Summary: Changes in cell composition of CBS 14 were monitored during growth of batch cultures with NHCl and glutamate as nitrogen sources. Carbohydrate was synthesized at the expense of lipid in NH -grown cells, whereas in glutamate-grown cells lipid accumulation was predominant. Total biomass and protein concentration were similar in both cultures. Uptake of [U-C], [1-C] and [6-C]glucose, and evolution of CO from these sources, by washed suspensions of cells grown on glutamate revealed the flux of carbon during glucose dissimilation was principally via the Embden-Meyerhof pathway (72%), with 28% being metabolized by the pentose phosphate pathway. Both urea and glutamate, when added to the cell suspensions, significantly stimulated glucose catabolism, with the flux of carbon via the former pathway increasing to about 89% of the total. Phosphofructokinase (PFK) was implicated as the likely controlling enzyme to explain these events.

PFK was only detected in extracts prepared from the yeast grown in a carbon-limited (nitrogen-excess) medium; no activity was detected in extracts of cells grown in nitrogen-limited medium. The presence of a protease in these latter extracts was revealed. PFK was purified 92-fold to a final specific activity (in the presence of 10 mM-NH ) of 4.2 units (mg protein) and exhibited one broad band on polyacrylamide gel electrophoresis. The apparent mol. wt () of the enzyme was approx. 700000. The major properties of the enzyme were examined to determine its regulatory role during lipid biosynthesis. Unlike the enzyme from no inhibition was found with 10 mM-ATP. ADP was not inhibitory either. NH ions increased activity 11-fold by increasing the affinity of the enzyme for both fructose 6-phosphate and ATP. K ions also stimulated activity but to a lesser extent. Activity was severely inhibited by citrate, isocitrate and -aconitate but this inhibition was dramatically alleviated by NH . Inhibition by citrate was competitive with fructose 6-phosphate in the absence of NH ions. The values for citrate were 1.0 mM (with no NH ) and 7.2 mM (with 10 mM-NH ). Long-chain fatty acyl-CoA esters had no significant inhibitory effect. It is concluded that the interplay between the prevailing intracellular concentrations of NH and citrate is the major determinant of the activity of PFK and thus governs the extent to which glucose is converted either to lipid or carbohydrate.


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