Center for Mitochondrial Diseases
Center for Mitochondrial Diseases
Minkler P.E.,Center for Mitochondrial Diseases |
Hoppel C.L.,Center for Mitochondrial Diseases |
Hoppel C.L.,Case Western Reserve University
Journal of Lipid Research | Year: 2010
An improved high-performance liquid chromatography-mass spectrometry method for the separation and characterization of cardiolipin molecular species is presented. Reverse-phase ion pair chromatography with acidi-fi ed triethylamine resulted in increased chromatographic retention and resolution when compared with chromatography without acidifi ed triethylamine. Using a hybrid triple quadrupole linear ion trap mass spectrometer to generate MS/MS spectra revealed three regions within each spectrum that could be used to deduce the structure of the cardiolipin molecular species: the diacylglycerol phosphate region, the monoacylglycerol phosphate region, and the fatty acid region. Cardiolipin standards of known composition were analyzed and exhibited expected chromatographic and mass spectral results. Two minor components in commercial bovine heart cardiolipin, (with the same molecular weight but different chromatographic retention times), were shown to differ by fatty acid composition: (C18:2) 2 (C18:1) 2 versus (C18:2) 3 (C18:0) 1. These compounds were then analyzed by HPLC-MS 3 to examine specifi c diacylglycerol phosphate generated fatty acid fragmentation. Also, two commercial sources of bovine heart cardiolipin were shown to have minor differences in cardiolipin species content. Cardiolipin isolated from rat liver, mouse heart, and dog heart mitochondria were then characterized and the relative distributions of the major cardiolipin species were determined. Copyright © 2010 by the American Society for Biochemistry and Molecular Biology, Inc.
Rosca M.,Center for Mitochondrial Diseases |
Rosca M.,Case Western Reserve University |
Minkler P.,Center for Mitochondrial Diseases |
Minkler P.,Case Western Reserve University |
And 2 more authors.
Biochimica et Biophysica Acta - Bioenergetics | Year: 2011
Mitochondrial dysfunction is a major contributor in heart failure (HF). We investigated whether the decrease in respirasome organization reported by us previously in cardiac mitochondria in HF is due to changes in the phospholipids of the mitochondrial inner membrane or modifications of the subunits of the electron transport chain (ETC) complexes. The contents of the main phospholipid species, including cardiolipin, as well as the molecular species of cardiolipin were unchanged in cardiac mitochondria in HF. Oxidized cardiolipin molecular species were not observed. In heart mitochondria isolated from HF, complex IV not incorporated into respirasomes exhibits increased threonine phosphorylation. Since HF is associated with increased adrenergic drive to cardiomyocytes, this increased protein phosphorylation might be explained by the involvement of cAMP-activated protein kinase. Does the preservation of cAMP-induced phosphorylation changes of mitochondrial proteins or the addition of exogenous cAMP have similar effects on oxidative phosphorylation? The usage of phosphatase inhibitors revealed a specific decrease in complex I-supported respiration with glutamate. In saponin-permeabilized cardiac fibers, pre-incubation with cAMP decreases oxidative phosphorylation due to a defect localized at complex IV of the ETC inter alia. We propose that phosphorylation of specific complex IV subunits decreases oxidative phosphorylation either by limiting the incorporation of complex IV in supercomplexes or by decreasing supercomplex stability. © 2011 Published by Elsevier B.V.
PubMed | Center for Mitochondrial Diseases and Case Western Reserve University
Type: Journal Article | Journal: The Journal of biological chemistry | Year: 2015
Mitochondria are dynamic organelles that continually undergo cycles of fission and fusion. Dynamin-related protein 1 (Drp1), a large GTPase of the dynamin superfamily, is the main mediator of mitochondrial fission. Like prototypical dynamin, Drp1 is composed of a mechanochemical core consisting of the GTPase, middle, and GTPase effector domain regions. In place of the pleckstrin homology domain in dynamin, however, Drp1 contains an unstructured variable domain, whose function is not yet fully resolved. Here, using time-resolved EM and rigorous statistical analyses, we establish the ability of full-length Drp1 to constrict lipid bilayers through a GTP hydrolysis-dependent mechanism. We also show the variable domain limits premature Drp1 assembly in solution and promotes membrane curvature. Furthermore, the mechanochemical core of Drp1, absent of the variable domain, is sufficient to mediate GTP hydrolysis-dependent membrane constriction.