Assaying poly(ADP-ribose) polymerase activity in plants by polarographic method

Abstract

A new method has been developed to assay poly(ADP-ribose) polymerase (PARP) activity in plant tissues through determining the content of nicotinamide (NIC) produced by enzymatic reaction by linear sweeping polarographic method. The detection limit of NIC was 0.03 μmol/L, the calibration graph was linear up to 5 μmol/L (r = 0.999). The recoveries were approximately in the range of 92% to 98% and the relative standard deviations were less than 6.6%. Moreover, NAD⁺ and other interference existing in the mixture after enzymatic reaction had been removed by simple pretreatment, thus PARP assays were not interfered. A rapid, simple, sensitive and reliable nonisotopic method is reported to assay PARP activity in plant tissues. The results show that the Km^NAD value of PARP in maize (Zea mays L.) seedlings is 59 and the optimum pH for PARP activity is 8.5. Moreover, physiological conditions affect PARP activity in plant tissues. which has not been reported previously. When tobacco (Nicotiana tobacum) suspension cells were stressed by NaCl at low concentrations (100, 200 mmol/ L), the PARP activity increased significantly; when the cells were stressed at high concentrations (400, 1 000 mmol/L), it decreased to or even below the control level. PARP activity in etiolated maize seedlings was higher than that in light-grown seedlings.