Subcellular distribution of pyruvate-degrading enzymes in Chlamydomonas reinhardtii studied by an improved protoplast fractionation procedure

  1. Karlheinz Kreuzberg*,
  2. Gerd Klock,
  3. Dorte Grobheiser

Article first published online: 18 FEB 2008

DOI: 10.1111/j.1399-3054.1987.tb09229.x

Issue

Physiologia Plantarum

Physiologia Plantarum

Volume 69, Issue 3, pages 481–488, March 1987

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How to Cite

Kreuzberg, K., Klock, G. and Grobheiser, D. (1987), Subcellular distribution of pyruvate-degrading enzymes in Chlamydomonas reinhardtii studied by an improved protoplast fractionation procedure. Physiologia Plantarum, 69: 481–488. doi: 10.1111/j.1399-3054.1987.tb09229.x

Author Information

  1. Botanisches Inst, der Univ. Bonn, Kirchallee 1, D-5300 Bonn 1, FRG. Dedicated to Prof. A. Betz on occasion of his 65th birthday.

* Botanisches Inst, der Univ. Bonn, Kirchallee 1, D-5300 Bonn 1, FRG. Dedicated to Prof. A. Betz on occasion of his 65th birthday.

Publication History

  1. Issue published online: 18 FEB 2008
  2. Article first published online: 18 FEB 2008
  3. Received 20 October, 1986; revised 10 November, 1986

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Keywords:

  • Algae;
  • chloroplasts;
  • mitochondria;
  • pyruvate dehydrogenase complex;
  • pyruvate formate-lyase;
  • starch fermentation

The subcellular distribution of pyruvate-degrading enzymes has been determined in Chlamydomonas reinhardtii (Dangeard) by protoplast induction with autolysine, dig-itonin lysis and further fractionation by differential centrifugation using a Percoll cushion. Mitochondrial and plastidic fractions contained intact and physiologically competent organelles - RC 1.7, ADP/O 2.7 and rate of malate oxidation 76 nmol O, (mg protein)-1min-1 for mitochondria, CO2; fixation 46.8 μmol (mg Chi)-1 h-1 for chloroplasts.

Results from protoplast fractionation were further confirmed by the determination of enzyme activities within trypsin-treated organelles. Mitochondria (formate fermentation) and chloroplasts (chlorofermentation) were shown to possess the capacity for anaerobic pyruvate degradation. Pyruvate dehydrogenase (NAD+, EC 1.2.4.1), pyruvate formate-lyase (EC 2.3.1.54) and lactate dehydrogenase (NADH, EC 1.1.1.27) showed equal distribution between mitochondria and chloroplasts, whereas activities of phosphotransacetylase (EC 2.3.1.8) and acetate kinase (EC 2.7.2.1) were only detectable in the mitochondrial fraction. NADH- and NADPH-dependent activities of both alcohol dehydrogenase (EC 1.1.1.1) and aldehyde dehydrogenase (acylating, EC 1.2.1.10) were localized in the mitochondrial and cytoplasmic or the plastidic and cytoplasmic fractions, respectively, whereas pyruvate decarboxylase (EC 4.1.1.1) was only detected in the cytoplasmic fraction.

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