Protein synthesis pulse labeling

Fig. 3. Protein synthesis in a maize primary root during ( ) one hr pulse labelling with [ HJleucine under aerobic conditions (b)-(e) pulse labelling with [ HJIeucine during the specified times under anaerobic conditions. The arrow labelled TPs indicates the position of the transition polypeptides. The unlabelled arrow indicates the position of alcohol dehydrogenase 1 (ADHl). From Sachs et al. (1980).

N]-, [ C]-, pHjleucine or p Sjmethionine in the case of proteins) for various periods, after which the cells are lysed and the protein of interest is purified (often by immunoprecipitation with specific antibodies). The time course of isotope incorporation gives information about the rate (slope of curve) and extent (amplitude of curve) of the proteins synthesis. To measure degradation, cells are first pulse-labeled (i.e., exposed to radiolabeled precursor for a fixed period, after which sufficient nonla-beled precursor is added to reduce the radiospecific activity of the precursor). Then, the cells are further incubated, and the radiospecific activity of a particular protein of interest is determined (again usually after immunoprecipitation or some other means for achieving its isolation from other cellular proteins). The key point is that the chase allows one to stop radiolabel uptake almost instantaneously, thereby permitting the kinetic... 

Alice et al studied the turnover kinetics of Listeria OTonocytogenex-secreted p60 protein (a murein hydrolase) by host cell cytosolic proteasomes. J774 cells, seeded in flasks and incubated overnight in culture medium, were infected with log-phase cultures of E. monocytogenes for 30 min, washed, and incubated in culture medium for 3 h, with gentamicin (50 tg/ml) added after the first 30 min to inhibit extracellular bacterial growth. Cells then were washed and placed in methionine-free medium with spectinomycin, gentamicin, the eukaryotic protein synthesis inhibitors [cycloheximide (50 tg/mL) and anisomycin (30 tg/ml),] and 25 dVI calpain inhibitor I. After 30 min, [ S]methionine was added, and the cells were pulse-labeled for periods of 20 to 60 min. Cells... 

Our kinetic studies of mutant PrPs synthesized in CHO cells suggest that individual steps in formation of PrP may take place in at least two different cellular locations (Fig. 5). Because mutant PrPs become PIPLC-resistant within minutes of synthesis in pulse-labeling experiments, this early step must take place in the ER. Consistent with this conclusion, acquisition of PIPLC resistance is not affected by treatment of cells with brefeldin A or by incubation at 18°C, manipulations that block exit of proteins beyond the Golgi (Daude et al, 1997). In contrast, detergent insolubility and protease resistance, which do not develop until later times of chase, and are reduced by brefeldin A and 18°C incubation, are likely to be acquired after arrival of the protein at the cell surface, either on the plasma membrane itself or in endocytic compartments. Raft domains may be involved in these changes (unpublished data). 

Fig. 2. Proteomic signatures of different stress/starvation conditions in Staphylococcus aureus. Comparison of the protein synthesis profile of exponentially growing cells (green) with that of stressed S. aureus cells (red) reveals changes in protein synthesis that are particular for the certain stress stimuli. Cells were cultivated in synthetic medium and exposed to the respective stimulus at an optical density at 500 nm (ODjqo) of 0.5. Protein synthesis was analyzed by [ S]-L-methionine labeling (5 min pulse) under control conditions and 10 min after imposition to stress. All proteins induced by one stimulus belong to a stimulon. (See Color Plate 1, following p. 46.)...

If protein synthesis is inhibited by CH considerable amounts of L-Phe from intracellular sources transit to the expandable pool. This leads to secondary labelling of alkaloids during pulse and labelling of proteins during chase even at lov/ concentrations of exogenous L-Phe ( <1 yug/ml). 

FIGURE 2. Fluorographs showing thylakoid protein synthesis durtrjg a 3h pulse labelling with methionine in 25 C-grown leaves at 25 C (track 1) and on transfer to 14 C for 3h (track 2), 3 days (track 3) and 6 days (track 4). Molecular mass markers in kDa are indicated. 

Figure 6. Effect of pactamycin on the distribution of radioactivity incorporated into the primary products of EMC-virus directed protein synthesis. At 5 iu 49 niin post infection, an infected cell suspension was exposed to pactamycin. Six min thereafter, samples were removed, pulse labeled with a (h)-amino acid mixture and electrophoresed on SDS-polyaciylamide gels (see ref. 57) Control, 6 min pulse (solid line) pactamycin, 6 min delay, 6 min pulse (dotted line). From Butterworth and Rueckert (57) ...

Till now we have considered the behaviour of thiols that are essentially foreign to the metabolism of the animal. However, perhaps the most sophisticated tracer techniques yet applied to the study of labelled thiols have been developed in the course of investigations of the utilization of a pulse-labelled cysteine in the on-going process of the synthesis of body proteins. After administration, S-cysteine quickly enters the various amino acid pools of the body and is incorporated along with naturally occurring cysteine into the polypeptides synthesized in various tissues. 

