PEP-Carboxylase Activities and the Regulation of CAM: Effects of Extraction Procedures and Leaf Age

Abstract

The amplitude of the diurnal oscillation of malic acid accumulation in leaves of Kalanchoe daigremontiana and Kalanchoe blossfeldiana varies considerably with leaf position on the shoot (leaf age) and growth conditions. The total activity of phosphoenolpyruvate carboxylase (PEP-c capacity) measured in desalted crude extracts shows a different dependence on the leaf position and in the majority of experiments no significant diurnal change. In contrast PEP-c capacity shows a clear diurnal rhythm in extracts prepared from freezedried (lyophylised) fully expanded leaves of K. daigremontiana or phyllodia of K. tubiflora, the enzyme being 10 times more active at the end of the light period than at the end of darkness. This diurnal rhythm in PEP-c capacity is negatively linearly correlated to the malic acid content of the leaves during lyophylisation. When acidification is prevented by applying C02-free air during darkness, lyophylisation does not cause loss in PEP-c capacity overnight. In extracts prepared from lyophylised material having high malic acid and therefore low enzyme capacity, the enzyme activity can not be restored by desalting of the extracts or increasing the amount of polyethylenglycol in the extraction buffer. The extractability of protein is constant and not effected by lyophylisation. We conclude that during the freeze-drying procedure the PEP-c undergoes an irreversible inactivation, the degree of which is dependent on the malic acid content of the leaves.