METHODSAdult and new-born Wistar rats were used throughout. New-born refers to animals 0-24 hr old and adult to animals at least 18 months old.Preparation of mitochondrial fraction. Brain suspensions in 0.32 M-SUCrOSe containing 1 m~-EDTA, were fractionated according to ADAMS, . After the density gradient separation, myelin, if present, was removed from the 0.32-0.8 M-SUCrOSe interface and discarded with some of the 0-8 M-sucrose. The tube was gently agitated and the loosely-packed portion of the pellet poured off. The remaining tightly-packed portion of the pellet constituted the mitochondrial fraction. This material was resuspended in 0-15 M-KCI to a known volume. Liver mitochondria were prepared by the same procedure, to allow for direct comparison. Enzyme assays. Succinafe dehydrogenase (succinate: tetrazolium oxidoreductase EC 1.3.99.1) was estimated colorimetrically using p-iodo-p-nitrophenyltetrazolium (INT) as the final acceptor (ADAMS et al., 1963). This method, assuming 100% trapping efficiency, gave rates of succinate oxidation of 0.171 and 0493 pmoles succinate/min/mg of protein in the neonatal and adult brain mitochondria respectively. ATPase (ATP phosphohydrolase, Mg stimulated EC 3.6.1.4) activity was estimated at 30" in tris buffer ( BRUNI and LUCIANI, 1962) in the absence and presence of 10 mM-dinitrophenol (DNP). Activity was measured on the day that the mitochondria were prepared.Cytochrome c oxidase (cytochrome c: oxygen oxidoreductase EC 1.9.3.1) was estimated colorimetrically (COOPERSTEIN and LAZAROW, 1951). The change in absorbancy at 550 mp was followed using the Perkin-Elmer (137 U.V.) spectrophotometer, with the output fed into a Kent 2 mv recorder, type 2 M. Maximum activity was obtained with Lubrol W., approximately 10 mg/100 mg of protein.Chemical estimations. Protein was estimated by an ultraviolet difference method (MURPHY and KIES, 1960), using Armour bovine serum albumin of known nitrogen content as the standard.Cytochromes Q, b and c + c1 were estimated from the reduced-oxidized difference spectrum obtained by the method of ROODYN, FREEMAN and TATA (1965). The spectrum was obtained with the Perkin-Elmer (137, U.V.) spectrophotometer using a Kent Mk 2 recorder as external amplifier and recorder. The cytochrome concentrations were estimated from the difference in extinction of the wavelength pairs, 605-625 m,u for cytochrome a, 562-575 mp for cytochrome 6, and 551-540 mp for cytochrome c + c l .Deoxyribonucleic acid was estimated in total, washed, trichloracetic acid-insoluble material from the homogenate. DNA was hydrolysed in 1 ~-perchloric acid at 90" for 20 min after hydrolysis of RNA by N-NaOH at 37" for 1 hr. The DNA in the hydrolysate was estimated from the extinction at 266 mp using EM(P) = 9930 (ALDRIDGE and JOHNSON, 1959).
