Mitochondria from the flight muscle of the blowfly Sarcophaga bullata undergo a dramatic decline in their capacity to rapidly take up calcium in a respiration linked manner as the muscle tissue undergoes a transition from development to maturity and senescence. The primary objective of this research proposal is to gain an understanding for the molecular basis of this decline. Calcium uptake (initial rate) studies (stopped flow apparatus) will ascertain the K0.5 and Vmax of the transport. Steady state calcium transport kinetics will be used to estimate the calcium exodus from the matrix of young and old mitochondrial membrane. Ruthenium red, a noncompetitive inhibitor of calcium transport, will be synthesized from 106RuCl3 and will be used to identify a change in the number of binding sites. By correlating the inhibition titer (106Ruthenium Red) with its binding to a (glyco) (lipo) (protein) of the solubilized mitochondria, a complex will be identified that is responsible for mitochondrial calcium transport. A change in its concentration per cytochrome aa3 is expected to account for the decline in calcium uptake rates with age. Since the ruthenium red binding (glyco) (lipo) (protein) may only be part of the calcium carrier, the decline in transport activity will also be sought in changes of the amount of water (in)soluble glycoprotein(s) and cardiolipin.