Reprinted from JouURNAL OF BacrERIOLOGY
Vol. 76, No. 1, pp. 1-14, July, 1958
Printed in U.S.A.

REGULATION OF ENZYMATIC ACTIVITY IN THE INTACT CELL: THE
B-D-GALACTOSIDASE OF ESCHERICHIA COLI

BORIS ROTMAN?

Institute for Enzyme Research and Department of Genetics, University of Wisconsin,
Madison, Wisconsin

Received for publication November 1, 1957

Large differences in enzymatic activity are
often found between intact cells and extracts.
Lacking a better explanation, it has frequently
been assumed that the penetration of the sub-
strate is the rate limiting step in the intact cells.
The experiments reported here were designed to
test several hypotheses which might account for
the discrepancy.

The @-galactosidase (lactase) in Escherichia
colt was sclected as a model system because of the
sensitivity and ease of assay, together with the
extensive knowledge of the induction, isolation,
and general properties of the system (Lederberg,
1950; Cohn and Monod, 1951; Kuby and Lardy,
1953; Rotman and Spiegelman, 1954). Deere
(1939) reported that drying HH. coli-mutabile
appeared to “activate” the lactase. Lederberg
(1950) studied the @-galactosidase of E. colt
strain K-12 with a chromogenic substrate and
found discrepancies of 10- to 47-fold between the
activity of the intact cells and of disrupted cells
or extracts. In addition, Lederberg found that the
activity of the intact cell was protected from pH
changes and inhibitory cations.

The hypotheses tested in this investigation
were: passive diffusion, inhibitors complexed
with the enzyme, and product inhibition of the
enzyme, All three were ruled out by the data
presented below. Consequently, a more com-
patible proposal is given which assumes the
existence of a specifically induced penctration
mechanism. During the course of this work,
Monod and his associates postulated an inducible
enzymelike mechanism for specific control over
galactoside penetration and termed the agent a
“permease” (see Cohen and Monod, 1957). The

! Paper no. 676 of the Department of Genetics.
Supported by an American Cancer Society Insti-
tutional Grant and by a grant from the United
States Public Health Service.

2 Present address: Instituto de Quimica Fisio-
logica, Borgoho 1470, Escuela de Medieina,
Santiago, Chile.

experiments described here were completed before
the data supporting the permease hypothesis
became available and therefore were not designed
to test this hypothesis. Nevertheless, when our
findings and the permease data were compared,
basic similarities and differences between the
two hypothetical penetration systems became
apparent, Possible ways of reconciling the two
views are discussed.

MATERIALS AND METHODS

Bacterial strains and culture conditions. To avoid
the additional variable of adaptation, the strain
W-1317, a constitutive mutant (K-12) of E. colt
was employed for most of the experiments. This
mutant, obtained by selection on neolactose
agar, synthesizes large amounts of 8-galactosiclase
in the absence of external inducer (Lederberg,
1951). Experiments involving adaptation were
performed with the parent K-12 stock. Davis’
(1949) minimal medium with 0.1 per cent glycerol
as the carbon source was used throughout. Unless
otherwise specified, the cells were grown in con-
tinuous culture in a modified chemostat (Rotman,
1955a@) with an average gencration time of 3 hr.
For most of the experiments, 12 ml of culture
were withdrawn from the growth tube. The cells
were then washed twice with 20 ml of cold 0.02
mM sodium phosphate buffer at pH 7.2 by centrif-
ugation at 14,000 x G for 3 min in the high
speed head of an International centrifuge. The
cells were resuspended in 12 ml of the same buffer
to give an average nitrogen content of 70 ug per
ml.

Some experiments required the use of bacteria
freed of exogenous salts. Repeated washing with
water proved inefficient in that the bacteria
ceased to pack in the centrifuge even at high
speed. Consequently, the cells were passed
through an ion exchange column as described by
Rotman (1956). The deionization procedure and
subsequent storage in distilled water at 0C did
not impair cell viability and recoveries were
2 ROTMAN

quantitative. After this treatment, less than
0.04 ppm of sodium or potassium were found to be
present in the supernatant by flame photometry.
Chemacal reagents? 0-Nitrophenyl-8-p-galacto-
pyranoside was synthesized according to the
method of Seidman and Link (1930) or procured
from Mann Laboratories. p-Nitrophenyl-6-p-
galactopyranoside, — p-nitrophenyl-a@-L-arabino-
pyranoside, and o-nitrophenyl-a-L-arabinopyran-
oside were kindly supplied by Dr. K. P. Link.
Methy1-8-p-thiogalactopyranoside was obtained
through the kind assistance of Dr. A. Novick.
Galactose (Pfanstichl) was recrystallized four
times from aqueous ethanol and dried at 60 C
under vacuum for 24 hr. Lysozyme and ribo-
nuclease were obtained from Armour Co. The
lactose was from Difco Laboratories, but. all
other reagents were analytical grade. Water
was deionized and then glass distilled.
Analytical methods. 6-p-Galactosidase was
measured with ONPG as a chromogenic substrate
(Lederberg, 1950). The test solution (0.05 to 0.2
ml) was added to 2 ml of m/1000 ONPG dissolved
in the buffer described above. The reaction was
stopped after 1 to 15 min incubation at 30 C by
addition of 10 ml of 0.1 m sodium carbonate.
The o-nitrophenol was determined colorimetri-
cally at 420 my. Enzymatic activity was expressed
as mumoles ONPG hydrolyzed per minute.
Benzene-treated cells were prepared by shaking
2 ml of a washed suspension of cells with 0.1 ml of
benzene. After incubation for 10 min at 30C,
0.05 ml was withdrawn from the tube and assayed
as indicated above. A 0.2 ml sample of the un-
treated suspension was used to assay the enzy-
matic activity of the intact cells. The cells were
centrifuged and removed after stopping the
reaction with sodium carbonate. Blanks were
prepared by adding the cells after the sodium
carbonate. The enzymatic activity obtained when
cells were treated with benzene was taken as the
standard. The activity of the intact cells showed
linear kinetics on a Michaelis-Menten plot up to a
concentration of 3.4 x 107? mM ONPG, thereby
confirming and extending Lederberg’s (1950)
original observations.
Ribose was determined by the orcinol test
(Kerr and Seraidarian, 1945) and desoxyribose

’ Abbreviations: ONPG = o-nitropheny!-6-p-
galactopyranoside, DNA = desoxyribose nucleic
acid, RNA = ribose nucleie acid, 8-galactosidase
= @-p-galactosidase.

