FEMS Microbiology Letters 16 (1983) 307-312 307 Published by Elsevier Biomedical Press Mitochondrial cytochromes of A canthamoeba castellanii: Oscillatory accumulation of haemoproteins, immunological determinants and activity during the cell cycle David Lloyd, Steven W. Edwards *, Judith L. Williams and J. Barbara Evans Department of Microbiology, UniversityCollege, Newport Road, Cardiff CF2 1TA, Wales, U.K. Received 6 August 1982 Accepted 18 August 1982 1. INTRODUCTION Although the molecular mechanisms underlying biogenesis of mitochondria are being elucidated in detail, insights into the overall control of mito- chondrial development in the growing organism are still lacking [14]. Sequences of events involved in the elaboration of mitochondrial membranes, the assembly and integration of their components, and the expression of their activities; requires the use of synchronous cultures [15]; accumulation of cytochromes and changes in activity of the respira- tory chain have been studied in several lower eukaryotes. In cultures of Saccharomyces cervisiae synchronized by starvation and refeeding, each of the respiratory chain cytochromes increased con- tinuously [3]. Discontinuous accumulation was ob- served in Schizosaecharomyces pornbe [18-20], where glucose-repressed cells showed two maxima per cell cycle for cytochromes a 3 and b-563; these maxima coincided with maxima in mitochondrial ATPase activity [5]. Oscillatory accumulation of mitochondrial ATPase has also been demonstrated during the cell cycle of Acanthamoeba castellanii [9]; enzyme * Present address: Department of Medical Biochemistry, Uni- versity Hospital of Wales, Heath Park, Cardiff CF4 4XN, U.K. assays in parallel with immunological determina- tions showed that changes in ATPase activity were accompanied by changes in ATPase protein, and also by activities of the naturally occurring inhibi- tor protein. Like total cellular protein and RNA in this organism [6], the oscillations in mitochondrial ATPase have a period of just over 1 h, so that in an 8-h cell cycle time at 30°C, seven maxima were observed. In the present study we have examined changes in spectrophotometrically detectable mitochondrial haemoproteins, cytochrome c oxidase activity and immunologically reacting cytochrome c oxidase protein, and show that all these components of the mitochondrial inner membrane oscillate with a period of approx. 1 h in cultures synchronized by a minimally perturbing selection method. 2. METHODS 2.1. Maintenance and growth of the organism A. castellanii was maintained and grown, with shaking at 30°C exactly as described previously [7]. Cells were counted in a Fuchs-Rosenthal haemocytometer (Baird and Tatlock, Chadwell Heath, Romford, Essex) after a suitable dilution in 50 mM MgC12, pH 7.4). 0378-1097/83/0000-0000/$03.00 © 1983 Federation of European Microbiological Societies Downloaded from https://academic.oup.com/femsle/article-abstract/16/2-3/307/493833 by guest on 21 May 2020