murein). Inspection of possible three-dimensional structures of Con A and its precursor show that maturation by transpeptidation is possible without unfolding of the polypeptide chain. 

NAD turnover studies. Pulse-chase experiments with [i C]-nicotinamide show that half of the labelled NAD in mature lymphocytes disappears during 8-12 hr of in vitro culture (Fig. 1). If 3-aminobenzamide is added, more than 50% of the radioactivity persists beyond 24 hr. These results indicate that most of die NAD turnover in non-dividing human lymphocytes is due to ADP-ribosylation reactions. Exposure of the cells to 10 xM dAdo causes a marked increase in the turnover of NAD (Fig. 1). The enhanced NAD consumption is associated with a 50% fall in cellular NAD pools after 24 hr (10). Thus, an increase in NAD utilization by lymphocytes for ADP-ribosylation, in response to accumulating DNA strand breaks, caimot be matched by a compensatory increase in NAD synthesis, and the NAD pool becomes depleted. 

Pulse-chase experiments with living cells show that this vesicle-mediated transport is quite rapid. The average time of displacement of cell wall matrix polysaccharides from dictyosomes and Golgi vesicles in plant cells is about 3 min. 

Bocher,M., Kluge, M. The C4-pathway of C-fixation in Spinacea oleracea. II. Pulse chase experiments with suspended leaf slices. Z. Pflanzenphysiol. 86,405-421 (1978)... 

Metabolic control analysis (MCA) assigns a flux control coefficient (FCC) to each step in the pathway and considers the sum of the coefficients. Competing pathway components may have negative FCCs. To measure FCCs, a variety of experimental techniques including radio isotopomers and pulse chase experiments are necessary in a tissue culture system. Perturbation of the system, for example, with over-expression of various genes can be applied iteratively to understand and optimize product accumulation. 

The biosynthesis of myeloperoxidase has been characterised by pulse-chase experiments. This is achieved by incubating cells with a radioactive... 

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. 

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