IvoL. 76

by the diphenylamine test (Sevag et al., 1940).
Separation of nucleosides, nucleotides, and free
bases was accomplished by the method of Cohn
(1950). Protein was measured by the biuret
reaction (Gornall e¢ a/., 1949).

EXPERIMENTAL RESULTS AND CONCLUSIONS

Tnerease in enzymatic activity by treatment with
solvents. (1) Benzenc—In agreement with other
investigators (Lederberg, 1950; Koppel et al.,
1953) treatment of the cells with benzene or
toluene in 0.02 Mm Na-phosphate buffer, pH 7 to
7.3, yielded enzymatic activity similar to that
obtained with cell-free extracts prepared by
sonic disintegration or drying in vacuo. Five
minute treatments with 5 per cent (v/v) benzene,
including shaking once or twice by hand, brought
the enzymatic activity of the suspension to its
maximal value without releasing more than 5 per
cent of the enzyme into the supernatant. The
bulk of the enzyme was retained by the “cells.”
Vigorous mechanical shaking provided maximal
activity im about 15 sec at room temperature.
The reaction appeared to be temperature inde-
pendent in the range 0 to 40 C.

Benzene-treated cells do not appear different
from normal cells under phase contrast micros
copy, but when Giemsa stain is used the treated
cells are less basophilic than the normal ones,
suggesting the loss of ribonucleic acid.

Under the electron microscope the difference
becomes evident as shown in figure 1. The
destruction of the cell wall in the benzenc-treated
cells is quite apparent but the possibility that the
drying process might have increased the damage
must be considered.

In contrast with the @-galactosidase of intact
cells, the benzenc-treated cells and the free
enzyme were similar in respect to pH optimum,
kK, value, and inhibition by alkali metal ions.
The only detectable difference was that in our
hands benzene-treated cells did not bind signifi-
eant amounts of specific rabbit antienzyme.
Throughout this work, therefore, benzenc-treated
cells were used as refcrence for assay of total
enzymatic activity.

(2) Other solvents—The low solubility in water
of benzene or toluene limits somewhat the useful-
ness of these solvents. Therefore a search for a
more suitable agent was conducted. Isoamy]
alcohol was chosen because of its higher solubility
at low temperatures and because it has 2 low
1958] REGULATION OF ENZYMATIC ACTIVITY IN THE INTACT CELL 3

 

Figure 1.* Top. Normal cells of Escherichia coli. Center. Cells after 10 min treatment with benzene.
Bottom. Benzene-treated cells as above but incubated 10 more min with substrate, alizarin-8-p-galacto-
pyranoside.

 

* Uranium shadowing. Electron photomicrographs courtesy of Dr. Paul J. Kaesberg.
4 ROTMAN

ultraviolet absorption and would not interfere
with the spectrophotometric estimation of
purines and pyrimidines. Table 1 shows that only
relatively insoluble hydrocarbons or aliphatic
alcohols give maximal activation under the con-
ditions employed.

Since isoamy! alcohol disappears during the
process of activation, a minimum of about 6 x
10-® g of isoamyl alcohol per cell was necessary
at 0 C for maximal enzymatic activity.

Phystological state of the cells influencing
enzymatic activity. From Lederberg’s (1950) work
and our earlier experiments it was evident that
the ratios between enzymatic activities of ex-
tracts and intact cells were not reproducible. As
shown in table 2, the variable was found to be the
age of the cultures. Stationary cells show nearly
the same enzymatic activity as their extracts,
whereas cells in the exponential phase of growth
exhibit only a small fraction of their potential
activity, e. g., the activity ratio between the
extract and the intact cells is 50 to 100. By using
a chemostat and keeping the culture in constant
growth it was possible to obtain reproducible
ralues for the activity ratio. Furthermore, it was
established that large variations in growth rate
in the chemostat did not influence this ratio
significantly (table 2). This observation supports

TABLE 1

Increase in enzymatic activity of cultures by
treatment with solvents

One ml of bacterial suspension, prepared in
phosphate buffer as described in Methods, was in-
cubated for 10 min with 0.2 ml of the solvent
tested. The 8-galactosidase activity is expressed
as per cent of the activity obtained with benzene.

 

 

B-Galacto-

 

Solvent sidase
Ye

No addition........0.....0..........,. 3.6
Benzene, toluene, xylene............. 100

Ethy)] aleohol.......00.....0..00...... ' 33
s-Butyl alcohol. ..2...0.00000.0...... —

t-Buty! aleohol....000000000000000.... 52
1-Pentanol......00000000000000000.... 97
Tsoamyl alcohol.........00000.00.00.... 96
Octyl aleohol. 0... 58
5% Phenol........................... | 382
5% Na Lauryl sulfate............... i 837
Ethyl ether....000000.....00...002... | 78
Acetone........0......000...00000.0... 8

 

[voL. 76

TABLE 2

Relationship between age of cultures and enzymatic
activity of whole cells
8-Galactosidase activity was determined in
washed cells with and without benzene treatment
as described in Methods. The activity is expressed
as the ratio between benzene-treated cell and in-
tact cell activities.

