Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADHcytochrome b5 reductase and cytochrome bs.

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1.The rotenone-insensitive NADH-cytochrome c reductase system of rat liver mitochondria and isolated mitochondrial outer membrane preparations is inactivated by trypsin. The trypsin sensitivity of the system is considerably higher than that of liver microsomes when compared on equal protein basis.2. The trypsin sensitivity of the mitochondrial system decreases with increasing ionic strength of the incubating medium, whereas that of the microsomal system increases with increasing ionic strength, except a t low protein concentrations (< 0.1 mg protein/ml), in which case the effect of ionic strength is similar to that found with mitochondria.3. Experiments with 2,6-dichlorophenolindophenol and ferricyanide as electron acceptors .indicate that trypsin does not inactivate the flavoprotein component of the mitochondrial system. The inactivation of the NADH-cytochrome c reductase by trypsin is accompanied by a release of cytochrome b,, similar to that found with microsomes.4. Trypsin does not inactivate the adenylate kinase of intact mitochondria, but readily inactivates this enzyme after brief exposure of the mitochondria t o a hypotonic medium containing EDTA. Respiration, respiratory control, and various translocator functions of the mitochondria which are not affected by this treatment, remain insensitive to trypsin.5. The results are discussed with regard to their bearing on the distinction between the mitochondrial (rotenone-insensitive) and microsomal NADH-cytochrome c reductase systems, as well as on the location of the mitochondrial NADH-cytochrome c reductase system and adenylate kinase in relation to the outer mitochondrial membrane.

The systemic availability of clomethiazole was assessed by comparing blood levels after intravenous and oral administration. Clomethiazole was rapidly absorbed after oral administration to volunteers, particularly when administered as syrup. The fraction of the given dose that reached the systemic circulation after 1 capsule of clomethiazole (192 mg clomethiazole) was 0.25±0.18, after 2 capsules (384 mg clomethiazole) 0.38±0.18, and after 15 ml syrup (480 mg clomethiazole) 0.42±0.20. The time‐blood concentration profiles were consistent with a two‐compartment open model and the mean elimination half‐lives of 3.6‐5.0 hrs. were found for the different formulations and administration routes. Elimination half‐lives showed little variation and a mean systemic clearance of 49 ml/min./kg was found for clomethiazole after intravenous administration. Clomethiazole is bound to human plasma proteins (63.4±1.6%, 37°), a binding which is not affected by Vacutainer® sample tubes. The blood/plasma distribution of clomethiazole was 0.76±0.02 at 37°. A sensitive mass fragmentographic assay for the determination of clomethiazole in blood/plasma down to levels of 1 ng/ml (6.2 nmol/l) is described.

Abstract. The morphological and biochemical properties of isolated mitochondrial inner and outer membranes are summarized and discussed in relation to the functional organization of the intact mitochondrion. The enzymatic composition of the mitochondrial inner compartment is compared to over-all mitochondrial function. The role of the mitochondrial outer compartment is discussed with reference to both the inner membrane-matrix fraction and the intact mitochondrion.The outstanding feature of the mitochondrion is its indigenous ability to perform integrated enzymatic activities. The mitochondrion is a self-sufficient work unit possessing all the necessary enzymes, coenzymes, and other cofactors required by the individual enzyme complexes catalyzing respiration and phosphorylation; it is a highly organized system orientating and uniting the individual energy-yielding processes into an integrated function aimed specifically at energy conservation; and it is distinctly structural-the structure-function relationship dictating the efficiency of the over-all mitochondrial work performance.Morphologically the mitochondrion appears to contain two distinct compartments-the outer and inner compartments-so delineated by two separate membrane systems.1 The outer membrane forms the outer surface of the mitochondrion and is separated from the inner membrane by an intermembrane space. The outer membrane and the intermembrane space make up the mitochondrial outer compartment. The inner membrane, comprised of highly invaginated and structurally distinct cristae, encloses the matrix space of the mitochondrion. The inner membrane and the matrix space constitute the mitochondrial inner compartment.As revealed electron microscopically,2 the inner membrane is characterized by distinct projections (80-90 A spheres) extending from its inside surface into the matrix space. This morphological characteristic imparts to the inner membrane not only a topological but also a functional directionality or sidedness. It is well established that the respiratory chain constitutes an integral part of the inner membrane.36 The functional sidedness of the inner membrane is illustrated by the fact that succinate7'8 and NADH910 interact with the respiratory chain only from the inside (matrix) surface of the inner membrane, whereas cytochrome c appears to be accessible preferentially by way of the outside sur-125

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