METHYLGLYOXAL, CREATINE AND MITOCHONDRIAL MICRO-COMPARTMENTS

___________________________________________________________________________ 67 Introduction ________________________________________________________________________ 68 Material and Methods_______________________________________________________________ 70 Animals __________________________________________________________________________ 70 Western blotting __________________________________________________________________ 70 Fixation of liver tissue and immunodecoration for electron microscopy _______________ 70 Tissue extracts and isolation of mitochondria _______________________________________ 70 Disintegration measurements ______________________________________________________ 70 Preparation of mtuCK containing microdomains ____________________________________ 71 Results______________________________________________________________________________ 72 Over-epressed mtuCK induces contact sites in vivo _________________________________ 72 Over-expressed mtCK increases mitochondrial structural integrity and stability________ 73 Biochemical isolation of contact sites ______________________________________________ 74 Discussion __________________________________________________________________________ 76 References _________________________________________________________________________ 79 NOVEL MITOCHONDRIAL CREATINE TRANSPORT ACTIVITY __________________________________________ 83 Abstract ____________________________________________________________________________ 85___________________________________________________________________________ 85 Introduction ________________________________________________________________________ 86 Experimental procedures ____________________________________________________________ 88 Materials _________________________________________________________________________ 88 Immunofluorescence of sections from rat ventricle__________________________________ 88 Immunoelectron microscopy of ventricle sections __________________________________ 88 Tissue extracts and isolation of mitochondria _______________________________________ 88 Western blotting __________________________________________________________________ 89 Isolation of outer and inner membrane from rat liver mitochondria___________________ 89 Immunolabeling and negative staining of isolated mitochondria ____________________ 89 Measurement of Cr transport into mitochondria ____________________________________ 90 Results______________________________________________________________________________ 92 Intracellular location of CRT in rat heart ____________________________________________ 92 Expression of CRT-related Polypeptides in Mitochondria from Different Organs ________ 93 Intramitochondrial location of CRT-related proteins _________________________________ 93 Cr Uptake Assay with Isolated Mitochondria ________________________________________ 94 Discussion __________________________________________________________________________ 98 CRT isoforms ______________________________________________________________________ 99 Mitochondrial location of the two major CRT-related protein species_________________ 99 Mitochondrial transport of Cr ______________________________________________________ 99 Implications for Cr compartmentation ____________________________________________ 100 Conclusions and perspectives ____________________________________________________ 101 References ________________________________________________________________________ 103 CREATINE TRANSPORTER ISOENZYMES: A REAPPRAISAL ____________________________________________ 105 Abstract ___________________________________________________________________________ 107__________________________________________________________________________ 107 Introduction _______________________________________________________________________ 108 Creatine metabolism ____________________________________________________________ 108 The creatine transporter__________________________________________________________ 108 Inborn errors in CrT _______________________________________________________________ 109 Generation of antibodies against CrT _____________________________________________ 109