The Intracellular Distribution of Glycolytic and Tricarboxylic Acid Cycle Enzymes in Rat Brain Mitochondrial Preparations

Abstract

IT WAS recently reported that the non-ionic detergent, Triton X-100, will solubilize over half of the proteins of the mitochondrial fraction obtained from rat whole brain honiogenates (BRUNNGRABER and AGUILAR, 1962). This extract contained several glycolytic as well as tricarboxylic acid cycle enzymes. It is well established that the usual preparation of rat brain mitochondria is a heterogeneous mixture of cell particulates ( HEBB and WHITTAKBK, 1958; WHITTAKER, 1959; DE ROBERTIS et nl., 1962) and that this preparation is capable of glycolysis (GALLAGHER et d., 1956; BKUNNGRAREK and ABOOD, 1960). In view of the effort in this laboratory directed toward fractionation of "insoluble" proteins derived from the particulate mitochondrial fraction, it was considered desirable to ascertain the origin of enzymes solubilized by Triton X-100. By using two methods of subfractionation, it was found that isocitrate dehydrogenase, fumarate hydratase, malate dehydrogenase, aspartate and alanine transaminases, and glutamate dehydrogenase in the Triton extract of the whole mitochondrial fraction originate from the "tan" particles (mitochondria), whereas the glycolytic enzymes originate mainly from the lightcr "white" particles (myelin fragments, nerve endings, synaptic vesicles and synaptic membranes). M E T H O D SSiih/iactionation of the mitochondriaI ,fraction: The mitochondrial fraction was prepared as previously described by BRUNNGRABER and AGUILAR (1962). Subfractionation was accomplished by two methods. Method 1 utilized the density gradient ultracentrifugation procedure of WmTrAKER (1959) with the No. 40 head of the Spinco Model L ultracentrifuge. Whittaker's fractions A, B, and C were obtained.An alternative procedure was also used (Method 2). The mitochondrial fraction was rcsuspended in 0.25 M-sucrose containing 5 x M-ethylenediamine-tetraacetic acid, pH 7.0, and centrifuged at 100,OOO g for 20 rnin. The presence of a white upper layer and a more densely packed tan lower layer was apparent. The upper white material (subfraction A) was teased off with a spatula carefully avoiding disturbance of the tan material. A subfraction B was obtained by removing next all of the remaining white, including tan, so that only pure tan (subfraction C) remained at the bottom of the centrifuge tube. Fractions obtained either by Method 1 or 2 were taken up in 0.25 Msucrose and recentrifuged at 100,000 g for 20 rnin. The appearance of the pellet gave some indication of the effectiveness of the separation in Method 2. A similar method based on separation of tan from white material was reported by DAHL et al. (1960). The subfractions A, B, and C obtained by Methods 1 and 2 and a portion of the original whole mitochondria1 fraction were homogenized by hand in 10 ml (final volume) of 0.5% Triton X-100 in 0,005 M-phosphate buffer, pH 7.6 and centrifuged at 100,000 g for 30 min. The clear supernatant was used for the enzyme assays. Enzyme assays: The following enzymes were assayed: Isocitrate dehydrogenase (Ociroa, 1955); fumarate hydratase...

Subcellular Distribution of Pyruvate Kinase (Ec 2.7.1.40) in Cerebral Cortex

“…Although pyruvate kinase is reported to be a cytoplasmic component in brain as well as in other tissues (JOHNSON, 1960;BRUNNGRABER, AGUILAR and OCCOMY, 1963), we have observed activity in a number of membrane fractions as well. To determine whether this enzyme was indeed associated with membranes, we thought it desirable to study its quantitative distribution in different subcellular fractions.…”

“…Since pyruvate kinase is mentioned in the literature as being present solely in the cytoplasm of nerve tissue (JOHNSON, 1960;BRUNNGRABER et al, 1963), we thought it advisable to secure further evidence that this enzyme is associated with neural membranes. The enzyme does not appear to be specific for the membranes in any particular subcellular fraction, since all membrane particles tested (microsomes, synaptosomes, mitochondria and synaptic vesicles) exhibited appreciable activity.…”

Glutamate and aspartate transport in rat brain mitochondria

“…The mitochondria exhibited rapid swelling in 250mosM solutions of ammonium phosphate or ammonium acetate, and in potassium phosphate in the presence of valinomycin and uncoupler, showing that they were capable of normal swelling. Glutamate is, however, metabolized in the mitochondrial matrix, since glutamate is readily oxidized (Brunngraber et al, 1963;Clark & Nicklas, 1970), and we have observed that glutamate is able to stimulate oxidation of malate, presumably by transamination with oxaloacetate in the mitochondrial matrix. Fig.…”

The quantitative histochemistry and cytochemistry of lactic dehydrogenase and NADH2‐tetrazolium reductase in human aortic wall

“…The histochemical reaction with all three enzyme systems (NADH2-TR, LDH+PMS and LDH-PMS) in unextracted fresh cryostat sections appears partly in the form of small smooth round granules, which could be interpreted as mitochondria. However, LDH is predominantly a cytoplasmic enzyme (see Brunngraber, Aguilar and Occomy, 1963;Johnson and Whittaker, 1963), so the LDH reaction product should not be located in mitochondria-particularly in the presence of the intermediate electron acceptor PMS that circumvents the need for mitochondria1 (or microsomal) " diaphorase ". Moreover, these smooth round granules are not seen in sections extracted with acetone before incubation and it may therefore be inferred that they are lipid droplets.…”