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Polymorphism of erythrocyte G-6-PD in the baboon.

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Polymorphism of Erythrocyte G-6-PD in the Baboon
D. GOURDIN, H. VERGNES, C. BOULOUX, J. RUFFIE
AND M. GHERARDI
Ceiztrr d’Hemotypologie dri Ceirtw Nntionnl de In Recherche Scieiztifiqzie
Hopital P u r p m i - Toulouse (France)
KEY WORDS
GS-PD . Baboon
,
Papio . Polymorphism
.
Erythrocyte.
Blood samples from several populations of baboons (genus
Papio) were examined for GB-PD variants. Several G - 8 P D phenotypes were
detected by starch gel electrophoresis. The so-called fast variant phenotypes of
G6-PD in baboons differ from human variant phenotypes in several physico-
ABSTRACT
chemical constants.
Glucose-6-Phosphate dehydrogenase
(G-6-PD, E.C. I, I, I, 41) is one of the best
known red-cell enzymatic systems of man,
both in its biochemistry and its genetics.
In Drosophila, Young et al. (‘64) put forward arguments in favor of genetic control of the enzyme by a locus situated on
the X chromosome; in the horse, donkey
and mule, Mathai et al. (’66) demonstrated the existence of a sex-linked character. Ohno et al. (’65) studying various
species of rats, showed that the trait is
linked to the X chromosome. Crawford et
al. (‘66) described a G-6-PD polymorphism
in chimpanzees and gorillas; single bands
similar in mobility to either the human A
or the human B bands were detected. But
in the other Old World Monkeys reported
by the same author, only one electrophoretic component similar to the human A
type was found.
The present paper will deal mostly with
the results obtained on the erythrocyte
G-6-PD of Old World cercopithecoids, genus
Papio: P. papio, P. cynocephalus and
P. anubis.
fig. l), Casamance, Eastern Senegal; 212
P. papio samples sent to us by Doctor
Naquet’s team whilst on a field study in
another part of Eastern Senegal (Damantan and Badi in Niokholo Koba National
Park, See fig. 1); 22 P. cynocephalus, 11
P. anubis samples and eight others difficult to classify, but resulting from a
cross between P. cynocephalus and P.
anubis collected at the Institute of Primatology and Ultracentrifugation of the
C.N.R.S., Villejuif, Paris (Doctor P. Dubouch now possesses a breeding colony
of about 140 with F, and F t generations).
The genitors in that group originate from
Kenya. It is interesting to point out here
that Doctor Naquet’s investigations relate
to epilepsy whilst one of his collaborators
(Professor Bert) studies sleep anomalies in
the baboon.
Blood was collected in 10 ml vacutainers containing ACD-A and shipped to our
laboratories by air in isothermic boxes.
They arrived in excellent condition four
to six days after collection.
(b) Analytical methods
MATERIAL AND METHODS
(a) Animals
Blood specimens were sent to our laboratories from the following sources:
143 P. pnpio samples from the Institute
of Neurophysiology of the Centre National de la Recherche Scientifique (Marseilles, France) (Doctor R. Naquet) and
originating from Sakar and Talito (see
AM. J. PHYS. ANTHROP., 37: 281-288.
G-6-PD was detected by electrophoresis
of each hemolysate by two methods: on
cellulose acetate gel (Rattazzi et al., ’67)
at 190 volts for 3 hours; on starch gel in
phosphate buffer 0.05 M , pH 7.5 (Jerome)
under continuous refrigeration (4”C), 4
voltslcm for 18 hours.
In certain animals possessing the electrophoretic phenotype “Common Baboon”
defined later and in those which showed
281
282
GOURDIN, VERGNES, BOULOUX, RUFFIE AND GHERARDI
OAK
Fig. 1 Map of Senegal and Gambia. Origin of the P. papzo samples. The Talito-Sakar group and
the Badi-Darnantan group.
I
a n enzymatic fraction of a more rapid
mobility we partly purified the enzyme by
column chromatography on DEAE cellulose. Using purified G-6-PD, we tried to
determine the physicochemical constants
of the enzyme - Michaelis constant (Km)
for the G-6-PD substrate, activity related
to the pH, thermostability, utilization of
a substrate analogue (2 deoxy. G-6-P),
electrophoretic mobility of purified fraction on starch gel in phosphate buffer
0.05 M, pH 7.50 (3) and in discontinuous
buffer TRIS, EDTA, Borate 0.1 M, pH 9.5
(Boyer et al., '62). The experimental protocol followed for G-6-PD purification and
the analysis of physico-chemical constants
of the molecule was performed according
to the techniques recommended by Kirkman ('62), Motulsky and Yoshida ('69)
and standardized by the W.H.O. ('67).
