Pergamon Int. J. Biochem. Vol. 26, No. 2, 229-233, 1994 pp. [614597]

Pergamon Int. J. Biochem. Vol. 26, No. 2, 229-233, 1994 pp.
Copyright 0 1994 Elsevier Science Ltd
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0020-71 IX/94 $6.00 + 0.00
DECREASE IN YEAST GLUCOSE-6-PHOSPHATE
DEHYDROGENASE ACTIVITY DUE TO OXYGEN FREE
RADICALS
IOAN FLOREA DUMITRU’* and MARINA TAMARA NECHIFOR’
‘Department of Enzymology and Biotechnology and *Department of Cell Biology, University of
Bucharest, P.O. Box 15-28, Bucharest, Romania [Fax (401) 31231701
(Received 8 July 1993)
Abstract-l. U.V. radiations and copper acetate, as free radical generating systems, determine
a significant diminishing of glucose-6-phosphate dehydrogenase activity in the homogenates
of Saccharomyces cerevisiae.
2. The inactivation is proportional to the concentration of the formed free radicals, existing
a direct dependence on the action time of the free radicals generating systems and on the
irradiation dose. The decrease of the enzyme catalytic activity is correlated with the increase
of the malondialdehyde concentration.
3. The affinity for the substrate of the enzyme under the action of free radicals does not
change significantly compared to the native enzyme: the K,,, value for NADP is halved, whilst
that for glucose-6-phosphate remains unchanged.
4. The electrophoretic study shows evidence of five electrophoretic bands with enzymatic
activity in the native extract and the disappearance of one molecular form under the free
radical action.
INTRODUCTION
Glucose-6-phosphate dehydrogenase (D-Glu-
case-6-phosphate: NADP oxidoreductase, EC
1.1.1.49), together with superoxide dismutase,
catalase, glutathione peroxidase, glutathione re-
ductase and glutathione S-transferase, is a part
of the antioxidant enzymatic system with an
important role in tissue protection against the
destructive action of oxygen free radicals (Ful-
bert and Cals, 1992; Hasegawa et al., 1992;
McElroy et al., 1992; Remacle et al., 1992).
Recent investigations have proved that the
free radicals can induce important protein
modifications-fragmentation of polypeptide
chains, as well as conformational and/or chemi-
cal changes of the constituent aminoacids
(Gillery et al., 1991; Fulbert et al., 1992; Gracy,
1992). In the case of enzymes, these modifi-
cations can result in the loss of catalytic activity
(Oliver et al., 1985; Stadtman et al., 1993). Thus,
the modifications of cysteine residues from the
mitochondrial creatine kinase structure deter-
*To whom correspondence should be addressed. Copper ions have a significant physiological
Abbreviation : lx = lux = cdsrm – * role but are also able to catalyze the production mine the decrease of catalytic activity (Yuan
et al., 1992); the oxidation of the cysteine
residue from triosephosphate isomerase un-
stabilizes the enzyme, increasing its sensitivity
to proteolytic attacks (Gracy et al., 1990); and
the inducement of disulfide bond formation, in
the aldolase structure, determine the enzyme
inactivation (Offerman et al., 1984). The free
radicals can modify the proteins and, indirectly,
initiating the lipid peroxidation, the products of
lipid peroxides decomposition (malondialde-
hyde, 4-hydroxynonenal) can block irreversibly
the -NH, and -SH groups in proteic structures
(Halliwell and Gutteridge, 1989).
Ultraviolet radiations induce the formation of
the active oxygen species and the lipid per-
oxides, or their disintegration products that
result in important structural modifications at
the level of nucleic acids and proteins (Ames
er al., 1982; Basu and Mamett, 1983; Kligman
and Gebre, 1991). Ultraviolet irradiation of
some proteins or cells also produce the photoox-
idation of tryptophan residues (Salmon et al.,
1992).
229

