Methylamine (methylammonium ion) entered Saccharomyces cerevisiae X2180-A by means of a specific active transport system. Methylamine uptake was pH dependent (maximum rate between pH 6.0 and 6.5) and temperature dependent (increasing up to 35 C) and required the presence of a fermentable or oxidizable energy source in the growth medium. At 23 C the Vmax for methylamine transport was-17 nmol/min per mg of cells (dry weight) and the apparent Km was 220 MM. The transport system exhibited maximal activity in ammonia-grown cells and was repressed 60 to 70% when glutamine or asparagine was added to the growth medium. There was no significant derepression of the transport system during nitrogen starvation. Ammonia (ammonium ion) was a strong competitive inhibitor of methylamine uptake, whereas other amines inhibited to a much lesser extent. Mutants selected on the basis of their reduced ability to transport methylamine (MeaR) simultaneously exhibited a decreased ability to transport ammonia.

A reduced nicotinamide adenine dinucleotide (NADH)-dependent glutamate synthase has been detected and partially purified from crude extracts of Saccharomyces cerevisiae . The enzyme is specific for NADH, glutamine, and α-ketoglutarate ( K m values of 2.6 μM, 1.0 mM, and 140 μM, respectively) and has a pH optimum between 7.1 and 7.7. The stoichiometry of the reaction has been determined as 2 mol of glutamate synthesized per mol of glutamine consumed. Glutamate synthase can be distinguished from either of the glutamate dehydrogenases of yeast on the basis of its substrate requirements and behavior during agarose gel and ion exchange chromatography. Variations in the specific activity of glutamate synthase, which occur in response to changes in the growth medium, are similar in character to those observed with the nicotinamide adenine dinucleotide phosphate-dependent (anabolic) glutamate dehydrogenase.

Yeast strains ,1278b and Harden and Young, which synthesize only an internal constitutive form of L-asparaginase, do not grow on D-asparagine as a sole source of nitrogen, and whole cell suspensions of these strains do not hydrolyze D-asparagine. Strains X2180-A2 and D273-1OB, which possess an externally active form of asparaginase, are able to grow slowly on i)-asparagine, and nitrogen-starved suspensions of these strains exhibit high activity toward the 1)-isomer. Nitrogen starvation of strain X2180-A2 results in coordinate increase of 1>-and L-asparaginase activity; the specific activity observed for the D-isomer is-20% greater than that observed for the I-isomer. It was observed, in studies with cell extracts, that hydrolysis of D-asparagine occurred only with extracts from nitrogen-starved cells of strains that synthesize the external form of asparaginase. Furthermore, the activity of the extracts toward the D-isomer was always higher than that observed with the L-isomer. A 400-fold purified preparation of external asparaginase from Saccharomyces cerevisiae X2180-A2 hydrolyzed r-asparagine with an apparent Km of 0.23 mM and a Vmax of 38.7 ,umol/min per mg of protein. D-Asparagine was a competitive inhibitor of Lasparagine hydrolysis and the Ki. determined for this inhibition was approximately equal to its Km. These data suggest that D-asparagine is a good substrate for the external yeast asparaginase but is a poor substrate for the internal enzyme.

Saccharomyces cerevisiae contains two distinct L-glutamate dehydrogenases. These enzymes are affected in a reciprocal fashion by growth on ammonia or dicarboxylic amino acids as the nitrogen source. The specific activity of the nicotinamide adenine dinucleotide phosphate (NADP) (anabolic) enzyme is highest in ammonia-grown cells and is reduced in cells grown on glutamate or aspartate. Conversely, the specific activity of the nicotinamide adenine dinucleotide (NAD) (catabolic) glutamate dehydrogenase is highest in cells grown on glutamate or aspartate and is much lower in cells grown on ammonia. The specific activity of both enzymes is very low in nitrogen-starved yeast. Addition of the ammonia analogue methylamine to the growth medium reduces the specific activity of the NAD-dependent enzyme and increases the specific activity of the NADP-dependent enzyme. Saccharomyces cerevisiae synthesizes two glutamate dehydrogenases (3, 4, 10, 11). One of these enzymes is specific for nicotinamide adenine dinucleotide (NAD)