Both N-methyl-N-nitrosourea and y-radiation lower cellular NAD in mouse leukaemia cells (L1210) in a dose-dependent way. The minimum NAD level is reached 2 h after a brief exposure to N-methyl-N-nitrosourea, but within 15 min of y-irradiation. The cells remain metabolically active ; they are able to recover their control NAD levels and are impermeable to trypan blue.Several inhibitors of poly(ADP-ribose) polymerase inhibit the drop in cellular NAD caused by these two agents: 2 mM 5-methylnicotinamide, 1 mM theophylline or 1 mM theobromine inhibit the effect of N-methyl-N-nitrosourea on cellular NAD level; 200 pM thymidine, 500 pM 5-methylnicotinamide, 500 pM theophylline and 500 pM theobromine prevent the lowering of cellular NAD by y-irradiation. The extent to which the drop in cellular NAD is inhibited is dependent on both the concentration of cytotoxic agent and of polymerase inhibitor. Caffeine will inhibit the drop in NAD but only at 10 mM, while nicotonic acid is ineffective even at this dose.The activity of poly(ADP-ribose) polymerase in permeabilized cells immediately after y-radiation increases with dose up to 12 krad, giving a maximal 3.4-fold stimulation of the enzyme activity, whereas the degradation of NAD under conditions optimal for NAD glycohydrolase does not change. The activity of the polymerase shows a close temporal correlation with the NAD drop following both y-radiation and N-methyl-N-nitrosourea. The enzyme activity is maximal when the NAD content is decreasing at the highest rate and has returned to normal levels when it ceases falling.In permeabilized cells we can distinguish poly(ADP-ribose) polymerase and NAD glycohydrolase activity by their differential response to inhibitors. The polymerase is sensitive to 5-methylnicotinamide, theophylline, theobromine and thymidine ; the NAD glycohydrolase is sensitive to 5-methylnicotinamide and theophylline, but not to theobromine and thymidine.We propose that the decrease in cellular NAD level produced by y-radiation and by N-methyl-Nnitrosourea is caused by an increased flux through poly(ADP-ribose) mediated by an increased activity of poly(ADP-ribose) polymerase. This consequently lowers the cellular NAD level. This hypothesis implies an involvement of (ADP-ribose), in the cellular response to cytotoxic drugs.In 1956, Roitt was the first to show that cytotoxic agents inhibit glycolysis due to a lowering of the cellular NAD level [l]. He used the ethyleneimines, a group of alkylating agents, but other workers have observed a marked decrease in cellular NAD following ionizing radiation [2 -41.Abbreviations. ADP-ribose, adenosine(S')diphospho(S)-P-D-ribose; (ADP-ribose),, mixture of ADP-ribose, oligo(ADP-ribose) and poly(ADP-ribose); EGTA, ethylenebis(oxoethylenenitri1o)tetraacetic acid ; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid; Mes, 4-morpholineethanesulphonic acid ; S.D., standard deviation of the mean.Enzymes. NAD glycohydrolase (EC 3.2.2.6); poly(ADP-ribose) polymerase (EC 2.7.7.-).Streptozotocin, the methylnitrosourea deri...

Our study not only supports the involvement of some of the formerly reported proteins in SCCE but also introduces additional proteins found to be lost in SCCE, including TMbeta.

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A halophilic α-amylase produced by Nesterenkonia sp. strain F was purified to homogeneity by 80% ethanol precipitation, Q-Sepharose anion exchange, and Sephacryl S-200 gel filtration chromatography. The purified amylase exhibited specific activity of 357 unit/mg protein that corresponds to twofold purification. The molecular mass of the amylase was determined to be 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. The optimal pH and temperature for enzyme activity were 6.5 and 45°C, respectively. The amylase was active over a wide range of salt concentrations (0-4 M) with maximum activity at 0.75-1 M NaCl. The α-amylase activity was stimulated by Ca(2+) and inhibited by ethylenediamine tetraacetic acid (EDTA), suggesting that this enzyme is a metalloenzyme. The purified enzyme showed remarkable stability towards surfactants (Tween 20, Tween 80, and Triton X-100), and its activity was increased by β-mercaptoethanol. The halophilic α-amylase was stable in the presence of various organic solvents such as benzene, chloroform, toluene, and cyclohexane. These properties indicate wide potential applications of this α-amylase in starch-processing industries.