Primate red cell enzymes Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase.код для вставкиСкачать
Primate Red Cell Enzymes: Glucose-6-phosphate Dehydrogenase and 6-Phosphogluconate Dehydrogenase JOHN BUETTNER-JANUSCH,' LAUREN DAME, GEORGE A. MASON AND DONALD S. SADE Departments of Anatomy and Zoology, Duke University, Durham, North Carolina; Caribbean Primate Research Center, Puerto Rico, a n d Department of Anthropology Northwestern University, Evanston, Illinois KEY W O R D S Erythrocyte enzymes morphism . M a c a c a . Prosimians . * Galago . Genetic polyPropithecus. ABSTRACT Glucose-6-phosphate dehydrogenase (E. C . : 126.96.36.199) phenotypes and 6-phosphogluconate dehydrogenase (E. C . : 188.8.131.52) phenotypes were determined by starch-gel electrophoresis of red cell hemolysates of Galago crassicaud a t u s subspp., Propithecus verreauxi, L e m u r spp., H a p a l e m u r griseus, and Macaca mulatta. A single glucose-6-phosphate dehydrogenase (G6PD) phenotype was found in each species. A single 6-phosphogluconate dehydrogenase (6PGD) phenotype was found in L e m u r spp., H a p a l e m u r griseus, and Galago crassicaud a t u s argentatus. I n a group of six Propithecus verreauxi, three GPGD phenotypes, PGDA, PGDAB, and PGDB, were found. Three phenotypes, PGDA, PGD AB, and PGD B, were found in 38 G . c. crassicaudatus. The three phenotypes in each species are apparently the products of two codominant autosomal alleles, PGDA and PGDE. The frequency of PGD" in G. c. crassicaudatus is 0.263. A population of 260 free-ranging macaques displays a polymorphism a t the GPGD locus. Three phenotypes, PGD A, PGD AB, and PGD B, were found. These also appear to be controlled by two codominant autosomal alleles, PGD" and PGD*; the frequency of PGDA = 0.913. Additional analysis of three well-defined troops within the macaque population indicated that there are no significant differences between the troops or within the population at the GPGD locus. Variant forms of red cell enzymes are well known in man, and some examples of variants of homologous enzymes have been reported among nonhuman primates (Schmitt et al.,'70; Barnicot and Cohen, '70; Tariverdian et al., '71; Kompf et al., '71; Barnicot and Hewett-Emmett, '71; Prychodko et al., '71). In order to increase our knowledge of these enzymes and their variants in the Primates, we have examined red cell hemolysates of many species, and we report here some electrophoretic and genetic properties of glucose6-phosphate dehydrogenase (E.C. : 184.108.40.206) and 6-phosphogluconate dehydrogenase (E.C.: 220.127.116.11) among seven species of prosimian primates at the Duke University Primate Facility (DUPF) and in 260 maAM. J. PHYS. ANTHROP.,41: 7-14. caques from the Caribbean Primate Research Center at Cay0 Santiago, Puerto Rico. Glucose - 6 - phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (GPGD) are red cell enzymes essential to the sequence of reactions in the hexosemonophosphate shunt pathway in aerobic glycolysis (Murphy, '60). The mature red cell, deprived of its nucleus, mitochondria, and Golgi apparatus, can no longer synthesize proteins or obtain energy from the Krebs cycle, It can use some of its remaining enzymes to make some simple compounds. At least 90% of red cell glycolysis, which provides energy for syn1 Present address: Department of Anthropology, N e w York University, New York, N.Y. 10003; all correspondence to this address. 