For aerobic growth, the mineral-salts medium A of
Davis and Mingioli (7) was used without citrate. For
anaerobic growth, the same medium was supplemented
with 2.4 mg of L-cystine per liter (11). Additions
to the minimal medium are indicated in the
legends to tables and figures. Tryptone, nutrient broth,
casein hydrolysate, and yeast extract were Difco
products.
Growth was measured turbidimetrically at a wavelength
of 570 nm with a Coleman Junior spectrophotometer.
One optical density unit corresponded to
0.93 mg of protein per ml, as determined, after lysis
of the cells, by the method of Lowry et al. (19) with
egg white lysozyme as standard. For determination of
asparaginase II, 15-ml samples were pipetted into ice;
the cells were collected by centrifugation at
14,000 X g, washed twice, and finally suspended in
0.8 ml of 0.1 M NaCl buffered at pH 7.8 with 50 mM
tris(hydroxymethyl)aminomethane-chloride. Cells
were lysed by treatment with ethylenediaminetetraacetic
acid (EDTA) and lysozyme. The suspension
was made 1 mm in EDTA and kept at 0 C for 8 min.
After the addition of magnesium acetate to a concentration
of 1 mm, 1 mg of egg white lysozyme (Worthington
Biochemical Corp.) was added to give a final
volume of 1 ml. The samples were incubated for 15
min at 37 C. Samples (0.01 ml) of these lysates were
assayed for asparaginase II either directly or after
dilution with buffered saline, in a final volume of 0.06
ml, by following the conversion of '4C-asparagine to
aspartic acid with rapid chromatography on ion-exchange
paper (Ecteola, Whatman ET81) (6). The
specific enzymatic activity is given as micromoles of
aspartic acid produced per hour per optical density
unit of the culture. At the low concentration of asparagine
used in this assay (0.1 mM), asparaginase I does
not contribute significantly to the products formed