Measurement of mitochondrial respiratory chain enzymatic activities in Drosophila melanogaster samples

Abstract

Mitochondrial respiratory chain (MRC) dysfunction is linked to mitochondrial disease as well as other common conditions such as diabetes, neurodegeneration, cancer, and aging. Thus, the evaluation of MRC enzymatic activities is fundamental for diagnostics and research purposes on experimental models. Here, we provide a verified and reliable protocol for mitochondria isolation from various D. melanogaster samples and subsequent measurement of the activity of MRC complexes I-V plus citrate synthase (CS) through UV-VIS spectrophotometry. For complete details on the use and execution of this protocol, please refer to Brischigliaro et al. (2021).

Keywords: Cell separation/fractionation; Metabolism; Model Organisms; Protein Biochemistry.

Graphical abstract

Figure 1

Expected outcomes for enzymatic activity assays (A) Complex I activity recordings of wild-type mitochondria in absence (Mito) or presence of the inhibitor (Mito + rotenone), plus recording of mitochondria from complex I subunit ND-18 (Ndufs4) gene knockdown D. melanogaster individuals (Mito CI KD), (Foriel et al., 2018). (B) Complex II activity recordings of wild-type mitochondria in absence (Mito) or presence of the inhibitor (Mito + Malonate). (C) Complex III activity recording of wild-type mitochondria in absence (Mito) or presence of the inhibitor (Mito + Antimycin A) plus recording of mitochondria from complex III assembly factor Bcs1 gene knockdown D. melanogaster individuals (Mito CIII KD), (Brischigliaro et al., 2021). (D) Complex IV activity recording of wild-type mitochondria in absence (Mito) or presence of the inhibitor (Mito + KCN) plus recording of mitochondria from complex IV assembly factor Coa8 gene knockdown D. melanogaster individuals (Mito CIV KD), (Brischigliaro et al., 2019). (E) Complex V activity recordings of wild-type mitochondria in absence (Mito) or presence of the inhibitor (Mito + Oligomycin). (F) CS activity recordings of wild-type mitochondria (Mito).