A method was developed to monitor continuously the matrix free Ca2+ concentration ([Ca2+]m) of heart mitochondria by use of the fluorescent Ca2+ indicators, fura-2 and quin2. The acetoxymethyl esters of fura-2 and quin2 were accumulated in and hydrolysed by isolated mitochondria. An increase of the mitochondrial Ca content from 0.3 nmol/mg of protein to 6 nmol/mg corresponded to a rise of [Ca2+]m from 30 to 1000 nM. The results indicate that physiological fluctuations of the mitochondrial Ca content elicit changes of [Ca2+]m in that range which regulates the matrix dehydrogenases.
Bovine anterior-pituitary microsomal fractions exhibit high-affinity, saturable and reversible binding of inositol 1,4,5-[32P]trisphosphate; 50%O of the labelled ligand is displaced by 3.5 nM-inositol 1,4,5-trisphosphate, 0.5/tM-inositol 1,4-bisphosphate and 10 gM-ATP. Inositol 1,4,5-trisphosphate induces the release of Ca2+ from the microsomal vesicles (half-maximal effect at 290 nM), and its action is potentiated by inositol tetrakisphosphate (half-maximal effect at 4,UM).
The entrapment of the Ca 2+-sensitive fluorescence indicators fura-2 or quin2 in the matrix space of isolated heart mitochondria renders possible the direct monitoring of the matrix free Ca z+ ([CaZ+]m) [(1987) Biochem. J. 248, 6094i13]. In this paper the correlation between the [Ca2+]m and the in situ activity of oxoglutarate dehydrogenase (OGDH) in fura-2-loaded mitochondria is shown. At the initial value of [Ca2+]m, 64 nM, which corresponded to 0.36 nmol/mg mitochondrial Ca content, the OGDH activity was 12% of the maximal. Half-maximal and maximal activation were attained at 0.8 and 1.6/zM [Ca2+]m, respectively. The results indicate that an increase of the mitochondrial Ca content in the physiological range enhances the OGDH activity by means of elevation of [Ca 2 ÷]m.
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