 

| Ratio 8-Ga-

 

Growth Conditions and Age of Cultures  lactosidase

Activity
Stationary, aerated, 12 hr............ 21
Stationary, not aerated, 24 hr........ 24
Stationary, not aerated, 37 hr........ 5
Stationary, not aerated, 47 hr........ 3
Chemostat, 1 hr generation time. ... | 173
Chemostat, 1.4 hr generation time... | 166
Chemostat, 7.7 hr generation time....) 124
| 161

Chemostat, 11.1 hr generation time...

 

the assumption that in the steady state the vast
majority of the cells are actively growing even at
low generation times.

Passive-diffusion hypothesis. Induced biosyn-
thesis of enzyme (adaptation) and cellular expres-
ston of enzymatic content. An opportunity to test
a simple passive diffusion hypothesis is offered by
the fact that the ratio of intact cell activity to
extract activity changes with the 8-galactosidase
content of the cell (Rickenberg et al., 1953). The
8-galactosidase content per cell can be altered
(Monod et al., 1951) by varying the concentration
of a suitable inducer of the enzyme in the wild
type E. colt.

The proposed diffusion hypothesis postulates
that the rate of penetration of the substrate is the
rate-limiting step when the cell expresses only
a fraction of its enzyme content. The hypothesis
was tested under the following assumptions:
(a) Inasmuch as passive diffusion is under test, it
can be assumed that the rate of penetration is
constant if conditions of “gratuity” are used
throughout the experiment, i. e., the medium has
a constant chemical composition except for the
concentration of the nonmetabolized inducer
(Monod and Cohn, 1952). Other possible penc-
tration mechanisms which fulfill this assumption
will not be discriminated by our test; (b) The
physical properties of the enzyme inside the cell
are similar to those of the enzyme in cell-free
extracts; and (ce) The concentration of substrate
around the cell remains practically constant,
1958]

i. e., diffusion rate in the medium is much larger
than the entry rate of the substrate. This assump-
tion is valid because benzene-treated cells exhibit
activities up to 170-fold larger than intact cells.
If we recall that a benzene-treated cell contains
the same amount of enzyme enclosed in practically
the same volume as an intact cell, we have to
conclude that the rate at which substrate reaches
the periphery of the cell is not the limiting step.
It should be noted that for the purpose of our
discussion “penetration of the cell by the sub-
strate” could mean also arrival of the substrate
at the enzyme site.

The same concentration of substrate was used
in all the assays and it remained practically con-
stant throughout the experiment.

The hypothesis thus formulated predicts the
curves illustrated in figure 2A. The predictions
are: (a) At low levels of induction, the intact cell
should express its full enzyme content until sub-
strate penetration becomes rate limiting, i. e.,
throughout this range, enzymatie activity of cells
and extracts should be the same, and, (b) After
substrate penetration becomes rate limiting as
indicated by the lower activity of cells compared
with extracts, the enzymatic activity of the
intact cells should remain constant. Therefore
the ratio of extract activity to cell activity should
increase progressively with the increase in the
enzyme content of the cell.

A test of these predictions is shown in figure
2. The experimental curves in figure 2B differed

    
 
 
  
    

A. Theoretical curves

wt
61]

e cells

Ww
9S

if

Enzymatic activity whol

 

B. Experimental curves

REGULATION OF ENZYMATIC ACTIVITY IN THE INTACT CELL 3

radically from the theoretical (2A). In no portion
of the former was either prediction satisfied. Thus
the intact cell expressed little or none of the
enzyme content when this was small and at
higher induction levels expressed a constant
fraction of the enzyme synthesized. The intact
cell activity did not reach a plateau but increased
steadily with increasing enzyme content. Finally,
the ratio of extract to cell activity reached a
maximum long before the enzyme content did.
Thus one must conclude that the passive diffusion
hypothesis as stated here is incompatible with the
facts. Any other hypotheses that do not involve a
change in the rate of substrate entry during
enzyme induction would be equally incompatible.

The same test was applied to the constitutive
strain which synthesizes as much enzyme in the
absence of inducer as does the parental stock in
the presence of optimal concentrations of potent
inducers. The enzyme content of the constitutive
strain was varicd by inhibition of the @-galac-
tosidase synthesis with lactose. It was found that
the lower the galactosidase content, the larger
was the fraction of enzymatic activity expressed
by the intact cells. Therefore passive diffusion
here too must be ruled out as a possible penetra-
tion mechanism.

The enayme complex hypothesis. The second
hypothesis to be tested was the existence in the
cell of an enzyme complex which reduced enzy-
matic activity. In this case, the increased activity
of extracts or benzene treated cells would result

8
enz act extract/enz.act.cell

8

8

 

 

0 300

300 1500" 2250

Enzyme per mi of culturefextracted) mpmoles ONPG/min

Figure 2. 8-Galactosidase activity of whole cells as a function of their 6-galactosidase content. Theo-
retical curves constructed according to the passive-diffusion hypothesis described in the text. Curve J.
B-galactosidase activity of whole cells in mpmoles of o-nitrophenyl-8-p-galactopyranoside (ONPG)
hydrolyzed per min per ml of culture. Curve IZ. Ratio 6-galactosidase activity of extract over 8-galac-
tosidase activity of whole cells. Increasing concentrations of lactose or methyl-8-p-thiogalactopyrano-

side were used to induce the cultures.
6 ROTMAN

from the dissociation of the complex. Dissociation
could oceur when the internal milicu of the cell
was no longer protected from external exchange.

