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Vol. 84, pp. 76-79, January 1987
Biochemistry
0 100
5 103
20 155 B E
30 365
50 15
100 0 C 16 G
*Activity in supernatant after solubilization and centrifugation for 1
hr at 105,000 X g. The solubilization solution was 0.1 M CHES, pH
9.0/2 mM dithiothreitol containing the concentration of Me2SO FIG. 3. EPR spectra of crude membranes and solubilized mem-
indicated. An activity of 100%o is equivalent to -8% of the total brane preparations of C. glycolicum. EPR conditions: microwave
membrane-bound enzyme activity. The reported pKa value for power, 5 mW; frequency setting, 9.16 GHz; temperature, -196°C;
CHES in 30%o Me2SO at 00C is 9.89 (13). scan range, 160 G; modulation amplitude 3.2 G (spectra A, B, and C)
or 8 G (spectra D and E); modulation frequency, 100 kHz; time
constant, 0.5 s; gain, 2.5 x 104 (spectra A, B, and C) or 4 x 104
superoxide dismutase to decompose active oxygen species or (spectra D and E). All spectra are centered around a g value of 2.02.
supplementation with additional reducing agents decreased Spectra: A, crude membrane preparations from 1,2-ethanediol-
recovery of active enzyme. Similar negative effects were grown cells in buffer A; B, as for spectrum A, but after a 15-min
observed with phosphoglycerides, dimethylformamide, and incubation with 10 mM hydroxyurea; C, crude membrane prepara-
tetrahydrofuran as additives. tions from glucose-grown cells in buffer A; D, membrane preparation
after solubilization with 0.1 M CHES, pH 9.0/2 mM dithiothreitol
In marked contrast to these results, sonication of mem- and centrifugation; E, as for spectrum D, but after incubation with
brane preparations in the presence of Me2SO greatly in- 10 mM hydroxyurea. The intensities of spectra A, B, and C can be
creased recovery of soluble active enzyme (Table 2). Addi- compared directly on a relative basis; also intensities of spectra D
tion of 30% Me2SO (vol/vol) gave a more than 3.5-fold and E can be compared directly. However, the absolute intensities
increase in activity recovered in the supernatant after cen- of the two groups are on different scales.
trifugation. Activity remaining in the pellet was negligible.
The further addition of phosphoglycerides to the solubiliza- brane preparations from cells grown on 1,2-ethanediol (Fig.
tion system containing 30% Me2SO/0.1 M CHES, pH 9.0/2 3, spectrum A) and that of membrane preparations of cells
mM dithiothreitol resulted in even greater improvement of grown on glucose (Fig. 3, spectrum C). The latter cells lack
soluble enzyme yields (Table 3). The more than 6-fold diol dehydratase activity, which is induced by growth on
increase in recovery of soluble enzyme observed when both diols (1), and the characteristic EPR signal at a g value of 2.02
30% Me2SO and lysophosphatidylcholine (0.15 mg/liter) is virtually absent in membrane preparations of these cells.
were added to the buffer system corresponds to an z50% Spectrum B shows the effect of a radical scavenger on the
recovery of the total diol dehydratase initially present in the amplitude of the EPR signal. Incubation of the membrane
crude membrane preparation. Addition of phosphoglycerides preparations from diol-grown cells with 10 mM hydroxyurea
alone decreased recovery of soluble diol dehydratase (Table for 15 min decreased the amplitude of the g = 2.02 signal by
1), but in the presence of Me2SO (Table 3), they increased 50% (compare spectra A and B). A comparable quenching of
recovery of the enzyme substantially. Unfortunately, stabil- the signal (spectrum E) was observed when hydroxyurea was
ity of the solubilized enzyme during storage, either at 40C or added to a solubilized diol dehydratase preparation. Addition
at - 196°C, was not improved by the additions of Me2SO and of 10 mM hydroxylamine to the membrane-bound enzyme
phosphoglycerides. Further studies to preserve or regenerate had a similar effect (data not shown). The solubilized enzyme
the possible enzyme-bound radical species (see below) may preparation in the absence of inhibitors exhibited spectrum
provide a solution to this problem. D. The decreases in signal amplitude observed in response to
As mentioned in a previous publication (1), membrane- the added radical scavengers was accompanied by virtually
bound diol dehydratase preparations exhibit a characteristic complete loss of diol dehydratase activity in both types of
EPR signal that correlates with enzyme activity. Moreover, enzyme preparations. Sulfhydryl modifying agents, which
a correlation was noted between diol dehydratase activity strongly inhibit diol dehydratase activity (1), also diminished
and the amplitude of this EPR signal in inhibition studies with the radical signal. Treatment of the enzyme with 2 mM
radical scavengers such as hydroxyurea. Data documenting p-chloromercuriphenylsulfonate for 15 min decreased the
these observations are shown in Fig. 3. Of particular impor- amplitude of the signal to 25% of the control. The EPR signal
tance is the difference between the EPR spectrum of mem- and the enzyme activity were also abolished by exposure to
oxygen (data not shown).
Table 3. Effect of phosphoglycerides in the presence of Me2SO These correlative effects suggest that a radical species may
on the recovery of diol dehydratase after solubilization be an integral property of the diol dehydratase from C.
Amount, Relative activity,*
glycolicum and that this serves as a substitute for a cobamide
coenzyme, which functions in diol dehydratases from other
Phosphoglyceride added mg/ml % sources. In the cobamide coenzyme-independent ribonucle-
None 100 otide reductase of Escherichia coli, a tyrosyl free radical with
Phosphatidylcholine 1 107 a g value of 2.0047, present in the protein B subunit of the
Lysophosphatidylcholine 1 149 enzyme, has been shown to be involved in the reaction
Lysophosphatidylcholine 0.35 152 mechanism (14). Ribonucleotide reductase activity and the
Lysophosphatidylcholine 0.15 171 EPR signal of the enzyme show a similar sensitivity to the
*Activity in supernatant after solubilization and centrifugation for 1 radical scavenger hydroxyurea.
hr at 105,000 x g. Solubilization solution was 30% Me2SO/0.1 M
CHES, pH 9.0/2 mM dithiothreitol containing the concentration of The authors are grateful to Dr. Hideo Kon for running the EPR
phosphoglyceride indicated. A relative activity of 171 represents spectra. M.G.N.H. thanks the Swedish Natural Science Research
50% of the activity originally present in the membranes. Council for their financial support.
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