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) 0 e m 9m 22 m In 0 8 e 5 c.l 0 s- ee 2: ee 22 3 3 3 e m ln c11 3 3 3 oa (9 m: r!E 2 3 0 3 0 N 3 U J 3 3 rl 22 E 22 3 ln 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. 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