Previous experiments (Rotman, 19555) had
suggested that RNA might be the complexing
agent. In the present work, the test of this sup-
position was confined to (a) attempts to isolate
the hypothetical enzyme-RNA complex from
the cells and to show its in vitro dissociation with
a comparable increase in enzymatic activity, and
(b) attempts to demonstrate the inhibitory power
of the RNA in vivo and in vitro.

The hypothesis could be extended to include
other complexing molecules such as the enzyme
itself (Kaplan, 1954; Bonner, 1955). A strict test
of the latter however would require methods to
analyze intracellular structure not available at
present.

Leakage of breakdown products of RNA. As
mentioned before, the lowered basophilia of
benzene-treated cells suggested a substantial
loss of nucleic acids. Spectrophotometrie and
chemical analysis confirmed that as much as 80
per cent of the total RNA content of the cell was
present outside, The RNA loss was accounted for
by comparable amounts of breakdown products
of RNA. Small amounts of free bases and poly-
merized material were among the breakdown
products. The relative composition of products
depended upon the eonditions of the benzene
treatment. No detectable amounts of DNA or
protein were found outside the benzene cells.
Iscamy! alcohol, like benzene, caused a rcleasc of
breakdown products of RNA.

RNA breakdown products were also found to
be released from the cells upon aging with a con-
comitant merease in enzymatic activity. The
cells remain viable throughout the aging process.
Borek et al. (1955) reported also that the viable
count of a cell suspension can remain constant
while nearly 40 per cent of the RNA is exereted
as breakdown products in the supernatant.

During aging, addition of Mg++ or Cat ions
in 8.2 X 107-4 m concentration prevented the
leakage of breakdown products of RNA as well
as the increase in enzymatic activity. Storage in
distilled water at 0 to 4.C was also effective in
preventing leakage.

The relationship between RNA release and
inerease in enzymatic activity of the intact cells
was tested further using lysozyme, polymyxin B,
and heating at 5! C. Polymyxin (Newton, 1956)

[vou. 76

and heating at 51 C (Califano, 1952) have been
previously shown to cause leakage of depoly-
merized form of RNA. Lysozyme alone has not
been reported to cause any direct effect on E. coli
at neutral pH, although recently it has been
shown to cause lysis at pH 7.6 in the presence of
Versene (Repaske, 1956). Treatment with lyso-
zyme released half of the RNA in polymerized
form precipitable by HClO, This effect of Lyso-
zyme was inhibited 70 per cent by 8 X 107° Mw
urany] acetate.

Furthermore, when the release of RNA by
polymyxin was inhibited by high concentrations
of the antibiotic or by divalent cations (Newton,
1956) the enzymatic activity was inhibited
proportionally. Cells treated with either lyso-
zyme or polymyxin exhibited about 15 per
cent of the maximal §-galactosidase activity. On
the other hand, when both agents were used in
the order indicated maximal activity was ob-
taincd. Reversing the order or adding the two
together resulted in reduced activity (table 3).
Cells heated at 51 C did not show additive effects
with either lysozyme or polymyxin. Lysozyme
treatment resulted in no detectable loss in viable
cells, whereas 14 min at 51 C killed 85 per cent
of the cells and polymyxin left only a few sur-
vivors.

As shown in figure 3, treatment with any of
these agents resulted in a release of RNA pro-
portional to the increase in 6-galactosidase.

Although this proportionality between RNA
excretion and 6-galactosidase activity was found
within every set of experiments involving a single
activating agent, the amount of RNA released
did not correspond to the same enzymatic activity
for experiments with different activators. For
instance, when isoamyl alcohol released 237
ug of RNA/mg dry cells with an increase in
enzymatic activity of 4520 mumoles ONPG/min
mg dry cells, aging in 0.02 m sodium phosphate
buffer for 8 hr released 23.6 ug of RNA/meg drv
cells with an increase of 119 mumoles ONPG /min
mg dry cells.

This lack of correlation between activating
agents suggests that the rclease of RNA and the
increase in enzymatic activity could be the result
of a more general effect, i. e., loss of permeability,
rather than a combination of RNA and enzyme
as postulated before (Rotman, 1955). Further
evidence of this hypothesis is given below.

Preparation of inhibited enzyme in vitro, It was
1958]

TABLE 3

Synergistic effect of lysozyme and polymyzin upon
the increase in enzymatic activity of whole cells

Chemostat-grown cells were washed and re-
suspended in phosphate buffer as described in
Methods. Three tubes containing 2 ml of cell
suspension plus the additions indicated below
were incubated 30 min at 37 C. After this first
incubation, 0.1 ml of each tube was used to deter-
mine @-galactosidase activity and the rest of the
cells were spun down and resuspended in 2 ml of
the same buffer. Aliquots of each tube, with and
without additions, were incubated at 37 C for
another 380 min. 6-Galactosidase was determined
in each aliquot at the end of this second period of
incubation. The results are expressed as per cent
of the 8-galactosidase activity of benzene-treated
cells. Activity of cells before incubation at 37 C
was 1.1 per cent. Activity of supernatants from
tubes 2 and 3 after the second incubation was 1.2
and 0.3 per cent, respectively.

