2 NAD(-ISOCITRATE DEHYDROGENASE FORMS IN PHYCOMYCES-BLAKESLEEANUS - INDUCTION IN RESPONSE TO ACETATE GROWTH AND CHARACTERIZATION, KINETICS,AND REGULATION OF BOTH ENZYME FORMS())

TWO forms of NAD(+)-isocitrate dehydrogenase, named ICDH-1 and ICDH-2, have been identified and purified in Phycomyces blakesleeanus NRRL-15 55(-). These enzyme forms may be separated by chromatography on DEAE-S ephacel. ICDH-2 induction was a response to the adaptation of Phycomyc es growth on acetate as the carbon source. Both enzyme forms were octa mers of 338 +/- 30 kDa with apparently identical subunits of 40.5 +/- 5 kDa, but they were distinguishable by their electrophoretic mobiliti es on polyacrylamide gel electrophoresis. Isoelectric pH values were 5 .28 and 4.96 for ICDH-1 and ICDH-2, respectively. ICDH-2 was more stab le to urea denaturation than ICDH-1. At pH 7.6, ICDH-1 showed a marked ly sigmoidal kinetic behavior with respect to isocitrate, However, ICD H-1 and ICDH-2 showed hyperbolic kinetics with respect to NAD(+). The tribasic form of isocitrate (I3-) and its magnesium complex (MI(-)) ar e the true substrates for both enzyme forms. Kinetic data obtained wit h Mg2+ as a divalent cation for both enzyme forms are compatible with the kinetic mechanism proposed by Cohen and Colman (1974) [Eur. J, Bio chem. 47, 35-45] but assuming some degree of interaction between bindi ng sites for the active form of isocitrate. This report describes for the first time the existence of two forms of NAD(+)-isocitrate dehydro genase in filamentous fungi. From the changes in activity levels for e ach form, during the adaptation of Phycomyces to growth on acetate and taking into account the kinetic and regulatory properties of both enz yme forms, we discuss the role of ICDH-1 and ICDH-2 in the control of isocitrate flux in Phycomyces.