It is known that vitamin B1 is a growth factor for numerous bacteria and fungi including the yeasts (see the summary in Koser and Saunders (1938)). It has also been demonstrated that vitamin B1 is essential for the growth of the isolated roots of higher plants (Bonner, 1937; Robbins and Bartley, 1937). Because of this general vitamin B1 requirement of living organisms, it would seem a priori probable that the vitamin plays a role in some basic cellular process. That this is indeed the case was shown conclusively by the work of Peters and coworkers (see Peters and O’Brien (1938)) and of Lohmann and Schuster (1937). The latter workers found that the prosthetic group of yeast carboxylase is vitamin B1 pyrophosphate. In the case of yeast, vitamin B1 is, then, a constituent of a respiratory enzyme and vitamin B1 pyrophosphate is hence commonly referred to as “cocarboxylase,” a terminology used throughout this paper. Although considerable information is available concerning the rôle of vitamin B1 as a growth factor for roots, there is little known about the carboxylase of such roots. The present work was undertaken with the hope of elucidating possible relationships between vitamin B1 and the carboxylase of pea roots

Heegaard, Erik

Horowitz, N. H.

English

Caltech Authors - Main

EXPERIMENTS ON THE CARBOXYLASE OF PEA ROOTS 
BY N. H. HOROWITZ AND ERIK HEEGAARD 
(From the William G. Kerckhof Laboratories of the Biological Sciences and 
the Gates and Crellin Laboratories of Chemistry, California Institute of 
Technology, Pasadena) 
(Received for publication, July 1, 1940) 
It is known that vitamin B1 is a growth factor for numerous 
bacteria and fungi including the yeasts (see the summary in Koser 
and Saunders (1938)). It has also been demonstrated that vita- 
min B, is essential for the growth of the isolated roots of higher 
plants (Bonner, 1937; Robbins and Bartley, 1937). Because of 
this general vitamin B1 requirement of living organisms, it would 
seem a priori probable that the vitamin plays a r&e in some basic 
cellular process. That this is indeed the case was shown conclu- 
sively by the work of Peters and coworkers (see Peters and O’Brien 
(1938)) and of Lohmann and Schuster (1937). The latter workers 
found that the prosthetic group of yeast carboxylase is vitamin B1 
pyrophosphate. In the case of yeast, vitamin Br is, then, a con- 
stituent of a respiratory enzyme and vitamin Br pyrophosphate is 
hence commonly referred to as “cocarboxylase,” a terminology 
used throughout this paper. Although considerable information 
is available concerning the r&e of vitamin B1 as a growth factor 
for roots, there is little known about the carboxylase of such roots. 
The present work was undertaken with the hope of elucidating 
possible relationships between vitamin B1 and the carboxylase of 
pea roots. 
The reaction system under consideration is defined by four 
experiments which show (1) that the respiration of whole pea 
roots is affected by the supply of vitamin B1 in the medium in 
which the roots are grown (Table I), (2) that pea roots contain an 
enzyme (carboxylase) capable of decarboxylating pyruvic acid, 
(3) that by proper treatment of the enzyme it can be made to lose 
the greater part of its activity and that the activity is restored by 
475 
476 Carboxylase of Pea Roots 
addition of vitamin B, pyrophosphate (Tables II and III), (4) 
that pea roots contain vitamin B1 pyrophosphate, as determined 
by the cocarboxylase activity of filtrates of boiled pea root car- 
boxylase with washed yeast. A once precipitated pea root prepa- 
ration was found to contain approximately 2.1 y of extractable 
cocarboxylase per gm. of dry weight. These findings indicate 
that pea roots contain a carboxylase similar in some respects to 
that found in yeast. 
Technical-Preliminary investigation showed that it is essential 
to extract pea roots at low temperatures if denaturation of the 
carboxylase is to be avoided. The roots used were from etiolated 
plants grown for 7 days in coarse sand at 25”. At the end of this 
time the plants were removed from the sand and cleaned with 
