THE
PHYSIOLOGICAL
IN RELATION
SIGNIFICANCE
TO GLUCOSE
BY HORACE
(From the Department
of Zymology,
(Received
for
OF DEAMINATION
OXIDATION.
B. SPEAKMAN.
University
publication,
of Toronto,
June
Toronto, Canada
)
18, 1926.)
In both plant and animal tissuesthe deamination of amino acids
is of wide spread occurrence and a variety of products are formed.
The exact distribution of the process and its chemical mechanism
in the animal body are still subjects of importance from the
physiological standpoint, but perhaps of greater importance is
the question of the influence of amino acid decomposition on the
total metabolism of the cell. The phenomenon of the “specific
dynamic action” of protein and amino acids has been established by several workers, and the literature has been fully reviewed by Lusk (1). Although the facts have been established
experimentally the mechanism by which total metabolism is
stimulated by deamination still remains a matter for hypothesis.
In connection with our work on the metabolism of bacteria experimental results have been obtained which support the view that
this stimulation is primarily due to the NH, liberated by deamination. Later in this report we shall consider more fully the possible bearing of our work on the problem of carbohydrate oxidation
in the animal body.
The organism used in this investigation was the typical form,
Bacillus
granulobacter
pectinovorum,
which produces acetone and
butyl alcohol in media containing utilizable carbohydrates. It is
a bacillus which hydrolyses starch to glucose, which is in turn
oxidised anaerobically.
In previous reports we have shown that
butyric acid a,nd acetic acid are intermediates in this process of
oxidation (2). Peterson, Fred, and Domogalla have demonstrated experimentally that during the fermentation the protein
present in the medium is hydrolysed as far as the amino acid stage
(3), and by the isolation of Lleucic acid have indicated the pos135
This is an Open Access article under the CC BY license.
136
Deamination
and Glucose Oxidation
sibility of a general deamination of the liberated amino acids (4).
Our experiments
were performed with the primary
object of
correlating
more closely (a) vegetative growth of the cells, (b)
oxidation of glucose and intermediate fatty acids, and (c) deamination of amino acids and the accumulation
or utilization
of the
products.
Relationship
between Growth and Oxidation.
Experiment I.-A
flask containing 1000 cc. of 5 per cent maize
mash was sterilized for 2 hours at 15 pounds steam pressure.
The medium was cooled and inoculated with 20 cc. of an active
culture of the bacillus in the same type of medium.
The flask
was incubated at 37”C., and at regular intervals samples of the
rABLE
Time after
inoculation.
0.1 N acid
in 10 cc. of
mash.
hrs.
cc.
0
2.0
4.0
6.0
10.5
14.5
24.5
29.5
48.0
0.45
0.60
0.80
1.30
3.20
4.35
1.70
1.80
2.30
I.
Morphological
observations.
per cent
3.5
6.0
11.0
60.0
100.0
Scattered
rods.
Long filaments.
Chains,
rods evenly
Rods, a few clostridia.
Clostridia,
granular
stained.
rods.
fermenting mash were taken under aseptic conditions.
Portions
of each sample were titrated with 0.1 N NaOH to obtain the acidity curve of the fermentation.
A standard
loopful of culture
was spread evenly over 2 sq. cm. on a slide, carefully fixed by
heat, and stained with methylene blue. Five fields in each preparation were counted, and at the same time observations
were
made regarding the general morphological
condition of the culture.
By combining the direct counts and these observations
we consider it possible to determine accurately the limits of the
period of vegetative growth.
The results from this experiment
are summarized in Table I. The counts are expressed as percentages of the highest figure.
H. B. Speakman
137
138
Dea.mination
and Glucose Oxidation
After about the 12th hour of the fermentation
period the
starch had become sufficiently
hydrolysed to pass into solution,
and the medium was slimy.
The organisms began to lose their
motility
and congregated in dense groups, a condition which
made bacterial counts by any method difficult and inaccurate.
That vegetative growth had ceased was made quite clear, however, by comparing slides made at this stage with earlier ones
showing the culture in a condition of vigorous cell division.
Furthermore, the appearance of the characteristic
spindle-shaped
cells or clostridia containing miniature endospores is an indication
of a cessation of cell division in a normal fermentation.
