Outer mitochondrial membrane preparation

Transport of newly synthesized PC from the ER to the mitochondria. Using conventional subcellular fractionation techniques, the transport of nascent PC to the mitochondria of baby hamster kidney cells was examined by pulse-chase experiments with a [ H]choline precursor (M.P. Yaffe, 1983). These experiments show that the newly made PC pool equilibrates between the outer mitochondrial membrane and the ER in approximately 5 min (Fig. 8). Similar studies performed in yeast (G. Daum, 1986) also revealed that the PC pool rapidly equilibrates between the ER and the mitochondria. Addition of metabolic poisons did not eliminate the PC radioequilibration in yeast. Studies with isolated mitochondria demonstrate that PC loaded into the outer mitochondrial membrane can be transported to the inner membrane in an energy-independent manner (M. Lampl, 1994). Consistent with this finding is the observation that PC rapidly moves across the membrane of vesicles derived from mitochondrial outer membranes prepared ifom either mammalian cells or yeast (D. Dolis, 1996 ... 

Monoamine oxidases are integral outer mitochondrial membrane proteins that catalyze the oxidative deamination of primary and secondary amines as well as some tertiary amines. MAO occurs as two enzymes, MAO-A and MAO-B, which differ in substrate selectivity and inhibitor sensitivity (Abell and Kwan, 2001 Edmondson et al., 2004 Shih et al., 1999). A number of MAO inhibitors have been developed for clinical use as antidepressants and as neuroprotective drugs. Clinically used drug substances include, among others, moclobemide, a relatively selective reversible MAO-A inhibitor, and L-deprenyl, an irreversible selective inhibitor of MAO-B. In vitro, clorgyline and L-deprenyl are used as selective irreversible inhibitors of MAO-A and B, respectively. (Note For in vitro studies using irreversible inhibitors, preincubation of the irreversible inhibitor with the enzyme prior to initiation of the substrate reaction is required for optimal inhibition.) Expressed MAO-A and MAO-B are not readily available via commercial resources however, MAO-A and MAO-B have been evaluated and are active in subcellular fractions. While monoamine oxidases are located in the mitochondria, many microsomal preparations are contaminated with monoamine oxidases during the preparation of the microsomal subcellular fraction and thus microsomes are sometimes used to evaluate monoamine oxidase activity in combination with selective inhibitors. 

Freshly prepared mitochondria contain ascorbate, as do mitoplasts, that lack the outer mitochondrial membrane (Li etal. 2001). Both mitochondria and mitoplasts rapidly take up oxidised ascorbate as dehydroascorbic acid and reduce it to ascorbate. Ascorbate concentrations in mitochondria and mitoplasts rise into the low micromolar range during dehydroascorbic acid uptake, although uptake and reduction are opposed by ascorbate efflux. Mitochondrial dehydroascorbic acid reduction depends mainly on GSH, but mitochondrial thioredoxin reductase may also contribute. Reactive oxygen species generated within mitochondria oxidise ascorbate more readily than they do GSH and a-tocopherol. 

If nuclear-encoded mitochondrial proteins are vectorially released into mitochondria from cytoplasmic ribosomes attached to the outer mitochondrial membrane in a manner analogous to the rough endoplasmic reticulum of secretory cells, then it should be possible to visualize (by electron microscopy) the attachment of cytoplasmic ribosomes to the outer mitochondrial membrane in situ, as well as in isolated mitochondrial preparations. 

While it is reasonable to assume that a significant portion of the cytoplasmic ribosomes which remain attached to purified mitochondria are those visualized by electron microscopy as attached to the outer mitochondrial membrane in situ, the extent to which adventitious association of cytoplasmic polysomes with the outer mitochondrial membrane occurs during cell fractionation is difficult to evaluate quantitatively. As an approach to this problem we determined conditions which would completely eliminate polysome binding to mitochondria in vitro, and then determined what effect these conditions had on the recovery of cytoplasmic rRNA with purified mitochondria. We showed that the binding of bound 80 S polysomes or free cytoplasmic polysomes to preparations of EDTA-washed mitochondria is completely inhibited by either 350 mM KCl or 10 m aurintricarboxylic acid (ATA). We found that when cells were opened and fractionated in buffer containing either 350 mM KCl or 10 M ATA, there was no significant reduction in the recovery of cytoplasmic rRNA with mitochondria when compared to mitochondria isolated in the absence of these components. These results support the notion that ribosomes which are attached to the outer mitochondrial membrane in isolated mitochondrial preparations are those which are seen attached to the outer membrane in situ. 

The second modified flavin of natural origin to be discovered was 8a-S-cysteinyl-FAD, the coenzyme of monoamino oxidase from liver and kidney outer mitochondrial membranes. Taking their departure from investigations of Yasunobu (8J) and Hellerman (SO), which indicated the presence of covalently bound flavin in preparations of this enzyme, Singer and his group (85, 185) isolated the flavinyl peptide by degradation of MAO with trypsin-chymotrypsin and identified cysteine as the amino acid residue bound next to the flavin moiety (184). The absorption spectrum of the flavin peptide from monoamino oxidase is readily differentiated from that of riboflavin by a hypsochromic shift of the second absorption band (360 nm, compare with 372 for riboflavin), in the neutral oxidized state (44, 184). It is similar to that of 8a-histidyl-riboflavin in the cationic state in that the band centered around 400 nm (abs. max. 375 nm, shoulder at 410 nm) is partially resolved. The fluorescence emission (4, 30) is only 10% of that of riboflavin, but oxidation with peracids raises it to 90% of riboflavin emission. 

Cytochrome c transfers electrons from complex III to complex IV via a heme prosthetic group. It is a small protein that resides on the outer face of the inner mitochondrial membrane. It is readily displaced during the experimental preparation of mitochondria, and this leads to lower than expected rates of oxygen consumption in such samples. 

On electron micrographs prepared from tissues fixed in osmic acid, the nuclear membrane appears as an envelope 250 A thick composed of three different layers an inner and an outer dense lining (each measuring 60 A in thickness) and a less dense median space (120 A thick). Unlike the cell or the mitochondrial membranes, the nuclear envelope (see Fig. 2-2) is not continuous, but is frequently interrupted by circular pores 1000 A in diameter [1-9]. 

Monoamine oxidase (MAO) serves as a marker enzyme for outer membrane. There is some MAO activity in the inner membrane and therefore also in SMPs however, a high level of monoamine oxidase in the SMP preparation indicates a large contamination by outer membrane. Mitochondrial monoamine oxidase is an FAD-dependent enzyme that catalyzes the oxidation of amines to aldehydes (Equation E10.2). A convenient assay for this enzyme uses benzylamine as substrate and monitors the rate of ben-zaldehyde production at 250 nm. 

---