Pär Gellerfors

Pore protein and the hexokinase-binding protein from the outer membrane of rat liver mitochondria are identical.

Hexoklnase of tumor cells [l] and certain normal tissues [2] are bound to the outer membrane of the mitochondrion. This binding is, at least in part, responsible for increased aerobic glycolysis, which characterizes rapidly growing tumor cells [3]. The rat liver outer membrane protein responsible for binding of hexokinase has been isolated and partially characterized [4]. The app. Mr reported for this peptide (31 000) is nearly identical to that of the outer membrane pore protein [5], purified and characterized in [6]. Since SDS electrophoresis of outer membranes reveals a single major band in the 3 1 000 Mr region, we have investigated the possibility that pore protein and the hexokinase-binding protein are identical. These results indicate that this is the case. [6,6’(n)-3H]Sucrose (l-5 Ci/mmol) was purchased from The Radiochemical Center (Amersham). [carboxyl-‘4C]Dextran (M, 70 000) with spec. act. 1 .l mCi/g was from New England Nuclear. Staphylococcus aureus V8 protease was purchased from Pharmacia (Uppsala). Chymotrypsin, soy bean lecithin and octyl-fl-D-glucopyranoside were all purchased from Sigma. Ampholines were from LKB (Stockholm).

Hydrodynamic properties of porin isolated from outer membranes of rat liver mitochondria.

The hydrodynamic properties of purified porin (Mr = 30 000), isolated from outer membranes of rat liver mitochondria has been studied. After gel filtration, active porin was eluted in a symmetrical peak with an estimated Stokes radius of 5.4 nm. The sedimentation coefficient (s) and partial specific volume (v) were found to be 2.6 S and 0.908 cm3/g, respectively, for the purified porin-Triton X-100 complex. Based on these determinations, a molecular weight of 170 000 for the porin-Triton X-100 complex was calculated. Correcting for bound Triton X-100, 1.8 g/g of protein, a molecular weight of 60 000 was estimated for the protein portion of the complex. Thus, isolated active porin appears to exist as a dimer.

Subcellular distribution of rat liver porin

The subcellular distribution of rat liver porin was investigated using the immunoblotting technique and monospecific antisera against the protein isolated from the outer membrane of rat liver mitochondria. Subfractionation of mitochondria into inner membranes, outer membranes and matrix fractions revealed the presence of porin only in the outer membranes. Porin was also not detected in highly purified subcellular fractions, including plasma membranes, nuclear membranes, Golgi I and Golgi II, microsomes and lysosomes. Thus, liver porin is located exclusively in the outer mitochondrial membrane.

The redox properties of the cytochromes of purified Complex III

Abstract Purified Complex III from beef heart contains two b cytochromes: a high-potential (Em 7.2 = +93 mV) cytochrome b-562 which can be enzymatically reduced, and a low-potential (Em 7.2 = −34 mV) cytochrome b-565 which is reduced only by dithionite. The two components each contribute approximately 50% to the total cytochrome b of Complex III. Cytochrome c1 of Complex III titrates with a half-reduction potential of +232 mV.

[13] Characterization and resolution of complex III from beef heart mitochondria

Publisher Summary This chapter describes the determination of peptide composition of complex III by sodium dodecyl sulfate (SDS) by gel electrophoresis. Complex III is dissolved in a mixture of 2% SDS, 5% 2-mercaptoethanol, and 10 mM Tris-chloride, pH 6.8, to a final concentration of 1–2 mg of protein per milliliter. Complex III (QH2-cytochrome c reductase) is isolated on the basis of its specificity toward short-chained ubiquinone homologs. Because the preferred substrates (Q 1 –Q 3 ) are not commercially available, several investigators have turned to duroquinol as an alternative substrate. The peptide pattern on SDS-gel is altered only slightly when the samples are prepared and electrophoresed in the absence of mercaptoethanol, indicating the absence of intermolecular disulfide bands. SDS gels stained for carbohydrate using periodic acid-Schiff reagent (PAS) show no positive staining reactions associated with the peptide bands. A positive reaction is observed near the dye front and probably represents carbohydrate-containing lipids.

A rapid method for the isolation of intact mitochondria from isolated rat liver cells.