running water. The roots were then cut off with a pair of scissors 
TABLE I 
Efect of Vitamin BI on Oxygen Consumption of Isolated Pea Roots Grown for 
4 Weeks on Medium Containing All Growth Substances Except 
Vitamin B1 As Noted 
Roota supplied with 
02 consumption 
per 100 mg. dry 
weight in 1 hr. 
e.mm. 
No vitaminB1........................................... 170 
Vitamin B1 (0.1 mg. per liter), 450 
and dried superficially. They were next transferred to a large 
porcelain mortar which was placed in an alcohol-dry ice mixture. 
After the mass had frozen solidly, it was ground, transferred to 
centrifuge tubes, and centrifuged for 10 to 20 minutes. The 
temperature of the material rose to 3-4’ during this process. The 
supernatant liquid was next transferred to a cold flask and the 
residue extracted with 0.1 M phosphate buffer of pH 6.2. This 
extract was combined with the preceding supernatant. To each 
100 cc. of the combined extract were added 35 gm. of ammonium 
sulfate. This addition precipitated proteins, among them the 
carboxylase, which could then be centrifuged off. The super- 
natant liquid possessed only slight carboxylase activity and was 
discarded. The precipitated proteins were redissolved in water 
and again precipitated by ammonium sulfate. Significant losses 
N. H. Horowitz and E. Heegaard 477 
of carboxylase activity occurred if the operations were carried 
out above 5’. 
The determinations of carboxylase activity were carried out with 
Warburg manometers. 2 cc. of the enzyme preparation suspended 
in phosphate buffer at pH 6.2 and containing 60 to 90 mg. of pro- 
tein were placed in each Warburg vessel, together with 0.5 cc. of 
coenzyme solution, containing approximately 100 y of vitamin B, 
pyrophosphate (Merck). 1 cc. of buffered 0.05 M solution of 
sodium pyruvate, containing 0.1 per cent MgC12, was placed in 
the side arm of the vessel and tipped in after attainment of tem- 
perature equilibrium. In some experiments, the coenzyme solu- 
tion was placed in the side arm, and the pyruvate added with the 
enzyme. Suitable controls for the CO, evolution and for the 
small 02 consumption of the preparation were run in every experi- 
ment. The CO2 evolution in the absence of pyruvate was in all 
cases zero or negligible. In a typical experiment, the CO2 evolved 
in 1 hour in the absence of pyruvate was 0.0 c.mm., and in the 
presence of pyruvate 106 c.mm. Quantitative determinations of 
vitamin B1 pyrophosphate were made according to the method of 
Lohmann and Schuster (1937) with yeast washed with alkaline 
buffer to remove the coenzyme. It may be noted here that 
neither the pea root preparation nor the yeast used in these ex- 
periments showed increased activity when 100 y of vitamin B, 
were added with the coenzyme (compare Ochoa and Peters 
(1938)). Total vitamin B, (phosphorylated and unphosphory- 
lated) was determined in a few experiments by the use of the 
Phycomyces assay (Schopfer and Jung, 1937; Bonner and Erickson, 
1938). It was found that under the conditions of the assay vita- 
min B1 pyrophosphate possessed an activity per mole equal to 
that of the vitamin itself. 
Removal of Prosthetic Group -Several different procedures were 
tried in attempts to free the pea root carboxylase preparation from 
a possible prosthetic group. If a once precipitated preparation 
was boiled and the denatured proteins filtered off, vitamin B1 
pyrophosphate (as determined by its activity in the yeast test) 
was found in the filtrate. This indicates that the preparation 
may contain a vitamin B, pyrophosphate-protein combination. 
Washing of the enzyme preparation with alkaline buffer (by sus- 
pending the preparation in 0.1 M Na2HP04 and reprecipitating 
478 Carboxylase of Pea Roots 
with ammonium sulfate) had no effect on the pea root enzyme, 
even though a similar procedure removed coenzyme from yeast 
carboxylase (Lohmann and Schuster, 1937). Repeated precipi- 
tation with ammonium sulfate (eight or more time+) or dialysis 
through a collodion membrane resulted in denaturation of a large 
