Biochemical Genetics, Vol. 17, Nos. 5/6, 1979
Transient Electrophoretic Forms of Red Cell 6PGD in American Buffalo (Bison bison) and Domestic Cattle (Bos taurus) Yoshiko Suzuki] Karen J. Auditore, 1,2 Bob Morris] and Clyde Stormont 1 Received 27 July 1978--Final 17 Nov. 1978
INTRODUCTION In a previous report in this journal, Naik and Anderson (1970) described three electrophoretic patterns of red cell 6-phosphogluconate dehydrogenase (6PGD) in American buffalo (Bison bison). Two of the phenotypes (PdAA and PdBB) were two-zone patterns and the third (PdAB) was composed of three zones. Based on a gene-frequency analysis of the distribution of the three phenotypes in 86 bison samples from the Wichita Mountains Wildlife Refuge in Cache, Oklahoma, they presumed that the three phenotypes are determined by codominant allelic genes, Pd A and Pd ~. In a much larger survey, including samples from 82 bison in the same population studied by Naik and Anderson, we found the bison red cell 6PGD to be monomorphic. Cattle red cell 6PGD is also monornorphic, but it occupies a different electrophoretic position from that of bison 6PGD. In this note we call attention to the oxidative storage effects on blood samples that result in the appearance of transient phenotypic patterns that resemble those described as variant forms of 6PGD in the aforementioned report. MATERIALS AND METHODS Blood samples were collected in commercial Vacutainer tubes (10 ml) containing either EDTA or ACD as anticoagulant and were shipped via air mail to 1 Serology Laboratory, Department of Reproduction, University of California, Davis, California 95616. 2 Present address: Massachusetts Institute of Technology, Center for Cancer Research, E 17-128, Cambridge, Massachusetts 02139. 445 0006-2928/79/0600-0445503.00/0 © I979PlenumPublishingCorporation
446
Suzuki, Auditore, Morris, and Stormont
our laboratory. Except for 82 bison samples, which were obtained from animals in the Wichita Mountains Wildlife Refuge, Cache, Oklahoma, and except for samples drawn from cattle in the University dairy herd, all samples were obtained from private bison and cattle herds throughout the United States. Hemolysates were prepared from washed, packed red cells by (1) rapid freezing and thawing, (2) sonication, and (3) addition of an equal volume of distilled water. Optimal diagnosis was obtained on samples hemolyzed with water. Horizontal starch gel electrophoresis and staining of the gel slices were performed according to the method described by Bengtsson and Sandberg (1973), with minor modifications relating mainly to the size of the gel frames, the cooling system, and the electrophoretic procedures. RESULTS AND DISCUSSION Altogether, 413 bison samples and 599 cattle samples were screened for red cell 6PGD phenotypes, with no evidence for intraspecific genetic variation in the electrophoretic forms of this enzyme. With respect to cattle, these results are in agreement with earlier data (Bender et al., 1970; Karadjole et aI., 1972; Amano et al., 1974). When hemolysates were prepared from freshly collected blood samples and were used soon after their preparation, 6PGD appeared as a single, monomorphic zone in each species, with that of bison, designated B, migrating to a position distinctly ahead of that of cattle, designated C (see Fig. 1). When, however, the hemolysates were stored frozen for longer than 24 hr, multiple, transitional zones of 6PGD began to make their appearance, as illustrated in Fig. 2. Similar effects were obtained when freshly prepared hemolysates were incubated for about 1 hr with oxidized glutathione, giving a final concentration of 20 mM. The addition of 2-mercaptoethanol (ME) to the hemolysates in a final concentration of 20 mM was effective in restoring the normal one-zone phenotype and in preventing the appearance of the artifactual zones. Because some of the samples reaching us by mail were already partially oxidized and because it was not always possible to predict the stability of such samples, it became a routine procedure to add ME to all bison and cattle hemolysates and to allow them to stand for ½ hr prior to electrophoresis. The appearance of transitional forms by addition of oxidized glutathione and the reversion to the original phenotypes by the addition of ME parallel the results obtained by Spencer et al. (1968) in their studies of adenosine deaminase. In light of our findings we believe that the three phenotypes observed by Naik and Anderson (1970) in their studies of bison erythrocytic 6PGD were probably artifactual types that came about from the use of oxidized
Transient Electrophoretic Forms of Red Cell 6PGD in American Buffalo
447
b l o o d samples. F i n a l l y , it s h o u l d be n o t e d t h a t when p r o p e r steps are t a k e n to p r e v e n t the a p p e a r a n c e o f s p u r i o u s p h e n o t y p e s , 6 P G D c a n serve reliably as a n o t h e r e l e c t r o p h o r e t i c m a r k e r in distinguishing between b i s o n a n d d o m e s t i c cattle.
+
--
C
B
C
Fig. 1. Relative positions of 6PGD-B of bison and 6PGD-C of cattle on a starch gel zymogram.
