Intracellular energetic units in red muscle cells

The kinetics of regulation of mitochondrial respiration by endogenous and e xogenous ADP in muscle cells in situ was studied in skinned cardiac and ske letal muscle fibres. Endogenous ADP production was initiated by addition of MgATP; under these conditions the respiration rate and ADP concentration i n the medium were dependent on the calcium concentration, and 70-80%, of ma ximal rate of respiration was achieved at ADP concentration below 20 muM in the medium. In contrast, when exogenous ADP was added, maximal respiration rate was observed only at millimolar concentrations. An exogenous ADP-cons uming system consisting of pyruvate kinase (PK; 20-40 units/ml) and phospho enolpyruvate (PEP; 5 mM), totally suppressed respiration activated by exoge nous ADP, but the respiration maintained by endogenous ADP was not suppress ed by more than 20-40%. Creatine (20 mM) further activated respiration in t he presence of ATP and PK + PEP. Short treatment with trypsin (50-500 nM fo r 5 min) decreased the apparent K,for exogenous ADP from 300-350 muM to 50- 60 muM, increased inhibition of respiration by PK + PEP system up to 70-80% , with no changes in MgATPase activity and maximal respiration rates. Elect ron-microscopic observations showed detachment of mitochondria and disorder ing of the regular structure of the sarcomere after trypsin treatment. Two- dimensional electrophoresis revealed a group of at least seven low-molecula r-mass proteins in cardiac skinned fibres which were very sensitive to tryp sin and not present in glycolytic fibres, which have low apparent K, for ex ogenous ADP. It is concluded that, in oxidative muscle cells, mitochondria are incorporated into functional complexes ('intracellular energetic units' ) with adjacent ADP-producing systems in myofibrils and in sarcoplasmic ret iculum, probably due to specific interaction with cytoskeletal elements res ponsible for mitochondrial distribution in the cell. It is suggested that t hese complexes represent the basic pattern of organization of muscle-cell e nergy metabolism.