 

 

 

8-Galacto-
Tube No. Additions Re
Rate
First
incubation ,
1 0.2 ml H.O 4.7
2 0.2 ml polymyxin B 22.6
(0.5 mg/ml)
3 0.2 ml lysozyme (12.5 14.9
mg/ml)
Second
incubation
j No additions 8.0
2 No additions | 22.6
2 0.1 ml lysozyme (50 57.5
mg/ml)
3 No additions - 20.0
3 0.1 ml polymyxin B 100.5
(0.5 mg/ml) |

 

possible to prepare extracts containing 6-galacto-
sidase in an inhibited form. The inhibition could
be overcome by addition of salts or by incubation
at 37 C (figure 4). The key to the preparation of
inhibited extracts is to avoid the use of salts
throughout the extraction procedure. The cells
were collected in a refrigerated Sharples centri-
fuge, and after three washings with water at
15,000 rpm in a Spinco (head no. 20), they were
ground with dry ice and lyophilized. The extracts
were prepared simply by grinding 0.5 g of dry
powder in a cold mortar with 5 ml of cold water

REGULATION OF ENZYMATIC ACTIVITY IN THE INTACT CELL ‘

S
ho
T

9
=

 

Increase in optical density at 260 my:

 

0 50 700
Increase in enzymatic activity mprmoles/min/ml

Figure 8. Relationship between release of RNA-
breakdown products in the supernatants and
activation of the 6-galactosidase of whole cells.
A. Cells aged in 0.02 m Na-PO, (pH 7.35 at 26 C)
for 3 hr at 37 C. B. Cells treated with polymyxin
B in decreasing concentration. The lowest increase
in activity corresponds to 830 gg of polvmyxin
per ml, the highest to 210 xg per ml. C. Cells heated
at 51 C in 0.02 mM Na-PO, (pH 7.2 at 23 C) for 15
min.

 

 

EI moximum activity”
€ - in the extract
~S
3 t
QL
&
AL
=
2 5
Ss
S |
2 L
2
=
a
0 "105 16° 104 10% — 10?

Molarity of Na-POQ; (pH 647)

Frgure 4. Increase in the 8-galactosidase activity
of inhibited cell-free extracts by addition of salts.
See the text for preparation and assay of inhibited
extracts. The pH of the buffer was measured at
0 C and it was chosen to match the pH of the
extract.

and eliminating the debris by 8 min centrifugation
at 10,000 rpm in the high speed head of an
International centrifuge.

These extracts were assayed at 0 C by adding
8 ROTMAN

0.05 ml of 0.05 m ONPG (previously equilibrated
at OC) to 0.1 ml of extract and stopping the
reaction with 10 ml of 0.l m Na,COs. The assay
was proportional to incubation time up to 6 min.

G-Galactosidase activity in these inhibited
extracts was not diminished by 60 min centrifu-
gation at 40,000 rpm in the no. 20 head of a
Spinco centrifuge. Thus, the association of the
enzyme with a particulate matter is ruled out as
a cause of inhibition.

That the inhibition in these extracts is not of
the known type produced by alkali metal ions
(Lederberg, 1950) was shown by the fact that
the initial salt concentration affected the enzy-
matic activity of aliquots of extract reaching
identical final conditions prior to assay (figure 4).

The relationship between the inhibited ex-
tracts and the physiological problem in question
is obscure because the cells whieh had been
treated previously with isoamyl alcohol, and
were therefore capable of expressing their entire
enzyme content, nevertheless yielded inhibited
extracts.

Inhibitors of 8-galactosidase released by the cells.
According to the enzyme-complex hypothesis the
complexing agent should be found free after
activation of the enzyme. An unsuccessful
search for the complexing agent was conducted
among the excretion products of cultures which
had leaked most of their RNA with a proportional
increase in 8-galactosidase activity. A 5-L culture
of the constitutive mutant was treated with
isoamyl alcohol and the supernatant was concen-
trated from the frozen state under vacuum.

Several fractions obtained by precipitation of
the concentrated supernatant with ethanol were
tested for inhibition of cell-free enzyme or ben-
zene-treated cells. None was found inhibitory.
Likewise yeast RNA was not inhibitory in either
system. The effect of highly polymerized RNA
from E. colt could not be determined. No reports
of a successful method of preparation could be
found in the literature. The difficulty probably
stems from the presence of active RNAase(s) in
the extracts (Manson, 1953).

The product inhibition hypothesis. This hypoth-
esis explains the reduced activity of the intact
ecll in terms of inhibition of the enzyme by the
reaction products. Galactose, one of the products,
is known to be a competitive inhibitor of the
enzymatic hydrolysis of ONPG. High concen-
trations of galactose could accumulate in the

[vou. 76

intact cell if its excretion rate happened to be
smaller than the hydrolysis rate of ONPG. The
increasing concentration of galactose would
inhibit the enzymatic reaction progressively
until a steady state had been reached in which the
rates of hydrolysis and excretion of galactose
remained constant. This hypothesis predicts that
the initial enzymatic activity of the intact. cell
before accumulation of reaction products would
be larger. The prediction was tested by following
the initial rates of hydrolysis of ONPG by a con-
centrated cell suspension in a recording spectro-
photometer of very high sensitivity. A double
beam apparatus which recorded the difference in
optical density between 440 and 480 my was
used (Chance, 1956). These experiments were
performed in collaboration with Dr. Britton
Chance at the University of Pennsylvania.

