Non-synaptic Mitochondria Research Articles

Subcellular distribution of cytochrome P-450 in the brain

The subcellular distribution of the monoogynase complexes in the brain was studied by using subcellular fractionation and characterization of these fractions by marker enzymes. Cytochrome P-450 was found to be mainly localized in both synaptic and non-synaptic mitochondria; only a small quantity of enzyme was also found in the microsomal fraction. Peeling off the outer membrane of mitochondria showed that the protein was retained in the inner membrane fraction. A comparative study among some other species confirmed the mitochondrial prevalence of cerebral cytochrome P-450. A partial purification of the rat brain mitochondrial P-450 was obtained.

Carnitine acyltransferase activities in rat brain mitochondria. Bimodal distribution, kinetic constants, regulation by malonyl-CoA and developmental pattern.

Carnitine palmitoyltransferase and carnitine octanoyltransferase activities in brain mitochondrial fractions were approx. 3-4-fold lower than activities in liver. Estimated Km values of CPT1 and CPT2 (the overt and latent forms respectively of carnitine palmitoyltransferase) for L-carnitine were 80 microM and 326 microM, respectively, and K0.5 values for palmitoyl-CoA were 18.5 microM and 12 microM respectively. CPT1 activity was strongly inhibited by malonyl-CoA, with I50 values (concn. giving 50% of maximum inhibition) of approx. 1.5 microM. In the absence of other ligands, [2-14C]malonyl-CoA bound to intact brain mitochondria in a manner consistent with the presence of two independent classes of binding sites. Estimated values for KD(1), KD(2), N1 and N2 were 18 nM, 27 microM, 1.3 pmol/mg of protein and 168 pmol/mg of protein respectively. Neither CPT1 activity, nor its sensitivity towards malonyl-CoA, was affected by 72 h starvation. Rates of oxidation of palmitoyl-CoA (in the presence of L-carnitine) or of palmitoylcarnitine by non-synaptic mitochondria were extremely low, indicating that neither CPT1 nor CPT2 was likely to be rate-limiting for beta-oxidation in brain. CPT1 activity relative to mitochondrial protein increased slightly from birth to weaning (20 days) and thereafter decreased by approx. 50%.

Denervation-induced decrease in mitochondrial calcium transport in rat hippocampus

Calcium accumulation by mitochondria and the activity and in vitro phosphorylation of pyruvate dehydrogenase were measured in control and partially denervated hippocampus. Calcium uptake was measured with a calcium-sensitive electrode and 45Ca2+ accumulation; both methods indicated that lesions of the entorhinal cortex produced a sizable reduction of calcium transport when mitochondria were fueled with pyruvate while much smaller changes were observed using succinate or ATP as energy sources. The decrease in calcium transport was evident by 24 hr after the lesion and was still present 6 months later. Synaptic and nonsynaptic mitochondria were similarly affected by the lesions. The activity and in vitro phosphorylation of pyruvate dehydrogenase were also significantly reduced following lesions of the entorhinal cortex, suggesting that denervation altered the endogenous state of phosphorylation of the mitochondrial enzyme. Commissural lesions but not septal lesions also resulted in a decrease in mitochondrial calcium transport when mitochondria were fueled with pyruvate. These findings suggest that denervation disturbs mitochondrial regulation of free calcium via an action on enzymes which regulate pyruvate dehydrogenase phosphorylation and activity. The potential relationship of this effect to degenerative changes associated with deafferentation and certain disease states is discussed.

Rat cortex synaptic and nonsynaptic mitochondria: enzymatic characterization and pharmacological effects of naftidrofuryl.

In order to investigate the in vivo pharmacological effects of the drug naftidrofuryl, we prepared populations of synaptic and nonsynaptic mitochondria from rat brain cortex. In these different mitochondrial populations the activities of citrate synthase, malate dehydrogenase, total NADH cytochrome c reductase, cytochrome oxidase, and glutamate dehydrogenase were evaluated. Except for glutamate dehydrogenase, the specific activities of the enzymes evaluated in the "free" mitochondrial fraction were higher than those observed in the "synaptic" SM1 and SM2 mitochondrial fractions, the difference between SM1 and SM2 fractions being significant. The in vivo administration of naftidrofuryl induced few and different changes in the various mitochondrial populations.

Externally disposed polypeptides of chick brain mitochondria

Externally disposed polypeptides of chick brain synaptic and non-synaptic mitochondria were identified using lactoperoxidase-catalyzed iodination and galactose oxidase—sodium boro [ 3H]hydride labeling (with or without neurominidase pretreatment). After surface labeling, polypeptides were separated by electrophoresis on sodium dodecyl sulfate—polyacrylamide gels and incorporation was detected by liquid scintillation counting. When intact synaptic or non-synaptic mitochondria were iodinated they gave quite similar labeling patterns with 10 major radioactive peaks. Most of the surface-incorporated iodine was removable by trypsin. Iodination of microsomes or synaptosomes gave incorporation profiles different from that of mitochondria. Without neuraminidase pretreatment, galactose oxidase labeling of mitochondria gave only near background incorporation. After neuraminidase treatment, galactose oxidase and sodium borotritide labeling of both synaptic and non-synaptic mitochondria produced 12 major radioactive polypeptides. Four were not iodinatable by lactoperoxidase while two components iodinated by lactoperoxidase showed only minor radioactivity peaks after galactose oxidase-sodium borotritide labeling. These results suggest that (a) chick brain mitochondrial membranes have a complex mixture of glycoproteins which are highly sialiated, (b) at least 14 polypeptides are exposed on the exterior surface of brain mitochondria, (c) the functional differences between synaptic and non-synaptic mitochondria are not reflected by the similarities of their major externally disposed polypeptides. Thus, surface labeled components may represent structural polypeptides common to both mitochondrial types.

