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A genetic variant of human erythrocyte glucose 6-phosphate dehydrogenase
Vol.
132,
November
BIOCHEMICAL
No. 3, 1985 15,
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
?ages 1151-1159
1985
A GENETIC VARIANT OF HUMAN ERYTHROCYTE GLUCOSE ~-PHOSPHATE DEHYDROGENASE Bahram
Haghighi,
Biochemistry
Received
September
Mohammad Suzangar, and Marzieh Mehnat Department, Isfahan,
25,
Afsaneh
University Iran
Yazdani,
of Isfahan,
1985
Human erythrocyte G6PD activity was measured in more than 500 subjects in Isfahan, Iran, and the percent of enzyme deficiency for males and femals are reported. Some properties of the abnormal enzyme is compared with its normal counterpart. Apparent Km values of glucose 6-phosphate for the variant and normal enzymes were 37 and 101 PM , respectively. The variant enzyme was less resistant to inhibition by 40 pM NADPH( 72% inhibition )than the normal enzyme(48% inhibition). The mode of inhibition for both enzymes was competitive with NADP+. ATP at 1.5 mM concentration also inhinbited normal and variant enzymes at 17% The inhibition was competitive with and lo%, respectively. glucose 6-phosphate. Polyacrylamide gel electrophores showed that normal enzyme has one major and another weak active bands, while the variant enzyme under identical conditions shows only one active band corresponding to the major band of the normal enzyme. Thermostability of variant G6PD was slightly lower that normal but no significant differences observed in their energy of activation. The activity pH profile of the variant enzyme was truncate. 0 1985
Academic
Press,
Inc.
Glucose 6-phosphate dehydrogenasec D-glucose 6-phosphate: NADP oxidoreductase, E.C. 1.1.1. 491, the initial enzyme of the pentose phosphate pathway,is of prime importance in generation of NADPH in human erythrocytes. NADPH is necessary for maintainance of adequate level of reduced glutathione (l-3). NADPH is also needed to optimize catalase activity. Catalase and reduced glutathione, in turn, are ABBREvIATIoNs:G6PD, glucose 6-phosphate dehydrogenase; NADP+ , nicotineamide adenine dinucleotide phosphate; NADPH , reduced nicotineamide adenine dinucleotide phpsphate ; ATP, adenosine 5Ltriphosphate; TEMED, N,N,N,N-tetramethylenediamine ; PMS, phenazine methosulfate; NBT, Nitroblue tetrazolium, G6P , glucose 6-phosphate. 0006-291X/85 1151
AN
Copyright 0 1985 rights of reproduction
$1.50
by Academic Press, Inc. in an-v form reserved.
Vol.
132,
No. 3, 1985
essential structural
for the integrity
deficiency concentration to anemia. More various hemolytic
of
than
enzyme(7). We are deficiency and the
BIOPHYSICAL
RESEARCH
140 variants
of human
COMMUNICATIONS
biochemical and (1,2). Severe genetic
G6PD is frequently associated of reduced glutathione with
with a low suseptibility
erythrocyte
G6PD from
have been reported(4,5). from abnormal variant
The forms of
be either spontaneous or induced by adminiscertain drugs(Q). Drug induced hemolgtic anemia
G6PD deficiency Over 80 variants
from each substrate
AND
protection of the of enthrocytes
parts of the world anemia resulted
G6PD could tration of in
BIOCHEMICAL
has been recentlv reviewed by Beutler(6). of G6PD have been distinguishable
other by their electrophoretic mobilities, specificity and kinetic characteristics not
aware
of
any
systematic
in Iran to date. In this degree of enzyme deficiency
Iranian males and females are the type of the enzyme variant and physicochemical enzymes have been
properties compared.
of human
study G6PD activity in red blood cells
reported, found, of
study
of
the G6PD of
To characterize some enzymological
normal
and abnormal
MATERIALS AND METHODS Reagens : NADP+, NADPH, glucose 6-phosphate, ATP, NBT, PMS, methylen blue, oxidized glutathione, acrylamide,bisacrylamide TEMED, ammunium persulfate, and sauonine were all obtained from Sigma Chemical Co.(U,S.A.). All other chemicals were reagent grade. Sereening Test : Blood samples were collected from randomly selected male and female sub.iects. The samples were collected on either EDTA or ACD (acid-citrate-dextrose) as anticoagulant. The fluorescent screening test described by Beutter et.al(ll) was used for the survev. For quantitative measurement of enzyme activity,the red blood cells of each sample were washed three times with 0.9% NaCl and hemolyzed in a solution containing 10 PM NADP+, 7mM, pmercaptoethanol and 2.7mM EDTA according to the WHO recomended method(8). The mixture was then centrifuged at 15OOOg for 20 minutes at 4pC using a Surval Model RC58 centrifuge and the supernatant was kept at 0-4°C nrior to the assav. Enzyme Assay : Enzyme activity was measured at 34Onm in a Beckman Model 25 reiording snectrophotometer with a thermostated cell compartment maintained at 30°C. The reaction mixture contained: O.lM Tris-KC1 buffer pH 3.0, O.OlM MgC12 ,0.2mM NADP+ and 0.6mM glucose-6-phosphate(9). One unit of enzyme activity was taken as the number of pmoles of NADP+ reduced per minute at 30°C in the reaction mixture used. 11.52
Vol.
