Rapid purification of pig heart NAD+-isocitrate dehydrogenase. Studies on the regulation of activity by Ca2+, adenine nucleotides, Mg2+ and other metal ions

Abstract

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.

Identification and Functional Characterization of a Novel, Tissue-specific NAD+-dependent Isocitrate Dehydrogenase β Subunit Isoform

“…Furthermore, the catalytically active recombinant H-IDH␣␤ 1 ␥ protein (316 kDa) most likely exists as an octamer of 2(2␣,␤,␥) (301 kDa) (32,33), although we cannot rule out other possible octameric structures, such as 2(␣,2␤,␥) or 2(␣,␤,2␥). However, our data clearly dispute the possibility of a heteropentamer of 2␣,2␤,␥ (25) or the mixture of other oligomeric structures (34).…”

“…B, optimal pH profiles for the mitochondrial IDH activity in mouse tissues. IDH activity was measured at 25°C as NADH production at 340 nm in the reaction mixture containing 50 mM MOPS buffer containing 5 mM threo-D S -ISOCITRATE, 35.5 MM triethanolamine, 2 mM NAD ϩ , 1 mM ADP, 2 mM MgCl 2 and 1 g/ml rotenone, as described by Rutter and Denton (25) . FIG.…”

Modulation of Oxidative Phosphorylation by Mg2+ in Rat Heart Mitochondria

“…A synergistic effect by Ca 2ϩ and adenine nucleotides has been described (28). Mg 2ϩ also activates 2-OGDH (30) (this work), but in contrast to other enzyme effectors, the mechanism of action is by enhancing the catalytic enzyme capacity (k cat ), rather than by increasing substrate affinity.…”

The Trypanosoma cruzi Enzyme TcGPXI Is a Glycosomal Peroxidase and Can Be Linked to Trypanothione Reduction by Glutathione or Tryparedoxin

“…Isopycnic centrifugation was then carried out using a SW40 rotor in a Beckman L8 -80 ultracentrifuge at 200,000 ϫ g for 150 min at 4°C. 0.75-ml fractions were collected, and the activities of trypanothione reductase (25), NADP ϩ isocitrate dehydrogenase (27), and hexokinase (28) were determined. For glutathione-dependent peroxidase (12) activity, the fractions were first dialyzed extensively against 100 mM HEPES, pH 8.0, 1 mM EDTA prior to analysis.…”