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January 1962
J. L. Peel
PC263
TABLE II
Radioactivity assay
of
washed crystalline precipitates from mixtures
of
dog and rat hemoglobins
The aqueous mixtures (pH -7.2) contained 84.6 mg of dog
hemoglobin, labeled with glycine-2-C14, and 71.2 mg of unlabeled
rat hemoglobin. Incubation, refrigeration, and recovery of
precipitates was as described in Table I. The specific activities,
counts per minute per milligram, were obtained for hemoglobin,
hemin, and apoprotein (cf. 11). The latter was prepared by the
HCl-acetone procedure and was thoroughly washed
and dialyzed
overnight.
Hemoglobin species
Dog*.......................
Mixture of dog and rat.
Mixture of dog and rat. 3 4.6
Mixture of dog and rat. 6
Mixture of dog and rat. 6
ncu-
ation
eriod
Washed precipitate
recovered
lOUIS w
6 0.0
0 3.9
4.9
10.0
Ratio of
hemin to
ipoprotein
c.p.m./mg
9.81
12.1
14.3
14.8
13.9
* Labeled dog hemoglobin (see text) used in the mixtures.
t This is a relatively closely reproducible ratio for dog hemo-
globin labeled with glycine-2-04 (12).
$ Values in parentheses are percentages of the rat hemoglobin
in the original mixture.
TABLE III
Distribution
of
radioactivity in a and @ polypeptide chains of dog
hemoglobin labeled with glycine-2-C14
Labeled globin, isolated from labeled dog hemoglobin, was
subjected to the fractional precipitation procedure of Wilson
and
Smith (6) for the separation of a and p polypeptide chains. The
analytical results are for precipitated fractions designated by
Wilson and Smith as Al/l and C3, a! and fi chains, respectively.*
Specific activity Ratio of hemin ta
apoprotein
Hemoglobin
Hemin
Globin.
or-chains
B-Chains .
c.p.m./mg
383
2795
1
284
403
192
c.p.m./mg
9.84
6.921
14.55t
* In the case of dog hemoglobin, radioactive assay of Wilson
and Smith’s subfraction C3/3 was not significantly different from
that of C3.
t Calculated on the presently reasonable assumption, pending
experimental verification, that the specific activity of each of the
four hemins in hemoglobin is identical.
reactions. It appears unnecessary to invoke the possibility of
hemin transfer (2) as also being involved.4
4 Excessively high ratios of hemin to apoprotein labeling were
obtained in incubation periods of 9 hours or longer, accompanied
Assuming the exchange of one /3 chain and taking into account
the unequal distribution of radioactivity, a maximum of 19%
of the total counts in the original dog hemoglobin may be ex-
pected in the presently studied hybrid.
On
this basis, the mean
recovery of radioactivity in the hybrid produced at neutral pH
in these experiments was 9.6%. Therefore, such molecular
hybridization of hemoglobins could be significant physiologically.
In itself, the dissociation of hemoglobin into smaller molecular
units has potentially important physical and functional implica-
tions.
REFERENCES
1. YEAS, M., AND DRABKIN, D. L., J. Biol. Chem., 224,921 (1957).
2. ROSSI-FANELLI, A., AND ANTONINI, E., J. Biol. Chem., 236,
PC4 (1960).
3. RHINESMITH, H. S., SCHROEDER, W. A., AND PAULING, L.,
J. Am. Chem. Sot., 79, 4682 (1957).
4. RHINESMITH, H. S., SCHROEDER, W. A., AND MARTIN, N., J.
Am. Chem. Sot., 80, 3358 (1958).
5. SINGER, S. J., AND ITANO, H. A., Proc. Natl. Acad. Sci., 46.
174 (1959).
6. WILSON, S., AND SMITH, D. B., Can. J. Biochem. and Physiol.,
37, 405 (1959).
7. VINOGRAD, J., AND HUTCHINSON, W. D., Nature, 187, 216
(1960).
8. ITANO, H. A., AND ROBINSON, E., Nature, 183, 1799 (1959).
9. INGRAM, V. M., Nature, 183, 1795 (1959).
10. PERUTZ, M. F., ROSSMANN, M. G., CULLIS, A. F., MUIRHEAD,
H., WILL, G., AND NORTH, A. C. T., Nature, 186,416 (1960).
11. MARSH, J. B., AND DRABKIN, D. L., J. Biol. Chem., 224, 909
(1957).
12. DRABKIN, D. L., AND WISE, C. D., Science (Proc. Natl. Acad.
hi.). 132, 1491 (1960).
13.
16. DRABKIN, D. L., Federation Proc., 16,740 (1957).
DRAB~~N, D. L., Science, 101,445 (1945).
14. DRABKIN, D. L., J. Biol. Chem., 164, 703 (1946); Arch. Bio-
them. Biophys., 21, 224 (1949).