132,
No. 3, 1985
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
: Protein concentration was determined Protein Determination bv Biuret method( Normal and variant G6PD, from human Enzyme Purification': erythrocytes of male individuals were partially purified. The packed cells obtained from 20ml of blood were washed The cells were hemolyzed and sub3 times with 0.9% NaCl. .jected to DEAE-Sephadex chromatography as described by Rattozzi(l2).The enzyme eluted from the column was precipitated with ammonium sulfate (4Og/lOO ml); the precipitate was separated,dissolved in Q.5M Tris-HCl buffer pH 8.0,containing lO@l NADP+, 0.27M EDTA (pH 7.0), lmP4 pmercaptoethanol and dialyzed against the same buffer for 24 hours with two changes. Polyacrglamide Gel Eletroohoresis: Disc gel electrophoresis was performed according to the method of Gabriel(lj)u a Pharmacia gel electrophoresis aparatus GE-Q. The gel concentration was 7% and 0.26%(W/V) for acrylamide and bisacrylamide respectively, The gel was prepared in 0.375M and ammonium persulfate at O.O7%(W/V),olus Tris-HCl pH 9.3, TEMED at 0.06% (W/V) were used for polymerization. The anodic buffer was 120mM Tris-HCl pH 8.1 and the cathodic buffer was 43mM Tris-glycine pH 8.9. Active bands were stained using the method of Dewey and Conklin(lQ), but omitting KCN; the staining, solution contained O.O016$(W/V) PMS,O.O26%(W/V) NET,O.ZmM NADPf,0.6mM glucose-6-phosnhat and 0.09M Tris-HC1 pH 7.8. Staining was terminated by placing the gels in 7% (V/V) acetic acid according to Richards and Hilf(l5). Kinetic Experiments: The steady state kinetic experiments were performed at 30°C. All coenzyme substrate or inhibitor solutions were prepared and if necessary neutralized, on the day of the experiments and ihe pH of each'solution was checked. All assays, were done in duplicate. The initial velocities were measured in all experiments. Data from these initial velocity experiments were plotted as Lineweaver-Burk plots. From with the apparent Km values were colculated( 16 ). Thermostability studies : Thermostability for normal and variant G6PDs were carried out under the identical conditions according to the report of WHO scientific group(8). Activation Energy studies : Activation energy was obtained based on the effect of temperature on the maximal velocity @f the reaction. The log (velocity) was plotted against l/T (When T is the absolute temperature in K"). Since the system followed Arrhenius Law, the activation energy obtained from the slope of the straight line. The enzyme in the assay mixture was incubated at temperatures of 25,30,35 and 400~ for 10 minutes and each time assayed in constant temperature cuvette chamber as described. RESULTS
AND DISCUSSION
G6PD Deficiency : Five hundred and three males, 260 females) collected randomly survey study. The enzymatic activity of was first qualitatively tested using the described above. The enzyme activities normal and the remaining samples showed 1153
blood samples (243 were used for the the red screening of
blood test
385 subjects either little
cells were
Vol. 132, No. 3, 1985
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE 1 Human erythrocgte Number of subjects
glucose-6-phosphate Partially
Deficient I
defi%ient
activity Normal 7i
More than Normal ( 150%)
Female
260
0.76
4.61
93.84
0.76
Male
243
9.37
0.41
89.3
0.41
For details
activity
see text.
Or were
close
to thenormalvalues..
