Inside-out Particles Research Articles

Some properties of mitochondria, mitoplasts and submitochondrial particles of different polarities from plant tissues

Irrespective of the starting material, i.e. washed mitochondria, purified mitochondria or mitoplast from Solanum tuberosum L., submitochondrial particles of well-defined polarities can be generated by French press treatment in low-salt medium or by sonication in high-salt medium. The first treatment will result in submitochondrial particles which are more than 80% right-side-out (right-side-out particles), the second in submitochondrial particles that are more than 80% inside-out (inside-out particles). The isoelectric point (p I = 4.0) of the inside-out particles measured by cross-partition is distinctly different from the isoelectric points of the other mitochondrial membranes which exhibit p I values between 4.5 and 4.7. The surface charge density measured by 9-aminoacridine fluorescence varies in the same order from −27 mC · m −2 for Percoll-purified mitochondria to −51 mC · m −2 for both right-side-out and inside-out particles. Even though the charge densities for the two surfaces of the inner membrane are similar, inside-out particles are much more negatively charged at pH 7.0, since they are 6-times larger. These results clearly demonstrate that it is possible to obtain submitochondrial particles of various polarities and sizes which in turn constitute valuable tools for the study of lateral and transverse asymmetry of the inner mitochondrial membrane.

Titration of the binding sites for the oligomycin-sensitivity conferring protein in beef heart submitochondrial particles

The binding parameters of the oligomycin-sensitivity conferring protein (OSCP) in inside-out particles from beef heart mitochondria have been tested by means of two assays, the oligomycin-sensitive ATP—P i exchange, and the oligomycin-sensitive ATP hydrolysis. The total number of OSCP binding sites in A particles was equal to 220 pmol/mg particle protein. Each mole of ATPase active site was able to bind 1.1±0.5 mol OSCP with K d 1.7 nM.

Kinetics of ubiquinol-1-cytochrome c reductase in bovine heart mitochondria and submitochondrial particles

A kinetic study on ubiquinol-cytochrome f reductase (EC 1.10.2.2) has been undertaken either in situ in KCN-inhibited mitochondria and submitochondrial particles, or in the isolated cytochrome b- c 1 complex using ubiquinol-1 and exogenous cytochrome c as substrates. The steady-state two-substrate kinetics of the reductase appears to follow a general sequential mechanism, allowing calculation of a K m for ubiquinol-1 of 13.4 μM in mitochondria and of 24.6 μM in the isolated cytochrome b- c 1 complex. At low concentrations of cytochrome c, however, the titrations as a function of quinol concentration appear biphasic both in mitochondria and in submitochondrial particles containing trapped cytochrome c inside the vesicle space, fitting two apparent K m values for ubiquinol-1. Relatively high antimycin-sensitive rates of ubiquinol-1-cytochrome c reductase have been found in submitochondrial particles: both the V max and the K m for ubiquinol-1 are, however, affected by the overall orientation of the particle preparation, i.e., by the reactivity of cytochrome c with its proper site. The turnover numbers corrected for particle orientation with respect to cytochrome c interaction are at least 2-fold higher in submitochondrial particles than in mitochondria. This is particularly evident using inside-out particles containing trapped cytochrome c in the vesicle space (and therefore reacting with its physiological site). A diffusion step for the quinol substrate appears to be rate limiting in mitochondria and can be removed by addition of deoxycholate, suggesting that the oxidation site of ubiquinol may be more exposed to the matrix side of the inner mitochondrial membrane.

4-Azido-2-nitrophenyl phosphate, a new photoaffinity derivative of inorganic phosphate. Study of its interaction with the inorganic phosphate binding site of beef heart mitochondrial adenosine triphosphatase.

4-Azido-2-nitrophenyl phosphate (ANPP) was synthesized and characterized. ANPP, unlabeled or labeled by 32P, was used as a photoreactive analogue of Pi to study the Pi binding site(s) in isolated F1-ATPase and inside-out particles from beef heart mitochondria. In the dark, the phosphate bond of ANPP was cleaved by alkaline phosphatase but not by mitochondrial F1-ATPase. ANPP bound reversibly to the phosphate site of F1-ATPase as shown by competitive inhibition of binding of Pi to F1-ATPase by ANPP in the dark; the Ki value was 60 microM. Upon photoirradiation with visible light, [32P]ANPP bound covalently to F1-ATPase and inactivated the enzyme. Part of the added ANPP was, however, photolyzed with release of Pi. By extrapolation, it could be calculated that complete inactivatin of F1-ATPase was accompanied by incorporation of 32P radioactivity corresponding to 1 mol of [32P]ANPP per mol of F1-ATPase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [32P]-ANPP-labeled F1-ATPase revealed only one radioactive peptide with a Mr of 50000. This peptide was characterized as the beta subunit of F1-ATPase by specific labeling with [14C]dicyclohexylcarbodiimide [Pougeois, R., Satre, M., & Vignais, P. V. (1979) Biochemistry 18, 1408-1413]. Photoirradiation of inside-out submitochondrial particles with [32P]ANPP resulted in the labeling of two peptides with a Mr of 50000 and 30000-32000; both labelings were significantly decreased by incubation of the particles with Pi prior to photoirradiation. The Mr 50000 peptide is most probably the beta subunit of F1-ATPase; the other peptide might be the Pi carrier protein.