15. DRABKIN, D. L., AND AUSTIN, J. H., J. Biol. Chem., 112, 51
(1935).
by evidence of protein denaturation.3 Under such circumstances,
the liberation of hemin and its recombination with native or
denatured protein may occur. However, this would not represent
“hemin transfer” in its present connotation
(cf.
2).
Vitamin B,, Derivatives and the CO,-
Pyruvate Exchange Reaction:
A Reappraisal
J. L. PEEL
From the Agricultural Research Council Unit for Microbiology,
The University, Shefield 10, England
(Received for publication, August 30, 1961)
Cell-free extracts of several bacterial species catalyze the
following exchange :
C*Oz + CH&OCOOH ti COz + CH&OC*OOH
This reaction has been studied extensively in
Clostridium butyri-
cum by Wolfe et al. (1, 2) who demonstrated a requirement for
coenzyme A and thiamine pyrophosphate. More recently,
by guest, on July 13, 2011www.jbc.orgDownloaded from
PC264 Vitamin B12 Derivatives
and
COz-Pyruvate Exchange Vol. 237, Xo. 1
Rabinowitz (3), with preparations from both C. butyricum and
Clostridium cylindrosporum, has obtained evidence that the re-
action also needs vitamin Brz or one of its derivatives, of which
Factor B was by far the most active. This additional require-
ment has now been studied further with preparations from the
rumen bacterium LC, strain 1, of Elsden et al. (4), a strict an-
aerobe with a pyruvate-oxidizing system closely resembling that
of C. butyricum (5) ; crude extracts of this organism have already
been shown to catalyze the exchange reaction (6).
Whereas most workers have studied the exchange reaction
under anaerobic conditions, Rabinowitz’s experiments were done
in tubes, with air as gas phase, and with 0.2
M
mercaptoethanol
present in the incubation mixture. When partially purified
preparations of pyruvic dehydrogenase from LC were tested for
exchange activity under conditions similar to those of Rabinowitz
but with 0.02
M
mercaptoethanol, a rapid reaction was observed.
These preparations were highly active in the absence of added
CoA or thiamine pyrophosphate but showed an absolute require-
ment for a vitamin Blz derivative. Preliminary data on the
relative activities of vitamin B12, hydroxocobalamin, and Factor
B were consistent with Rabinowitz’s results. Further, it was
found that the mercaptoethanol was essential for activity, that
it was more active at 0.2
M
and essentially inactive at 0.002
M,
and that it could not be replaced by 0.02
M
cysteine or thiogly-
colate. The apparent specificity of the thiol requirement and
the high concentration needed seemed rather unusual, and the
possibility that the mercaptoethanol might react nonenzymically
TABLE I
E$ect of vitamin
BIG derivatives
on auto-oxidation
of
%mercaptoethanol
Warburg manometers contained in a total reaction volume
of 2.0 ml: 0.21 M phosphate buffer (KH2POa-NaOH mixture)
pH 7.0, 0.2 M 2-mercaptoethanol. Sufficient vitamin Biz or
other supplement to give the final concentrations indicated,
together with 20 pmoles of the phosphate buffer were originally
placed in a volume of 0.1 ml in the side arm and tipped at zero
time. Temperature 37”; gas phase, air. Rates of oxygen uptake
were measured over the period of 5 to 20 minutes. Vitamin Biz
and hydroxocobalamin were gifts from Dr. E. Lester Smith;
Factor B was a gift from Dr. H. R. V. Amstein. 2-Mercapto-
ethanol, Eastman Grade (Kodak Ltd., Liverpool) was redistilled
before use.
Additions
None.
Vitamin B,z..
Hydroxocobalamin.
Factor B. . . . .
Co(NOe)z..
Concentration 02 uptake
M
1 x 10-5
1 x 10-e
1 x 10-T
3 x 10-G
1 x 10-G
1 x 10-r
1 x 10-T
1 x 10-a
I x
10-Q
1 x 10-b
rl/hr
28
652
234
48
1416
860
80
1308
680
142
24
* Values from Rabinowitz (3).
t Author’s observation.