230 IOAN FLOREA DUMITRU and MARINA TAMARA NECHIFOR
of cytotoxic radicalic species (Romandini et al.,
1992; Floyd and Carney, 1993).
This work concentrates on the finding of
biochemical criteria for the estimation of the
oxygen free radical (generated by U.V. radiations
and copper acetate) effect on the action of the
glucose-6-phosphate dehydrogenase from Sac-
charomyces cerevisiae, an enzyme initiating a
major metabolic way for NADPH generation.
MATERIAL AND METHODS
The homogenate with enzymatic activity was
obtained from beer-yeast (Saccharomyces cere-
uisiae), by grinding it with quartz sand, followed
by an extraction in a buffer solution Tris-HCI
0.055 M, pH 7.8, for 1 hr at 40°C and continued
overnight at 4°C. The homogenate was cen-
trifuged at 2000g for 10 min and the supernatant
was again centrifuged at 10,OOOg for 20 min. All
the experiments were made on the final super-
natant.
The determination of glucose-6-phosphate
dehydrogenase activity was made by the Nolt-
mann and Kuby method (1963). A unit of
enzymatic activity is defined as that quantity of
enzyme that catalyzes the reduction of 1 pmol
of NADP in 1 min in optimum reaction con-
ditions. The protein content was determined by
the method of Lowry et al. (1951).
The measurement of malondialdehyde, pro-
duced through the decomposition of the lipid
peroxides, was performed by the thiobarbituric
acid reaction (Ohkawa et al., 1979).
The systems generating free radicals were
formed of lamps with u.v.-A emission
(320-400nm), equal to 6 and 70 lx, and of a
50 PM copper acetate solution. The lamps were
located 25 cm above the samples.
The electrophoretic study was made in gel of
7.5% polyacrylamide, in a vertical system, using
a Midget-Pharmacia LKB electrophoresis ap-
paratus. Volumes of 30 pi/sample were applied,
having a proteic ~on~ntration of 3.93 mg/ml,
the electrophoretic migration being achieved at
lO”C, at 30 mA/plate, for 60 min. The electro-
phoretic bands with enzymatic activity were
made evident by incubating the gel in a buffer
solution Tris-HCl 0.055 M, pH 7.8, containing
0.03% glucose-6-phosphate, 0.01% NADP,
0.02% Nitro BT, 0.002% phenazine methosul-
fate, for 1 hr at 37°C.
~hemi&als
All reagents were of the best grade commer-
cially available and were used without further purification. NADP and glucose-6-phosphate
were supplied by Merck and the Tris buffer by
Serva. Nitro BT and phenazine methosulfate
were purchased from Aldrich.
RESULTS AND DISCUSSIONS
In a first stage, the effects of free radicals on
the glucose-6-phosphate activity were studied by
observing the modification of the enzymatic
activity after the irradiation with U.V. and incu-
bation with copper acetate of the extract ob-
tained from a culture of S. cerevisiue. Figure 1
shows the inactivating effect of these radical
generating systems on the enzymatic activity, at
different time periods, using three experimental
alternatives: irradiation with U.V. (70 lx), incu-
bation with 50 PM copper acetate and ir-
radiation with U.V. in the presence of copper
acetate. After 90min of the free radical source
action only 58.8, 57.3 and 27.8% out of the
initial enzymatic activity was noticed,
suggesting that the glucose-6-phosphate dehy-
drogenase inactivation percentage is pro-
portional to the concentration of the formed
free radicals. This statement is supported by the
results of other similar experiments, in which a
U.V. radiation source of 6 lx was used, where the
enzymatic activities remaining after 90min of
irradiation are signi~cantly higher: 84.3% in the
extract irradiated with the U.V. lamp of 6 lx and
47.0% in the alternative with U.V. irradiation of
6 lx in the presence of 50 p M copper acetate.
The values represent the average of the signifi-
cant statistic experiment sets.
The dynamics of the oxygen free radicals
concentration, controlled by the formation of
the malondialdehyde in the irradiated and
6
a I 1 I
0 30 60 90
Time (min)
Fig. I. The modification of glucose-&phosphate dehydro-
genase activity at different time periods, after the irradjation
with U.V. (70 Ix) (O), incubation with 50 PM copper acetate
(x) and irradiation with u.v. (70 Ix) in the presence of copper
acetate (@).

