Metabolism of Pyruvate by Starved Cells of Chlorella elipsoida

Abstract

The metabolism and function of the organic acids of the tricarboxylic acid (TCA) cycle in plants and microorganisms is not clearly understood. Evidence from the effects of inhibitors, the incorporation of radioactivity from specifically labeled acids, and the isolation of the acid intermediates and the active enzymes (3, 4, 11, 24, 26, 30) indicate that the cycle can operate. By feeding acids as a sole source of carbon to Chlorella cells depleted of their polysac-charide reserves the functions of the TCA cycle can be isolated from those of the glycolytic and pentose phosphate pathways. Their efficiency in promoting growth or the synthesis of reserve material will be a measure of the ability of the TCA cycle in supplying the carbon skeletons, energy, and reducing power need-ed for the synthetic reactions. In a few instances it has been demonstrated that the acids of the TCA cycle support neither growth (7) nor the incorpora-tion of NH3 (16, 23) as efficiently as glucose. Con-versely, it has been shown that in a number of micro-organisms the disappearance of the acids from the medium is greatly increased with the change from resting to growing conditions (18). In this report the kinetics of pyruvate utilization. the fates of the various carbons of pyruvate, and the factors limiting its utilization are described in an attempt to indicate the importance of the TCA cycle in synthetic and energy producing reactions. Materials & Methods Cells of Chlorella clipsoida (culture supplied by Prof. A. Pirson) were grown in a complete mineral nutrient solution in liter flasks in the light (14). Air supplemented with 5.0 % COO was constantly bubbledl through the flasks. The cells were harvest-ed 3 to 4 (lays later, centrifuged, washed twice with distille(l w-ater, ancl allowed to shake in the dark for 16 to 24 hours. Before the experiment they were recentrifuged and enough distilled water was added to give a specific density which was checked photo-metrically. Aliquots of cells, 1 or 2 ml, together with potas-siumil phosphate buffer (final concentration 0.017 MI,