Biochemical Genetics, Vol. 15, Nos. 5/6, 1977
Purine Nucleoside Phosphorylase Polymorphism in Sheep Erythrocytes P. G. Board 1 and J. E. Smith ~
Received 30 July 1976--Final 11 Nov. 1976
A polymorphism of purine nucleoside phosphorylase is described in sheep erythrocytes. Two isozymes were distinguished eleetrophoretically, one with high activity (NP-!) and one with low activity (NP-2). Breeding data suggest that the two isozymes are the product of two eodominant alleles, N P 1 and N P z. The Km's for inosine did not differ between NP-1 and NP-2; however, NP-2 had a lower p H optimum and was relatively unstable when incubated at 48 C. KEY WORDS: purine nucleoside phosphorylase; polymorphism; erythrocyte; sheep.
INTRODUCTION Purine nucleoside phosphorylase (NP; E.C. 2.4.2.1) is found in many tissues and may catalyze both the synthesis and degradation of purine nucleosides (Friedkin and Kalckar, 1961). Inherited variants of N P from erythrocytes have been reported in humans (Edwards et al., 1971) and cattle (Ansay and Hanset, 1972; Ansay, 1975). Previous studies have demonstrated many biochemical polymorphisms in sheep erythrocytes (Blunt, 1975). This article reports a biochemical and genetic study of a new NP polymorphism in sheep erythrocytes. Contribution No. 421-J, from the Department of Pathology, Kansas Agricultural Experiment Station, Manhattan, Kansas. Supported in part by USPHS Grants HL-70119 and HL 12072. 1 Department of Pathology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas. 439 © 1977 Plenum Publishing Corp., 227 West 17th Street, N e w York, N.Y. 10011. To promote freer access to published material in the spirit o f the 1976 Copyright Law, Plenum sells reprint articles f r o m all its journals. This availability underlines the fact that no part o f this publication m a y be reproduced, stored in a retrieval system, o r transmitted, in any f o r m or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission o f the publisher. Shipment is p r o m p t ; rate per article is $7.50.
440
Board and Smith METHODS
Red cell hemolysates were prepared by the method described by Ansay and Hanset (1972). NP activity was determined using inosine as a substrate and by recording the formation of uric acid at 293 nm by the method of Kalckar (1947). The activities were expressed as enzyme units (/~Muric acid formed per gram of hemoglobin per minute at 37 C). Electrophoresis was carried out on 12~ starch gels with the following buffers: tris, 200 mM; EDTA, 20 mM; boric acid, 162 mM, pH 8.3. The gel buffer was made by diluting the bridge buffer ten-fold. Enzyme activity was localized on sliced gels by the method of Edwards et al. (1971). NP was partially purified and the optimum pH, heat stability and Michaelis constant (Kin) for inosine were determined by the methods used to compare erythrocyte glucose-6-phosphate dehydrogenase variants (Beutler et al., 1968).
RESULTS Electrophoresis of sheep hemolysates and subsequent localization of NP activity indicated two phenotypes. In most sheep, NP migrates as a large band immediately in front of the hemoglobin zone. In some, NP activity was much weaker and was localized as a very faint zone (Fig. 1). We called the two phenotypes NP-1 and NP-2, respectively. Quantitative determination
4"
Fig. 1. Starch gel electrophoretic pattern of the two NP phenotypes: NP-1 (left)and NP-2 (right).
Polymorphism in Sheep Erythroeytes
441
15
c
LL
0
1
2
t 3
I 4
i
I 5
]
I 6
L 7
8
9
r-"] 1 10 11 12
pMlgHb/min Fig. 2. Distribution o f erythrocyle N P Suffolk ewes.
activities in
of NP activity in a group of Suffolk ewes demonstrated three subpopulations (Fig. 2). The animals with lower NP activities ( < 1 unit) correspond to animals with the weakly staining phenotype (NP-2). The animals from the two populations with the higher activities ( > 1 unit), correspond to animals with the strongly staining electrophoretic phenotype (NP-1). The data suggested the existence of two alleles, N P 1 and N P z, animals with NP activities of 7 units or greater being homozygous ( N P 1-1) and those with less than 1 unit of activity being homozygous for the alternate allele ( N P Z - 2 ) . With this scheme, sheep with intermediate values (1-6 units) may be considered as heterozygotes ( N P I - 2 ) . To test that possibility, we examined breeding data from small flocks of Suffolk, Hampshire, and Southdown sheep. Table I shows the available breeding data and the expected occurrence of each genotype, assuming a codominant relationship between the two alleles. There were more N P ~-~ progeny than expected on the basis of a two coTable I. T h e Inheritance o f N P G e n o t y p e s Progeny" Matings
1-1
1-2
1-1 x 1-1
8 (8) 31 (22.5) 7 (4.75) 2 (0) o (o) 0 (0)
0 (0) 14 (22.5) 8 (9.5) 5 (7) 6 (3) 0 (0)
1-2 x 1-1 1-2 x 1-2 2-2 x 1-1 2-2 x 1-2 2-2 × 2-2
" E x p e c t e d n u m b e r in parentheses. C
2-2
0 (0) 0 (0) 4 (4.75) 0 (0) o (3) 1 (1)
442
Board and Smith
.15
1 V (u'l)
.1
.05
J
-I
I
.05
I
.1
.15
I
.2
! (pM"1) S Fig. 3. Lineweaver-Burk NP-1 ; - - , NP-2.
