Raman spectroscopic investigation of the interaction of gramicidin A with dipalmitoyl phosphatidylcholine liposomes.

The interaction of gramicidin A with dipalmitoyl phosphatidylcholine liposomes is investigated by Laser-Raman spectroscopy. As revealed by the methylene C-H stretching mode the phase transition of the hydrocarbon chains near 40 degree C is eliminated in the presence of gramicidin A. Liposomes prepared from a mixture of lecithin and cholesterol seem to be unaffected by gramicidin A and show only the normal broadened phase transition.     

Inhibition of adenine nucleotide transport through the mitochondrial porin by a synthetic polyanion

The effect of a synthetic polyanion of Mr 10,000 (a copolymer of methacrylate, maleate and styrene in a 1:2:3 proportion) was studied on isolated rat liver mitochondria and on mitochondrial porin reconstituted into lipid bilayer membranes. Increasing concentrations of the polyanion inhibited the adenyl kinase located between both mitochondrial membranes in a dose-dependent fashion. Upon addition of the detergent digitonin in increasing concentrations the adenyl kinase activity was fully reversible. In reconstitution experiments with mitochondrial porin the polyanion increased the voltage dependence of the pore in such a way that the pore is switched into the closed state at much smaller voltages than in the absence of the polyanion. The asymmetric addition of the polyanion resulted in an asymmetric shift of the voltage-dependence of the pore. If the voltage is negative at the cis-side (the side of the addition of the polyanion) the pore closed rapidly whereas it was always open for potentials of opposite polarity. The results are discussed on the basis of a modification of the gate properties of the mitochondrial porin by the polyanion and by the assumption that the closed state of the pore is not permeable for nucleotides.     

Mitochondrial boundary membrane contact sites in brain: points of hexokinase and creatine kinase location, and control of Ca2+ transport

The location of hexokinase at the surface of brain mitochondria was investigated by electron microscopy using immuno-gold labelling techniques. The enzyme was located where the two mitochondrial limiting membranes were opposed and contact sites were possible. Disruption of the outer membrane by digitonin did not remove bound hexokinase and creatine kinase from brain mitochondria, although the activity of outer membrane markers and adenylate kinase decreased, suggesting a preferential location of both enzymes in the contact sites. In agreement with that, a membrane fraction was isolated from osmotically lysed rat brain mitochondria in which hexokinase and creatine kinase were concentrated. The density of this kinase-rich fraction was specifically increased by immuno-gold labelling of hexokinase, allowing a further purification by density gradient centrifugation. The fraction was composed of inner and outer limiting membrane components as shown by the specific marker enzymes, succinate dehydrogenase and NADH-cytochrome-c-oxidase (rotenone insensitive). As reported earlier for the enriched contact site fraction of liver mitochondria the fraction from brain mitochondria contained a high activity of glutathione transferase and a low cholesterol concentration. Moreover, the contacts showed a higher Ca2+ binding capacity in comparison to outer and inner membrane fractions. This finding may have regulatory implications because glucose phosphorylation via hexokinase activated the active Ca2+ uptake system and inhibited the passive efflux, resulting in an increase of intramitochondrial Ca2+.     

Mitochondrial intermembrane inclusion bodies: The common denominator between human mitochondrial myopathies and creatine depletion due to impairment of cellular energetics

Mitochondrial inclusion bodies are often described in skeletal muscle of patients suffering diseases termed mitochondrial myopathies. A major component of these structures was discovered as being creatine kinase. Similar creatine kinase enriched inclusion bodies in the mitochondria of creatine depleted adult rat cardiomyocytes have been demonstrated. Structurally similar inclusion bodies are observed in mitochondria of ischemic and creatine depleted rat skeletal muscle. This paper describes the various methods for inducing mitochondrial inclusion bodies in rodent skeletal muscle, and compares their effects on muscle metabolism to the metabolic defects of mitochondrial myopathy muscle. We fed rats with a creatine analogue guanidino propionic acid and checked their soled for mitochondrial inclusion bodies, with the electron microscope. The activity of creatine kinase was analysed by measuring creatine stimulated oxidative phosphorylation in soleus skinned fibres using an oxygen electrode . The guanidino propionic acid-rat soleus mitochondria displayed no creatine stimulation, whereas control soleus did, even though the GPA soled had a five fold increase in creatine kinase protein per mitochondrial protein. The significance of these results in light of their relevance to human mitochondrial myopathies and the importance of altered muscle metabolism in the formation of these crystalline structures are discussed. (Mol Cell Biochem 174: 283–289, 1997)     

Changes in freeze-fractured mitochondrial membranes correlated to their energetic state. Dynamic interactions of the boundary membranes

Structural changes of mitochondria in correlation to their energetic state have been observed as matrix expansion and condensation. In this communication we describe a morphological correlation in freeze-fractured mitochondrial membranes which is also dependent on the metabolic state of the organelle: the frequency by which the fracture plane following the inner or outer boundary membrane deviates by jumping from one membrane to the other is higher in phosphorylating mitochondria when compared to freshly isolated or energized mitochondria. These deflections of the fracture plane occur mostly in minimal, short steps showing close apposition of the two boundary membranes. We therefore conclude that the observed change in morphological appearance is produced by a change in interactions between the inner and outer membranes correlated to the different functional states of the inner membrane.     

The influence of the cytosolic oncotic pressure on the permeability of the mitochondrial outer membrane for ADP: implications for the kinetic properties of mitochondrial creatine kinase and for ADP channelling into the intermembrane space

Summary Cytosolic proteins as components of the physiological mitochondrial environment were substituted by dextrans added to media normally used for incubation of isolated mitochondria. Under these conditions the volume of the intermembrane space decreases and the contact sites between the both mitochondrial membranes increase drastically. These morphological changes are accompanied by a reduced permeability of the mitochondrial outer compartment for adenine nucleotides as it was shown by extensive kinetic studies of mitochondrial enzymes (oxidative phosphorylation, mi-creatine kinase, mi-adenylate kinase). The decreased permeability of the mitochondrial outer membrane causes increased rate dependent concentration gradients in the micromolar range for adenine nucleotides between the intermembrane space and the extramitochondrial space. Although all metabolites crossing the outer membrane exhibit the same concentration gradients, considerable compartmentations are detectable for ADP only due to its low extramitochondrial concentration. The consequences of ADP-compartmentation in the mitochondrial intermembrane space for ADP-channelling into the mitochondria are discussed.