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Analysis of mitochondrial function is central to the study of intracellular energy metabolism, mechanisms of cell death and pathophysiology of a variety of human diseases, including myopathies, neurodegenerative diseases and cancer. However, important properties of mitochondria differ in vivo and in vitro. Here, we describe a protocol for the analysis of(More)
Regulation of mitochondrial outer membrane (MOM) permeability has dual importance: in normal metabolite and energy exchange between mitochondria and cytoplasm and thus in control of respiration, and in apoptosis by release of apoptogenic factors into the cytosol. However, the mechanism of this regulation, dependent on the voltage-dependent anion channel(More)
In this chapter we describe in details the permeabilized cell and skinned fiber techniques and their applications for studies of mitochondrial function in vivo. The experience of more than 10 years of research in four countries is summarized. The use of saponin in very low concentration (50-100 microg/ml) for permeabilisation of the sarcolemma leaves all(More)
Relative diffusivities of ADP and creatine in cardiomyocytes were studied. The isolated rat cardiomyocytes were lysed with saponin (40 micrograms/ml) to perforate or completely disrupt sarcolemma that was evidenced by leakage of 80-100% lactate dehydrogenase. In these cardiomyocytes mitochondria were used as 'enzymatic probes' to determine the average local(More)
The kinetics of in vivo regulation of mitochondrial respiration by ADP was studied in rat heart, slow-twitch skeletal muscle (soleus) and fast-twitch skeletal muscle (gastrocnemius, plantaris, quadriceps and tibialis anterior) by means of saponin-skinned fibres. Mitochondrial respiratory parameters were determined in the absence and presence of creatine (20(More)
The efficiency of stimulation of mitochondrial respiration in permeabilized muscle cells by ADP produced at different intracellular sites, e.g. cytosolic or mitochondrial intermembrane space, was evaluated in wild-type and creatine kinase (CK)-deficient mice. To activate respiration by endogenous production of ADP in permeabilized cells, ATP was added(More)
The kinetics of regulation of mitochondrial respiration by endogenous and exogenous ADP in muscle cells in situ was studied in skinned cardiac and skeletal muscle fibres. Endogenous ADP production was initiated by addition of MgATP; under these conditions the respiration rate and ADP concentration in the medium were dependent on the calcium concentration,(More)
The current problems of regulation of myocardial energy metabolism and oxidative phosphorylation in vivo are considered. With this purpose, retarded diffusion of ADP in cardiomyocytes was studied by analysis of elevated apparent Km for this substrate in regulation of respiration of saponin-skinned cardiac fibers, as compared to isolated mitochondria.(More)
Possible reasons for retarded intracellular diffusion of ADP were investigated. The isolated skinned cardiac fibers were used to study apparent kinetic parameters for externally added ADP in control of mitochondrial respiration. Participation of myosin-ATPase in binding of ADP within cells as it was supposed earlier (Saks, V.A., Belikova, Yu.O. and(More)
The history of the development of the ideas and research of organized metabolic systems during last three decades is shortly reviewed. The cell cytoplasm is crowded with solutes, soluble macromolecules such as enzymes, nucleic acids, structural proteins and membranes. The high protein density within the large compartments of the cells predominantly(More)