7 8 BUETTNER-JANUSCH, DAME, MASON AND SADE thesis of such compounds, e.g., glutathione, occurs through the anaerobic pathway, 10% occurs through the aerobic pathway (Harris, '63). GGPD, in the presence of a hydrogen ion acceptor, catalyzes the conversion of D-glucose-6-phosphate to Dglucono-&lactone phosphate (6-phosphogluconate). GPGD, also in the presence of a hydrogen ion acceptor, catalyzes the oxidation of 6-phosphogluconate to D-ribulose-5-phosphate (pentose phosphate) and C 0 2 ; the pentose phosphate then enters the Embden-Meyerhof pathway to be converted to fructose-l,6-diphosphateor glyceraldehyde-3-phosphate. G6PD occurs in two active forms, tetramers and dimers made up of subunits, each of which has a molecular weight of about 50,000 (Cohen and Rosemeyer, '68, '69; Bonsignore et al., '71a). The interconversion of these two forms in solution is influenced by pH, ionic strength, and the presence of M g + + and other divalent cations (Wrigley et al.,'72; Bonsignore et al., '71b, '71c). Stability and enzymatic activity of G6PD are dependent upon the presence of nicotinamide adenine dinucleotide phosphate (NADP), Removal of NADP leads to the dissociation of G6PD into inactive monomers (Kirkman et al., '64; Bonsignore et al., '71a). Electrophoretically detectable variants have been reported in human populations, which suggests there is molecular heterogeneity of G6PD in man (Porter et al., '64; Kirkman et al., '64; Dern et al., '66). The active form of GPGD in man is a dimer, consisting of identical subunits each of which has a molecular weight of about 40,000 (Kazazian, '66). Three phenotypes have been reported in m a n as well as among several species of macaques (Prychodko et al., '71). After starch-gel electrophoresis, each of the two homozygous human phenotypes (PGD A, the more anodal on starch gels, and PGD B, the more cathodal) appears as a single band of GPGD activity. The two homozygous phenotypes of Macaca mulatta are labeled in exactly the opposite manner. PGD A is the more cathodal, PGD B the more anodal (Prychodko et al., '71). I n both man and macaque, the heterozygous phenotype, PGD AB, produces three bands of GPGD activity on starch gels. Two of these are presumably identical to the bands of activity produced by each of the homozygous phenotypes; the third band, of intermediate mobility, is apparently a hybrid molecule containing a subunit from each allele. These observations support the view that two identical subunits form a single GPGD dimer in homozygotes, each of the dimers being the product of alleles at the same locus. G6PD is inherited as an X-linked trait in man, and GPGD appears to be inherited as the product of codominant autosomal alleles at a single locus. MATERIALS AND METHODS Hemolysates were prepared from fresh blood samples, taken in anticoagulant, from the following prosimians at DUPF : 64 Galago crassicaudatus subspp., 50 Lemur fulvus subspp., 11 L . catta, 8 L . macaco, 6 L. variegatus, 6 Propithecus verreauxi coquereli, and 4 Hapalemur griseus. The macaque hemolysates were obtained from 260 free-ranging Macaca mulatta living on the island of Cayo Santiago, Puerto Rico (Altmann, '62; Sade, '67). These 260 animals consist of members of seven distinct troops and 17 peripheral males, or they were members of troops when the samples were taken. For three of the troops currently remaining on the island, troops labeled F, I, and L, we have sampled almost all of the members. G6PD and GPGD phenotypes were determined from fresh hemolysates by starchgel electrophoresis in phosphate buffers. The gel buffer was 0.01 M, pH 7.0; the buffer i n the electrode trays was 0.1 M, pH 7.0. Electrophoresis, either vertical or horizontal, was for 18 hours at 4", 5 volts/ cm. Because the reactions of G6PD and 6PGD are similar in vitro and because the methods of staining starch gels are sensitive, we believe it is useful to present some details of the techniques used in our laboratory for detecting both enzymes. These techniques are adaptations of those reviewed by Giblett ('69), Motulsky and Yoshida ('69), and Brewer ('69). Generally, when we determined only GGPD, we added 2.5 mg NADP to the starch before the hot buffer was added and 3.5 mg NADP to the buffer in the cathodal G6PD AND GPGD OF PRIMATES electrode tray (Giblett, '69). NADP was added to demonstrate G6PD if the samples were not run within 72 hours after they were prepared. We omitted NADP if the gels were to be used for determining both G6PD and 6PGD. Gels that contain NADP have extra cathodal bands on the