Brown-adipose-tissue mitochondria possess an energy-dissipating ion uniport which is inhibited by purine nucleotides. The regulatory nucleotides bind to a high-affinity site on the outer face of the inncr membrane which is independent of the adenine nucleotide translocator. A direct correlation between affinity for the regulatory site and ability to inhibit the ion uniport is demonstrated for a numbcr of nucleotidc analogues. 8-Azido-adenosine 5'-triphosphate, a photoaffinity label, also competes with GDP f o r thc binding site and induces respiratory control. 8-Azido-adenosine [~-"'P]tripliospl~ate was prepared and covalcntly bound to hamster brown-adipose-tissue niitochondria by neur-ultraviolet irradiation. Two major radioactive bands were identified of apparent molecular weight 30000 and 32000, representing 6'%, and 10~

The proton conductance of the inner membrane of hamster brown adipose tissue mitochondria can be regulated in vitro by exogenous purine nucleotides, which bind-ro-a-GOmponent on the outer face of the inner membrane.This unique mechanism has been proposed to represent the molecular site of nonshivering thermogenesis in this tissue.Using a photo-affinity analogue of ATP, we have identified the nucleotide binding component as a protein of 32,000 daltons.

1. A method is described for establishing steady-state conditions of calcium transport across the inner membrane of rat liver mitochondria and for determining the current of Ca2+ flowing across the membrane, together with the Ca2+ electrochemical gradient across the native Ca2+ carrier. These parameters were used to quantify the apparent Ca2+ conductance of the native carrier. 2. At 23 degrees C and pH7.0, the apparent Ca2+ conductance of the carrier is close to 1 nmol of Ca2+-min-1-mg of protein-1 mV-1. Proton extrusion by the respiratory chain, rather than the Ca2+ carrier itself, may often be rate-limiting in studies of initial rates of Ca2+ uptake. 3. Under parallel conditions, the endogenous H+ conductance of the membrane is 0.3 nmol of H+-min-1-mg of protein-1-mV-1. 4. Ruthenium Red and La3+ both strongly inhibit the Ca2+ conductance of the carrier, but are without effect on the H+ conductance of the membrane. 5. The apparent Ca2+ conductance of the carrier shows a sigmoidal dependence on the activity of Ca2+ in the medium. At 23 degrees C and pH7.2, half-maximum conductance is obtained at a Ca2+ activity of 4.7 muM. 6. The apparent Ca2+ conductance and the H+ conductance of the inner membrane increase fourfold from 23 degrees to 38 degrees C. The apparent Arrhenius activation energy for Ca2+ transport is 69kJ/mol. The H+ electrochemical gradient maintained in the absence of Ca2+ transport does not vary significantly with temperature. 7. The apparent Ca2+ conductance increases fivefold on increasing the pH of the medium from 6.8 to 8.0. The H+ conductance of the membrane does not vary significantly with pH over this range. 8. Mg2+ has no effect on the apparent Ca2+ conductance when added at concentration up to 1 mM. 9. Results are compared with classical methods of studying Ca2+ transport across the mitochondrial inner membrane.

No abstract