DPN-linked isocitrate dehydrogenase from fly thoracic muscle mitochondria

Abstract

Both TPN- and DPN-linked isocitrate dehydrogenases have been identified in the thoracic muscle mitochondria from the face fly and the American cockroach. The DPN-linked enzyme from these two insects is closely related on the basis of chromatographic pattern.

DPN-linked IDH has been isolated from face fly thoracic muscle mitochondria and purified 50-fold from the initial mitochondria acetone powder extract in good yield (22%). The enzyme may be stored without loss of activity as a suspension in 0.3 sat. (NH₄)₂SO₄ containing 20 mM potassium phosphate, pH 7.0, for three weeks at room temperature. It was found that the enzyme requires mg⁺⁺or Mn⁺⁺ for activity, but the optimal metal ion concentration depends upon the Mg⁺⁺ or Mn⁺⁺ to substrate ratio. The optimal stimulation effect of ADP on the enzyme activity was found to be 2 mM in correlation with the concentration of DPN⁺ (4mM), isocitrate (6mM), and Mg⁺⁺(6mM) .

The pH optimum varied depending on the concentrations of the reaction cofactors involved. As a result, the activity of DPN-linked IDH is pH dependent. Kinetic studies on the forward reaction revealed several interesting properties of the DPN-linked IDH. At lower pH values, the stimulatory effect of ADP on the enzyme was observed to be less than at higher pH. It has been clarified in the text that in the presence of ADP, the maximal pH shifts slightly higher". The results indicate that the stimulation provided by ADP is not solely due to the amount of ADP which participates in the reaction but is rather controlled by H+ concentration in a specific fashion. The effect of pH on the activity of DPN-linked IDH is dependent on the concentrations of isocitrate and DPN+. The enzyme is inhibited by low concentration (0.16 mM) of p-chloromercuribenzoate.

Comparative kinetic studies at pH 7.0 and 6.0 reveal that the enzyme is inhibited by the reaction products. With DPNH as the variable substrate, Lineweaver-Burk plots reveal a pattern of competitive inhibition for isocitrate. Furthermore, with a-ketoglutarate as the variable substrate, Lineweaver-Burk plots reveal a pattern of noncompetitive inhibition for both DPN and isocitrate. The reaction catalyzed by purified enzymes is inhibited by ATP and EDTA. DPN-linked IDH was activated by citrate.