half stained for 6PGD; these are nongenetic artifacts. We found no problem with extraneous bands of GGPD activity if the gels were run shortly after the samples were drawn, but after electrophoresis of older samples we generally found nongenetic artifacts. Although GPGD activity is demonstrable after electrophoresis of relatively old, properly stored hemolysates, unambiguous phenotypes cannot always be determined for such samples, The results reported here were obtained from samples no older than 96 hours. The two enzymes were usually demonstrated on separate halves of the same gel, and different staining procedures were used for each enzyme. After the gel was sliced, strips of excess gel were used to make a box or fence to enclose the part of the gel surface to be stained. The boundaries of the box were the origin (site of insertion of samples), a line about 10 cm anodal to the origin, and the outer edges of the gel. The stain for G6PD is made as follows : 12.0 mg disodium glucose-6-phosphate (G6P), 2.4 mg tetrazolium salts MTT [ 3- ( 4,5-dimethyl thiazolyl-2 ) -2,5-diphenyl tetrazolium bromide], 2.4 mg MgCl,.H,O, and 0.5 mg phenazine methosulfate (PMS) are dissolved in 6 ml of a 0.2 M tris-HC1 buffer, pH 8.0. NADP, 2.5 mg, is included if it was omitted from the gel and electrode buffers. About 10 ml of warm ( 6 0 " ) 2% melted aqueous agar was added to the staining solution and this mixture was immediately poured into the box on the gel surface. The stain and overlay for GPGD are similar except that 12.0 mg of 6-phosphogluconate (6PG) is used in place of disodium G6P, and 2.5 m g NADP is added to the stain whether or not it was in the gel. The gels with the stain overlay were incubated at 37" for 30 minutes, or until purple-blue bands appeared, a t which time the agar overlay was removed. Prolonged incubation was avoided. Gels that showed G6PD and 6PGD activity were photo- 9 graphed or sketched shortly after the bands developed, because the stain is fugitive and will not remain distinct unless the gels are kept frozen at about -20". Removing the agar overlay often lifted part of the stain from the middle of the colored bands. Fuzzy smears close to some of the more distinct bands of 6PGD are, by general opinion, artifacts that result from the use of NADP (Giblett, '69). RESULTS AND DISCUSSION Except in human control samples, we found no intraspecific variation of G6PD after electrophoresis of hemolysates (table 1). The electrophoretic mobility of the enzyme from Galago crassicaudatus crassicaudatus is indistinguishable from that of G. c. argentatus; and the enzyme from each of the subspecies of Lemur fulvus and from L. macaco has the same electrophoretic mobility. We have no evidence for or against the hypothesis that G6PD is X-linked in these nonhuman primates. Zones of G6PD activity from prosimian blood samples do not migrate as far anodally as do human controls (fig. 1 j . A single band that migrates more anodally than the common human enzyme was found in samples from all Macaca mulatta (fig. 1) . 6PGD variants were demonstrated among Galago crassicaudatus crassicaudatus, Propithecus uerreauxi coquereli, and Macaca mulatta (tables 1, 2, 3, figs. 1, 2, 3 ) . The patterns on the gels are comparable to those found in human subjects (Fildes and Parr, '63; Parr, '66; Davidson, '67) and in several species of macaques (Prychodko et al., '71 ). Three phenotypes were found in each of the three species. In order to avoid a complicated system of nomenclature and a proliferation of abbreviations for each species, we have simply named the more cathodal bands PGD A and the more anodal PGD B (Prychodko et al., '71). It is important to point out that PGD A of Galago, PGD A of Propithecus, and PGD A of Macaca have different electrophoretic mobilities; the bands of PGD B activity for each species are also different. The distribution of the phenotypes in the three species is shown in tables 2 and 3. In each species the three phenotypes, PGD A, PGD