The Oxidation of Malate by Isolated Turnip (Brassica napusL.) Mitochondria

Washed and purified turnip mitochondria oxidize malate with respiratory control and ADP: O values approaching 3.0 and produce pyruvate as the principal product. Oxaloacetate is also produced in significant amounts but is removed by an endogenous mechanism. Malate dehydrogenase appears to be important to the oxidation of malate but requires the removal of oxaloacetate. During malate oxidation the mitochondria undergo configurational changes similar to those observed in animal mitochondria. Both ‘right side-out’ and ‘inside-out’ submitochondrial particles have been prepared. Right side-out particles oxidize malate in the same way as intact mitochondria, whereas the inside-out particles have a biphasic oxidation, the first phase producing oxaloacetate and the second, NAD-requiring phase producing pyruvate. It is concluded that malate oxidation is a complex process using several enzymes located in the matrix compartment with a minor component possibly on the outer face of the inner membrane.

Photoaffinity labeling of the adenine nucleotide carrier in heart and yeast mitochondria by an arylazido ADP analog

▪ 1. 1. Arylazido analogs of ADP and ATP ( N-4-azido-2-nitrophenyl-aminobutyryl-ADP and N-4-azido-2-nitrophenylaminobutyryl-ATP have been prepared in radioactive form and used in photolabeling experiments to identify the adenine nucleotide carrier in mitochondria and sonic submitochondrial particles. 2. 2. When added in the dark to beef heart mitochondria, azidonitrophenyl-aminobutyryl-ADP binds to the adenine-nucleotide carrier. It is not transported across the membrane to the matrix space, but it inhibits ADP transport in mito-chondria. The inhibition is of a mixed type with a K i value of about 10 μM. 3. 3. The nitrene derivative formed upon photoirradiation of tritiated azidonitrophenylaminobutyryl-ADP or -ATP binds to a polypeptide of apparent molecular weight 30 000 in beef heart mitochondria and 37 000 in Saccharomyces cerevisiae mitochondria. The photolabeling is prevented by preincubation of the mitochondria with atractyloside or carboxyatractyloside. 4. 4. Photoirradiation of sonic submitochondrial particles from beef heart (inside-out particles) with tritiated azidonitrophenylaminobutyryl-ADP or -ATP results in the labeling of the 30 000-dalton polypeptide and also in the labeling of higher molecular weight peptides (50 000–55 000) probably belonging to F 1-ATPase. Addition of bongkrekic acid specifically decreases the photolabeling of the 30 000-dalton polypeptide. 5. 5. An arylazido derivative of atractyloside ( N-4-azido-2-nitrophenylaminobutyryl atractyloside) binds upon photoirradiation to the 30 000-dalton polypeptide in beef heart mitochondria and to the 37 000-dalton polypeptide in S. cerevisiae mitochondria. 6. 6. Since the adenine nucleotide carrier is readily damaged by ultraviolet light, nitro-arylazido analogs of ADP and ATP or of atractyloside, which are photoactivated in visible light, were used in preference to other azido analogs, which require ultraviolet light for photoactivation. 7. 7. Data presented in this paper support the view that the same mitochondrial protein belonging to the adenine nucleotide transport system is able to bind ADP (or ATP) and atractyloside.

Two types of vesicles from the erythrocyte-ghost membrane differing in surface charge. Separation and characterization by preparative free-flow electrophoresis.

Vesicles of erythrocyte ghosts were produced by treatment with Primaquine or hexokinase. The resulting vesicles could be separated from each other and characterized by preparative free‐flow electrophoresis. One population consisted of outside‐out vesicles carrying the same sialic acid distribution as the original ghosts. The other represented homogeneous inside‐out particles with a different electrophoretic mobility carrying most of their sialic acid at their inner side. The two kinds of vesicles could not be separated by density gradient centrifugation.