Poly polymerase activity assays

It is clear from these graphs that the modified polynucleotide, MPC, significantly inhibits the DNA polymerases present in both viral extracts furthermore, the inhibitory activity of MPC is very nearly the same in the two systems when poly (dA-dT) is used as the template (50% inhibition at 18 jxg/r.mix.), but in the presence of poly rA (dT)j4 as the template, MPC acts as a much more potent inhibitor of 3H-TMP incorporation in the MSV-M assay system (50% inhibition at 4 /rg/r.mix.) than in the FLV system (50% inhibition at 35 fig/r. mix.). In contrast, the unmodified polynucleotide, PC, stimulates DNA polymerase activity in both viral systems with poly (dA-dT) as the template, and it shows slight inhibitory activity (only 25 % inhibition, at 20-40 /ug/r.mix.) in the presence of the poly rA (dT)14 template. 

Caspase activation assays and other molecular read-outs for apoptosis Commercially available caspase antibodies, as well as caspase activity assays, allow the rapid measurement of caspase activity in cells. Caspase activity correlates well with the apoptotic program. Examples of other molecular markers used for the indication of apoptosis are Bax, Bcl-2, BCL-XL and PARP (poly (ADP-ribose) polymerase). 

The main steps of the activity gel assay are outlined in Table 1. The in situ detection of poly(ADP-ribose) polymerase activities includes gel electrophoresis in SDS, renat-uration of proteins with appropriate buffers, incubation of the intact gel with [ P]-NAD, removal of nonincorporated precursor by TCA, washing, and autoradiography. The method was developed by using extracts and partially purified fractions of the poly(ADP-ribose) polymerase. The catalytic peptides of the enzyme are identified as activity bands and their Mj. determined by referring to protein markers [1]. 

Fig. 1. Effect of RNase A on free mRNP poly(ADPR) polymerase activity. The enzymatic assay was performed as described in [4] with the addition of variable amounts of RNase A...

Poly(ADP-ribose) polymerase activity was assayed according to Berger al. (10) in peimeabilized cells and Chemey etal. (11) cellular homogenates. Total (alkali-labile) protein-bound mono(ADP-ribose) levels were determined as described in this report while sample preparation for RIA was as reported by Bredehorst et al. (9). NAD assay was done by the cycling assay of Bemofsky et al. (12). 

Chicken testis cells at different stages of spermatogenesis, separated by centrifugal elutriation as described in (7), and spermatozoa obtained from the vas deferens, were treated with bleomycin for 1 hr and the poly(ADP-ribose) polymerase activity was assayed in nuclei isolated from the different cell t)rpes. Incubation with bleomycin resulted in stimulation of the enzymatic activity in premeiotic and meiotic cells, round spermatids and in elongated spermatids and was ineffective in spermatozoa (Fig. 1). 

Fig. 1. Transfection of COS cells with pcD-12 produces enhanced expression of polymerase activity and immunoprecipitable polymerase protein. 1 x 10 COS cells in duplicate flasks were treated in the presence or absence of plasmid DNA. A. Sonicated samples were assayed for activity for 10 sec with 2.8 pCi P pjnAD by electrophoresis on 7.5% NaDodS04/polyacrylamide gels. B. The sonicated samples were separated by electrophoresis in 7.5% NaDodS04/polyacrylamide (containing 100 pg/ml sonicated salmon sperm DNA). The gels were subsequently renatured and the gel assayed directly with for in situ poly(ADP-ribose) polymerase activity. The washed gels were...

Figure 2 Stability of /3-poly(L-malate) measured by its activity to inhibit purified DNA polymerase a of P. polyceph-alum. The relative degree of inhibition is shown (100 rel. units refer to complete inhibition). The DNA polymerase assay was carried out in the presence of 5 /tg/ml /S-poly(L-malate) as described [4]. The polymer was preincubated for 7 days at 4°C in the following buffer solutions (50 mM) KCl/HCl (—A—). Citrate (—V—). 2-(A/-Morpholino)-ethanesulfonic acid, sodium salt (—O—). Sodium phosphate (— —). N-(2-Hydroxyethyl)piperazine-N -(2-ethanesul-fonic acid), sodium salt (— — ). N,N-b s (2-Hydroxyethyl)-glycine, sodium salt (—T—). Tris/HCl (— —). 3-(Cyclo-hexylamino)-l-propanesulfonic acid, sodium salt (— —).

DNA pol a with primase was assayed in a reaction mixture (0.2 ml) containing 50 mM Tris-HQ buffer, pH 7.4, 10 mM MgCU, 1 mM ATP, 2.5 Mg of poly(dT), 20mM[ H]-dATP (35 cpm pmoF ) and an appropriate amount of the enzyme. The reaction was carried out at 37°C for 1 h. DNA polymerase a and i3 activity was assayed with activated DNA as template-primer. Details of the assay will be described elsewhere [30]. The assay of DNA ligase [12,13], Ca ", Mg -dependent endonuclease [14], and terminal deoxynucleotidyl transferase [15] was carried out according to the respective reported method. 