part of the enzyme, while the activity of the undenatured portion 
was not enhanced by addition of vitamin B, pyrophosphate. It 
was not found possible to destroy the activity of the enzyme 
preparation by treatment with sulfite, although such treatment is 
known to destroy vitamin B, itself (Williams et al., 1935). 
The simplest procedure found for reversible inactivation of the 
enzyme was the action of the enzyme on pyruvat,e itself. The 
enzyme preparation, suspended in phosphate buffer of pH 6.2, 
was added to a 0.05 M pyruvate solution (containing 0.1 per cent 
MgClJ in the ratio of 2 volumes of enzyme to 1 volume of 
pyruvate solution. This mixture was allowed to stand at room 
temperature for 2 hours and then placed at 0” overnight. Prepa- 
rations made according to the above procedure, which will be re- 
ferred to hereafter as “partially inactivated,” lost as much as 70 
per cent of their original carboxylase activity. Upon addition 
of crystalline vitamin B, pyrophosphate the original activity was 
completely restored, indicating that no significant denaturation 
occurred. The presence of pyruvate was essential for the inacti- 
vation. 
Activating Effect of Pyrophosphate -It was found that vitamin 
B1 pyrophosphate can be replaced by pyrophosphate alone in the 
reactivation of the pea root enzyme. The action of pyrophosphate 
is not augmented by addition of the vitamin. This is shown in 
Table II. Similar experiments with washed yeast showed that 
pyrophosphate is without cocarboxylase effect on our yeast 
carboxylase. 
That pyrophosphate is actually removed in the pyruvate in- 
activation of the enzyme is indicated by one experiment in which 
the supernatant fluid (concentrated to a small volume) from the 
pyruvate treatment for removal of the coenzyme was found to 
contain no cocarboxylase (was incapable of activating washed 
yeast), but this concentrated supernatant was still capable of 
reactivating treated pea root enzyme to some extent, a property 
possessed by pure pyrophosphate (Table II). 
N. H. Horowitz and E. Heegaard 479 
Nature of Pyrophosphate Efect-On the assumption that pea 
root carboxylase contains vitamin B1 pyrophosphate as its func- 
tional group, it would seem likely from the above that treatment 
of the enzyme removed only the pyrophosphate, leaving the 
vitamin B1 residue bound to the protein carrier in such a manner 
as to permit a resynthesis upon addition of pyrophosphate. The 
following experiment bears upon this hypothesis: 160 cc. of raw 
juice were precipitated once with ammonium sulfate and taken 
up in 50 cc. of phosphate buffer. This was divided into two 
portions. One portion, referred to as Fraction I, was immediately 
assayed for (a) carboxylase activity, (b) total vitamin B1 (phos- 
phorylated and unphosphorylated) by the Phycomyces method, 
TABLE: II 
Reactivation of Partially Inactivated Pea Root Enzyme 
All the flasks contained pyruvate in addition to the indicated reagents. 
T 
Cxperi. Experi- Experi- 
merit ment ment 
5 6 16 
Nothingadded...................................... 18.9 29.7 52.4 
85 y  vitamin Bt pyrophosphate.. . . . . . . . . . . . . . 52.4 60.3 
200 y  Na pyrophosphate.lOHzO.. . . . . . . . . . . . . 40.4 
2000 y  ‘I . . . . . . . . . . . . . . . . . . . . 57.6 64.0 100 
2000 y  “ + 100 y  vitamin B1., 54.8 62.7 
0.5 cc. supernatant from inactivated pea root car- 
boxylase........................................... 69.4 
COe in 1 hr., cmm. 
(c) vitamin B1 pyrophosphate extracted by boiling for 3 minutes 
on a water bath, (d) total vitamin B1 remaining in the denatured 
proteins following this extraction. The remaining portion was 
reprecipitated once and partially inactivated with pyruvate, as 
described previously. This fraction, known as Fraction II, was 
again precipitated and assayed in the same ways as Fraction I. 
The supernatants resulting from the reprecipitations of Fraction 
II were mixed together to give Fraction III. Fraction III was 
analyzed for total vitamin B1 and for vitamin B1 pyrophosphate 
as a check on the preceding assays. The results of this experiment 
are presented in Table III. The carboxylase activity measure- 
