Preparation and properties of mitochondria derived from synaptosomes

A method has been developed whereby a fraction of rat brain mitochondria (synaptic mitochondria) was isolated from synaptosomes. This brain mitochondrial fraction was compared with the fraction of “free” brain mitochondria (non-synaptic) isolated by the method of Clark & Nicklas (1970). (J. Biol. Chem. 245, 4724-4731). Both mitochondrial fractions are shown to be relatively pure, metabolically active and well coupled. 2. The oxidation of a number of substrates by synaptic and non-synaptic mitochondria was studied and compared. Of the substrates studied, pyruvate plus malate was oxidized most rapidly by both mitochondrial populations. However, the non-synaptic mitochondria oxidized glutamate plus malate almost twice as rapidly as the synaptic mitochondria. 3. The activities of certain tricarboxylic acid-cycle and related enzymes in synaptic and non-synaptic mitochondria were determined. Citrate synthase (EC 4.1.3.7), isocitrate dehydrogenase (EC 1.1.1.41) and malate dehydrogenase (EC 1.1.1.37) activities were similar in both fractions, but pyruvate dehydrogenase (EC 1.2.4.1) activity in non-synaptic mitochondria was higher than in synaptic mitochondria and glutamate dehydrogenase (EC 1.4.1.3) activity in non-synaptic mitochondria was lower than that in synaptic mitochondria. 4. Comparison of synaptic and non-synaptic mitochondria by rate-zonal separation confirmed the distinct identity of the two mitochondrial populations. The non-synaptic mitochondria had higher buoyant density and evidence was obtained to suggest that the synaptic mitochondria might be heterogeneous. 5. The results are also discussed in the light of the suggested connection between the heterogeneity of brain mitochondria and metabolic compartmentation.

A ZONAL ROTOR METHOD FOR THE PREPARATION OF MICROPEROXISOMES FROM EPITHELIAL CELLS OF GUINEA PIG SMALL INTESTINE

A method is described for the preparation of catalase particles from homogenates made from suspensions of epithelial cells of the small intestine of the guinea pig. Electron microscope examination of the preparations revealed the presence of small diaminobenzidine-positive particles measuring 0.1–0.3 nm in diameter and resembling the microperoxisomes observed by Novikoff and Novikoff (1972. J. Cell Biol. 53:532.). Analytical data upon which the method is based are presented. The technique consisted of a rate zonal separation of microperoxisomes from large particles followed by an isopycnic separation from less dense organelles. Application of the method yielded microperoxisomes purified between 20- and 30-fold.

Dissociation Reassociation and Phenylalanine Incorporation by Chloroplast and Cytoplasmic Wheat-Leaf Ribosomes

Ribosome preparations from wheat seedlings were separated by zonal centrifugation to yield monomers of 80 S (cytoplasmic) and 70 S (chloroplast). When centrifuged through gradients containing high KCl-to-MgCl2 buffer, the 80 S ribosomes dissociated into subunits of 61 S, 49 S, and 42 S, while the 70 S monomers gave subunits of 50 S and 30 S. After zonal separation of the subunits, they could be reassociated into parent ribosomes in the presence of MgCl2. Both 80 S and 70 S ribosomes actively synthesized polyphenylalanine in the presence of poly U. The rate of synthesis was similar for both species. Incorporation of phenylalanine by 80 S and 70 S ribosomes was inhibited by puromycin, was not inhibited by cycloheximide, and only the 70 S species was affected by chloramphenicol, indicating that the latter was of chloroplast origin.