Kinetic and electrophoretic characterization of NADP dependent dehydrogenases from root tissues of Norway spruce [Picea abies (L.) Karst.] employing a rapid one-step extraction procedure

Summary The NADPH generating enzymes glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), and isocitrate-dehydrogenase (NADP dependent; EC 1.1.1.42) have been characterized in spruce Picea abies (L.) Karst.] roots. Interference from inherent phenolic compounds was minimized by complexation with borate and insoluble polyvinylpyrrolidone in the presence of 2-mercaptoethanol and NADP. A further addition of protective substances had no (bovine serum albumin) or even an inhibitory effect (ascorbate) on the enzyme activities. The enzymes were shown to be strictly NADP specific. The optimal pH values and the apparent Michaelis constants from spruce roots are in good agreement with data from different photosynthetic organisms and gametophytic tissues of conifers. Native electrophoresis and subsequent activity staining showed the same banding patterns for enzymes both from root and needle tissues. In addition, the applicability of a highly sensitive dot-blot assay for the accurate quantification of the extracted protein is shown.