The data summarized in figure 5 show that a
steady rate of hydrolysis had been attained by the

0.07;-

0.005;-

- substrate

| added \

poner Amnon

Difference in optical density 440-480 my

 

 

40

 

| \ ! !
0 10 20-30 50

time (sec)
Figure §. Hydrolysis of o-nitrophenyl-@-p-
galactopyranoside (ONPG) by intact cells meas-
ured in a double-beam recording spectropho-

tometer. The instrument recorded the difference
between optical densities at 440 and 480 mx.
1958]

cells as early as one second after substrate addi-
tion. Extrapolation to zero time permitted the
estimate that the concentration of o-nitrophenol
(and therefore of galactose) in the total suspen-
sion at the end of the first second could not have
exceeded 1 X 1076 mM. Since the product inhibition
hypothesis would demand that a steady state of
enzymatic inhibition should already have been
established at this time one may ask if the
amount of galactose that was present was
sufficient to have brought this about. The intra-
cellular volume of the suspended cells was deter-
mined to be 6.3 X 10-3 ml per ml suspension.
Thus even were all the galactose that had been
produced in one second still within the cells,
its concentration could have been no more than
(1 x 10-8)/ (6.3 XK 107%) = 16 X 104M. But
the data of Kuby and Lardy (1933) for the i
vitro inhibition of highly purified 6-galactosidase
showed that 0.4 m galactose is required for 95
per cent inhibition in the presence of 2.28 X 1074
mM ONPG (the amount used in the present experi-
ments). It follows then that this concentration of
galactose is (0.4)/0.6 xX 1074) = 2500-fold
greater than could have been generated in the
cells in one second. In other words, the amount
of galactose that actually was present would
have had to be crammed into 34590 of the total
cell volume to have been inhibitory. (Assuming
a significant effusion of galactose from the cells
during this time, the fraction would be even
lower.) But this is patently impossible when it

REGULATION OF ENZYMATIC ACTIVITY IN THIr INTACT CELL 9

is considered that the highly purified 8-galac-
tosidase represents 0.5 to 1 per cent of the total
cell protein (Rotman and Spiegelman, 1954) so
that the volume of enzyme itself is greater than
14309 of the total cell volume.

It should be noted that the only assumptions
underlying this argument are that the enzyme
inside the cell behaves as the purified enzyme
and that the substrate concentration inside the
cell is the same as that outside.

Penetration mechanism hypothesis. p-
o-Nitrophenyl-a-L-arabinopyranoside have also
been found to act as substrates for the B-galac-
tosidase (Lederberg, private communication).
These compounds have the same ring structure
as the @-p-galactopyranosides. They differ
strikingly in that intact cells exhibited the same
activity against them as did extracts (table 4).
The full expression of enzyme content by the
intact ecll excludes immediately the idea of an
altered (Kaplan, 1954), inhibited (Rotman,
1955b), or stacked enzyme (Bonner, 1955). It is
not likely that the enzyme would be compounded
in such a form that binding sites for the arabi-
nosides would be free but those for the galacto-
sides would be obstructed. The same reasoning
applies to the stacked enzyme.

In addition, the difference in behavior of the
intact cell toward the two classes of substrates
cannot readily be ascribed to different rates of
passive diffusion because they are so similar struc-
turally. It therefore seems more likely that a se-

and

TABLE 4

Effect of Na and K ions in the hydrolysis of 6-p-galactosides and a-L-arabinosides by B-galactosidase

g-Galactosidase activity was determined as described in Methods at 1073 M substrate concentration.
Na or K phosphate (0.02 M) buffer pH 7.2 (25 C) were used in the assay mixture. The results on the
effect of ions are given as the ratio @-galactosidase activity in Na* buffer over 6-galactosidase activity
in K+ buffer. The values for induced wild type whole cells are the results of a single experiment.

 

| Ratio Nat Activity/K* Activity

mumoles of Substrate Hydrolyzed per
min per wg N in Na~ Butfer

 

 

 

 

 

Culture | —_
i Substrate Substrate

ONPG* ONPA* ONPG* | ONPA*
Constitutive whole cells. .............. 1.08 3.30 1.73 | 0.72
Wild type induced whole cells.......... 1.01 3.35 1.79 | 0.62
C,He-treated constitutive.............. 1.75 5.55 66.7 1.69
C.H¢-treated induced wild type........ 1.83 4.10 57.8 1.36
Noninduced wild type whole cells...... 1.35 _— 0.03 Undetectable
Purified B-galactosidase................ 1.78 5.60 960 27.9

 

*ONPG = o-nitrophenyl-6-p-galactoside; ONPA =

o-nitropheny|-a-L-arabinoside.
10 ROTMAN

lective penetration mechanism is involved. To ac-
count for the increase in enzymatic activity of the
intact cell with increase in enzyme content, one
would have to assume that the penctration mech-
nism is induced together with the enzyme (figure
2B). Because of its inducibility and specificity,
one might visualize the penetration mechanism as
enzymatic in nature although it could equally
well act as a specific “keyhole” at the cell surface.

Further evidence in support of the above
hypothesis was obtained by a study of metal ions
known to inhibit the galactosidase in vitro.
Lederberg (1950), using ONPG as a substrate,
reported that Na ions did not affect the intact
cell activity although they could activate
B-galactosidase in extracts as compared with K
ions. We have extended these results using
p-nitropheny|-@-p-galactopyranoside. Again, no
cffect. of ions upon intact cell activity toward
this substrate could be noted but the effects of
Na and K were precisely reversed in extracts.
When the arabinosides were employed, metal
ions were found to influence both intact cells
and extracts (table 4). A similar situation was
encountered in noninduced cells with only back-
ground enzyme.

Regardless of the nature of the substrate,
intact. cells did not exhibit any 8-galactosidase
activity whatever when assayed in distilled water.
The activity was restored upon addition of
maleic or phosphoric buffers. Although Rb, K,
Na, Li, ete. were innocuous toward intact cells
when they were acting upon 6-galactosides,
triethanolammonium ions were potent inhibitors.
Kuby and Lardy (1953) reported that this ion
inhibits the purified @-galactosidase.