The latter
samples
subjected to quantitative analysis for enzyme activity and results obtained were classified according to standard procedures (8). The activity of normal enzyme was found to be 2000 f 200 m.i.u per ml of packed cells. Thus, the mean value of 2000 m.i.U./ mplc was chosen for comparison between normal and deficient or partially deficient enzymes. These results are summerized in Table 1. Ten percent of the males and less than 1% of the females had G6PD activities of less than 25% About 5% of females showed mild enzyme of normal enzyme. activity having 25-65% of normal value. Male subjects with mild enzyme activity were less than 1%. The data alSO indicated that the enzyme with greater than 150% activity existed in less than 1% of both male and female individuals tested. These observations are consistant with the fact that there is a single stractural gene for G6PD which is located on the X chromosome (17). A large number of mutant G6PD have indentified in human erythrocytes from various parts of the world with altered enzymological properties (5). Attempts were made to characterize the enzyme variant Lack of sufficient amount of G6PD found In this study. deficient blood did not alow us to obtain homogenous enzyme. Thus, normal and variant G6PD were partially purified under identical conditions using 20ml of blood for comparative studies. Normal and deficient enzyme with spectic activities of 74 and 35 m.i.u./mg protein were obtained, respectively, with a yeild of about 74%. The major inhibitors for G6PD in red cells Kinetic studies: are NADPH and ATP(18). Figure 1 indicates that the variant
were
1154
Vol.
132,
No. 3, 1985
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1 12
B
4
0
I
I
Q
I
16
12
20
Time lDayd
Fig.
when
1. Effect of temperature and iron-limiting conditions on growth and sfderophore production by C. albicans. (A) Iron-binding activity of cultare fluids indicating presence of siderophoree In Lee’s medium; control at 37 C ( 0 ) and at 41 C ( 0 ); in the deferrated medium at 37’C ( n ), andat41°C( 0 ). Ferric chloride (0.06 M, pH 2.0) was used to form the colored iron-siderophore complex and absorbance at 480 nm was recorded. Uninoculated Lee’s medium with or without L,lOphenanthroline was used as a blank. (B) Growth of 5. albicans measured by total cell count. Stationary phase cells (48 h) were released into Lee’s medium; control at 37’C ( 0 ) and at 41°C ( 0 ); in the deferrated medium at 37’C ( n ), and at 41’C ( 0 ).
compared
affect
to
the amount
However,
(Figure
less
at
secreted
of siderophore than
the
For
both
was directly
41’C.
Elevated
in the
control
produced
amount
that
culture associated
with
medium
in the control
accumulated
media,
temperature
in
regardless the
total
did
(Figure
media
not 1A).
was
the deferrated
media
at
of temperature,
the
length
siderophore
production
1A).
Elevated (Figure
1B).
observed time,
temperature
in the
rate
was followed
in the deferrated
not
affect
the growth
suppression
deferrated
media
of candidal
when compared
temperatures.
did
A significant
the growth
recovery
37'C
produced
of siderophore
temperatures.
of incubation
growth
quantity
the quantity
comparatively both
the
to the
Although medium,
greater
at both cells
growth
by an increase
of growth
in
the
deferrated
siderophore
and 41'C. 1162
control
medium
to the controls 1B).
medium This
production
of siderophore difference
in
(Figure
of the controls.
quantities
no significant
compared
temperatures
rate in
of cells
was
However showed recovery
with
partial in
at both were
was apparent
produced in
at 37'C
the growth
at
Vol.
132,
No. 3, 1985
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
-1 -j x ,03 @q i 1
03
NAfd
Figure Figure
2,
BIOCHEMICAL
3. Effect of NADPH on reaction rate of normal G6PD. Symbols and the reaction mixture are the same as those in Figure 2. 4. Effect of ATP on reaction rate of the variant G6PD. 0, In the presence of 1.5mM ATP; without ATP. Reaction mixture contained 0.1 M Tris-HCl pH 8.0, 0.01 M MgC12 , 80 PM NADP+ and various concentrations of glucose 6-phosphate.
50 + 5,
40 and 1500 PM, respectively (18,lg). Therefore, the resistance of the present variant G6PD to ATP inhibition and higher affinity for glucose 6-phcsphate binding may compensate for may only occur
Figure
its low activity in the cell and the when oxidant compounds are present.
5. Effect of ATP on reaction Symbols and the reaction those in Figure 4. 1156
hemolysis
rate of normal G6PD. mixture are the same as
Vol.
132,
No.
3, 1985
Characteristics
G6PD Normal Variant
BIOCHEMICAL
of normal
and
Apporent Km Activity (;6P %Normal (uM)
Number active
101 37
2 1
1.0 0 58
AND
BIOPHYSICAL
TABLE 2 variant glucose
Electrophoretic
RESEARCH
6-phosphate
mobility
of bands
COMMUNICATIONS
dehydrogenase
Heat stability
PH optima
Energy of Activation
R, of major active bands 0.22 0.22
i 0.009 f 0.025
KCal/mol Normal Slightly low
8-9 Truncate
5.34 5.21
: In polyacrylamide gel electrophoresis Gel Electrophoresis the normal G6PD showed one major active band with relative mobility (R,) of 0.22 + 0.009 and another weak active band with Rm of 0.39. However, the mutant enzyme under the identical conditions showed only one active band corresponding to the major band of the normal enzyme (Rm of 0.22 + 0.025) in the gel pattern (Table 2). Elimination of glucose 6phosphate form the staining of the active bands, rulling of other
dehydrogerases.
