Gcg Ata Gct Gct Ggc Agt Gct Gca Aag Gca Ata Ttc Aag Cca Gct Ctc Ctc Tgc Cgt Cct Tgg Gag Gtt Ctg Gct Gcc Cat Gag Gcc Ccc Cga Agg

NAD+-isocitrate dehydrogenase (NAD+-ICDH) is one of three enzymes which catalyse the oxidation of threo-Ds-isocitrate to oxoglutarate and carbon dioxide in eukaryotic cells, the other two enzymes being the cytoplasmic and mitochondrial forms of NADP+-isocitrate dehydrogenase (NADP+-ICDH). NAD+ICDH is located within mitochondria and is a component of the citrate cycle. Although mitochondria from many sources contain NADP+-ICDH activities which can greatly exceed that ofNAD+ICDH, NAD+-ICDH appears to be the sole important source of reducing equivalents for the respiratory chain (Plaut, 1970; Colman, 1975; Haselbeck and McAlister-Henn, 1993). NAD+-ICDH has complex regulatory properties which are consistent with it contributing to the control of the citrate cycle. In particular, it exhibits sigmoidal kinetics with respect to isocitrate and is activated by increasing ADP/ATP ratios (which decrease the Km for isocitrate) (Plaut, 1970; Gabriel et al., 1985; Rutter and Denton, 1989a). In addition, the enzyme from vertebrates, but not from invertebrates, plants or yeast, is activated by Ca2+ (Denton et al., 1978; McCormack and Denton, 1981, Rutter and Denton, 1989a; B. J. Nichols and R. M. Denton, unpublished work). Pyruvate dehydrogenase phosphatase and oxoglutarate dehydrogenase from vertebrates are also activated by Ca2 . Parallel activation of the three dehydrogenases is an important means whereby ATP production is accelerated in cells stimulated by hormones or other extracellular agents which act through increases in cell Ca2+ (McCormack et al., 1990; Denton and McCormack, 1990). Previously, the only available cloned NAD+-ICDH subunits were from the yeast Saccharomyces cerevisiae (Cupp and McAlister-Henn, 1991, 1992). NAD+-ICDH from S. cerevisiae is