Fig. 3A-C. Inhibition of DNA ligase II, DNA polymerase jS, and terminal deoxynucleotidyl transferase by poly(ADP-ribos)ylation. Highly purified DNA ligase II (5 Mg, A), DNA polymerase ]3 (5 Mg, B), and terminal deoxynucleotidyl transferase (0.65 Mg, Q from bovine thymus were incubated in a poly(ADP-ribos)ylating reaction mixture as described in Sect. 2 except that the concentration of NAD was changed as indicated. After incubation, respective enzyme activity was assayed. The activity of a control sample incubated without NAD in each experiment was set at 100%...

Fig. 2. (right) Effect of poly(ADP-ribosyl)ation on replicase-stimulating activity of PSF and replicase activity of DNA polymerase a-primase. Phnified PSF and DNA polymwase a-primase were separately incubated in poly(ADP-ribosyl)ating enzyme system (1) in the presence of the indicated concentration of NAD+. Replicase assay was carried out principally as described by Yagura et al. (8) in the presence of 10 ng of purified PSF. 

Fig. 4. (right) Inhibition of DNA polymerase a and primase activities by poly(ADP-ribosyl)ation reaction. DNA polymerase a-primase was incubated in poly(ADP-ribosyl)ating enzyme system in the presence of the indicated concentration of NAD+ and the DNA pol5nnerase a ( , A) and primase (O, A) activities were assayed as described in the legends of Fig. 3 and Fig. 1, respectively. A, A the sample was incubated in the presence of 20 mM nicotinamide and 1 mM NAD+ in poly(ADP-ribosyl)ating reaction mixture. 

Inhibition of primase and DNA polymerase a activities. Purified DNA polymerase a-primase complex was incubated in a reconstituted poly(ADP-ribosyl)ating enzyme system and, after the incubation, the DNA polymerizing activity with activated DNA as template-primer and the primer synthesizing activity with poly (dT) as template were separately assayed. Both activities decreased almost in a parallel manner with increasing concentration of NAD+ and reached to approximately 20% of control at 1 mM NAD+ (Fig. 4). An inhibition of poly(ADP-ribose) polymerase (20 mM nicotinamide) blocked the suppression of both activities. 

Fig. 2. Inhibition of NAD depletion in Ca + depleted medium by 3-aminobenzamide. CF-3 cells were placed in Ca + depleted medium containing the indicated concentrations of 3-aminobenzamide and 3 hr later were extracted and assayed for NAD (16). Control cultures without 3-aminobenzamide had a 40% reduction in NAD content. Values shown (O) are the percentage of NAD depleted in the absence of 3-aminobenzamide and represent the mean of duplicate determinations. The relative activities of poly(ADP-ribose) polymerase (A) and mono(ADP-ribosyl) transferase ( ) are also shown as a function of 3-aminobenzamide concentration. Partially purified poly(ADP-ribose) polymerase was obtained from beef thymus (12) and purified mono(ADP-ribosyl)transferase was obtained from turkey erythrocytes (13) and was provided by Dr. Joel Moss (NIH). Reprinted with permission from ref. 14.

The results of the biosynthesis experiments suggest that NAD depletion is due to an increased rate of NAD consuming reactions. NAD can be consumed by ADP-ribosyl transfer reactions which can be inhibited by 3-aminobenzamide (10). When 5 mM 3-aminobenzamide was added to the culture medium, total inhibition of NAD depletion was observed (data not shown). Since ADP-ribosyl transfer reactions can be catalyzed by nuclear poly(ADP-ribose) polymerase and by cytoplasmic mono(ADP-ribosyl) transferases (11), the inhibition of these enzymes by 3-aminobenzamide was quantitatively compared. Fig. 2 shows dose response curves for the inhibition of purified poly(ADP-ribose) polymerase (12) and mono(ADP-ribosyl) transferase (13) by this compound. The activity of poly(ADP-ribose) polymerase was much more sensitive to inhibition with a 50% inhibitory concentration (IC50) of approximately 5.5 xM under the assay conditions used compared to an IC50 of 2000 iM for the mono(ADP-ribosyl) transferase. The effect of different concentrations of 3-aminobenzamide on NAD depletion in CF-3 cells incubated with Ca + depleted medium was also determined (Fig. 2). These cell experiments indicated an IC50 similar to that of mono(ADP-ribosyl) transferases and argue that NAD depletion in CF-3 cells was due to a stimulation of cellular mono(ADP-ribosyl)ation. 

Escherichia coli RNA-polymerase hole- and core enzymes were 95% pure by sodium dodecyl sulfate (SDS) gel electrophoresis. Enzyme activity was measured as the amount of [ C]AMP or [ C]UMP incorporated into acid-insoluble material after a 10-min incubation at 37°. The assay mixture contained in 0.1 ml 40 mM Tris-chloride at pH 8.0, 8 mM MgCl2, 5 mAf dithioerythritol, 0.2 Azoo unit of poly[d(AT)], 0.05 M KCl, 1 mAf ATP, and 1 mAf [ C]UTP. For kinetic studies, a fixed concentration of ATP (0.4 mAf) was used and the concentration of [ C]UTP was varied. Enzyme activity was measured as the amount of [ C]UTP incorporated into acid-insoluble material after 5 min. 

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