Adaptive Enaynes
September - November, 1950

Preface
After working three montha on adaptive enzymes of U. Coli and making very little

headway, it seems worthwhile to summarize the reasons for the study and the propress
made. This summary may then provide a basis for deciding what experimenta, if any,
should be done when it is possible to resume work in biophysics.

Reasons for program

Adaptive enzynes appeared to be an attractive point of attack on the nore
general problem of protein synthesis because in the adaptive process the cell could
te directed to synthesize one particular protein as contrasted to the duplication
of all cellular components that occurs during growth. Furthernere, adaptation is
generally possible even in a nitrogen deficient medium where norial growth stops.
Yor this reason it appeared that perhaps the process of adaptive enzyne fornation
involved only certain phases of protein synthesis and these particular steps might
be more understandable than the complete process.

It is known that the formation of adaptive enzymes requires (with certain
exceptions) the presence of the appropriate gene (Spiegelman, “onod, Lwoff) and
the substrate. However, once initiated, the synthesis can continue in the absence
of the gene (Spiegelman) or in the absence of the substrate (Hinshelwood). Fur-
thermore, most enzymes show some adaptive behaviour as they are synthesised in
greater quantity in the presence of their substrate. Consequently, adaptive
enzynes are not unusual types of proteins and any insight into their formation
should apply quite generally.

As adaptive enzyne fornation proceeds without external nitrogen or sulfur
sources, the origin of the nitrogen and sulfur was of interest. In the case of
yeast, Splegelman found that one adaptive enzyme was forsed as another diminished

indicating perhaps that one particular enzyme was transformed into another. If,
=2=
in this process, the enzyme was broken down into amino acids and those were
then resynthesized, an exchange of labelled amino acida might be observed.

Under various conditions the concentration of RNA and DNA in the cell varies,
if these are involved in the adaptation process, then some correlation might be
observed between their concentration in the cell and the rate of adaptation.

Finally, the intensive study of any particular process using all the available
techniques (particularly P and 3") usually yields sone new information. on this
basis the study of adaptive enzyzes was atarted.

Choice of Gell

KB. Coli was used because of our previous experience and because adaptation
is much more rapid than in yeast.

Choice of insyne

The sugars available in the laboratory were tested as substrates using
glucose adapted cells and neasuring oxygen uptake in the warburg. The results
Table I show that lactose and arabinose are the nost suitable az there is a very
low initial rate, a rapid adaptation and a high final rate.

Table I
Initial Rate Final Rate Adapting Tine

Glucose 1co 400 . ~
Mannose 100 160 -
Galactose 50-100 120 30 minutes
Arabinose 10 LOO 40 minutes
Ribose 30 30 3 hours +
Xylose 12 75 2 hours
Sucrose 10 3 -

Lactose 5 190 4) minutes

Maltose 100 130 -

 

 
-3-

Sffect of “ediunm
i£ffect of NH, on glucose netacolism

The oxygen uptake with glucose as substrate is roughly doubled by the
addition of NH 4 to the nedium, This has been ascribed to the increased energy
renuirenents due to growth which affects giucose metabolism by using up ATP
and saking ADP available as a phosphate acceptor.

This theory is not acceptable because the effect of HH A is present with
1) cells in a lag phase that are not growing. 2 ) celis of such high density that
there is no growth. 3) Lysine deficient cells which cannot grow because of the
absence of lysine.

There are probably sone synthetic processes occurring in the presence of NH,
which account for the increased uptake of oxygen (and a comparable increase in
uptake of PO 42° One such process could. be the synthesis of amino acids which then
diffused out into the mediun. in one experinent we looked for radioactive organic
sulfur in the supernatent of lysine deficient cells incubated with glucose, NH 4
and $"0 4, but without lysine. After two hours incubation a small quantity of
non-sulfate sulfur was found, some of which would precipitate with an excess
of cystine. These experiments should be followed up.

Effect of NH, on adaptation. |

The same effect of NH, is found with lactose or arabinose as substrates,

4. @e, the final rate is about double in the presence of NH, but the tims
required for adaptation is the same. NH, does not appear to be involved in the
formation of the adaptive enzyné.

. Other ingredients of the medium

The adaptation time was not affected by the addition of methionine using
normal cells or b: the addition of lysine with lysine deficient cells. The

effect of K, Mg, and $0, and PO, on adaptation should be tested. However ,

 
~4=
me eve not Scand any vequire-ents for the adaptation rrecers similar to the
comparable requirement for growth, It appears that the cell containg the
material for the new protein within itself and only the substrate and energy
(derived from the substrate or from cellular reserves) are required to initiate
the transformation.
State of the Cell
Growing cells, cells grown on lew glucose and cells grown on Pull glucose
show increasing times for adaptation similar to the increasing tine required
for the initiation of growth. This would be expected if one of the rate limite
ing factors in growth is a process sixilar to adaptation,
Low K and ig cells
Cells were grown in a low K low ig nedium, then washed and put in media
containing Mg only K only and a control containing K and Mg for further growth.
This was clearly linited in both cases, linear rrowth being observed in the K
deficient sedium and a levelling off in the Mg deficient uodiun, After the
density had doubled the cel]Swere washed and tested for adaptation to lactose
NH, in a medium with adequate K and Mg. The adaptation tine was doubled by this
pretreatzent in deficient media. Growth on Clucoge in a complete medium was
also delayed. These delays were probably not due to the time required to take
up the lacking K or Mg as the oxygen uptake (with glucose) returned to nornal
a8 scon as the Kf\Mg was restored. The main object of this experinent was to
test adaptation of cells with different DNA/RNA ratios. However, this failed
a3 neasurevents of RNA and DNA (by oJ showed that both the DNA and RNA were
normal.
Nitrogen deficient cells
If cells contain protein precursors (peptides?) these night be the source
of nitrorcen for the adaptive enzyzes, This idea was tested by incubating the
=

