Abstract— The distribution of a series of enzymes in the post‐nuclear supernatant of rat brain homogenates was investigated following continuous density‐gradient centrifugation. The enzymes studied were acetyl coenzyme A synthetase, glutamic dehydrogenase, glutamine synthetase, glutaminase I, succinic dehydrogenase and monoamine oxidase. Each of these enzymes with the exception of glutamine synthetase appears predominantly in the mitochondrial region of the gradient. Although about 20 per cent of this enzyme is present in the crude mitochondrial pellet, on density gradient centrifugation no special association of glutamine synthetase with any of the mitochondrial fractions was observed. Each of the other enzymes studied was found to have a characteristic distribution in the gradient; this suggests that brain mitochondria may be heterogeneous both in buoyant density and in their enzyme content. Three principal fractions are described: (i) dense particles containing high concentrations of acetyl coenzyme A synthetase and glutamic dehydrogenase; (ii) a fraction comprising the bulk of the mitochondria with high levels of monoamine oxidase, succinic dehydrogenase and glutaminase I; and (iii) particles in the synaptic ending region of the gradient characterized by relatively high levels of monoamine oxidase and succinic dehydrogenase and containing only small amounts of the other enzymes studied. If the mitochondrial heterogeneity that is observed on centrifugation reflects the existence within brain cells of mitochondria with specialized function, a partial explanation may be available for multiple pools of tricarboxylic acid cycle intermediates which have been postulated from isotopie labelling experiments.

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Changes in protein components of purified myelin were measured following incubation in vitro with purified intra- and extracellular enzymes. Incubation with calf brain cathepsin D did not result in a significant relese of acid-soluble peptides as measured by ninhydrin analysis but was accompanied by a large loss of myelin proteins as determined on SDS-acrylamide gels. After 5 hr at 37°C there was a loss of about 25% for fast and slow basic proteins and the Agrawal proteolipid, but only a 5-10% loss for the Folch-Lees and Wolfgram components. Rat brain cathepsin D prepared by affinity chromatography gave a 30-60% breakdown of basic proteins and proteolipids. In general, breakdown using lyophilized myelin was increased over two-fold as compared to experiments with fresh myelin. Breakdown induced by cathepsin D was completely inhibited by the pentapeptide pepstatin. Incubation of myelin at physiological pH resulted in an endogenous breakdown of about 12% for basic proteins in freshly prepared, and 50% for lyophilized material. Addition of a soluble neutral proteinase that splits hemoglobin did not induce additional breakdown except for a small change in the Folch-Lees component. The extracellular enzymes pepsin and TPCK-treated trypsin resulted in a larger breakdown of all components as compared to brain enzymes. Present results demonstrate that all protein components of myelin are accessible to hydrolases and vulnerable to breakdown to varying extents by brain enzymes. These facts are consistent with the known rates for myelin protein turnover and may have a bearing on changes associated with demyelinating diseases.

The pentapeptide pepstatin obtained from culture filtrates of actinomycetes completely inhibited brain acid proteinase (cathepsin D) at exceedingly low concentrations. Among the brain enzymes tested, the effect is specific for acid proteinase because addition of 1000-fold higher concentrations was without effect on neutral proteinase, aminopeptidase, and arylamidases. Pepstatin also inhibits pepsin as tested with hemoglobin or with N-acetylphenylalanyl-L-diiodotyrosine as substrate. Pepstatin must be regarded as the most powerful agent yet described that inhibits intracellular acidic proteolytic enzyme in brain.

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