This
point was reached about 4 hours previous to the peak of the
acidity curve.
In Chart 1 the results obtained in this experiment have been
correlated
with previously
established
facts regarding glucose
oxidation (2). The curves in Chart 1 demonstrate
very clearly
the existence of two distinct phases of the fermentation
period.
During the first 10 to 12 hours the cells added to the medium
divide rapidly and the acidity rises.
At the same time roughly
30 per cent of the carbohydrate
is utilized.
During the remainder
of the fermentation
the cells no longer multiply but follow one
of two paths, changes leading to spore formation or slow autolysis
and disintegration.
These morphological
changes are accompanied by (a) a rapid primary oxidation of glucose, (b) a marked
increase in the rate of oxidation of intermediate fatty acids leading to a fall in the free acidity of the medium, and (c) the formation of characteristic
neutral end-products,
butyl alcohol and acetone. The true fermentation
period is that during which the
cells are passing into the resting state or breaking up. Crey has
shown that during a period of cell disintegration
Bacillus coli
communis oxidises glucose with great rapidity (5).
Process of Deamination.
In addition to the volatile acids which are formed
an acid fraction accumulates,
particularly
during
phase of the fermentation,
which is non-volatile.
fraction Schmidt, Peterson, and Fred (4) isolated
and prepared its zinc salt which they submitted to
analysis.
They concluded that it was derived from
from glucose
the second
From this
I-leucic acid
a combustion
I-leucine and
139
H. B. Speakman
suggested that a more general deamination
occurs during the
fermentation.
I propose to confirm very briefly these observations and to support their suggestion.
Esperiment 11.-100
liters of maize mash were sterilized and
fermented through to completion.
The material was filtered
through muslin and evaporated down to a thick syrup under
reduced pressure at a temperature
of 50°C.
This treatment
removed volatile acids and neutral products.
The syrup was
divided into 250 cc. portions, which were acidified with H&304,
Distilled Hz0
and extracted continuously
for 4 days with ether.
was added to the ether in the receiver, and the ether was removed.
The residue was an amber-coloured
liquid containing acid products.
This solution was made slightly alkaline with Ba(OH)2,
and extracted
again with ether to remove colouring matter.
The mixture of barium salts in aqueous solution was boiled for
TABLE
II.
Material.
Experimental
(a) ........................
“
(b) ........................
Schmidt, Peterson, and Fred .............
Zn hydroxyisocaproate
...................
gm.
0.2045
0.2689
Zn
-per cent
C
per cent
per
20.25
20.43
19.93
19.96
42.81
42.70
44.15
43.97
6.412
6.493
6.66
6.60
HZ
cent
2 hours with ZnCOa under a reflux condenser. The solution of
zinc salts was reduced in volume and allowed to crystallize.
The
largest portion obtained in a pure form by recrystallization from
Hz0 was composed of long rhombic crystals. Two portions of
this material were submitted to ultimate analysis by my colleague Professor L. Rogers. His results are given in Table II.
It was not considered necessary to repeat further the methods
adopted by Schmidt, Peterson, and Fred in their identification
of I-leucic acid.
In addition to I-leucic acid we obtained a small amount of an
acid product which had the following chemical properties. Its
barium salt was decomposed in aqueous solution by COz. With
Millon’s reagent a dilute solution formed a deep red colouration
when the two were shaken together in the cold, indicating the
presence of a phenolic ring. Koessler and Hanke (6) diazo rea-
140
Deamination
and Glucose Oxidation
gents plus a trace of a solution containing the acid gave a reddish
brown colour, which developed to its maximum intensity in 10
minutes and remained constant for several days.
This colour
was not changed by the addition of hydroxylamine
hydrochloride
and NaOH, showing that the side chain of the benzene ring, if
any, does not contain an amino group.
Solutions of the acid
decolourized bromine water and alkaline potassium permanganate solution.
The free acid crystallized
from water in radially
arranged groups of needles.
In view of the above chemical data
and the isolation of Fleucic acid by independent observers we
conclude that tyrosine is also deaminated during the fermentation, and that p-hydroxyphenyllactic
acid is produced.
Our
residue of salts contained traces of at least two other acids which
is further evidence of a general deamination
of amino acids.
Correlation
of Deamination,
Ammonia
Production,
and Oxidation.