Liu
solution resulted out the probable et.
a1.(20)
in disappearance interferance
reported
insoelecric fucusing, G6PD is separated in 7 active and on sebsequent two-dimensional gel electrophoresis band is resolved into 2 components, the band and appeared to be a dimeric form.
first Thus,
that
in
bands each
being the major the fast moving
band of normal enzyme observed in this study could be a monomeric form or the digest product of slow component. As isolation no effect
and storage procedures of human blood G6PD has on the enzyme (21), different bands observed may
represent polymorphism of the enzyme. Table 2 also shows that heat stabiliy
of
the variant
G6PD is slightly lower than that of the normal enzyme (slopes of the line obtained by plotting activity versus time of incubation were -0.67 and -0.7, respectively). No significant change was observed in the energy of activation for normal and abnormal enzymes. The determination of G6PD reaction rates at various pHs permits discrimination between some G6PD variants. The reaction rates of a normal enzyme and a partially deficient enzyme was measured at different pHs but at constant conditions such as ionic strength and buffer. The pH profiles 11.57
Vol. 132, No. 3, 1985
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PH
Figure
6. Dependence of G6PD activity on pH. The activities of normal ( 0 ) and variant ( Cl ) enzymes were measured in 0.2 mMNADP+, 0.6 mM glucose 6phosphate. 0.01 M MgC12 and 0.1 M Tris-HCl buffer at various pHs.
shown in Figure 6 indicates that normal enzyme is between 8-9 but
the the
optimum pH for the pH curve of the variant
enzyme is much more truncate with no clear peack maximum which consistent with many other G6PD variants previously reported(l8). The altered properties observed for the abnormal enzyme could be due to a conformational change brought
by the genetic
about
defect. REFERENCES
1. 2. 3.
4. 2: 7.
a. 9. Ill I”.
11.
Eaton,J.W., and Brewer, G.J.(1974) pentose phosphate metabolism. In "The Red Blood Cell". Surgenor ,D.M., editor .,2nd ed., ~01.1, PP 435-471, Academic Press , New York. Keller,D.F.(1971),Glucose-6-phosphate dehydrogenase deficiency, CRC press, Celeveland. Beutler, E.(1978)Glucose-6-phosphate dehydrogenase dieficiency. In,"The Metabolic Basis of Inherited Diseases",Stanbury J.B., Wyngaarden J.B., and Fredrickson D.S.,editors, 4th ed., Chap. 60,McGrawHill Book Co. Inc. New York. Yoshida, A.(1973) Science, 179, 532-537. Advan. Enzymol. 48,97-192. Levy, H.R.(1979) Beutler, E.(1984) Banbury Rep. 16, 205-11. Yoshida, A.,Beutler, E., Motulsky, A.G.(1971)Bult. World Health Organ. 45,243-253. Report of a WHO Scientific Group(l967)Wld. Hlth. Techn. Rep. Ser. 366, 5-53. Kahn, A,, and Dreyfus, J.(1974)Biochim. Biophys. Acta, 334,257-265. __ Weichselbaum, T.E.(1946)Am.J.Clin.Pathol.Tech.Sect.,lO, 16. Beutler,E., Blume, K.G., Kaplan, J.C., Lohr, G.W., Ramot, B ., and Walentine W.M.(1979) Brit. J. Haemat. 43, 465-467. 1158
is
Vol.
132.
12. 13. 14. 15. 16. 17. 18. 19 -
20. 21.
No. 3, 1985
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Rattazzi, M.C.(1969)Biochim. Biophys. Acta, 181, l-11. Gabriel 0.(1971) Meth. Enzymology, 22, 565-578, Dewey, M.M., and Coklin, J.L.(1960) Proc. SOC. Exptl. Biol. Med., 105, 492-494. Richards, A.H. and Hilf, R.(1972) Endocrinology, 91 , 287-295. W.W.(1967)Advan, Enzymol. 29, l-32. Cleland, Kirkman, J.N., and Hendrickson, E.n'I.(1963)Ame.J.Human. Genet., 15, 241-258. Yoshida, A., and Lin, M.(1973) Blood 41, 877-891. Minakami, S., Suzuki, C., Saito, T, Yoshikawa, H,(1965) J.Biochem.(Tokyo) 58,543-550. Liu, M.S., Liou, R.F., Chen. Y.H., Chen, S.H.(1984) Sci. Count. ,Repub China, Part B 8,155-160. Proc. Natl. Shatskayo, T.L.(1983) Mol. Genet. Microbial. Virusol. g, 18-22.
