1. A new procedure for purifying pig heart NAD+-isocitrate dehydrogenase from mitochondrial extracts has been developed. This relies on the use of f.p.l.c. techniques and exploits the hydrophobic properties of the gel-filtration medium Superose 6 at high ionic strength. A 300-fold purification to apparent homogeneity is achieved within 5 h and with a yield of greater than 20%. 2. The enzyme had an apparent native molecular mass on gel filtration of 320 kDa. In agreement with previous studies [Ramachandran & Colman (1980) J. Biol. Chem. 255, 8859-8864], three subunits (all close to 38 kDa) were separable by isoelectric focusing 3. This preparation was used to investigate the effects of adenine nucleotides, KCl and the required bivalent metal ions, Mg2+ and Mn2+, on the regulation of the enzyme by Ca2+. 4. In the presence of 1.5 mM-ADP, increasing the concentration of Mg2+ from 20 microM to 6.0 mM raised the concentration of Ca2+ required for half-maximal effect (K0.5 value) from 1.2 microM to 232 microM. Similarly, in the presence of 2.5 microM-Mn2+, a K0.5 value for Ca2+ of 3.3 microM was obtained, and this value was increased to 8.9 microM in the presence of 100 microM-Mn2+. In the presence of 1 mM-Mg2+ and 1.5 mM-ADP, the K0.5 value for Ca2+ was raised from 4.7 microM to 10 microM by 75 mM-KCl.

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