The non-volatile
acids are formed almost entirely when vegetative growth has ceased; i.e., during the period of most vigorous
oxidation.
If we could assume that the whole of this acid material
is derived from protein it would be possible to conclude without
further experimentation
that deamination is associated in time
with vigorous intracellular
oxidation and not with vegetative
growth.
A part of the non-volatile acid material may, however.
be derived from carbohydrate,
but there is no possibility
that
p-hydroxyphenyllactic
acid has this origin.
We therefore followed the course of deamination by measuring the rate of production of this acid in the fermentating
mash, and, at the same time
we observed the rate and extent of ammonia formation.
Experiment III.-A
flask containing
1500 cc. of 5 per cent
maize mash was sterilized and inoculated with 20 cc. of an active
culture.
The titratable
acidity curve of the fermentation
was
obtained in the usual way.
At intervals ammonia determinations
were made by the aeration method, using 50 cc. samples.
The
estimations
of p-hydroxyphenyllactic
acid were made in the
following manner.
To 10 cc. of mash add 25 cc. of ether in a separating funnel
and shake mechanically
for 10 minutes.
Remove the aqueous
layer and add 5 cc. of 1.1 per cent Na2C03 to the ether.
Shake
for 10 minutes.
Run the carbonate solution into a cup of the
H. B. Speakman
141
calorimeter and add 2 cc. of the Koessler and Hanke diazo reagent
with 1 cc. of distilled HzO. Shake and allow to stand for 10
minutes.
Compare with a standard of Congo red and methyl
orange.
When the acidity of the fermenting mash was at its maximum
a large sample was withdrawn
and incubated with an excess of
toluene in order to arrest any endoenzyme activity.
From this
point onwards readings were made on the fermenting
and nonfermenting mash.
The results from the experiment are given in
Table III.
TABLE
Deamination
Time after
inoculation.
hrs.
13
16
20
25
37
43
68
92
Normal
0.1 Nacid
10 cc.
cc.
4.0
4.4
4.0
3.5
2.1
2.3
2.8
in
_-
III.
and NH3
Production.
T
fermentation.
-
p-hydroxyE~henyllactic
acid.
(1.1 N NHa
50 cc.
_-
nzm.
in
Mash
f
-
ID.l~acidin
10 cc.
.-
-
cc.
cc.
toluene.
p-hydroxyI ,henyllactic
acid.
mm.
0
2.0
3.0
23.0
74.0
75.0
0
0
0.1
0.2
-
4.0
0
3.9
3.9
0
0
-
The experimental results lead to the following conclusions: Deamination is an endocellular process and it occurs mainly during
the second phase of the fermentation period; i.e., when the cells
are passing into the spore form or disintegrating and the oxidation of glucose and intermediate fatty acids is most vigorous.
During this period the hydroxy acids formed from the amino
acids accumulate in the medium, but none of the liberated NH3
diffuses out from the cells. When the fermentation has practically ceased a trace of NH3 can be detected. At the end of a
normal fermentation similar to that in Experiment III 50 cc. of
mash contain about 5 cc. of 0.1 N acid which is non-volatile and
only 0.2 of 0.1 N NH,.
The question then arose as to whether the deamination process and carbohydrate oxidation are connected physiologically
142
Deamination
in addition to
tion to exist,
effect of NH,
when the cells
and Glucose Oxidation
running parallel in time. Assuming some connecit seemed logical to enquire more fully into the
utilization
during the period of the fermentation
are in the resting state.
Ammonium
Phosphate as a Catalyst of Oxidation.
Before attempting
to investigate the influence of NH3 on the
oxidation of glucose it was necessary to determine whether NH3
was a suitable source of N for the organism during t.he period of
protoplasmic
synthesis
and vegetative
growth.
We inoculated
a large number of different media containing
glucose, mineral
salts, and different ammonium salts in sufficient concentration
to
equal the N present in maize mash.
To each flask containing 200
cc. of medium we added 2 cc. of an active culture of the bacillus.
We observed no signs of vegetative growth, and the media were
not fermented.
A similar amount of inoculum in media containing protein, peptone, or a mixture of amino acids gives rise
to a vigorous and complete fermentation.
We concluded from
these experiments that NHs in the form of salts will not support
vegetative growth.
