The isolation of coupled mitochondria from Physarum polycephalum and their response to Ca2+.

A method for the isolation of coupled mitochondria from the acellular slime mould Physarum polycephalum is described. The mitochondria oxidize respiratory substrates at rates comparable to those of mitochondria from other microorganisms and show similar responses to respiratory inhibitors. ADP/O values approach similar values to those obtained with mitochondria from higher organisms: 3 with NAD-linked substrates, 2 with succinate, and 1 with ascorbate-TMPD. Mitochondria actively take up low concentrations of Ca2+ with stimulation of their respiration. With succinate or pyruvate-malate as substrates respiratory responses are depressed by Ca2+ concentrations in excess of 200 micron in the presence or absence of phosphate. Exogenous NADH is unique in supporting the uptake of large amounts of Ca2+ in the presence of phosphate and in showing an unusual 'uncoupled' response in the absence of phosphate. A sigmoidal relationship occurs between initial velocity of Ca2+ uptake and Ca2+ concentration with a maximum velocity of approx. 15 nmol/s per mg protein and half maximum velocity occurring at approx. 50 micron Ca2+.