Apparent K,
in exchange*
M
2.5 X IO-
1.8 x 10-T
4.2 X lo-i0
Inactive+
TABLE II
Effect of Factor B
and
Smercaptoethanol
on exchange
reaction
under
aerobic
and anaerobic
conditions
Reaction volumes of 1 ml contained: 0.2 M phosphate buffer
(KH2P04-NaOH mixture) pH 7.0, 50 pmoles of sodium pyruvate,
0.1 ml of enzyme preparation containing 0.064 mg of protein,
10.5 pmoles of KHC03 (= 3150 c.p.m. assayed as BaCOa and cor-
rected for self-absorption), and, where added, 0.2 M 2-mercapto-
ethanol and 1O-4 wmole of Factor B. The reaction was carried out
in closed tubes of 1 cm internal diameter. Factor B (where
present), enzyme, and bicarbonate were added last in that order
at 30.second intervals after equilibrating all other components
for 5 minutes. In tubes under Nt, the reaction mixture and tube
were flushed during equilibration with Nz from a Pasteur pipette
passing through a rubber cap which rested loosely in place on the
top of the tube. The pipette was withdrawn to above the liquid
surface and flushing continued during addition of the last com-
ponents. The cap was then pressed home and the pipette with-
drawn. Mixtures were incubated for 15 minutes at 37” after
which the procedure of Rabinowitz (3) was followed, with minor
modifications, for the isolation and plating of the pyruvate 2,4-
dinitrophenylhydrazone. The thickness of the plated material
was approximately constant (9.2 to 10.9 mg total weight in the
different samples), and no correction was applied for self-absorp-
tion.
The enzyme preparation was obtained from dried cells of the
organism LC. Crude extract of the dried cells, prepared as de-
scribed previously (5), was acidified to pH 5.0 with acetic acid
and the precipitate centrifuged off and discarded. The super-
natant solution was fractionated with ammonium sulfate, and
the fraction precipitating between 45yo and 70% saturation was
treated further. The ammonium sulfate was first removed by
applying the material to a column of Sephadex G-25 and eluting
with water, after which the preparation was adsorbed on to DEAE
cellulose* from 0.025 M potassium phosphate buffer, pH 6.5.
The DEAE-cellulose was next washed with 0.04 M potassium
phosphate, pH 5.6, and the active material then eluted by in-
creasing the phosphate concentration to 0.08
M at the same pH.
This fraction was stored under vacuum at -20” until used.
Additions
In air Under N,
C.p.?%/p?Z&
None......................................... 0.1 0.4
2-Mercaptoethanol. 0.3 15.1
Factor B..................................... 0.1 0.4
2-Mercaptoethanol + Factor B. 14.4 16.0
* Prepared according to Peterson and Sober (IO).
with the vitamin Blz derivatives was therefore explored. It was
observed that when 0.02
M
mercaptoethanol was incubated at
20” with hydroxocobalamin at pH 7.0 (the pH used in the enzyme
experiments), the spectrum changed within a few minutes to
one closely resembling that of vitamin Bltr (7), a readily auto-
oxidizable reduction product of vitamin B,g. Analogous spectral
changes were observed with Factor B. Vitamin Blz, or some
similar auto-oxidizable product is formed when vitamin Bia is
treated with thioglycolate in alkaline solution, and Smith has
pointed out that under these circumstances, the vitamin Blz
catalyzes the auto-oxidation of the thioglycolate (8).
Manometric tests showed that vitamin Bls derivatives greatly
by guest, on July 13, 2011www.jbc.orgDownloaded from
January 1962
T. Abramsky, L. P. Rowland, and D. Shemin
PC265
accelerate the auto-oxidation of mercaptoethanol at pH 7.0
(Table I). In the absence of any Biz derivative, the rate of
auto-oxidation was very low but was increased more than 20-
fold by 1 x 1O-5
M
vitamin Blz; hydroxocobalamin gave a some-
what greater effect at 1 X 1OV
M
and a 50-fold stimulation at
3 X 1OV
M.
Factor B was much more active than either of
these two compounds; 1 x 1O-8
M
produced roughly the same
effect as 1 X 1O-5
M
vitamin B12, and a significant stimulation
of the auto-oxidation was observed with 1 x 1O-g
M
Factor B.
Cobaltous ions were inactive at 1 X 1O-5
M.
Rabinowitz’s values for the apparent
K,
of these substances
in the exchange reaction are given for comparison in Table I,
and it is seen that their relative activities roughly parallel their
ability to promote the auto-oxidation of the mercaptoethanol.
It had already been noticed that the LC preparations were com-
pletely inactivated by prior incubation for 10 minutes at 37” in
the presence of all the reaction components save the thiol or the
Blz derivative. Moreover, an apparent sensitivity to oxygen of
a Cot-pyruvate exchange system has been reported in
Clostridium
kluyveri (9). These findings suggested that the Biz derivatives,
by catalyzing the rapid auto-oxidation of the mercaptoethanol,
may serve to remove oxygen from the incubation mixture and
so protect the enzyme. This idea is supported by more direct
experiments in which the requirements for mercaptoethanol and
Factor B were compared in air and under nitrogen (Table II).
In air, both the thiol and the Blz derivative were essential,
whereas under nitrogen, the reaction proceeded with the thiol
alone and as rapidly as with both thiol and Factor B in air. The
further addition of Factor B under nitrogen produced no signifi-
cant enhancement, although the thiol was still essential for ac-
tivity. In other experiments, considerable activity has been
observed under nitrogen with much lower thiol concentrations;
for instance in one experiment the radioactivity incorporated
into pyruvate in the presence of 0.001
M
mercaptoethanol was
44% of the value observed in air with 0.02
M
mercaptoethanol
and lo-’
M
Factor B.