+
l
m
0
1
3
6
10
15
21
days frozen Fig. 2. Transient 6PGD phenotypes exhibited by cattle and bison hemolysates which have been stored frozen.
448
Suzuki, Auditore, Morris, and Stonnont
ACKNOWLEDGMENTS We thank Linda Nickel for drawing Fig. 2. We are grateful for the contribution of blood samples by breeders of American buffalo and by others having access to special bison herds. The contributors were Gene Bartnicki, Wichita Mountains Wildlife Refuge, Cache, OK; Ralph Bartinelli (Gene Harlan, D.V.M.), Petaluma, CA; James H. Burnett, Luther, MT; Jack T. Errington, Durham Meat Co., San Jose, CA; Norman L. Ginther, Livermore, CA; Robert Gorrindo, D.V.M., Minden, NV; Hayes Product, Inc., Seattle, WA; Dr. Dale Lott, University of California, Davis, CA; Joe Miller, Twin Mills, ID; Ed Newquist, Hartsel, CO; John Salmon, Choteau, MT; D. L. Wallace, Winchester, KS; and Fred A. Wolken, Box Elder, SD. REFERENCES Amano, T., Stormont, C., and Suzuki, Y. (1974). Survey of 6PGD, PGM, ACP and PHI phenotypes in red cells of cattle, horse and rabbits. Anita. Blood Grps. Biochem. Genet. 5:21 (Suppl. 1). Bender, K., Op't Hof, J., and Engel, W. (1970). Zur Genetik der 6-Phosphogluconate-Dehydrogenase (EC 1.1.1.44) bei Saugern. Humangenetik 11:59. Bengtsson, S., and Sandberg, K. (1973). A method for simultaneous electrophoresis of horse red cell enzyme systems. Anon. Blood Grps. Biochem. Genet. 4:83. Karadjole, I., Safarova, P., Larsen, B., and Hyldgaard-Jensen, J. (1972). Studies on electrophoretic variation of bovine phosphoglucomutase (PGM) and 6-phosphogluconate dehydrogenase (6-PGD). Anita. Blood Grps. Biochem. Genet. 3:35 (Suppl. 1). Naik, S. N., and Anderson, D. C. (1970). Study of glucose 6-phosphogluconate dehydrogenase and 6-phosphogluconate dehydrogenase in the American buffalo (Bison bison). Biochem. Genet. 4:651. Spencer, N., Hopkinson, D. A., and Harris, H. (1968). Adenosine deaminase polymorphism in man. Ann. Human Genet. 32:9.
Transient Electrophoretic Forms of Red Cell 6PGD in American Buffalo (Bison bison) and Domestic Cattle (Bos taurus) Yoshiko Suzuki] Karen J. Auditore, 1,2 Bob Morris] and Clyde Stormont 1 Received 27 July 1978--Final 17 Nov. 1978
INTRODUCTION In a previous report in this journal, Naik and Anderson (1970) described three electrophoretic patterns of red cell 6-phosphogluconate dehydrogenase (6PGD) in American buffalo (Bison bison). Two of the phenotypes (PdAA and PdBB) were two-zone patterns and the third (PdAB) was composed of three zones. Based on a gene-frequency analysis of the distribution of the three phenotypes in 86 bison samples from the Wichita Mountains Wildlife Refuge in Cache, Oklahoma, they presumed that the three phenotypes are determined by codominant allelic genes, Pd A and Pd ~. In a much larger survey, including samples from 82 bison in the same population studied by Naik and Anderson, we found the bison red cell 6PGD to be monomorphic. Cattle red cell 6PGD is also monornorphic, but it occupies a different electrophoretic position from that of bison 6PGD. In this note we call attention to the oxidative storage effects on blood samples that result in the appearance of transient phenotypic patterns that resemble those described as variant forms of 6PGD in the aforementioned report. MATERIALS AND METHODS Blood samples were collected in commercial Vacutainer tubes (10 ml) containing either EDTA or ACD as anticoagulant and were shipped via air mail to 1 Serology Laboratory, Department of Reproduction, University of California, Davis, California 95616. 2 Present address: Massachusetts Institute of Technology, Center for Cancer Research, E 17-128, Cambridge, Massachusetts 02139. 445 0006-2928/79/0600-0445503.00/0 © I979PlenumPublishingCorporation
446
Suzuki, Auditore, Morris, and Stormont
our laboratory. Except for 82 bison samples, which were obtained from animals in the Wichita Mountains Wildlife Refuge, Cache, Oklahoma, and except for samples drawn from cattle in the University dairy herd, all samples were obtained from private bison and cattle herds throughout the United States. Hemolysates were prepared from washed, packed red cells by (1) rapid freezing and thawing, (2) sonication, and (3) addition of an equal volume of distilled water. Optimal diagnosis was obtained on samples hemolyzed with water. Horizontal starch gel electrophoresis and staining of the gel slices were performed according to the method described by Bengtsson and Sandberg (1973), with minor modifications relating mainly