RESULTS
I. Polymorphism
The G-6-PD typing by electrophoresis
revealed several phenotypes characterized
by the presence of only one enzyme fraction with a variable mobility and activity.
We did not find a series of multiple isoenzymes as known to exist in other active
molecules of the erythrocyte (acid phosphatases, LDH, PGM, AK). G-6-PD of that
species of Primates behaves as a molecular model identical to that described in
man and other mammals.
In the baboon, the erythrocyte G-6-PD
is electrophoretically polymorphic.
(1) In 396 animals, the electrophoretic
separation of the red cell homogenate indicates the existence of a unique fraction
with a greater mobility than that of
G-6-PD B + in man. On comparative zymograms the band with the enzymatic
activity has a mobility coefficient of 111%
compared to human G-6-PD B (100%).
Baboon G-6-PD has a mobility analogous
to that of the A + Negroid variant (equal
to 110% in relation to B type). That fast
moving band, similar to human G-6-PD A
has been detected in 378 animals. The
283
G-6-PD POLYMORPHISM IN THE BABOON
Fig. 2 Horizontal starch gel electrophoresis of hemolysates. Phosphate buffer at pH 7.5. (1) Human
G-6-PD B (normal). ( 2 ) , (3), (4), and (5) common phenotypes of baboon G-6-PD.
TABLE 1
G-6-PD variants in various baboon species
G-6-PD type frequencies
Number of
Species
animals
Origin
studied
P. anubis
P. cynocephnlus
Cross P. cyno X P. anubis
Paris
Paris
Paris
P. papio
Senegal
143
P. papio
Senegal
212
11
22
8
electrophoretic diagrams reproduced in
figure 2 illustrate that phenomenon.
(2) G-6-PD variants were encountered
only in the P. papio (table 1). In 17 animals we found an enzyme fraction more
rapid than the preceding one. Its mobility
was 124% of the human enzyme. An example of this fast G-6-PD type is found in
figure 3 which shows a common baboon
phenotype, a rapid variant, and a human
phenotype A - Negroid variant. The frequency of the fast G-6-PD variant in the
animals studied is about 4.29%.
There is no apparent association of the
G-6-PD type with sex. The presence of
both fast variant and normal enzyme in
female baboons is compatible with X
linkage of G-6-PD in that species. P. papio
512 is a male sampled twice. It had two
G-6-PD fractions. It died before we could
check its karyotype.
( 3 ) We do not have evidence in this
sample of mutations with diminished enzyme activities. The enzyme on starch gel
Common
1113
Rapid
1248
n o = 512
l l l c I f124%
1009
100%
100%
137 animals
95.8%
200 animals
94.3%
5 animals
3.5%
12 animals
5.7%-
1 animal
0.7%
Fig. 3 Horizontal starch gel electrophoresis
of hemolysates. Phosphate buffer at pH 7.5. Slot
1 : common phenotype of baboon’s GS-PD. Slot 2:
fast variant. Slot 3: negroid variant A - of human G-6-PD.
284
GOURDIN, VERGNES, BOULOUX, RUFFIE AND GHERARDI
Fig. 4 Horizontal starch gel electrophoresis of hemolysates. Phosphate buffer at pH 7.5.
Slot 1: common phenotype of baboon G-6-PD. Slot 2: variant with double band. Slot 3 : human
phenotype A f B . Slot 4: common phenotype. Slot 5: fast variant.
+
TABLE 2
Origin a n d repartition of the G-6-PD rrnrinnt in n rnndom srtmple of baboon species
-
G-6-PD electrophoretic
Indentification
of a 111 m d 1
51 1
512
1001
523
002
503
0009
Gunther
Superfamily
P pnp1o
P
P
P
P
pup10
pap10
pnp1o
cynocepliulus
P pnp1o
P plZJ3lO
P pnplo
or on cellogel has normal activity. We
found enzyme fractions with weak activity
in only two baboons from Senegal (12 and
35) but loss of activity due to poor conservation of the sample is one likely explanation. The volume of blood available
in those two cases was not adequate to
determine the physico-chemical constants
of purified G-6-PD necessary to confirm
the mutation.
(4) In the three groups of baboons which
we studied, P. papio, P. cynocephalus and
P. anubzs, we found the rapid variant only
in the first group. I n the others only the
usual phenotype with slower mobility was
found (fig. 3). Were it not for the relatively
small number of P. a n u b i s and P. cynocephalus studied compared to P. papio,
we would be tempted to consider the
G-6-PD rapid variant as a marker gene.