Experiment
IV.-We
next attempted
by the use of larger
volumes of inoculum, and therefore a larger number of active
cells, to study the effects of ammonium phosphate on the oxidative processes of the cells. Six 300 cc. flasks containing 200 cc.
of medium were sterilized in the usual manner.
The basis of
the medium was a mineral
salt solution
of the following
composition.
KzHPOd..
.
.
KH2P04..
. . .
MgS04 . . . . . . . . . .
MnS04.
...
.. ..
.. ...
... ..
.. .
.O. 5 gm.
. .O. 5
“
. . 0.2
“
.O. 01 “
FeSOa..
. . . . . . . . .O. 01 gm.
NaCl.................
0.01
“
C~H1206..
. . . .30.00
“
HzO..
. . . . .lOOO.OO cc.
Two flasks, A and B, contained the above medium, Flasks C and D
the same with the phosphates doubled in amount, and Flasks E and
F the same plus 0.5 gm. of NHJ.H,POh
and 0.5 gm. of (NH&HP04
per liter. Each flask was inoculated with 10 cc. of an active
culture of the bacillus in maize mash, and an Atwood valve containing H&S04 was inserted in ea.ch in place of the cotton plug.
The flasks were weighed before and during the period of incuba-
H. B. Speakman
143
tion.
The losses in weight due to gas production are indications
of the rate and extent of glucose utilization.
The results from
the experiment are given in Table IV and in Chart 2.
Experiment
Y.-Four
Erlenmeyer
flasks each containing 500
cc. of medium were prepared and sterilized.
Two contained
TABLE
Ammonium
Flask.
22 hrs.
A.. .........
B.. .........
c.. .........
Il...........
E.. .........
F.. .........
gm.
0.17
0.16
0.18
0.18
0.17
0.17
--
Loss
44 hrs.
68 hrs.
0.43
0.36
0.41
0.39
0.32
0.31
gm.
-
0.67
0.56
0.63
0.61
0.52
0.48
TABLE
Time after
inoculation.
hrs.
0
24
48
76
100
120
144
168
192
220
240
Flask
A.
Flask
B.
and
Oxidation.
in weight
after:
9G hrs.
--
gm.
-
IV.
Phosphate
I
115 hrs.
--
140 hrs.
164 hrs.
gm.
c7m.
gm.
gm.
0.86
0.68
0.80
0.77
0.89
0.77
96
0.72
0.88
0.86
1.19
1.09
1.02
0.78
0.98
0.94
1.52
1.41
1.09
0.82
1.08
1.02
1.80
1.65
0
V.
Waak
T
C.
188 hrs.
gm.
1.16
0.84
1.15
1.10
2.08
1.80
Flask
D.
Acid.
Acid.
Acid.
NHs
Acid.
cc.
cc.
cc.
cc.
cc.
cc.
0.8
2.3
3.4
3.4
3.4
3.3
3.3
3.2
3.2
3.2
3.1
0.8
2.3
3.3
3.4
3.4
3.4
3.3
3.3
3.3
3.3
3.4
1.2
2.4
3.4
3.7
3.8
3.8
3.7
3.7
4.2
3.7
3.2
1.1
2.4
3.6
3.7
3.7
3.7
4.2
3.7
3.0
2.8
3.0
1.2
1.2
1.2
1.2
1.2
0.9
0.75
-
NH3
1.2
1.0
0.8
0.7
-
0.5
mineral salts and glucose but no source of nitrogen, and the other
pair contained a similar medium with the addition of ammonium
phosphate equal to the concentration
used in Experiment
IV.
The flasks were inoculated with 20 cc. of an active culture and
incubated at 37°C.
At regular intervals determinations
of the
total free acidity and ammonia were made. The residual sugar
144
Deamination
and Glucose Oxidation
8
N
H. B. Speakman
146
Deamination
and Glucose Oxidation
in each flask was also determined.
The results from this experiment are given in Table V and Chart 3.
During the first 3 days of the incubation period the fermentations were almost’ identical.
The accumulation of free acid, i.e.,
primary oxidation products, proceeded at the same rate in all
cases, and at the end of this period the acidity curves were running horizontally.