ceils with -lucose in a ‘free aediuna before adaptation. 'o effect xas found
on the rate of adaptation. As a more stringent test cells were adapted to
arabinose in N~free nediun and then adapted te lactose. This pre~adantation
did not affect the rate of the second adaptation. Consequently, there is no
evidence that the nitrogen of adaptive enpynes is forned from any limited
supply of precursors,
Losa of adaptive enzynes

Unlike Spiegelnan we have not been able to reverse the adaptation in any
of the treatments tried. ‘These include incubation up to 24 hours with glucose,
galactose, arabinose, = Ni, and in buffer only. The lactose adaptation remaing
after 5 generations of growth on glucose. We have not attempted the prolonged
reversion nethods of Ninshelwood, Ina few cases a slight loss (20 4) has been
found but this has not bean repeatable.

isolation of the lactose enzyne |

it was found that supernatents fron lactose cells and extracts of lactose
grown cells speeded up the adaptation. This ras traced to the presence of the
lactase enzyne. As & quick test 2 ml of glucose grown cells (cld) 1/2 ml of
lactose and 1 drop of ethylene blue were used. With glucose instead of lactose
the 1. B. was decolorized in 4 minutes instead of 40. A strong enzyme solution
added woul: also decolorize in 4 minutes. This test was used to follow the
lactose activity of various preparations. Roughly 50 of of the enzyne activity
could be extracted from the cells or an acetone power of the cells by shaking
at 60 cycles with glass beads. The enzyme could be precipitated with CHIL, ) 2
SO, or 75 Op ethyl alcohol, it was stable in solution, could be dialyzed, and
was destroyed by boiling. No activity was found in sinilar extracts of glucose
grown cells. At this point Lederberg's article appeared which confirmed vhat we

Knew but did not help in further purification.

 
on

Cells were rown with P* and S* and these tracers used together were

helpful in following purification. The ~ethods used were not sutisfactory out

some little crogress was nade.

i.

3.
4e

de

be

Te
&.

Very little can be extracted from acetone powder with PO, buffer .

H 4.5-8.

About 50 is extracted on shaking with glasa beads (cells or acetone
powder )«

A second extraction does no good,

The extract is cloudy and can be cleared by repeated centrifugating
without loss of activity.

Pregipitation by alcohol and NH sO depends on concentration and
previous purification and is not reliable.

Precipitation by “iH, 50 23 + saturated does improve the $/P ratio but
causes a loss of activity.

No improvenent was achieved in the enzyne /S ratio.

The spectrum of the active extract looks like pure nucleic acid and

no way was found to renove the NA and leave the enzyze.

Work on purification was interrupted. As a pure enzyne is highly desir~

able (see below) further trials may be attempted later, particularly if Lederberg

has any further information,

Uptake of radioactivity during adaptation

Cells incubated with glucose and lactose one hour in N free nediun with

radioactive cystine,

Total
‘TCH Sol

TCA ppt

Glucose Lactose
37. 22.
24 1 ,
5.8 | 5.3

This uptake corresponds to .03 ng of sulfur per ul of cells (in the TCA ppt) or

 
—ja

1/100 of the total sulfur content. As there is no {fference between glucose

acd lactose no conclusions can be drawn. Furthermore, the lonses from both groups
of celis on further incubation were negligible whether the Second incubation

was carried out in glucose or lactose. This is not surprising as the lactose

activity is not renoved.

Uptake of Sx0,

Cells incubated 1 hour + NH, with glucose and lactose

Glucose Lactose
+, oNH, +NH, muHy
Total 546 el0 1.5 12
Protein 2 el 0037 47 0045

In presence of ', there is such nore SO, uptake vdth glucose due to more growth,
ete. ‘without NH, there is slight excess with lactose possibly due to formation
of adaptive enzyn7e. |
4s the effects are small it will be possible to detect incorporation of
tracers into the adaptive enzymes (which may be 1/10 of of the total protein)
only bys
4) Preferentially removing the enzyne.
b) Purifying the enzyme.
The enzynes studied so far cannot be removed. Catalase (studied by
Abelson) does decrease and might offer possibilities. Purification seems difficult.
Discussion
Most all the indications obtained so far are negative. The most consistent
picture is that some cellular protein (probably an enzyme) is slightly altered
by contact with lactose to form the lactase enzyne.
E> EY x

 
Once : is converted to , ¥* hot E is gynthesized by the cell even in the
absence of lactose. As 5 is a slightly wre stable configuration, reversion
z”. . B may occur after many generations (Hinshelwood). The gene may not be
involved in this transfornation at all. In the absence of the gene an alternate
form of E aay occur which camot transform to E* « Aacording to this picture
there would be no uptake of naterial from the medium and no exchange of
amino acids. The only hope of proof would be in obtaining highly purified
lactase entyne and denonstrating sinilarity to a protein extracted by the sane
process from glucose adapted celis which did not have lactase activity.

This transformation might conceivably be induced in vitre.

Rurther work requires either a good zethod for purification or a fresh
approach.e

Richard B. Roberts