1159
132,
November
BIOCHEMICAL
No. 3, 1985 15,
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
?ages 1151-1159
1985
A GENETIC VARIANT OF HUMAN ERYTHROCYTE GLUCOSE ~-PHOSPHATE DEHYDROGENASE Bahram
Haghighi,
Biochemistry
Received
September
Mohammad Suzangar, and Marzieh Mehnat Department, Isfahan,
25,
Afsaneh
University Iran
Yazdani,
of Isfahan,
1985
Human erythrocyte G6PD activity was measured in more than 500 subjects in Isfahan, Iran, and the percent of enzyme deficiency for males and femals are reported. Some properties of the abnormal enzyme is compared with its normal counterpart. Apparent Km values of glucose 6-phosphate for the variant and normal enzymes were 37 and 101 PM , respectively. The variant enzyme was less resistant to inhibition by 40 pM NADPH( 72% inhibition )than the normal enzyme(48% inhibition). The mode of inhibition for both enzymes was competitive with NADP+. ATP at 1.5 mM concentration also inhinbited normal and variant enzymes at 17% The inhibition was competitive with and lo%, respectively. glucose 6-phosphate. Polyacrylamide gel electrophores showed that normal enzyme has one major and another weak active bands, while the variant enzyme under identical conditions shows only one active band corresponding to the major band of the normal enzyme. Thermostability of variant G6PD was slightly lower that normal but no significant differences observed in their energy of activation. The activity pH profile of the variant enzyme was truncate. 0 1985
Academic
Press,
Inc.
Glucose 6-phosphate dehydrogenasec D-glucose 6-phosphate: NADP oxidoreductase, E.C. 1.1.1. 491, the initial enzyme of the pentose phosphate pathway,is of prime importance in generation of NADPH in human erythrocytes. NADPH is necessary for maintainance of adequate level of reduced glutathione (l-3). NADPH is also needed to optimize catalase activity. Catalase and reduced glutathione, in turn, are ABBREvIATIoNs:G6PD, glucose 6-phosphate dehydrogenase; NADP+ , nicotineamide adenine dinucleotide phosphate; NADPH , reduced nicotineamide adenine dinucleotide phpsphate ; ATP, adenosine 5Ltriphosphate; TEMED, N,N,N,N-tetramethylenediamine ; PMS, phenazine methosulfate; NBT, Nitroblue tetrazolium, G6P , glucose 6-phosphate. 0006-291X/85 1151
AN
Copyright 0 1985 rights of reproduction
$1.50
by Academic Press, Inc. in an-v form reserved.
Vol.
132,
No. 3, 1985
essential structural
for the integrity
deficiency concentration to anemia. More various hemolytic
of
than
enzyme(7). We are deficiency and the
BIOPHYSICAL
RESEARCH
140 variants
of human
COMMUNICATIONS
biochemical and (1,2). Severe genetic
G6PD is frequently associated of reduced glutathione with
with a low suseptibility
erythrocyte
G6PD from
have been reported(4,5). from abnormal variant
The forms of
be either spontaneous or induced by adminiscertain drugs(Q). Drug induced hemolgtic anemia
G6PD deficiency Over 80 variants
from each substrate
AND
protection of the of enthrocytes
parts of the world anemia resulted
G6PD could tration of in
BIOCHEMICAL
has been recentlv reviewed by Beutler(6). of G6PD have been distinguishable
other by their electrophoretic mobilities, specificity and kinetic characteristics not
aware
of
any
systematic
in Iran to date. In this degree of enzyme deficiency
Iranian males and females are the type of the enzyme variant and physicochemical enzymes have been
properties compared.
of human
study G6PD activity in red blood cells
reported, found, of
study
of
the G6PD of
To characterize some enzymological
normal
and abnormal
MATERIALS AND METHODS Reagens : NADP+, NADPH, glucose 6-phosphate, ATP, NBT, PMS, methylen blue, oxidized glutathione, acrylamide,bisacrylamide TEMED, ammunium persulfate, and sauonine were all obtained from Sigma Chemical Co.(U,S.A.). All other chemicals were reagent grade. Sereening Test : Blood samples were collected from randomly selected male and female sub.iects. The samples were collected on either EDTA or ACD (acid-citrate-dextrose) as anticoagulant. The fluorescent screening test described by Beutter et.al(ll) was used for the survev. For quantitative measurement of enzyme activity,the red blood cells of each sample were washed three times with 0.9% NaCl and hemolyzed in a solution containing 10 PM NADP+, 7mM, pmercaptoethanol and 2.7mM EDTA according to the WHO recomended method(8). The mixture was then centrifuged at 15OOOg for 20 minutes at 4pC using a Surval Model RC58 centrifuge and the supernatant was kept at 0-4°C nrior to the assav. Enzyme Assay : Enzyme activity was measured at 34Onm in a Beckman Model 25 reiording snectrophotometer with a thermostated cell compartment maintained at 30°C. The reaction mixture contained: O.lM Tris-KC1 buffer pH 3.0, O.OlM MgC12 ,0.2mM NADP+ and 0.6mM glucose-6-phosphate(9). One unit of enzyme activity was taken as the number of pmoles of NADP+ reduced per minute at 30°C in the reaction mixture used. 11.52
Vol.