AB, and PGD 8 , 10 BUETTNER-JANUSCH, DAME, MASON A N D SADE TABLE 1 Summary .of observed GGPD and GPGD phenotypes G6PD Species Lorisiformes Galago crassicaudatus crassicaudatzts Galago crassicaudatus argentatus Lemuriformes Lemur f u l v u s subspp.1 L . catta L. macaco L. variegatus Propithecus verreauxi coquereli Hapalemur griseus Cercopithecoidea Macaca mulatta Number of animals GPGD Number of phenotypes Numberof animals Numberof phenotypes 38 1 38 3 26 1 26 1 50 11 8 6 1 1 1 1 50 11 8 6 1 1 1 1 6 1 1 6 4 4 3 1 260 1 260 3 ~________ 1 Includes 19 L. f . f u l v u s , 7 L. f. albifrons, 3 L. f . collaris, and 21 L. f . r u f u s . are probably the products of two codominant, autosomal alleles, PGDA and PGD". An analysis of pedigrees of Galago crassicaudatus crassicaudatus (Nute e t al., '69) and Propithecus verreauxi coquereli supports this assumption (table 4 ) . Thus our data are interpreted exactly as those from studies of 6PGD in man. Tests of the hypothesis of codominant inheritance of GPGD activity in our samples of Galago crassicaudatus crassicaudatus and Macaca mulatta produced the following xz values, calculated from estimates of allele frequencies and fit to Hardy-Weinberg expectations: Galago, x; = 0.09, 0.70 < P < 0.80; Macaca, xi = 0.66, 0.30 < P < 0.50. These tests support the hypothesis. To our knowledge, this is the first report of a polymorphism at the GPGD locus in Galago and Propithecus. It should be noted that the single phenotype found in Galago crassicaudatus argentatus has the same pattern as PGD B of G. c. crassicaudatus. The electrophoretic mobilities of PGD of Lemur spp. and Hapalemur griseus are identical (fig. 1 ) . We do not believe it appropriate to subject the data from the DUPF prosimians to further statistical analysis. The sample is small, and the animals are highly inbred (Nute et al., '69). Because of this, we cannot determine whether the founder principle, positive selection, or some other mechanism is acting at the GPGD locus in these animals. The distribution by troop of GPGD phenotypes of Macaca mulatta and the results of statistical analysis are presented in tables 3 and 5. For purposes of analysis we divided the population into four groups : troop F, troop I, troop L, and all other troops. The sample of 260 macaques is in Hardy-Weinberg equilibrium, and each of the three troops sampled almost in their entirety, F, I, and L, is also in equilibrium (table 3 ) . Tests of homogeneity (table 5) reveal no significant differences between the macaque troops or within the population of troops. The frequency of PGD', 0.913, in the sample of 260 M . mulatta is similar to the frequency reported by Prychodko et al. ('71) for a sample of 58 animals from Pakistan, India, and Thailand. These investigators found that continental populations of M . mulatta, as well as continental populations of M . cyclopis, M . fascicularis [= M . irus], M . nemestrina, and M . speciosa were polymorphic at the GPGD locus, Three alleles and five phenotypes were demonstrated in M . fascicularis [= M . irus], and two alleles and three phenotypes in the other species. Island populations from Taiwan, the Philippines, and Japan were monomorphic (Prychodko et al., '71). The polymorphism for the alleles at the PGD locus in the Cay0 Santiago popula- 11 G6PD AND GPGD OF PRIMATES GGPD rinin 6 PGD + = D t Origin 1 2 3 4 5 6 7 8 9 1 0 1 1 1 Fig. 1 Relative electrophoretic mobilities of G6PD and 6PGD; conditions of electrophoresis and methods of staining are given in the text. Bands of G6PD are shown for (1) Lemur catta, ( 2 ) L. macaco and L. fulvus subspp., ( 3 ) L. variegatus, ( 4 ) Hapalemur griseus, ( 5 ) Propithecus uerrefluxi coquereli, ( 6 ) Galago CTaSSiCaudatus subspp., ( 7 ) Homo sapiens G6PD A, and ( 8 ) Macaca mulatta. Bands of 6PGD are shown for (1) Galago crassicaudatus crassicaudatus PGD A, ( 2 ) G. c . crassicaudatus PGD AB, ( 3 ) G . c. crassicaudatus and G. c. argentatus PGD B, ( 4 ) Propithecus uerreauxi coquereli PGD A, ( 5 ) P . v . coquereli PGD AB, ( 6 ) P . v . coquereli PGD B, ( 7 ) Macaca mulatta PGD A, ( 8 ) M . mulatta PGD AB, ( 9 ) M. mulatta PGD B, (10) Lemur spp. and Hapalemur griseus, and (11) Homo sapiens PGD A. TABLE 2 Distribution of GPGD phenotypes i n Galago and Propithecus Phenotype Species 1 2 1 PGDA PGDAB PGDB Total Galago crassicaudatus crassicaudatus 2 3 14 21 38 Propithecus uerreauxi coquereli 3 2 1 6 See text for explanation of nomenclature. The frequency of PGDA = 0 . 