Show simple item record Koncsos, Gábor Varga, Zoltán Baranyai, Tamás Ferdinandy, Péter Schulz R Giricz, Zoltán Boengler K 2018-08-30T09:11:46Z 2018-08-30T09:11:46Z 2018
dc.identifier 85041604065
dc.identifier.citation pagination=50-58; journalVolume=91; journalTitle=JOURNAL OF PHARMACOLOGICAL AND TOXICOLOGICAL METHODS;
dc.identifier.uri doi:10.1016/j.vascn.2018.01.004
dc.description.abstract INTRODUCTION: In the heart, subsarcolemmal (SSM), interfibrillar (IFM) and perinuclear mitochondria represent three subtypes of mitochondria. The most commonly used protease during IFM isolation is the nagarse, however, its effect on the detection of mitochondrial proteins is still unclear. Therefore, we investigated whether nagarse treatment influences the quantification of mitochondrial proteins. METHODS: SSM and IFM were isolated from hearts of mice and rats. During IFM isolation, nagarse activity was either stopped by centrifugation (common protocol, IFM+N) or inhibited by phenylmethylsulfonyl fluoride (PMSF, IFM+N+I). The amounts of proteins located in different mitochondrial compartments (outer membrane: mitofusin 1 (MFN1) and 2 (MFN2); intermembrane space: p66shc; inner membrane (connexin 43 (Cx43)), and of protein deglycase DJ-1 were determined by Western blot. RESULTS: MFN2 and Cx43 were predominantly in SSM isolated from mouse and rat hearts. MFN1 and p66shc were present in similar amounts in SSM and IFM+N, whereas the level of DJ-1 was higher in IFM+N compared to SSM. In IFM+N+I samples from mice, the amount of MFN2, but not that of Cx43 increased. Nagarse or nagarse inhibition by PMSF had no effect on oxygen consumption of SSM or IFM. DISCUSSION: Whereas the use of the common protocol indicates the localization of MFN2 predominantly in SSM, the inhibition of nagarse by PMSF increases the signal of MFN2 in IFM to that of in SSM, indicating an underestimation of MFN2 in IFM. Therefore, protease sensitivity should be considered when assessing distribution of mitochondrial proteins using nagarse-based isolation.
dc.relation.ispartof urn:issn:1056-8719
dc.title Nagarse treatment of cardiac subsarcolemmal and interfibrillar mitochondria leads to -artefacts in mitochondrial protein quantification
dc.type Journal Article 2018-08-28T18:04:18Z
dc.language.rfc3066 en
dc.identifier.mtmt 3328031
dc.identifier.wos 000430275600007
dc.identifier.pubmed 29378341
dc.contributor.department SE/AOK/I/Farmakológiai és Farmakoterápiás Intézet
dc.contributor.institution Semmelweis Egyetem
dc.mtmt.swordnote Giricz Z; and Boengler K authors contributed equally to this work.

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