Characterization of the effects of Ca 2+ on the intramitochondrial Ca 2+-sensitive dehydrogenases within intact rat-kidney mitochondria

Abstract

The regulatory properties of the Ca 2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD +-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat kidney mitochondria were found to be essentially similar to those described previously for other mammalian tissues; in particular each enzyme could be activated severalfold by Ca 2+ with half-maximal effects ( K 0.5 values) of about 1 μM and effective ranges of approx. 0.1–10 μM Ca 2+. In intact mitochondria prepared from whole rat kidneys incubated in a KCl-based medium containing respiratory substrates, the amount of active, nonphosphorylated pyruvate dehydrogenase could be increased severalfold by increases in extramitochondrial [Ca 2+]; these effects could be blocked by ruthenium red. Similarly, Ca 2+-dependent activations of NAD +-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase could be demonstrated in intact, fully coupled, rat kidney mitochondria by either following O 2 uptake (in the presence of ADP) and NAD(P)H reduction (in the absence of ADP) on presentation of non-saturating concentrations of either threo-D s-isocitrate or 2-oxoglutarate, respectively, under appropriate conditions, or for the latter enzyme only, also by following 14CO 2 production from 2-oxo[1- 14C]glutarate (in the absence or presence of ADP). Effects of Na + (as a promoter of egress) and Mg 2+ (as an inhibitor of uptake) on Ca 2+-transport by rat kidney mitochondria could be readily demonstrated by assaying for the Ca 2+-sensitive properties of the intramitochondrial Ca 2+-sensitive dehydrogenases within intact rat kidney mitochondria. In the presence of physiological concentrations of Na + (10 mM) and Mg 2+ (2 mM), activation of the enzymes was achieved by increases in extramitochondrial [Ca 2+] within the expected physiological range (0.05–5 μM) and with apparent K 0.5 values in the approximate range of 300–500 nM. The implications of these results on the role of the Ca 2+-transport system of kidney mitochondria are discussed.