The first measurements of protein turnover in a human subject were performed in 1949. Since that time several models have been proposed to give true estimates of protein turnover, or at least permit intra-subject comparison of synthesis and catabolism. The great majority of estimates of protein turnover in the human subject have involved the use of [ N]glycine administered either as a pulse label or by constant infusion, and measurement of the resultant N label in the urine ( N total, [ N]urea or [ N]ammonia). Detailed treatment of the underlying theory of these models has been presented in 2 excellent reviews [399,400]. Initial studies used [ N]glycine, administered as a single dose (pulse labelling) and the rate of protein turnover was calculated from the curve of N excretion over 48—60h [401]. The excretion curve obtained was fitted to the equation ... 

Finally, both constant infusion and pulse label techniques using isotopic labels to estimate whole body protein turnover share the common premise that there is a homogeneous metabolic nitrogen pool of which the plasma constitutes an integral part. That this is in fact an oversimplification, has been shown from animal studies [438]. Despite these, and other objections, work will continue in the search for a reliable method for the estimation of protein synthesis, catabolism and turnover in man. To the clinician such a method would provide information about nitrogen loss from the body resulting from malnutrition, postoperative trauma, burns or severe infection and perhaps more importantly an indication of the success or otherwise of the specific therapeutic regime implemented. 

We next determined if this increase was due to a change in the rate of synthesis or degradation of the ADA protein. Parental and dCF cells were pulse-labeled with 3H-leucine and l C-leucine, respectively. Labeled cell extracts were mixed and the ratio of 3h/ C present in total soluble protein determined. ADA protein was immunoprecipitated from mixed cell extracts with purified IgG. The immunoprecipitates were run on SDS polyacrylamide gels, the gels sliced, and slices containing ADA assayed for radioactivity. Ratios of 3h/14c for total protein synthesis and for ADA protein were compared. Table 3 shows that the relative rate of ADA synthesis clearly paralleled the relative enzyme levels in 3 resistant cell lines with different levels of ADA activity. Degradation rates of ADA, on the other hand, did not differ significantly in the sensitive or resistant cells. 

In an experiment designed to follow the synthesis and secretion of zymogen, a radioactive amino acid was Injected into the pancreas of a guinea pig to label proteins undergoing synthesis (pulse). Since most proteins in pancreas are synthesized as zymogens, the radioactive amino acid would be incorporated primarily in gmiogens. Three minutes after the pulse, all incorporated radioactivity was found to be present in the rough endoplasmic reticulum (ER). 

Our studies of non-canonical amino acids were motivated initially by an interest in making proteins with novel properties. Our interest broadened when our colleague Daniela Dieterich suggested that pulsed metabolic labeling of cellular proteins with non-canonical amino acids might provide a method for time-resolved analysis of protein synthesis in neurons. With Daniela and Erin Schuman, we developed this idea into the BONCAT (bio-orthogonal non-canonical amino acid tagging) method shown in Scheme 2 [33]. 

The phenomenon of protein synthesis inhibition in VSV-infected cells has been well described for various other virus-host systems. The characteristics of inhibition appear to depend in part on the host cell studied and on the multiplicity of infection. From pulse-labeling experiments, Mudd and Summera (1970) reported a 90% inhibition of total protein synthesis at 4 hr postinfection in HeLa cells by the Indiana serotype of VSV. These studies were done on HeLa cells grown in suspension infected at a multiplicity of infection of 85. Only a 40%... 

Pulse-labeling of cells followed by measurement of total isotope incorporation at various time postinfection cannot differentiate between an effect on cell-specific vs. viral-specific protein synthesis. In an effort to determine to what extent cell-specific protein synthesis was being affected, McAllister and Wagner (1976) compared, by SDS-polyacrylamide gel electrophoresis, the synthesis of specific cellular proteins from uninfected and infected L cells and determined that VSV inhibited the synthesis of cellular proteins by about 80% at an MOI of 10 by 5 hr postinfection. In a similar manner, Lodish and Porter (1980) reported a 35% inhibition of BHK cell total protein synthesis by 4 hr postinfection at an MOI of 10, but noted that cell-specific proteins were being synthesized at a rate that was only 25% of that in uninfected cells. 

Fig. 3. Effect of reovirus types 1, 2, and 3 on L cell protein synthesis. Subconfluent monolayers of mouse L cells were infected with 80 PFU/cell of reovirus type 1 ( ), type 2 (A), or type 3 ( ). At various times postinfection, the cells were pulse-labeled with [ SJmethionine. Mock-infected L cells (O) were included as controls. The capacity of reovirus to inhibit host cell protein synthesis maps to the S4 dsRNA segment. From Sharpe and Fields (1982), by permission of Virology.

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