Glycerol oxidation in rat brain: subcellular localization and kinetic characteristics.

The oxidation of [1,3-14C] glycerol to 14CO was measured in slices, whole homogenates, and subcellular fractions of rat brain. In all of these tissue preparations, the Lineweaver-Burk plots of glycerol oxidation were biphasic, yielding two apparent Km and V values. Similar kinetic characteristics were obtained with brain homogenates from guinea pig, mouse, rabbit, monkey, and pig. In other tissues of the rat, including heart, kidney, liver, and skeletal muscle, the Lineweaver-Burk plots for glycerol oxidation were not biphasic but were linear. Heating the brain homogenates for five minutes at 5 degrees C caused a 50% decrease in the rate of oxidation of glycerol without a change in the biphasic double reciprocal plot. The addition of purified glycerol kinase (EC 2.7.1.30) to the homogenate caused an increase in the rate of oxidation and resulted in linear Lineweaver-Burke plot. Brain mitochondria were prepared by two different methods, both of which yielded an enrichment of glycerol oxidation. In contrast, the rate of glucose oxidation was higher in homogenates than in mitochondria, and glucose competed with glycerol as substrate only extramitochondrially. The effects of various metabolic inhibitors suggested the participation of intact, coupled mitochondria, of glycolytic enzymes, and of electron transport in the oxidation of glycerol. The data support the primary localization of glycerol oxidation in nonsynaptic mitochondria in brain and the presence in that organelle of enzymes of the Embden-Meyerhoff pathway or an as yet unidentified system for oxidizing this compound.

The synthesis of glutamate by rat brain mitochondria.

Abstract—The synthesis of glutamate from 2‐oxoglutarate generated by the citric acid cycle and ammonium acetate has been studied in brain mitochondria of synaptic or non synaptic origin.Non synaptic brain mitochondria synthesise glutamate at twice the rate (1.3 nmol. min−1. mg protein−1) of synaptic mitochondria (0.65 nmol. min−1. mg protein−1) when pyruvate is the precursor for 2‐oxoglutarate, but at a similar rate (0.9 and 0.7 nmol. min−1, mg protein−1) when 3 hydroxybutyrate is the precursor.Glutamate synthesis from ammonium acetate and extramitochondrially addcd 2‐oxoglutarate (5 mM) by both synaptic and nonsynaptic mitochondria was 5‐fold higher (5‐6nmol. min−1. mg protein−1) than glutamate synthesis from endogenously produced 2‐oxoglutarate. In the uncoupled state (or un‐coupler + oligomycin) the rate was reduced by half. (2.5‐3 nmol. min−1. mg protein−1) as compared to mitochondria synthesising glutamate in states 3 or 4 (± oligomycin).The changes in brain mitochondrial nicotinamide nucleotide redox state have been monitored by fluorimetric, spectrophotometric and enzymatic techniques during glutamate synthesis and compared with liver mitochondria under similar conditions. On the instigation of glutamate synthesis by NH+4 addition a significant NAD(P)H oxidation occurs with liver mitochondria but no detectable change occurs with brain mitochondria.Leucine (2 mM) causes a doubling of glutamate synthesis by both synaptic and non synaptic brain mitochondria with no detectable change in the NAD(P)H redox state.The results are discussed with respect to the control of glutamate synthesis by mitochondrial redox potential and the possible intramitochondrial compartmentation of this process.

Synaptic and non-synaptic mitochondria from rat brain: isolation and characterization.

Abstract— A method has been developed where by three distinct populations of metabolically active, well coupled and relatively pure mitochondria from rat brain may be prepared. Two mitochondrial populations are derived from synaptozomes and the third consists of ‘free’ (i.e. non‐synaptic) mitochondria. These mitochondrial populations have been characterized with respect to both enzyme content and ability to oxidize substrates. The results indicate that these mitochondrial populations are heterogeneous with respect to maximal activities of certain enzymes concerned with the citric acid cycle and glutamate and 4‐aminobutyrate metabolism as well as their ability to utilize various substrates. The data reported here also confirm that brain mitochondria are very heterogeneous and suggest that synaptic mitochondria may contain at least two sub‐populations. The relations between the heterogeneity of brain mitochondria and the metabolic compartmentation of the citric acid cycle and related metabolites such as glutamate, aspartate and 4‐aminobutyrate are briefly discussed in the light of two proposed models of metabolic compartmentation in the mammalian brain.

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.