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EnzymeActivityOrganellarMarkers.md 1.47 KiB

2.4 Measurements of enzyme activity of organellar marker enzymes

The distribution of peroxisomes, mitochondria, and plastids within the four organellar fractions were examined using enzymatic marker proteins. All enzyme assays were performed photospectrometrically in a plate reader (SynergyH1, BioTek) at room temperature. For each sample three technical replicates were measured. Total protein concentration of the fractions was determined using the Pierce BCA protein assay kit (ThermoFisher Scientific). Enzyme activity was expressed as units per mg total protein. The activities of the following marker enzymes were analyzed: Catalase for peroxisomes (Breidenbach et al., 1968), fumarase for mitochondria (Nishimura and Beevers, 1981), and phosphoglycerate dehydrogenase for plastids (Benstein et al., 2013).

  • Breidenbach, R. W., Kahn, A., and Beevers, H. (1968). Characterization of glyoxysomes from castor bean endosperm. Plant Physiol. 43, 705–713. doi: 10.1104/ pp.43.5.705
  • Nishimura, M., and Beevers, H. (1981). Isoenzymes of sugar phosphate metabolism in endosperm of germinating castor beans. Plant Physiol. 67, 1255–1258. doi: 10.1104/ pp.67.6.1255
  • Benstein, R. M., Ludewig, K., Wulfert, S., Wittek, S., Gigolashvili, T., Frerigmann, H., et al. (2013). Arabidopsis phosphoglycerate dehydrogenase1 of the phosphoserine pathway is essential for development and required for ammonium assimilation and tryptophan biosynthesis. Plant Cell 25, 5011–5029. doi: 10.1105/tpc.113.118992