ments show that the pyruvate treatment caused inactivation of 
Carboxylase of Pea Roots 
the preparation to the extent of 51 per cent. This was completely 
reversed on addition of pyrophosphate, showing that no appre- 
ciable denaturation of the enzyme occurred. That reactivation 
with pyrophosphate is possible in spite of the removal of the 
greater part of the vitamin B1 (Table III, 5th line) may indicate 
that this part of the vitamin B, is free; i.e., not combined as co- 
carboxylase. This view is supported by the failure to find vita- 
min B, pyrophosphate in the supernatants (Fraction III). The 
portion of the vitamin B1 which is not removed by the pyruvate 
treatment, but which remains in the proteins, is so strongly held 
TABLE III 
Fate of Vitamin B1* and Vitamin B1 Pyrophosphate during Treatment of Pea 
Root Carboxylase with Pyruvic Acid 
The results are expressed per 100 mg. of the dry weight of the solids. 
Dry weight, mg.. . . . . . . . . . . 
CO2 per hr. 
No additions, c.mm.. . . . 
104 y  vitamin Br pyrophosphate, c.mm... 
2000 y  Na pyrophosphate, c.mm.. 
Total vitamin Br, y. _. . . 
Extractable vitamin Br pyrophosphate, y.. 
Total residual vitamin B1, y.. 
FrYtion 
990 
122 60 
140 116 
151 151 
1.02 0.545 1.45 
0.30 0.00 0.00 
0.42 0.49 
605 335 
* The vitamin B1 determinations by the Phycomyces method represent 
the total of the phosphorylated and unphosphorylated vitamin present in 
the sample. 
that it is not extracted even after denaturation by boiling. This 
evidence, then, supports the hypothesis that pyruvate inactiva- 
tion removes only pyrophosphate from the enzyme and that re- 
synthesis occurs upon addition of the latter. 
The fact that inactivated pea root enzyme preparations can be 
reactivated by pyrophosphate suggests that it might be possible 
to detect a significant cocarboxylase synthesis under these condi- 
tions. Experiments to test this possibility were carried out, but 
a synthesis could not be demonstrated with certainty. One 
negative result of particular interest follows. A pea root prepa- 
ration was made and treated to remove pyrophosphate. It was 
N. H. Horowitz and E. Heegaard 481 
then pipetted into a series of Warburg vessels in the side arms of 
which had been placed previously water, cocarboxylase, and pyro- 
phosphate solutions, respectively. The measurements were 
started and the contents of the side arms tipped in. When the 
next reading indicated an increased rate of CO2 evolution (and, 
hence, presumably cocarboxylase synthesis) in the vessel contain- 
ing pyrophosphate, the vessels were removed and immersed in 
boiling water. After cooling, 1 cc. of washed yeast was put into 
the side arm of each vessel and the vessels were replaced on the 
manometers. After equilibration the yeast was tipped in and the 
TABLE IV 
Attempt to Demonstrate Synthesis of Vitamin B1 Pyrophosphate by Pea Root 
Preparation 
Vessels 2, 3, and 4 contained, during the first run, partially inactivated 
pea root carboxylase, pyruvic acid, and the additions noted below. The 
contents of each vessel were then boiled, cooled, and 1 cc. of yeast added 
for the second run. Vessel 6 contained onlv vitamin B1 pyrophosphate and 
pyruvate during the first run. 
Vessel No. Additions 
2 Nothing 
3 Vitamin Br pyrophosphate (IO. 4 y) 
4 Na pyrophosphate (2000 y) 
6 Vitamin B1 pyrophosphate (10.4 y) 
CO2 in 30 min., omm. 
1st run 
64.6 
74.0 
113.5 
0.0 
2nd run 
7.3 
23.6 
7.3 
171.3 
cocarboxylase in each vessel was assayed. The results are shown 
in Table IV. 
The failure to demonstrate a synthesis of cocarboxylase in the 
presence of pyrophosphate under the conditions of this experiment 
may be attributable to the fact noted earlier, that vitamin B1 is 
held very firmly in the protein portion of the pea root carboxylase. 
Vitamin (and probably cocarboxylase) remains in the protein 
even after the latter has been denatured by boiling; as shown in 
the experiment of Table III, 90 per cent of the vitamin present in 
the partially inactivated preparation was retained by the de- 
natured protein. It is possible that in the experiment of Table IV 
