Conditions in vitro which affect respiratory control and capacity for respiration-linked phosphorylation in brown adipose tissue mitochondria.

Oxidative phosphorylation in brown adipose tissue mitochondria is greatly influenced by variations in the in vitro environment. Factors found to be of particular importance were the concentrations of serum albumin, of magnesium and EDTA, and the osmolality and the pH of the medium. The maximal P/O ratios obtained in an isoosmolar medium at pH 6.3, containing 2% serum albumin, 7.5 mM MgCl2, and 7.5 mM EDTA with pyruvate-malate as substrate, and using the glucose-hexokinase trapping system, were 2.8 for newborn guinea-pigs and 2.6 for unweaned rats. Under otherwise equal conditions the P/O ratios obtained with 4-week-old guinea-pigs were higher than those with the newborn. EDTA was a requirement for demonstration of respiratory control in brown adipose tissue mitochondria. Respiration with pyruvate-malate or succinate (+ rotenone), and stimulated by inorganic phosphate and ADP, was found to be sensitive to oligomycin and to carbonyl cyanide phenylhydrazone. However, the small extent of stimulation by the phosphate acceptor system, the incomplete inhibition by oligomycin, and the fact that the respiratory rate after exhaustion of added ADP was higher than that prior to the addition of ADP, suggest that brown adipose tissue mitochondria are loosely coupled. The measured respiratory control ratios all increased from the newborn to the 3-week-old guinea-pigs. Mitochondria from newborn and 6-day-old guinea-pigs exhibited the phenomenon of “reversed acceptor control”. Thus, in the absence of added inorganic phosphate, ADP inhibited the respiration with pyruvate-malate or with succinate. The reversed ADP control was abolished by carbonyl cyanide phenylhydrazone, but was not suppressed by treatment of the mitochondria with oligomycin. Inorganic phosphate affects respiration in a composite manner. With all of the preparations tested, inorganic phosphate stimulated respiration whether exogenous ADP was present or not. In the absence of added ADP, the respiration initially stimulated by inorganic phosphate entered a subsequent inhibitory phase. The implications of these findings to the control of brown adipose tissue thermogenesis are discussed.