It is during this earlier part of the total fermentation period that we should expect vegetative reproduction
to take place, and the results show that if cell division and synthesis took place at all they did not involve the utilization of
measurable amounts of NH3 in Flasks C and D. On the 10th
day the concentration
of NHs in Flask D began to fall, and this
continued until the close of the experiment.
Its effect on the
general character of the fermentation
was very striking.
The
medium until this point had remained transparent,
and only
occasional bubbles of gas could be seen rising from the strips of
filter paper at the bottom of the flask, but in a short time the
supernatant
liquid became slimy and charged with gas, capped
by a deep layer of foam.
The acidity rose slightly and then fell
to a level below that of the controls, an indication of increased
primary and secondary oxidation,
In Flask C the utilization of
NH3 commenced 3 days later than in Flask D, and the period of
vigorous fermentation
was correspondingly
delayed.
This fermentation was still in progress when our last observations
were
made. The results regarding glucose utilization
confirm our
results in Experiment
IV in which the extent of oxidation was
measured by gas production.
Both experiments have been repeated several times, and we consider that they supply convincing evidence of a stimulation of intracellular oxidation by a simultaneous utilization of free NHS.
E$ect of Tyrosine
on Oxidation.
We have observed that the bacillus used in our experiments is
unable to grow and multiply from a few cells in a medium containing a single amino acid as the sole source of N. We repeated
Experiment
V but substituted
for the ammonium
phosphate
various amounts of pure tyrosine.
Experiment
VI.-Four
300 cc. Erlenmeyer
flasks each containing 200 cc. of medium were prepared and sterilized.
Each
H. B. Speakman
147
flask contained the usual salts, 6 gm. of glucose, and strips of
filter paper.
Two flasks contained no source of N, and two contained 0.2 gm. and 0.1 gm. respectively
of tyrosine.
After inoculation with 10 cc. of an active culture the flasks were equipped
with Atwood traps containing
concentrated
HzS04, and they
were then incubated.
During the fermentation
period the losses
in weight due to gas production
were determined.
When gas
production had ceased in all the flasks a 10 cc. sample from each
was used to determine the concentration
of p-hydroxyphenyllactic acid. The colour value of the controls was 7.8 and 7.4 mm.,
and of the flasks containing tyrosine 30.6 and 40.8 mm. respectively.
The losses in weight due to gas production are given in
Table VI.
TABLE
Zn,$uence
of Twosine
VI.
on Rate
Loss
Flask.
22 hrs.
---gm.
Control..
0.1 pm.
0.2
“
. . . . . . . . . . . 0.27
tyrosine..
0.35
“
. . 0.31
of Oxtdation.
in weight
114
hrs.
after:
gm.
gnz.
gm.
140
hrs.
~--Bnz.
0.35
0.68
0.62
0.52
1.01
0.92
0.70
1.22
1.11
0.74
1.42
1.32
46 hrs.
70 hrs.
168
hrs.
216
hrs.
288
hrs.
gn.
*Tn.
gm.
0.78
1.64
1.53
0.95
2.00
1.83
1.16
2.20
2.01
The results from this experiment
confirm our previous observation that the bacillus is able to deaminate tyrosine, and also
that as a result the oxidation of glucose by the cells is catalyzed.
DISCUSSION.
It is unnecessary to discuss further the evidence for the existence in these anaerobic bacilli of a biochemical mechanism for
the deamination of amino acids and the production of NH, and
hydroxy acids.
The question which merits more detailed consideration is the physiological
function of this process in the
general metabolism of the cells. Koessler and Hanke (7) in their
study of the decomposition
of histidine by bacteria suggest three
possible functions for deamination,
and these we shall consider
in the light of our own experimental findings.
1. Nitrogen is removed from an amino acid owing to its absence from the medium in more easily utilizable forms, e.g. am-
148
Deamination
and Glucose Oxidation
monium salts or nitrates,
in order to support the vegetative
growth of the organism.
Such is the case when Bacillus coli
communis grows in a medium containing
carbohydrate
and a
a single amino acid (8). The decomposition
of the amino acid
ceases when the NH8 has been liberated.
This explanation
is
rendered untenable in connection with our results by the following facts.
The organism does not grow and multiply when the
N supply consists of ammonium salts or a single amino acid. In
maize mash the organism grows rapidly, but deamination
is
chiefly, if not entirely, active when growth has ceased.