132,
No. 3, 1985
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
: Protein concentration was determined Protein Determination bv Biuret method( Normal and variant G6PD, from human Enzyme Purification': erythrocytes of male individuals were partially purified. The packed cells obtained from 20ml of blood were washed The cells were hemolyzed and sub3 times with 0.9% NaCl. .jected to DEAE-Sephadex chromatography as described by Rattozzi(l2).The enzyme eluted from the column was precipitated with ammonium sulfate (4Og/lOO ml); the precipitate was separated,dissolved in Q.5M Tris-HCl buffer pH 8.0,containing lO@l NADP+, 0.27M EDTA (pH 7.0), lmP4 pmercaptoethanol and dialyzed against the same buffer for 24 hours with two changes. Polyacrglamide Gel Eletroohoresis: Disc gel electrophoresis was performed according to the method of Gabriel(lj)u a Pharmacia gel electrophoresis aparatus GE-Q. The gel concentration was 7% and 0.26%(W/V) for acrylamide and bisacrylamide respectively, The gel was prepared in 0.375M and ammonium persulfate at O.O7%(W/V),olus Tris-HCl pH 9.3, TEMED at 0.06% (W/V) were used for polymerization. The anodic buffer was 120mM Tris-HCl pH 8.1 and the cathodic buffer was 43mM Tris-glycine pH 8.9. Active bands were stained using the method of Dewey and Conklin(lQ), but omitting KCN; the staining, solution contained O.O016$(W/V) PMS,O.O26%(W/V) NET,O.ZmM NADPf,0.6mM glucose-6-phosnhat and 0.09M Tris-HC1 pH 7.8. Staining was terminated by placing the gels in 7% (V/V) acetic acid according to Richards and Hilf(l5). Kinetic Experiments: The steady state kinetic experiments were performed at 30°C. All coenzyme substrate or inhibitor solutions were prepared and if necessary neutralized, on the day of the experiments and ihe pH of each'solution was checked. All assays, were done in duplicate. The initial velocities were measured in all experiments. Data from these initial velocity experiments were plotted as Lineweaver-Burk plots. From with the apparent Km values were colculated( 16 ). Thermostability studies : Thermostability for normal and variant G6PDs were carried out under the identical conditions according to the report of WHO scientific group(8). Activation Energy studies : Activation energy was obtained based on the effect of temperature on the maximal velocity @f the reaction. The log (velocity) was plotted against l/T (When T is the absolute temperature in K"). Since the system followed Arrhenius Law, the activation energy obtained from the slope of the straight line. The enzyme in the assay mixture was incubated at temperatures of 25,30,35 and 400~ for 10 minutes and each time assayed in constant temperature cuvette chamber as described. RESULTS
AND DISCUSSION
G6PD Deficiency : Five hundred and three males, 260 females) collected randomly survey study. The enzymatic activity of was first qualitatively tested using the described above. The enzyme activities normal and the remaining samples showed 1153
blood samples (243 were used for the the red screening of
blood test
385 subjects either little
cells were
Vol. 132, No. 3, 1985
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE 1 Human erythrocgte Number of subjects
glucose-6-phosphate Partially
Deficient I
defi%ient
activity Normal 7i
More than Normal ( 150%)
Female
260
0.76
4.61
93.84
0.76
Male
243
9.37
0.41
89.3
0.41
For details
activity
see text.
Or were
close
to thenormalvalues..
