Nagarse treatment of cardiac subsarcolemmal and interfibrillar mitochondria leads to artefacts in mitochondrial protein quantification

Gábor Koncsos, Zoltán V. Varga, Tamás Baranyai, Péter Ferdinandy, Rainer Schulz, Z. Giricz, Kerstin Boengler

Research output: Contribution to journalArticle

3 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)50-58
Number of pages9
JournalJournal of Pharmacological and Toxicological Methods
Volume91
DOIs
Publication statusPublished - May 1 2018

Fingerprint

Subtilisins
Mitochondria
Mitochondrial Proteins
Artifacts
Connexin 43
Rats
Peptide Hydrolases
Phenylmethylsulfonyl Fluoride
Membranes
Centrifugation
Mitochondrial Membranes
Oxygen Consumption
Proteins
Western Blotting
Oxygen

Keywords

  • Connexin-43
  • Heart
  • IFM
  • Methods
  • Mitochondria isolation
  • Mitofusin
  • Nagarse
  • SSM
  • Subtilisin BPN’

ASJC Scopus subject areas

  • Toxicology
  • Pharmacology

Cite this

Nagarse treatment of cardiac subsarcolemmal and interfibrillar mitochondria leads to artefacts in mitochondrial protein quantification. / Koncsos, Gábor; Varga, Zoltán V.; Baranyai, Tamás; Ferdinandy, Péter; Schulz, Rainer; Giricz, Z.; Boengler, Kerstin.

In: Journal of Pharmacological and Toxicological Methods, Vol. 91, 01.05.2018, p. 50-58.

Research output: Contribution to journalArticle

Koncsos, Gábor ; Varga, Zoltán V. ; Baranyai, Tamás ; Ferdinandy, Péter ; Schulz, Rainer ; Giricz, Z. ; Boengler, Kerstin. / Nagarse treatment of cardiac subsarcolemmal and interfibrillar mitochondria leads to artefacts in mitochondrial protein quantification. In: Journal of Pharmacological and Toxicological Methods. 2018 ; Vol. 91. pp. 50-58.
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T1 - Nagarse treatment of cardiac subsarcolemmal and interfibrillar mitochondria leads to artefacts in mitochondrial protein quantification

AU - Koncsos, Gábor

AU - Varga, Zoltán V.

AU - Baranyai, Tamás

AU - Ferdinandy, Péter

AU - Schulz, Rainer

AU - Giricz, Z.

AU - Boengler, Kerstin

PY - 2018/5/1

Y1 - 2018/5/1

N2 - 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.

AB - 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.

KW - Connexin-43

KW - Heart

KW - IFM

KW - Methods

KW - Mitochondria isolation

KW - Mitofusin

KW - Nagarse

KW - SSM

KW - Subtilisin BPN’

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