cocarboxylase was synthesized, but was not liberated by boiling. 
The pea root preparation is capable of rapid destruction of 
482 Carboxylase of Pea Roots 
cocarboxylase. This is shown in Table IV by comparing the CO2 
production during the second run in Vessel 3 (pea root enzyme 
present) with that in Vessel 6 (pea root enzyme absent). This 
effect may be due to removal of cocarboxylase from the solution 
by the enzyme or to hydrolysis of the cocarboxylase into vitamin 
and pyrophosphate, followed by resynthesis from the liberated 
pyrophosphate and the vitamin bound in the protein. 
DISCUSSION 
It is shown in the experiments reported above that pea roots 
contain a carboxylase capable of decarboxylating pyruvic acid. 
The enzyme preparation contains appreciable amounts of vitamin 
B1 and vitamin B1 pyrophosphate. Treatments designed to 
dissociate vitamin B1 pyrophosphate from the preparation rever- 
sibly were, however, unsuccessful, as described above. In these 
experiments it was found that by allowing the enzyme to act on 
pyruvic acid it could be partially inactivated, and that reactiva- 
tion could be accomplished by the addition of either vitamin B1 
pyrophosphate or sodium pyrophosphate. It seems highly prob- 
able, from analogy with yeast carboxylase, that pea root carbox- 
ylase contains vitamin B1 pyrophosphate as the prosthetic group. 
On this assumption, the evidence suggests that pyruvate inactiva- 
tion is due to the splitting of pyrophosphate from the cocarboxylase 
molecule, and that resynthesis occurs upon addition of large 
amounts of pyrophosphate to the preparation. 
A possible interpretation of the pyruvate inactivation is that 
a phosphorylation is linked with decarboxylation in the pea root 
enzyme (compare Lipmann (1939)). Attention should be called 
to the work of Peters and Sinclair (1933) which showed that 
addition of sodium pyrophosphate promotes oxidative decarbox- 
ylation by brain tissues. 
Generous support was accorded this investigation by the Re- 
search Corporation. The authors are indebted to Dr. James 
Bonner and Dr. E. R. Buchman for their interest in the work. 
SUMMARY 
1. A protein preparation from pea roots contains a carboxylase 
capable of decarboxylating pyruvic acid in the presence of Mg++. 
X. H. Horowitz and E. Heegaard 
Heat denaturation of the preparation liberates cocarboxylase 
(vitamin B1 pyrophosphate). 
2. The pea root enzyme is partially inactivated by allowing 
the preparation to act on pyruvate. Vitamin B1, but not co- 
carboxylase, is found in the supernatant following precipitation 
of the pyruvate-inactivated enzyme. Heat denaturation fails 
to liberate cocarboxylase from the pyruvate-inactivated enzyme. 
3. Reactivation of partially inactivated enzyme is accomplished 
by the addition of cocarboxylase, pyrophosphate, or the concen- 
trated supernatant from the pyruvat.e inactivation. 
4. The reactivation by pyrophosphate suggests that the essen- 
tial feature of pyruvat#e inactivation is the removal of inorganic 
pyrophosphate from the enzyme. 
BIBLIOGRAPHY 
Bonner, J., Science, 86, 183 (1937). 
Bonner, J., and Erickson, J., Am. J. Bot., 26, 685 (1938). 
Koser, S., and Saunders, F., Bact. Rev., 2, 99 (1938). 
Lipmann, F., Nature, 144, 381 (1939). 
Lohmann, K., and Schuster, P., Biochem. Z., 294, 188 (1937). 
Ochoa, S., and Peters, R. A., Biochem. J., 32, 1501 (1938). 
Peters, R. A., and O’Brien, J., in Luck, J. M., Annual review of biochem- 
istry, Stanford University, 7, 305 (1938). 
Peters, R. A., and Sinclair, H. M., Biochem. J., 27, 1910 (1933). 
Robbins, W., and Bartley, M., Science, 85, 246 (1937). 
Schopfer, W. II., and Jung, A., Compt. rend. V Cong. internat. tech. et 
chim. ind. agric., Scheveningen, 1, 22 (1937). 
Williams, R. R., Waterman, R. E., Keresztesy, J. C., and Buchman, E. R., 
J. Am. Chem. Sot., 67, 536 (1935). 


Experiments on the carboxylase of pea roots

Annual review of biochemistry,

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Experiments on the carboxylase of pea roots

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