2 6 3 . 12 BUETTNER-JANUSCH, DAME, MASON AND SADE TABLE 3 Distribution of 6PGD p h e n o t y p e s a n d alleles in Macaca mulatta Phenotype Troop F I L Total X I 0.80 125 125.00 2.13 10 10.50 1 0.73 49 49.00 0.13 7 6.30 0 0.35 35 35.00 0.43 0 0 0 0 0 0 4 1 12 17 17 51 AB Obs EXP 107 105.80 16 18.40 2 Obs EXP 38 37.77 Obs EXP 28 28.35 Other A E H 4 J 1 10 16 PM Total 45 Total Obs EXP 1 B A PM 1 14 218 216.71 39 41.31 3 1.98 0.920 0.878 0.900 0.941 260 260.00 Frequency of PGDA 0.913 0.66 peripheral m a l e s . Fig. 2 Photograph of starch gel stained for 6PGD. The samples are, from left to right, h u m a n PGD A, Galago crassicaudatus crassicaudatus PGD AB, G . c . crassicaudatus PGD B, Propithecus verreauxi coquereli PGD B, P. v . coquereli PGD A, P. v . coquereli PGD A; a n d Macaca mulatta PGD A. The origin is at the bottom, the anode is a t the top; electrophoretic conditions a n d method of staining a r e described in the text. 13 GGPD A N D GPGD O F PRIMATES TABLE 4 GPGD genotypes of progeny of irzatings in Galago and Propithecus Progeny Matings PGD '/PGD* PGDq/PGDn PGDB/PGDB Total 0 1 1 0 1 0 2 0 2 4 1 3 4 4 0 2 0 2 Galago crassicaudatus crassicaudatus PGD"/PGD" X PGDB/PGDB PGDA/PGDB x PGDA/PGDB PGDA/PGDB x PGDB/PGDB PGDB/PGDB x PGDB/PGDB Propithecus verreauxi coquereli PGDA/ PGDA x PGDB/PGD3 1 1 Distribution of genotypes 1 1 0 Data from pedigrees of a n i m a l s a t DUPF variants of malate dehydrogenase occur in these animals; and albumin and lactate dehydrogenase are monomorphic (unpublished observations). These data suggest that we should also consider selection, mutation, and inbreeding as processes contributing to the maintenance of the GPGD polymorphism. Additional analyses of the proteins currently being studied in our laboratory may allow us to specify some of the reasons for the presence of the polymorphism in this group of macaques. ACKNOWLEDGMENTS Fig. 3 Photograph of a portion of a starch gel stained for GPGD of Macaca mulatta. The samples are, from left to right, PGD A, PGD AB, PGD A, and PGD A. The origin is at the bottom, the anode is at the top. Conditions of electrophoresis and staining are described in the text. The faint leading band, pronounced in the sample on the right, occurs only in samples that are more than 48 hours old. tion is possibly the result of a mixture of populations; when the Cay0 Santiago population was founded in 1940 it consisted of groups of macaques imported from several parts of India (Carpenter, '72). The Cay0 Santiago macaques are also polymorphic at the transferrin locus; rare We acknowledge with thanks the cooperation and assistance of the staff of the Caribbean Primate Research Center and Dr. Jan Bergeron, Managing Director, Duke University Primate Facility. The work reported here was supported in part by National Science Foundation Grant GS 30657X and National Institutes of Health Grants RR 00388, GM 16722 (Research Career Development Award to J. B-J.), and GM 02007 (Training Grant to G.A.M.). We also acknowledge the assistance of TABLE 5 T e s t s o f homogeneity of allele frequencies at the GPGD locus in macaques Troops compared F/I F/L F/ Other I/L I/Other L/Other Total (x: ) 2 x 1 1.52 0.28 0.47 0.21 2.47 1.01 2.89 14 BUETTNER-JANUSCH, DAME, MASON AND SADE Eastern Airlines personnel who usually delivered blood samples without undue delay. LITERATURE CITED Altmann, S . A. 1962 A field study of the sociobiology of rhesus monkeys, Mncaca mulatta. Ann. N. Y. Acad. 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