The latter
samples
subjected to quantitative analysis for enzyme activity and results obtained were classified according to standard procedures (8). The activity of normal enzyme was found to be 2000 f 200 m.i.u per ml of packed cells. Thus, the mean value of 2000 m.i.U./ mplc was chosen for comparison between normal and deficient or partially deficient enzymes. These results are summerized in Table 1. Ten percent of the males and less than 1% of the females had G6PD activities of less than 25% About 5% of females showed mild enzyme of normal enzyme. activity having 25-65% of normal value. Male subjects with mild enzyme activity were less than 1%. The data alSO indicated that the enzyme with greater than 150% activity existed in less than 1% of both male and female individuals tested. These observations are consistant with the fact that there is a single stractural gene for G6PD which is located on the X chromosome (17). A large number of mutant G6PD have indentified in human erythrocytes from various parts of the world with altered enzymological properties (5). Attempts were made to characterize the enzyme variant Lack of sufficient amount of G6PD found In this study. deficient blood did not alow us to obtain homogenous enzyme. Thus, normal and variant G6PD were partially purified under identical conditions using 20ml of blood for comparative studies. Normal and deficient enzyme with spectic activities of 74 and 35 m.i.u./mg protein were obtained, respectively, with a yeild of about 74%. The major inhibitors for G6PD in red cells Kinetic studies: are NADPH and ATP(18). Figure 1 indicates that the variant
were
1154
Vol.
132,
No. 3, 1985
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1 12
B
4
0
I
I
Q
I
16
12
20
Time lDayd
Fig.
when
1. Effect of temperature and iron-limiting conditions on growth and sfderophore production by C. albicans. (A) Iron-binding activity of cultare fluids indicating presence of siderophoree In Lee’s medium; control at 37 C ( 0 ) and at 41 C ( 0 ); in the deferrated medium at 37’C ( n ), andat41°C( 0 ). Ferric chloride (0.06 M, pH 2.0) was used to form the colored iron-siderophore complex and absorbance at 480 nm was recorded. Uninoculated Lee’s medium with or without L,lOphenanthroline was used as a blank. (B) Growth of 5. albicans measured by total cell count. Stationary phase cells (48 h) were released into Lee’s medium; control at 37’C ( 0 ) and at 41°C ( 0 ); in the deferrated medium at 37’C ( n ), and at 41’C ( 0 ).
compared
affect
to
the amount
However,
(Figure
less
at
secreted
of siderophore than
the
For
both
was directly
41’C.
Elevated
in the
control
produced
amount
that
culture associated
with
medium
in the control
accumulated
media,
temperature
in
regardless the
total
did
(Figure
media
not 1A).
was
the deferrated
media
at
of temperature,
the
length
siderophore
production
1A).
Elevated (Figure
1B).
observed time,
temperature
in the
rate
was followed
in the deferrated
not
affect
the growth
suppression
deferrated
media
of candidal
when compared
temperatures.
did
A significant
the growth
recovery
37'C
produced
of siderophore
temperatures.
of incubation
growth
quantity
the quantity
comparatively both
the
to the
Although medium,
greater
at both cells
growth
by an increase
of growth
in
the
deferrated
siderophore
and 41'C. 1162
control
medium
to the controls 1B).
medium This
production
of siderophore difference
in
(Figure
of the controls.
quantities
no significant
compared
temperatures
rate in
of cells
was
However showed recovery
with
partial in
at both were
was apparent
produced in
at 37'C
the growth
at
Vol.
132,
No. 3, 1985
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
-1 -j x ,03 @q i 1
03
NAfd
Figure Figure
2,
BIOCHEMICAL
3. Effect of NADPH on reaction rate of normal G6PD. Symbols and the reaction mixture are the same as those in Figure 2. 4. Effect of ATP on reaction rate of the variant G6PD. 0, In the presence of 1.5mM ATP; without ATP. Reaction mixture contained 0.1 M Tris-HCl pH 8.0, 0.01 M MgC12 , 80 PM NADP+ and various concentrations of glucose 6-phosphate.
50 + 5,
40 and 1500 PM, respectively (18,lg). Therefore, the resistance of the present variant G6PD to ATP inhibition and higher affinity for glucose 6-phcsphate binding may compensate for may only occur
Figure
its low activity in the cell and the when oxidant compounds are present.
5. Effect of ATP on reaction Symbols and the reaction those in Figure 4. 1156
hemolysis
rate of normal G6PD. mixture are the same as
Vol.
132,
No.
3, 1985
Characteristics
G6PD Normal Variant
BIOCHEMICAL
of normal
and
Apporent Km Activity (;6P %Normal (uM)
Number active
101 37
2 1
1.0 0 58
AND
BIOPHYSICAL
TABLE 2 variant glucose
Electrophoretic
RESEARCH
6-phosphate
mobility
of bands
COMMUNICATIONS
dehydrogenase
Heat stability
PH optima
Energy of Activation
R, of major active bands 0.22 0.22
i 0.009 f 0.025
KCal/mol Normal Slightly low
8-9 Truncate
5.34 5.21
: In polyacrylamide gel electrophoresis Gel Electrophoresis the normal G6PD showed one major active band with relative mobility (R,) of 0.22 + 0.009 and another weak active band with Rm of 0.39. However, the mutant enzyme under the identical conditions showed only one active band corresponding to the major band of the normal enzyme (Rm of 0.22 + 0.025) in the gel pattern (Table 2). Elimination of glucose 6phosphate form the staining of the active bands, rulling of other
dehydrogerases.
