October .0, 1948.

Dr. M. tie Doudoroff,
Dept. Bacteriology,
University of California,
Berkeley, California.

Dear Mike:

I trust that you received those cultures OK which I sent you sone time
age. These fermentative mtants are not the most stable things in the world,
especially those involving glucose assimilation, and they really have to be
repurified occasionally by streaking them out on FMB agar and repicking white
colonies.

What's the latest on phosphorolysis in F. coli? I've gotten aut » very active
cell free prep. of b-galacteeidase from K-l2, using cells grown on lactoseesynthetic
medium and geingting thé in the Booth Green Mill. Assays with O-nitropheyl galactoside
which 4s working beautifully. The enzyme does not respgnd to phosphate, and is
active in ita absencs (14.6. considerably less than 107? YU), and I think must be
taken as hydrolytic. Na” up to M/50 or higher stimulates about 100%, but is not
essentially. Taking the "H enzyme" as the base, (i.e. low salt concentration),

K and Cs are inert, aa is WH,; Li and especially Rb are inhibitory, the inhibition
being reversed by either K of Na. A number of substituted NH, Lons are also inhi-
bitory, iy conclusion is that the enzyme can function with tarying efficiency

with i, metal ions, etc., but that all of these compete with each other for the
enzyne surface, Mg doea not seem to be necessary; nevertheless, F ds inhibitory
only in the presence of phosphate, and the inhibition 1s slightly aggravated by

the addition of Mg. Pyrophosphate is also inhibitory, but gitrate is not. I win-
der whether the fluoro-phosphate complex can tie up other non-'ig enzymes. On the
other hand, ig may be present, but with an exceedingly low dissociation constant.
The enzyme preps can be purified to some extent with Ammonium sulfate pptn., and
can be dried down with acetone without mich L.activation. with the chromogenic
substrate is about 2x 107%, with reasonably good linear 1/8— 1/v plots. Most re-
ducing sugara tie up the enzyme, whether they are utilized by the organism or not,
presumably competitively, but In still working out the details. The enzyme 19 formed
in measurbble amounts only after adaptation in lactose or other galactosides. It is
not produced by any of the several different mutants tested. “he one-to-one theory
does not seem to apply here. For comparative purposes, I've been running some
eomparative tests on Snell's Lactobacillus bulgaricus, His growth observations are
verified (i.c., lactose/, glucose;galactose-). By grinding, I ve gotten out a lactase
which i9 much less active than the one from coli, is also stimulated by Na, but also
needs Mg and probably phosphate, alth-ough aybprepa aren't clean enough yet to be
certain. The bulgaricus lactase is not nearly so rugged as the coll, and I'm having
much more trouble with it.

let's hear from you, best rega‘dsz