PYRUVATE HOLOCARBOXYLASE AND APOCARBOXYLASE CONTENT OF BIOTIN-DEFICIENT BAKERS-YEAST AND THE CHARACTERISTICS OF THE HOLOENZYME FORMATION IN PERMEABILIZED CELLS

The holo- and apocarboxylase proteins in baker's yeast grown in a chemostat at different biotin concentrations (from 0.1-200 μg/l) and on different carbon sources were assayed. The growth and the type of metabolism are considered with respect to the activity of the enzymes involved in oxaloacetate regeneration (pyruvate carboxylase and isocitrate lyase activities). In order to assay the level of apocarboxylase protein in the cells the characteristics of the pyruvate holocarboxylase formation in permeabilized cells were studied and thereby an assay method was developed. The pyruvate carboxylase activity of the cells grown in a medium with 4% glucose as the carbon source was almost constant from the lowest biotin concentration up to a biotin concentration of 10 μg/l, after which it rose and obtained a maximum at a biotin concentration of about 50 μg/l. The content of the apocarboxylase protein was maximal at 0.5 μg/l biotin, and then exceeded the level of the active pyruvate carboxylase protein by a factor of about 2.5. With increasing biotin concentration in the medium the content of apocarboxylase protein decreased and was negligible in cells grown at biotin concentrations higher than 100 μg/l. The total content of pyruvate carboxylase protein (i.e. apo- + holoenzyme) was roughly constant over a wide biotin concentration range (from 0.5-15 μg/l), the maximum being only double the minimum. At a biotin concentration 50 μg/l, where the maximum yield was reached, the cells still contained pyruvate apocarboxylase. The rapid increase in yield found around a biotin concentration of 10 μg/l correlates, on the basis of measured enzyme activities, more with the appearance of activity of glyoxylate cycle enzymes than with the increase in the activity of pyruvate carboxylase. When cells were growing on ethanol with biotin as the growth limiting factor, the cells still used biotin for the formation of pyruvate holocarboxylase, and proportionally more of the total content of pyruvate carboxylase protein was in the form of holoenzyme than in the cells growing on glucose under biotin limitation. The existence of urea amidolyase apoprotein in yeast cells grown with urea as the sole nitrogen source under biotin deficiency is reported. The presence of acetyl-CoA apocarboxylase in biotin-deficient cells could not be demonstrated. © 1979 Springer-Verlag.