The simplest explanation of these results would appear to be
that in the case of the LC preparations at least, the Blz deriva-
tives are not specific coenzymes of the COz-pyruvate exchange
reaction but under aerobic conditions are required in conjunction
with mercaptoethanol to protect the enzyme against oxygen. The
effectiveness of this combination of reagents in protecting the en-
zyme concerned in these experiments suggests that it may find a
more general application in the handling of other enzymes that
are sensitive to oxygen.
REFERENCES
1. WOLFE, R.
S.
AND O’KANE, D. J., J. Biol. Chem.,
216, 637
(1955).
2. MORTLOCK, R. P., VALENTINE, R. C., AND WOLFE, R. S., J.
Biol. Chem., 234, 1653 (1959).
3. RABINOWITZ, J. C., J. Biol. Chem., 236, PC50 (1960).
4. ELSDEN, S. R., VOLCANI, B. E., GILCHRIST, F. M. C., AND
LEWIS, D., J. Bacterial., 72, 681 (1956).
5. PEEL, J. L., Biochem. J., 74, 525 (1960).
6. LADD, J. N., Biochem. J.,
71, 16
(1959).
7. DIEHL,
H.,
AND MURIE; R., Iowa State Coil. J. Sci., 26, 555
(1952).
8. SMITH, E. LESTER, Vitamin B,z,
Methuen &Co., Ltd., London,
1960, p. 51.
9.
SCHUSTER, C.
W.,
AND LYNEN,
F.,
Biochem. and Biophys.
Research Communs., 3, 350
(1960).
10. PETERSON, E. A., AND SOBER,
H. A.,
J.
Am.
Chem. Sot., 73,
751 (1956).
The Formation of Isoleucine from
P-Methylaspartic Acid in
Escherichia coli
W. *
TESSA ABRAMSKY, LEWIS P. ROWLAND,
AND DAVID SHEMIN
From the Department of Biochemistry,
Columbia
University, New
York 32, New York
(Received for publication, September 28, 1961)
The discovery of Barker, Weissbach, and Smyth (1) that the
rearrangement of glutamic acid to P-methylaspartic acid is cata-
lyzed by a coenzyme Biz-dependent enzyme has opened up the
interesting possibility of uncovering the role of this coenzyme
in other metabolic processes. Although the relationship between
Blz function and thymine in red cell maturation is far from clear,
the above finding in
Clostridium ktanomorphum
has independ-
ently led us and others (2-4) to the suggestion that P-methyl-
aspartic acid might be directly converted to thymine. This con-
version would be analogous to the formation of uracil from
aspartic acid and would obviate the need of subsequent methyla-
tion.
In order to test this possibility we investigated the occurrence
of the formation of ureidomethylsuccinate from P-methylaspar-
tate and carbamyl phosphate in Escherichia coli extracts, the
growth of thymine-requiring and Blz-requiring mutants on @-
methylaspartate, and the incorporation of
m-three
Cl4-methyl-
labeled /3-methylaspartic acid (5) into thymine of the nucleic
acids in growing cultures of
Escherichia coli.
In none of these
experiments was evidence found for the conversion of P-methyl-
aspartate into any pyrimidine or its possible intermediates.
These latter results do not agree with the report by Woolley
(4).
However, in the experiments in which C14-methyl-labeled p-
methylaspartate was added to the growth medium of a growing
culture of
E.
coli, the isolated protein fraction was found to be
very radioactive. The amino acid mixture obtained on hydroly-
sis was partially separated by two-dimensional paper chromatog-
raphy (butanol-acetic acid-water; phenol-ammonia-water). An
autoradiogram revealed one radioactive area. On elution of this
area with water and rechromatographing of the concentrat,ed
eluate on Whatman No. 1 paper in either a secondary butanol-
ammonia (3 %) mixture (3 : 1) (6) or in a pyridine-n-amyl alcohol-
water mixture (35 : 35 : 30) (7) the radioactive ninhydrin-positive
spot had an
RF
corresponding to an authentic sample of isoleu-
tine. In order to obtain further evidence that the radioactive
compound was isoleucine, the following procedures were carried
out. The radioactive area was eluted with water and the amount
of the amino acid determined by a quantitative ninhydrin reac-
tion (8). An aliquot of the solution was mixed with an equal
amount of nonradioactive isoleucine, another sample with an
equal amount of leucine. The two samples were chromato-
graphed on Whatman No. 1 paper with the secondary butanol-
* This work was supported by grants from the National In-
stitutes of Health, United States Public Health Service (A-1101),
from the National Science Foundation, and from the American
Cancer Society.
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