Origin
phenotype
Casamance
Casamance
Casamance
Casamance
Kenya
Casamance
Niokholo Koba
Casamance
Common
Common
Common
Common
Common
Fast
Fast
Fast
11. T h e physico-chemical characteristics
of t h e purified e n z y m e
The physico-chemical parameters of
purified G-6-PD, from hemolysates, was
determined on eight random animals with
at least one representative of each origin:
five baboons with the “common phenotype,” three with the rapid variant. Table 2 gives the origin and G-6-PD types of
the samples. The results obtained are
compared with the values of human conand A
in table 3. The
trols G-6-PD B
biochemical profile of the enzyme of those
animals is not markedly different from
that of the human enzyme.
We have noted only two special features:
(a) N o 002, P. cynocephalus (common
phenotype) presented a very important
diminution of enzyme thermostability. The
+
+
G-6-PD POLYMORPHISM I N THE BABOON
0)
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In
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ee
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e
m
ln
c11
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m:
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285
normal enzyme of baboons is characterized by a loss of 20% of its activity after
20 minutes of incubation a t 47°C. In that
animal under similar conditions, we found
about 8 0 % diminution of activity. The
G-6-PD molecule of that animal seemed
to be very sensitive to denaturation by
heat. Rapid G-6-PD variants from two P.
papio (Gunther and No BA 0009) showed
a reduced thermostability, but to a lesser
degree (60% loss after 20 minutes at
47°C). No mixing experiments of the normal and labile enzymes were carried out.
(b) The utilization of 2 deoxy-G-6-PD, a
structural analogue of the normal substrate of the enzyme, is very close to-that
of human G-6-PD, sometimes a t the higher
limits of the normal (10%-12%) but the
variations noted are not significant.
The values for the Michaelis constant
(Km G-6-P) are close to those of human
G-6-PD.
The electrophoretic mobility of the
purified and dialysed enzyme is always
greater than that of human fraction B +
(figs. 5 and 6), on PO-' buffer, on E.D.T.A.
borate (3,l). In both buffer systems used,
the mobility of the purified enzyme makes
it possible to differentiate the common
phenotype from the rapid variant. As an
example, we compare the electrophoresis
of dialysates of a normal baboon (common
phenotype, N o 522) and the rapid variant
of P. papio ( N o 503) (fig. 6).
3
=I
+ 22
d
3 3
0
+ 022
m
DISCUSSION
As in man, baboon G-6-PD is polymorphic. This heterogeneity of the enzyme is
of genetic origin. Family studies carried
out only on P. anubis support this hypothesis. As we have gathered information, so
far only on the common G-6-PD phenotype
of baboons, it has not been possible for us
to study the transmission of the rapid
variant. The locus controlling the biosynthesis of the G-6-PD in baboons, has a t
least two alleles: the common allele seen
in most animals, which we provisionally
designate Gd( )C, the allele responsible
for the synthesis of the rapid variant
Gd( +)R and, lastly, we suspect the existence of the mutant Gd( -)C gene, responsible for the phenotype with reduced enzymatic activity found i n some animals.
+
286
GOURDIN, VERGNES, BOULOUX, R U F F I E AND GHERARDI
+
Fig. 5 Electrophoretic comparison of partially purified h u m a n G-6-PD B
and baboon G-6-PD.
Slot 1 : h u m a n G-6-PD B +. Slot 2: baboon. Slot 3 : h u m a n sample. Slot 4 : baboon. Horizontal starch
gel electrophoresis TEB buffer pH 9.
Fig. 6 Horizontal starch gel of partially purified G-6-PD. Phosphate buffer at pH 7.5. Slot 1: h u m a n
enzyme B +. Slot 2: baboon enzyme, fast variant B A 503. Slot 3: baboon enzyme, common phenotype B A 522. Slot 4 : h u m a n enzyme B
+.
However, a n artefact cannot be excluded
on the basis of our present data.
The physico-chemical properties of the
normal enzyme molecule and the rapid
variant imply no notable differences in the
kinetic characteristics of these two types
of G-6-PD. The enzyme of baboons resembles that of man except that in three of
them thermal stability is greatly reduced.
The Michaelis constant for G-6-P and the
activity of the enzyme as a function of pH
(truncate between pH 7 and pH 9) are not
different from those of man. These biochemical parameters of G-6-PD in mail
and the baboon are remarkably similar.
Crawford et al. (‘66) found single bands,
similar in mobility to the negroid A variant in chimpanzees and gorillas.
