NAD + kinase activity, calmodulin levels during the growth of isolated cells from Lycopersicon pimpinellifolium and kinetic constants of the calmodulin-dependent NAD + kinase

Abstract

NAD + kinase activity and levels of active calmodulin (CaM), i.e. CaM able to activate NAD + kinase, were determined during the culture growth of isolated cells from Lycopersicon pimpinellifolium. Two peaks of CaM-dependent NAD + kinase activity occurred at times when the cells were not actively dividing: the first one, a few hours after medium inoculation and the second one, at the end of the exponential growth phase. These alterations in CaM-dependent NAD + kinase activity were not related to changes in active CaM levels, which remained nearly constant throughout the culture and were found to be sufficient enough to fully activate NAD + kinase in vivo in the presence of Ca 2+. Thus, the increases in NAD + kinase activity observed in the cell extracts during the culture growth, may result either from a de novo synthesis of the enzyme or from the presence of an additive effector, other than the Ca 2+–CaM complex. The CaM-dependent NAD + kinase from tomato cells was partially purified (870-fold). This enzyme displays a sequential addition of the substrates and a K m of 0.20 mM for NAD + and 0.08 mM for MgATP 2− were determined in the presence of tomato CaM, also purified during the course of this study.