2. The acid produced by deamination is utilized as a source of
C during synthesis or in respiration owing to the absence of carbon compounds such as glucose or glycerol.
An example of this
type of deamination is to be found in Raistrick’s
work on the
utilization
of histidine by various bacterial species in solutions
containing only minera. salts and the amino acid (9). In our
experiments there was in the medium an abundance of readily
utilizable carbohydrate,
and there is no evidence that the bacteria utilize the hydroxy acids formed by deamination.
3. Deamination
may be resorted to by the cells as a method
whereby the hydrogen ion concentration
of the medium can be
controlled.
The acid products of glucose oxidation are neutralized by the liberated NH,.
This involves the decomposition
of
the acid products of deamination, otherwise the NH3 would have
little effect.
This cannot be the physiological
explanation
of
our results.
The product of deamination which is utilized by the
cells is the NH, and not the acid.
I propose, therefore, to ascribe to bacterial deamination
an
During the anaerobic
additional possible physiological function.
respiration
of carbohydrates
and fatty acids the rate of oxidation is stimulated,
directly or indirectly, by a simultaneous
deamination of amino acids within the cell. This effect is directly
associated with the utilization of the liberated NH,, and the hydroxy acids are secreted into the surrounding
medium.
The
cycle through which the NH3 passes, and the precise mechanism
by which its effect on oxidation is brought about are unknown.
At the present time I know of no direct evidence from biological
experiments which supports my conclusion, but some support is
afforded by the in vitro experiments of Dakin (10) and Witzemann
H. B. Speakman
149
(11) on the oxidation of butyric acid by means of HZOz. They
found that the process of oxidation is catalyzed by the hydroxides of Na, K, and NHs.
Witzemann
(12) in a later paper
pointed out that the most marked effect is obtained by NHhOH,
and that it is not directly attributable
to changes in the reaction
of the substrate.
He suggested that NHIOH
functions
as a
catalyst by giving rise to some unstable peroxide, but he also
states that this peroxide is not formed from ammonium phosphates.
On the basis of these experiments he advanced the hypothesis that NH3 acts as a catalyst of oxidation in the animal
body, and called attention to the association in the liver of constant supplies of NH3 and a peculiar facility for the oxidation of
such compounds
as acetoacetic acid. Ray (13) has recently
shown that the oxidation of lactic acid by HZOn is catalyzed by
the presence of glycine, and he attributes
this effect to the amino
group.
Allowing
for numerous
differences there still remain many
points of resemblance between the bacterial cells used in our experiments and the tissue cells of the animal body, and the question arises as to whether our results throw any new light on the
mechanism of carbohydrate
utilization
in such cells. The phenomena relating to the “specific dynamic action” of protein and
of single amino acids are sufficiently established, but the mechanism by which the total metabolism of the cells is stimulated
remains a matter for hypothesis.
Lusk (14) has suggested that
the stimulus is due primarily to the hydroxy acids formed during
deamination.
Glycollic acid or lactic acid have not, however,
the same quantitative
influence as glycine or alanine in equivalent
concentrations,
and Lusk is led to postulate that “it may be that
if deamination takes place within them with the production
of
glycollic acid, this substance may then play quite a different role
from that which it plays when brought to the cell from without.”
If our results have any bearing on this aspect of the problem they
provide a more definite answer to such questions.
According to
our view deamination takes place in the tissue cells, and hydroxy
acids and NH3 are formed.
The acids may or may not be further
oxidised; if so, then they have a definite glucose value, but exert
no specific dynamic effect. The NH3 in our experiments passed
through some unknown cycle, but in the animal body the end of
150
Deamination
and Glucose Oxidation
the corresponding
cycle is known, namely urea. According
this view we should expect to find the cause of the action
amino acids on total metabolism in the ammonia-urea
cycle.
to
of
BIBLIOGRAPHY.
1. Lusk,
G., The elements
of the science
of nutrition,
Philadelphia
and
London,
3rd edition,
1917.
2. Speakman,
H. B., J. Biol.
Chem.,
1920, xii, 319; xliii,
401; 1923-24,
Iviii,
395.
3. Peterson,
W. H., Fred, E. B., and Domogalla,
B. P., J. Am. Chem. Sot.
1924, xlvi, 2086.
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