The primary structure of the catalytic
site of the baboon’s enzyme could well be
GB-PD POLYMORPHISM I N THE BABOON
identical to that of man, on the basis of
these physico-chemical constants. However, some amino acid substitutions in
the non-active zone of the molecule are
highly probable in light of the difference
in electrophoretic mobility. Such phenomena are common in man. The most usual
rapid variant of human G-SPD, the negroid type A , possesses the same physico-chemical constants as the enzyme B .
It differs by only one amino-acid substitution: in the polypeptide chain of the B
form asparagine is replaced by aspartic
acid (Yoshida, ’68).
Other fast variants have been identified
by their electrophoretic mobility on starch
gel: G-6-PD Levadia and Attica among the
Greeks (Rattazzi et al., ’69; Stamatoyannopoulos et al., ’70), Kings County and
Lourenzo Marques (Reys et al., ’70) among
the blacks. The investigation of other
physico-chemical parameters does not
show any difference from the common
type G-6-PD B. However, in these cases, as
in the rapid variant isolated in baboons,
only sequential analysis of amino-acids
in the polypeptide chains of the molecule
will make it possible to explain the polymorphism observed in this primate species.
+
+
CONCLUSION
Red cell G-6-PD polymorphism is shown
in groups of baboons. There appears to be
a molecular heterogeneity of the enzymes
in the several populations of baboons
tested; 212 animals examined were studied in their natural habitat and the blood
was collected from natural populations.
Physico-chemical properties of the normal
enzyme molecule and of the variant molecule were examined.
ACKNOWLEDGMENTS
We thank Dr. J. Ruffie, R. Naquet, and
P. Dubouch for their interest and support
of this project. We also appreciate the
helpful suggestions of Dr. John BuettnerJanusch.
28 7
LITERATURE CITED
Boyer, S. H., I . H. Porter and R. G. Weilbacher
1962 Electrophoretic heterogeneity of glucose6-phosphate dehydrogenase and its relationship
to enzyme deficiency in man. Proc. Nat. Acad.
Sci., 48: 1868-1876.
Crawford, M. H., A. C. Morrow and A. G. Motulsky 1966 Red cell glucose-6-phosphate
dehydrogenase variation in primates. Amer. 3.
Phys. Anthrop. 25: 207.
Jerome, H. Personal communication.
Kirkman, H. N. 1962 Glucose-6-phosphate dehydrogenase from human erythrocytes. I. Further purification and characterization. J. Biol.
Chem., 237: 2364-2370.
Mathai, C. K., S . Ohno and E. Beutler 1966 Sexlinkage of the glucose-6-phosphate dehydrogenase gene in the family Equidae. Nature,
210: 115-116.
Motulsky, A. G., and A. Yoshida 1969 Methods
for the study of red cell glucose-6-phosphate
dehydrogenase. In: Biochemical Methods in
Red Cell Genetics. J. Yunis, ed. Academic Press,
New York, pp. 51-93.
Ohno, S., J. Poole and I. Gustavsson 1965 Sexlinkage of erythrocyte glucose-6-phosphate dehydrogenase in two species of wild hares. Science, 150: 1737-1738.
Rattazzi, M. C., L. F. Bernini, P. M. Mannucci
and G. Fiorelli 1967 Electrophoresis of glucose-6-phosphate dehydrogenase: a new technique. Nature, 213: 79-80.
Rattazzi, M. C., L. Lenzerini, P. Meera Khan and
L. Luzzatto 1969 Characterization of glucose-6-phosphate dehydrogenase variants. 11.
G-6-PD Kephalonia, G-6-PD Attica, and G-6-PD
“Seattle-like” found in Greece. Amer. J. Hum.
Genet., 21 : 154-167.
Reys, L., C. Manso and G. Stamatoyannopoulos
1970 Genetic studies on south eastern Bantu
of Mozambique. I. Variants of glucose-6-phosphate dehydrogenase,. Amer. J. Hum. Genet.,
22: 203-215.
Stamatoyannopoulos, G., P. Kotsakis, V. Voigtlander, A. Akrivakis and A. G. Motulsky 1970
Electrophoretic diversity of glucose-6-phosphate
dehydrogenase among Greeks. Amer. J. Hum.
Genet., 22: 587-596.
W.H.O. 1967 Normalisation
des techniques
d’Btude de la glucose-6-phosphate dehydrogenase. Geneve, Bul. 366.
Young, W. J., J. E. Porter and B. Childs 1964
Glucose-6-phosphate dehydrogenase in Drosophila: X-linked electrophoretic variants. Science, 143: 140-141.
Yoshida, A. K. 1968 The structure of normal
and variant human glucose-6-phosphate dehydrogenase. In: Hereditary Disorders of Erythrocyte Metabolism. E. Beutler, ed. Grune and
Stratton, New York, p. 146.
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