From these results it seems that the metal ions
arc inert to the intact cell activity on B-galac-
tosides not because the ions cannot reach the
enzyme site, but because they do not interfere
with the penctration mechanism. This idea is
reinforced by the inhibitory effect of Tris buffer
which should not exist if the ions cannot reach
the enzyme site. The a@-L-arabinosides would
have such affinity for the B-galactoside penetra-
tion mechanism as to permit a relatively rapid
entrance. Thus, for the arabinosides, the
B-galactosidase would be the rate limiting step
an vivo. The possibility of a separate penetration
mechanism specific for the arabinosides can not
be excluded. Nevertheless, its postulation adds
nothing to our hypothesis. On the contrary,

[voL. 76

to set aside the 6-galactoside penetration
mechanism, it would be necessary to accept the
existence of a specific penetration mechanism for
arabinosides but not for galactosides.

The fact that intact cells placed in a mixture
of ONPG and 0.1 4 Na.COs still ean hydrolyze a
minute amount of ONPG is additional evidence
in favor of the penetration mechanism. The
experiment indicates that ONPG penetrates the
cell faster than OH™ ions do, Similarly, HCl,
trichloracetic acid, or formaldehyde do not stop
the hydrolysis in vive completely.

The inference from the a-arabinoside experi-
ments is valid only if the @-galactosidase is the
main enzyme capable of splitting a@-L-arabi-
nosides. This assumption is strongly supported!
by two lines of experimental data: (a) The ratio
between the rates of hydrolysis of o-nitropheny1-
e-L-arabinopyranoside and ONPG, — under
constant conditions, is the same for benzene-
activated cells, crude extracts, or purified
B-galactosidase. The average ratio at 10-3 1
concentration of substrate for benzene-activated
cells was 39.4 and for purified enzyme, 34.3. The
ratio given by Kuby and Lardy (1953) for
highly purified 8-galactosidase is 38, (b) Cells
induced with L-arabinose (a poor inducer) show
traces of @-galactosidase and no detectable
a-L-arabinosidase (to be expected because of the
30-fold difference in sensitivity). Extracts of
B-galactosidase constitutive mutants grown in
the presence of succinate and t-arabinose show
the same hydrolysis ratio of arabinosides and
galactosides as given above. If an active a.
arabinosidase exists it would have to be in-
duced by galactose but not arabinose, a very
unlikely event.

Another possible objection to the induced
penctration mechanism hypothesis stems from
the fact that a-L-arabinosides are hydrolyzed by
extracted enzyme at a smaller rate than 8-galac-
tosides. The results with arabinosides could then
be interpreted as if the B-galactosidase becomes
the rate limiting step at low enzymatic activities,
This interpretation is incorrect because whole
cells with the same enzyme content, in terms of
moles of substrate hydrolyzed per min whether
it was a galactoside or an arabinoside, show re-
duced activity only when B-galactosides were
used.

An analysis of the data obtained in the previous
experiments from the standpoint of the inducible
1958]

penetration mechanism is warranted. Moreover,
such analysis vields a forecast of the properties
of the hypothetical penetration mechanism which
should be useful for its future isolation.

The increase in enzymatic activity, using
ONPG, which occurs with organic solvents,
lysozyme, polymyxin, heat, or aging of the cell
would have to be explained by a nonspecific
increase in permeability. This assumption would
fit well with the proportional excretion of RNA-
breakdown products which occurs concomitantly,
indicating a loss of the ability to retain cytoplasm.

Interpreting the results shown in figure 2B
under the inducible penetration mechanism
hypothesis one can conclude: (a) The data con-
forms with the hypothesis. (b) The @-galacto-
sidase is induced at lower inducer concentration
than the penetration mechanism to account for
the carly part of the curve in which the enzyme
content increases more rapidly than the intact
cell activity, (c) The penetration mechanism,
after a certain period, is induced linearly with
inducer concentration, at the same rate as the
enzyme. This accounts for the proportional
increase of the intact cell activity.

The inhibited enzyme found in extracts cannot
be readily explained under any hypothesis
because of its presence in extracts of cells which
could display activity. The possibility of an
artifact is not unlikely.

The experiments with short reaction times
performed at Dr. Chance’s laboratory do not
contradict the inducible penetration mechanism
hypothesis. They would indicate that the entry
of the substrate by way of the penetration
mechanism follows zero order kinetics from its
initial velocity.

The inertness of alkali metal ions for the en-
zyme in vivo indicates that, in contrast with the
6-galactosidase, the penetration mechanism,
which in vivo is the rate limiting step, is not
influenced by alkali metal ions. Nevertheless,
the penetration mechanism needs some salts to
function since it is inhibited by triethanolamine
buffer.

DISCUSSION

Four hypotheses which could account for the
partial expression of the galactosidase in vive
have been proposed. Of them, only the one which
postulates an inducible penetration mechanism
specific for 8-galactosides was found compatible

REGULATION OF ENZYMATIC ACTIVITY IN THIS INTACT CELL 11

with all the data. The experiments which rule
out the other three hypotheses are conclusive
and the few assumptions used in them were sup-
ported by subsequent experimental evidence.

In other instances, specific induction of trans-
port systems, required to bring substrate to the
enzyme site, have been postulated. It was used
to explain (Barret eé al., 1953) the process of
citrate utilization in Pseudomonas fluorescens and
in Aerobacter aerogenes (Davis, 1956). Thus, in
A. aerogenes intracellular accumulation of citrate
was shown to oceur only after specific induction,
Monod and his collaborators have demonstrated
directly in Escherichia colt a number of specific
systems which accumulate amino acids (Cohen
and Rickenberg, 1955) and an inducible one which
accumulates @-thiogalactosides (Rickenberg et
al., 1956). They have postulated an enzvmcelike
“permease”? which controls the entrance of the
substance. The exit rate of the substance was
assumed to depend upon the internal concentra-
tion. Thus it is explained how an accumulation
occurs and how a steady state between entry and
exit is obtained. The ‘permease’? which accu-
mulates 6-thiogalactosides has been shown to be
induced specifically by the same inducers as the
B-galactosidase although it is clearly different
from the latter. The different substances accu-
mulated by the system compete with each other
in the same fashion as substrates compete for the
enzyme,

According to Monod the ‘permease’ mecha-
nism of accumulation can explain the reduced
B-galactosidase activity of the whole cell of
E. coli, The “permease” would be the rate limit-
ing step in the process of bringing the substrate
to the @-galactosidase site.

