The activation of isocitrate dehydrogenase (NAD+) by Ca2+ within intact uncoupled rat brown adipose tissue mitochondria incubated in the presence and absence of albumin [proceedings].

There are three mammalian, exclusively intramitochondrial, enzymes that, in mitochondrial extracts or aRer purification, are activated by Ca2+ with a k,, (concentration required for half-maximal effect) for Ca'+ of about 1 p ~ . These are pyruvate dehydrogenase phosphate phosphatase (Denton el al., 1972), ismitrate dehydrogenase (NAD+) (Denton et al., 1978) and the oxoglutarate dehydrogenase complex (McCormack & Denton, 1979). The possibility is thus raised that Ca2+ may be an important regulator of oxidative metabolism in mammalian mitochondria, perhaps mediating hormonal effects (see Denton & Halestrap, 1979). However, it is clearly important to demonstrate that these effects of Ca2+ can occur within intact mitochondria. We have therefore compared the kinetics of oxidation of threo-D,-isocitrate by brown-fat mitochondria, in the presence and absence of albumin, to those of the extracted isocitrate dehydrogenase (NAD+). Freshly isolated brown-fat mitochondria are uncoupled (Cannon & Lindberg, 1979) and have little or no pH gradient (ApH) or membrane potential (Aty) across the inner membrane (Nicholls, 1979). Thus it is reasonable to expect that the concentration of Ca2+ in the matrix will be close to that outside the mitochondria. Albumin addition can create a small ApH and Ay without establishing respiratory control (Nicholls, 1979). Sources of all materials, and the preparation of threoD,-isocitrate, were as described by Denton el al. (1978). Mitochondria were prepared from cold-adapted rats (McCormack & Denton, 1979) by a modification of a method for white fat (Severson et al., 1976). Mitochondria were extracted as described by McCormack & Denton (1979) and isocitrate dehydrogenase (NAD+) was assayed (at 3OoC and pH 7.2) as described by Denton et al. (1978). Mitochondria (0:5mg of protein/ml) were incubated at 3OoC in 125 mu-KC1/2Omu-Tris (PH 7.2)/5 mM-KH,POJl mu-malate, saturated with air and with appropriate additions (of substrates, EGTA, Ca2+ and Sf'+). 0, uptake was monitored with a small Clark-type oxygen electrode. Concentrations of Ca2+ were maintained with 5 mM-calcium EGTA buffers; calculation of Ca2+ concentrations and kinetic parameters were as given previously (Denton et al., 1978). Ca*+ activates isocitrate dehydrogenase (NAD+) (in the presence of ADP) by decreasing its K , for threo-D,-isocitrate in extracts of rat heart mitochondria (Denton et al., 1978). The enzyme shows sigmoidal kinetics and has a k,, for Ca2+ of about 1 pu. Sr2+ mimics the effect of Ca2+ with a k,, about 10 times higher. Table 1, column 1, shows that the brown-fat enzyme has similar properties. A comparison of the kinetic parameters for the enzyme with those for the oxidation of threo-D,-isocitrate by intact brown-fat mitochondria (Table 1, column 2) reveals a strikingly close similarity in K,, sigmoidicity and the effects of CaZ+. The major end-product of threo-D,-isocitrate oxidation is probably succinate. This would explain why the maximum rate of oxidation is equivalent to about twice the Vmu. for the separated enzyme. The addition of albumin to the mitochondrial incubation results in a decrease in the K , values for threo-D,-isocitrate (both in the presence and absence of CaZ+) and the ko.5 values for Ca2+ and Sr2+ (Table 1, column 3). This is compatible with the establishment of a small ApH (leading to accumulation of substrate) and a small Aty (leading to some accumulation of CaZ+) across the inner membrane. Succinate oxidation by these mitochondria was employed as a control. No effects of Ca2+ or Sr2+ on succinate oxidation were noted. Albumin addition also caused a decrease in the K,,, for succinate oxidation. These studies demonstrate that isocitrate dehydrogenase "AD+) may be sensitive to CaZ+ activation within mitochondria. Parallel studies on oxoglutarate oxidation and pyruvate dehydrogenase activity indicate that both pyruvate dehydrogenase phosphate phosphatase and oxoglutarate dehydrogenase also exhibit similar sensitivity to Caz+ within intact mitochondria (Denton & McCormack, 1980). Indeed, these enzymes can be used in coupled mitochondria as a means of exploring the relationship between extraand intra-mitochondrial CaZ+ (Denton & McCormack, 1980).