Hydrogen cyanamide on triphenyltetrazolium chloride reduction, sulfhydryl group binding, and catalase activity in bromegrass (Bromus inermis Leyss) cells Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/w37638854

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  • Hydrogen cyanamide (CY) has been used to break rest in temperate crops, but levels used to overcome rest are sometimes toxic to plants. The rest breaking effect of CY is thought to involve peroxide metabolism. Inhibition of catalase by CY has been proposed as a mechanism for overcoming dormancy in plants. The objectives of this study were to determine effects of CY on: (1) the viability of bromegrass cells; (2) the reduction of SH groups from SH-containing compounds, and the concentration of SH groups in bromegrass cells; and (3) the relationship between TTC reduction and the amount of SH groups; and (4) the mode of inhibition of cyanamide on both bovine liver catalase from Sigma and on catalase extract from bromegrass cells. The 2,3,5-triphenyltetrazolium chloride (TTC) method was used to determine cell viability. The TTC test indicates that the effect of cyanamide on cell viability is both concentration and time dependent. Relatively low concentrations of CY (1 and 5 mM) and short exposure times increased the reduction of TTC above the level of the controls, but reduction decreased with increasing exposure or higher concentrations. Sulfhydryl (SH) groups are known to function as an antioxidant in cells and, therefore, have been proposed to protect cells against physical and chemical stresses. Hydrogen cyanamide reduced the titratable SH groups of SH-containing compounds in in vitro assays and the effect on SH groups depended on the ratio of CY concentration and the concentration of SH-containing compounds: the rate of reduction of titratable SH groups was fastest in dithiothreitol (DTT) followed by glutathione (GSH) and lastly by cysteine. Hydrogen cyanamide also stimulates the production of nonprotein SH compounds in bromegrass cells in vivo. The effect of CY on levels of SH compounds is concentration and time dependent. The concentration of SH compounds increased following relatively short exposure time (4 hours) to low concentrations of CY and then decreased with additional time. The level of TTC reduction was positively correlated with the concentration of SH groups in bromegrass cells. Hydrogen cyanamide inhibited catalase activity and catalase activity was restored to near the level of the untreated control after the removal of CY by dialysis suggesting that hydrogen cyanamide is a reversible inhibitor of catalase. Enzyme kinetic studies indicate that the inhibition of catalase by cyanamide is of the mixed-type inhibition.
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