Glucose-6-phosphate dehydrogenase and free radicals 231
Control “V.
Fig. 2. The correlation between the level of glucose-6-phos-
phate dehydrogenase activity (0) and the malondialdehyde
(MDA) concentration (tZ4) in the native extract and under
the action of free radicals, formed after 90min of U.V.
irradiation (701x), incubation with 50pM copper acetate
and U.V. irradiation in the presence of copper acetate.
non-irradiated extracts is shown in Fig. 2 and
describes a good correlation between the glu-
cose-6-phosphate dehydrogenase inactivation
degree and the increase of malondialdehyde
concentration. By relating our data to the ones
in the literature, we suggest the possibility that
the inactivation can be determined even by the
malonylation of the amino groups from the
enzyme structure. By using the same free radical
generating sources, U.V. and Cu2 + , Salmon et al.
(1992) demonstrated high-density lipoproteins’
malondialdehyde-modification resulting in an in-
adequate recognition of these lipoproteins chemi-
cally modified by the cellular receptors for HDL.
To confirm our supposition, the affinity of the
native enzyme and of that under the action of
free radicals for NADP and glucose-6-phos-
phate were studied. Figures 3(A) and (B) shows
the Lineweaver-Burk graphic processing of the
saturation curve in those substrate. For the
enzyme in non-irradiated extract, a value of
K,,, = 3.6 x 10 -‘M NADP was recorded in com-
parison with K,,, = 1.8 x 10m5M for the enzyme
under the action of free radicals, formed by
irradiation with U.V. 70 lx in the presence of
50 PM copper acetate. These values show a
high affinity of glucose-6-phosphate dehydro-
genase for the coenzyme, even in oxidizing
conditions, the Km NADP values having the
same order of magnitude as that reported by
other authors (Lowry et al., 1961; Crans et al.,
1992). The K,,, value for glucose-6-phosphate
remained practically unmodified in the presence
of free radicals, of 2.05 x 10m4M glucose-6-
phosphate respectively, in comparison with
2.20 x lop4 for the enzyme from non-irradiated
extract [Fig. 3 (B)]. The low energy radiations are also reflected in
the dynamics of the glucose-6-phosphate dehy-
drogenase’s molecular forms. Figure 4 shows
the electrophoresis pattern achieved for the
native extract and for the three above men-
tioned experimental alternatives. Five elec-
trophorectic bands were made evident in the
initially non-treated extract [Fig. 4 (A)]. This
result agrees with the model presented for glu-
cose-6-phosphate dehydrogenase from erythro-
cytes (Yoshida, 1966). This author asserts the
existence of five molecular forms of the enzyme
that can be converted, with different molecular
weight and catalytic activity. Under the action
of the free radicals generated in those three
experimental alternatives (u.v. irradiation, U.V.
irradiation in presence of 50 PM copper acetate
and incubation with 50 PM copper acetate),
with the time of action of the radical generating
source of 60 min, one can notice the disappear-
ance of a molecular form [Figs 4(B), (C), (D)],
perhaps as a result of the modification of the
interactions among the subunits or of the mal-
ondialdehyde-modification of some constitutive
aminoacid residues and consequently of the
>
>
9 0
l/[NADPI (mM_‘)
0 IO
l/Cglucose-6-PI (rnM_‘1
Fig. 3. The Lineweaver-Burk graphic processing of the
saturation curve in NADP (A) and glucose-6-phosphate (B)
for the glucose-6-phosphate dehydrogenase in the native
extract (0) and after 60 min of U.V. irradiation (70 lx) in the
presence of 50pM copper acetate (x).

232 IOAN FLOREA DIJMITRU and MARINA TAMARA NECHIFOR
(A) (El) (Cl (II)
(B) Absorbance
Absorbonce (A-sln:OO4 A-moxr0691 A-0x1s 1s norrnd~zed Bose lme 01 0032 AU $5 (A-sm-O.O3A-mm-0.701
252626 30 32 34 36 36 40 42 44 46 46
Absorbance
0.76
1 (Cl 070
CI60 Y-positton 7 Y-start=25 y-step-3
(m) Y-stw-so l~120micronsI
A-0x1s: 1s normalized
IA-s,n = 0.03 A-mar: 0.761 Absorbance iA-sin:O.O4A-maxz069)
069
a60
0.70
0.60
Osb
040
i (D) 4
2526 26 30 32 34 36 36 40 42 44 46 46 2526 26 30 32 34 36 36 40 42 44 46 46
Y-POSItIon – Y-start : 25 Y-step: 3 Y-poshon v Y-stort=25 v-step:3
trn) y-stop: 50 (: I20 microns) lm) Y-stop=50 ~:120mlcrons)
Fig. 4. (a) The electrophoresis pattern of glucose-6-phosphate dehydrogenase in the native extract (A) and
under the action of free radicals, formed by U.V. irradiation (70 lx) (B), U.V. irradiation in the presence
of 50 PM copper acetate (C) and incubation with 50 PM copper acetate (D); (b) the corresponding
densitograms of the lanes A, B, C, D.
glucose-6-phosphate dehydrogenase subunits’ (Gracy, 1992; Stadtman et al., 1993). Recently,
structure. Jeffery et al., 1993) have shown that possible
Numerous authors have shown that free rad- active site arrangements in 8 glucose-6-phos-
ical generating irradiations are reflected in the phate dehydrogenases involve in classical a-he-
modification of some aminoacid residues lix His-200, Tyr-201, Gly-203 and Lys-204,

Glucose-&phosphate dehydrogenase and free radicals 233
namely the aminoacids that are easily attacked
by the free radicals, result from cell and organ-
ism irradiation (Amici et al., 1989; Fulbert et al.,
1992).
The investigation of the action of the oxygen
free radicals, generated by various ionizing radi-
ation sources, has been carried out to satisfy
medical demands, usually using the periods and
doses adequate for the recommended treat-
ments. In a lesser degree the radicals’ generating
factors and systems with a daily action on
organism have also been considered.
The experimental results described in this
work justify the hypothesis that glucose-6-phos-
phate dehdyrogenase beside other used methods
or enzymes sensitive to radiations can be a
sensor for the estimation of low energy ir-
radiation, or of the insignificant concentration
degrees of certain metals.
Acknowledgements-This work was supported by the
Romanian Ministry of Education and Science, grant
48/1991-1993. The authors would like to thank Professor R.
Mester and Dr. A. Dinischiotu for their constructive review
of this manuscript.
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