Bovine milk-associated calcium as an uncoupling factor of mitochondrial respiration

Attempts to obtain coupled mitochondria from lactating bovine mammary tissue proved unsuccessful. To ascertain the nature of the uncoupling factor(s) responsible, we chose mitochondria of rat liver as a test system for their established isolation with retention of coupled respiration. Isolated, coupled rat liver mitochondria were uncoupled immediately by addition of microliter amounts of bovine milk and aqueous milk fractions. Dialysis of whey resulted in loss of the uncoupling factor, whereas boiling did not prevent the uncoupling effect. Inclusion in whey of the calcium chelator ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid protected rat liver mitochondria from the uncoupling effect. Mitochondrial swelling paralleled whey-induced respiratory uncoupling. When monitored by both oxygen consumption and swelling experiments, loss of respiratory control was correlated with the ionic calcium content of bovine milk. These findings suggest that high ionic calcium in bovine milk caused uncoupling. Extensive washings to remove or chelate calcium or inhibit mitochondrial calcium uptake were ineffective in preventing bovine mammary mitochondrial uncoupling. The implications of these findings suggest large amounts of compartmentalized intracellular calcium in bovine mammary tissue.