Abstract A method is described for the preparation of intact mitochondria from isolated hepatocytesby sonication. Sonication of a suspension of rat liver cells for 10–30 s yields a homogenate from which tightly coupled mitochondria can be isolated. These mitochondria exhibit high respiratory control ratios and normal ADP:O ratios using glutamate plus malate, β -hydroxybutyrate, succinate, or ascorbate plus N,N,N′,N′ -tetramethyl- p -phenylendiamine as substrates. The yield of mitochondrial protein is approximately 100–120 mg starting from 5 g of liver tissue. The mitochondrial fraction is essentially free of contaminating plasma membrane and microsomes and contains only small amounts of peroxisomes and lysosomes.

Synthesis of mitochondrial proteins in isolated rat hepatocytes.

Mitochondrial synthesis has been extensively studied in lower eucaryotic cells [l-3]. Comparatively little, however, is known about this process in intact higher eucaryotic cells, and the bulk of this information is restricted to rapidly growing, often poorly differentiated, cells in culture [ 1,4-71. Until recently, few systems were available with which to study the biosynthesis of mitochondrial membranes in the intact, well differentiated, normal mammalian cell. Such a system is required if we are to understand the nucleocytoplasmic interactions in normal higher eucaryotic cells, or the possible influence of hormones and growth factors [8,9] on biosynthesis. As such an experimental system, the isolated hepatocyte offers the following advantages: (1) Much is already known about the synthesis of rat liver mitochondrial membranes,,based primarily on in vitro labeling of isolated mitochondria [1,10,113; (2) Hepatocytes can be maintained in suspension for several hours, and can be manipulated with substrates and inhibitors; (3) Large quantities of cells are available for subcellular fractionation; (4) Nucleo-cytoplasmic relationships can be studied; (5) The cells retain full sensitivity to hormones and other growth factors [9]. In contrast to other mammalian cell types which have been studied in tissue culture [4-71, growth of freshly isolated hepatocytes is slow, or non-existent [9]. Mitochondrial synthesis could, thus, also prove so

Biogenesis of the cytochrome bc1 complex in isolated rat hepatocytes

Abstract Isolated rat hepatocytes were labeled with [35S]methionine in the absence or presence of cycloheximide or chloramphenicol. The cytochrome bc 1 complex was isolated from labeled cells by a micromethod and analyzed by SDS-polyacrylamide gel electrophoresis and fluorography. All subunits except the two smallest, subunits VII and VIII, were labeled in the absence of translational inhibitors. In the presence of cycloheximide only subunit III (molecular weight, 30 000) was labeled. This polypeptide, identified as an apo-cytochrome b, was weakly labeled with [35S]methionine in the presence of cycloheximide, indicating a strict dependence of cytoplasmically synthesized products for its assembly. In the presence of chloramphenicol, labeling was inhibited only in subunit III.

Biogenesis of the outer mitochondrial membrane in isolated rat hepatocytes

The outer membrane from rat liver mitochondria was first isolated in 1966 [ 1,2]. Since then very little attention has been paid to the synthesis and assembly of the outer membrane. Today synthesis and assembly of the outer membrane is becoming increasingly important, particularly in view of its possible role in import of cytoplasmically made matrix proteins [3]. For example, to explain the specific recognition of imported proteins by the mitochondrion, several investigators have proposed the presence of receptor proteins in the outer membrane [3-S]. If such a receptor exists, however, one must solve the paradox as to how the receptor protein recognizes the mitochondrial membrane. One obvious possibility is that such protein might be coded for by mitochondrial DNA. This is also suggested by earlier experiments, which showed a small but significant labeling of the outer membrane in in vitro labeled mitochondria [6,7]. Here, we have studied biosynthesis of the outer mitochondrial membrane in isolated rat hepatocytes. Our results show that all of the peptides of the outer membrane, including the pore protein [8], are translated on cytoplasmic ribosomes.

Alkali-induced reduction of the b-cytochromes in purified Complex III from beef heart mitochondria

Approx. 40-50% of the cytochrome b in purified Complex III is reduced by ascorbate plus N,N,N,N-tetramethyl-p-phenylenediamine or phenazine methosulfate at neutral pH. The remaining cytochrome b, including cytochrome b-565, is reduced by increasing the pH. The apparent pK for this reduction is between pH 10 and 11, and is more than two pH units higher than a similar alkali-induced transition in Mg-ATP particles. Alkali-induced reduction of cytochrome b occurs concomitantly with the exposure of hydrophobic tyrosine and tryptophan residues to a more hydrophilic environment. The relationship of these findings to the presence of a substrate accessibility barrier in Complex III is discussed.