to the size of the gel frames, the cooling system, and the electrophoretic procedures. RESULTS AND DISCUSSION Altogether, 413 bison samples and 599 cattle samples were screened for red cell 6PGD phenotypes, with no evidence for intraspecific genetic variation in the electrophoretic forms of this enzyme. With respect to cattle, these results are in agreement with earlier data (Bender et al., 1970; Karadjole et aI., 1972; Amano et al., 1974). When hemolysates were prepared from freshly collected blood samples and were used soon after their preparation, 6PGD appeared as a single, monomorphic zone in each species, with that of bison, designated B, migrating to a position distinctly ahead of that of cattle, designated C (see Fig. 1). When, however, the hemolysates were stored frozen for longer than 24 hr, multiple, transitional zones of 6PGD began to make their appearance, as illustrated in Fig. 2. Similar effects were obtained when freshly prepared hemolysates were incubated for about 1 hr with oxidized glutathione, giving a final concentration of 20 mM. The addition of 2-mercaptoethanol (ME) to the hemolysates in a final concentration of 20 mM was effective in restoring the normal one-zone phenotype and in preventing the appearance of the artifactual zones. Because some of the samples reaching us by mail were already partially oxidized and because it was not always possible to predict the stability of such samples, it became a routine procedure to add ME to all bison and cattle hemolysates and to allow them to stand for ½ hr prior to electrophoresis. The appearance of transitional forms by addition of oxidized glutathione and the reversion to the original phenotypes by the addition of ME parallel the results obtained by Spencer et al. (1968) in their studies of adenosine deaminase. In light of our findings we believe that the three phenotypes observed by Naik and Anderson (1970) in their studies of bison erythrocytic 6PGD were probably artifactual types that came about from the use of oxidized
Transient Electrophoretic Forms of Red Cell 6PGD in American Buffalo
447
b l o o d samples. F i n a l l y , it s h o u l d be n o t e d t h a t when p r o p e r steps are t a k e n to p r e v e n t the a p p e a r a n c e o f s p u r i o u s p h e n o t y p e s , 6 P G D c a n serve reliably as a n o t h e r e l e c t r o p h o r e t i c m a r k e r in distinguishing between b i s o n a n d d o m e s t i c cattle.
+
--
C
B
C
Fig. 1. Relative positions of 6PGD-B of bison and 6PGD-C of cattle on a starch gel zymogram.
+
l
m
0
1
3
6
10
15
21
days frozen Fig. 2. Transient 6PGD phenotypes exhibited by cattle and bison hemolysates which have been stored frozen.
448
Suzuki, Auditore, Morris, and Stonnont
ACKNOWLEDGMENTS We thank Linda Nickel for drawing Fig. 2. We are grateful for the contribution of blood samples by breeders of American buffalo and by others having access to special bison herds. The contributors were Gene Bartnicki, Wichita Mountains Wildlife Refuge, Cache, OK; Ralph Bartinelli (Gene Harlan, D.V.M.), Petaluma, CA; James H. Burnett, Luther, MT; Jack T. Errington, Durham Meat Co., San Jose, CA; Norman L. Ginther, Livermore, CA; Robert Gorrindo, D.V.M., Minden, NV; Hayes Product, Inc., Seattle, WA; Dr. Dale Lott, University of California, Davis, CA; Joe Miller, Twin Mills, ID; Ed Newquist, Hartsel, CO; John Salmon, Choteau, MT; D. L. Wallace, Winchester, KS; and Fred A. Wolken, Box Elder, SD. REFERENCES Amano, T., Stormont, C., and Suzuki, Y. (1974). Survey of 6PGD, PGM, ACP and PHI phenotypes in red cells of cattle, horse and rabbits. Anita. Blood Grps. Biochem. Genet. 5:21 (Suppl. 1). Bender, K., Op't Hof, J., and Engel, W. (1970). Zur Genetik der 6-Phosphogluconate-Dehydrogenase (EC 1.1.1.44) bei Saugern. Humangenetik 11:59. Bengtsson, S., and Sandberg, K. (1973). A method for simultaneous electrophoresis of horse red cell enzyme systems. Anon. Blood Grps. Biochem. Genet. 4:83. Karadjole, I., Safarova, P., Larsen, B., and Hyldgaard-Jensen, J. (1972). Studies on electrophoretic variation of bovine phosphoglucomutase (PGM) and 6-phosphogluconate dehydrogenase (6-PGD). Anita. Blood Grps. Biochem. Genet. 3:35 (Suppl. 1). Naik, S. N., and Anderson, D. C. (1970). Study of glucose 6-phosphogluconate dehydrogenase and 6-phosphogluconate dehydrogenase in the American buffalo (Bison bison). Biochem. Genet. 4:651. Spencer, N., Hopkinson, D. A., and Harris, H. (1968). Adenosine deaminase polymorphism in man. Ann. Human Genet. 32:9.