Liu
solution resulted out the probable et.
a1.(20)
in disappearance interferance
reported
insoelecric fucusing, G6PD is separated in 7 active and on sebsequent two-dimensional gel electrophoresis band is resolved into 2 components, the band and appeared to be a dimeric form.
first Thus,
that
in
bands each
being the major the fast moving
band of normal enzyme observed in this study could be a monomeric form or the digest product of slow component. As isolation no effect
and storage procedures of human blood G6PD has on the enzyme (21), different bands observed may
represent polymorphism of the enzyme. Table 2 also shows that heat stabiliy
of
the variant
G6PD is slightly lower than that of the normal enzyme (slopes of the line obtained by plotting activity versus time of incubation were -0.67 and -0.7, respectively). No significant change was observed in the energy of activation for normal and abnormal enzymes. The determination of G6PD reaction rates at various pHs permits discrimination between some G6PD variants. The reaction rates of a normal enzyme and a partially deficient enzyme was measured at different pHs but at constant conditions such as ionic strength and buffer. The pH profiles 11.57
Vol. 132, No. 3, 1985
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PH
Figure
6. Dependence of G6PD activity on pH. The activities of normal ( 0 ) and variant ( Cl ) enzymes were measured in 0.2 mMNADP+, 0.6 mM glucose 6phosphate. 0.01 M MgC12 and 0.1 M Tris-HCl buffer at various pHs.
shown in Figure 6 indicates that normal enzyme is between 8-9 but
the the
optimum pH for the pH curve of the variant
enzyme is much more truncate with no clear peack maximum which consistent with many other G6PD variants previously reported(l8). The altered properties observed for the abnormal enzyme could be due to a conformational change brought
by the genetic
about
defect. REFERENCES
1. 2. 3.
4. 2: 7.
a. 9. Ill I”.
11.
Eaton,J.W., and Brewer, G.J.(1974) pentose phosphate metabolism. In "The Red Blood Cell". Surgenor ,D.M., editor .,2nd ed., ~01.1, PP 435-471, Academic Press , New York. Keller,D.F.(1971),Glucose-6-phosphate dehydrogenase deficiency, CRC press, Celeveland. Beutler, E.(1978)Glucose-6-phosphate dehydrogenase dieficiency. In,"The Metabolic Basis of Inherited Diseases",Stanbury J.B., Wyngaarden J.B., and Fredrickson D.S.,editors, 4th ed., Chap. 60,McGrawHill Book Co. Inc. New York. Yoshida, A.(1973) Science, 179, 532-537. Advan. Enzymol. 48,97-192. Levy, H.R.(1979) Beutler, E.(1984) Banbury Rep. 16, 205-11. Yoshida, A.,Beutler, E., Motulsky, A.G.(1971)Bult. World Health Organ. 45,243-253. Report of a WHO Scientific Group(l967)Wld. Hlth. Techn. Rep. Ser. 366, 5-53. Kahn, A,, and Dreyfus, J.(1974)Biochim. Biophys. Acta, 334,257-265. __ Weichselbaum, T.E.(1946)Am.J.Clin.Pathol.Tech.Sect.,lO, 16. Beutler,E., Blume, K.G., Kaplan, J.C., Lohr, G.W., Ramot, B ., and Walentine W.M.(1979) Brit. J. Haemat. 43, 465-467. 1158
is
Vol.
132.
12. 13. 14. 15. 16. 17. 18. 19 -
20. 21.
No. 3, 1985
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Rattazzi, M.C.(1969)Biochim. Biophys. Acta, 181, l-11. Gabriel 0.(1971) Meth. Enzymology, 22, 565-578, Dewey, M.M., and Coklin, J.L.(1960) Proc. SOC. Exptl. Biol. Med., 105, 492-494. Richards, A.H. and Hilf, R.(1972) Endocrinology, 91 , 287-295. W.W.(1967)Advan, Enzymol. 29, l-32. Cleland, Kirkman, J.N., and Hendrickson, E.n'I.(1963)Ame.J.Human. Genet., 15, 241-258. Yoshida, A., and Lin, M.(1973) Blood 41, 877-891. Minakami, S., Suzuki, C., Saito, T, Yoshikawa, H,(1965) J.Biochem.(Tokyo) 58,543-550. Liu, M.S., Liou, R.F., Chen. Y.H., Chen, S.H.(1984) Sci. Count. ,Repub China, Part B 8,155-160. Proc. Natl. Shatskayo, T.L.(1983) Mol. Genet. Microbial. Virusol. g, 18-22.
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