Although Monod’s scheme apparently agrees
with the results obtained here there are serious
diserepancies which must be clarified before
drawing conclusions. The accumulation of
methyl-8-p-thiogalactoside is inhibited by 2,4-
dinitrophenol, azide or by the absence of a source
of energy; whereas, the hydrolysis of ONPG is
not affected at all under the same conditions. The
main evidence obtained by Rickenberg et al.
(1956) to show the role of the “permease” in the
hydrolysis of 6-galactosides is the correlation
between “permease” and §-galactosidase in vivo
obtained upon aging of the cells. Both are shown
to decrease proportionally. The results obtained
here are quite opposite inasmuch as the 6-galac-
12 ROTMAN

tosidase activity in the intact cell increases upon
aging (table 2).

More recently additional evidence (Cohen and
Monod, 1957) for the role of the permease in the
hydrolysis of 6-galactosides was presented. It is
based on the correlation between the two systems
in cells with different permease activity as a result
of inhibition of the permease synthesis with
p-fluorophenylalanine.

Whether the discrepancies between Monod’s
results and ours are real or duc to differences in
strains or conditions will have to be tested. In
view of these differences, the constitutive mutant
strain used in our experiments was subsequently
examined for the presence of permease and was
found to have a constitutive one inasmuch as
noninduced cells accumulate radioactive methyl-
B-p-thiogalactoside. The strain can be distin-
guished from the “cryptic” constitutive because
it is Inctose positive and because of the kinetics
of ONPG hydrolysis (Cohen and Monod, 1957).
Therefore, the possibility that we have studied
a different penetration system than did Monod
seems very unlikely.

Assuming that the discrepancies between
Monod’s results and ours are real, they could be
reconciled by postulating two different enzyme-
like systems. One would govern the entry of
specific substrates into the cell; the second would
cause the entered material to accumulate in high
concentration. The permeation system would be
specifically induced but would not require ap-
preciable energy to function. The substrate
brought in by this system could reach the
8-galactosidase site or could contact the aceumu-
lation system. The accumulation system, though
requiring energy, need not be inducible or highly
specific in its function to fit our scheme.

A support for this hypothesis comes from recent
experiments of Monod (1956). He reported a
competition between inducer and inhibitor for a
site or substance which remains constant in
amount per cell during induction. He concludes
that the site or substance must be distinct from
cither @-galactosidase itself or the permease
uptake system. In our scheme this site or sub-
stance would correspond to the second enzyme-
like system and it would indicate that it is non-
inducible.

Off hand, this hypothesis with two enzymelike
systems appears very complex. However, if the
accumulation system is represented by the cell

[voL. 76

membrane which could control the exit of the
substrate and therefore its accumulation, the
hypothesis becomes amenable to test. If the
assumption is correct, in the presence of azide or
dinitrophenol the rate of exit of an accumulated
B-galactoside should inerease appreciably. The
rate of exit could be determined easily in the
absence of external substrate.

To postulate, as Cohen and Monod (1937
have done, that azide or dinitrophenol inhibits
“only the energy coupling which allows the
permease reaction to function as a pump” serves
only to describe the facts.

Thus, the utilization of 8-galactosides in
Escherichia coli would be regulated by the induc-
ible penetration mechanism, a flexible system. It
permits the full expression of the enzyme in cells
with very little of it or in cells which have entered
the stationary phase. Such cells might need the
use of greater synthesizing ability to start growing
again as is illustrated by the faster rate of RNA,
DNA, or protein synthesis in the lag-phase cells.
Furthermore, it would indicate that the normal
cell synthesizes simultaneously the enzyme and
the mechanism which will control its activity.

The smaller quantity of RNA present in
stationary cells (Morse and Carter, 1949) can
now be explained by the large excretion of RNA
in the form of depolymerized products which
occurs with age. The remarkable fact that cells
can lose most of their RNA and still remain
viable poses new questions as to the role of RNA.

For the enzyme chemist it should be of interest
that the activating effects of Na and K ions
depend upon whether o-nitrophenyl-8-p-galac-
toside or p-nitrophenyl-8-p-galactoside is the
substrate. With the analogous a-L-arabinosides,
the same phenomenon is not observed. For both
of these substrates, Na ions activate the enzyme
as compared with K ions, although to a different
extent in cach case.

ACKNOWLEDGMENTS

The author is most grateful to Professors H. A.
Lardy and J. Lederberg for their helpful discus-
sions and criticism, and to Professor B. Chancv
for his valuable cooperation in some of the experi-
ments.

SUMMARY

Four hypotheses were tested to explain the
mechanism of the reduced @-galactosidase
1958]

activity of whole cells of Escherichia coli. Passive
diffusion, product inhibition, and inhibitors com-
pounded with the enzyme were ruled out. The
data are compatible with the presence of an
inducible penetration mechanism. The penetra-
tion mechanism is said to be specific for B-galac-
tosides and to be induced together with the
G-galactosidase. Its properties differ from those
of an inducible system which accumulates
@-thiogalactosides in #. coli. The penctration
mechanism hypothesis offers a flexible regulation
system for the enzymatic activity of the cell.
The assay of whole cells with a-L-arabinosides
permits the bypass of the penetration mechanism.

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