plots of N P
activity. - - -
dominant allele system. However, g z analysis using the Yates correction term for small classes (Strickberger, 1968) indicated that the difference from the expected was not statistically significant. Partial purification of NP from presumed N P 1-1 and N P z - 2 animals was carried out to increase the specific activity of NP-2 and to facilitate the characterization of both enzymes. The Km's for inosine were calculated from double reciprocal plots of velocity against inosine concentration. The Km's of the NP-1 and NP-2 enzymes did not differ. The data presented in Fig. 3, giving a Km of 1.8 x 100'
80
~ %
60
~
~ ",o\ \
._~
40
20-
00
I 10
I 20
I 30
[ 40
I 50
I 60
Minutes Fig. 4. Effect of incubation at 48 C on the activity o f NP-1 ( - - - ) and NP-2 ( ).
Polymorphismin Sheep Erythrocytes
443
140
120
loo I ~6
\
80
g >,
.>
60
< 40
20--
0
I 6
I 7
I 8
I 9
I 10
t 11
pH Fig. 5. Effect o f p H variation on the activity o f NP-1 ( - - -) and NP-2 ( ).
10 . 5 M for NP-1 and 1.9 x 10 -5 M for NP-2 are representative of data obtained on several occasions from different sheep. When the partially purified enzymes were incubated at 48 C (Fig. 4), NP-2 was relatively unstable. The pH optimum was lower for NP-2 (6.5-7.0) than for NP-1 (7.5-8.0) (Fig. 5). Electrophoresis of the partially purified NP-1 and NP-2 showed little difference in mobilities of the two enzymes.
DISCUSSION The erythrocyte NP polymorphism in sheep appears to be similar to the twoallele system described in cattle (Ansay and Hanset, 1972). A third erythrocyte NP allele reported recently in cattle results in extremely high NP activities (Ansay, 1975). A similar allele in sheep may explain the difference of our breeding data from the pattern expected for a two-allele system. We have observed two animals with NP activities exceeding" 18 units. Such animals cannot be distinguished electrophoretically from N P 1-1 animals with NP activities of 7-10 units. Further biochemical and
444
Board and Smith
genetic analyses will be necessary to determine if they represent a third (NP-3) variant. The differences in p H optima and the decreased heat stability of the NP-2 variant suggest that the lower activity, of this enzyme in erythrocytes may result from instability of the enzyme and a shortened activity span in erythrocytes. NP polymorphism was observed in all the sheep breeds examined in this investigation. Further study of the inheritance of the two enzymes may permit them to be used as easily identified genetic markers. ACKNOWLEDGMENTS The authors thank Mrs. Kateri Moore and Mrs. Margret Lee for technical assistance. REFERENCES
Ansay, M. (1975). Note on a third allele in the erythrocytic NP system of cattle. Anhn. Blood Grps. Biochem. Genet. 6:121. Ansay, M., and Hanset, R. (1972). Purine nucleoside phosphorylase (NP) of bovine erythrocytes : Genetic control of electrophoretic variants. Anim. Blood Grps. Biochem. Genet. 3:219. Beutler, E., Mathai, C. K., and Smith, J. E. (1968). Biochemical variants of glucose-6phosphate dehydrogenase giving rise to congenital nonspherocytic hemolytic disease. Blood 31:131. Blunt, M. H. (1975). The Blood of Sheep, Springer-Verlag, New York. Edwards, Y. H., Hopkinson, D. A., and Harris, H. (1971). Inherited variants of human nucleoside phosphorylase. Ann. Hum. Genet. 34:395. Friedkin, M., and Kalckar, H. (1961). Nucleoside phosphorylases. In Boyer, P. D., Lardy, H., and MyrNick, K. (eds.), The Enzymes, Vol. 5, Academic Press, New York, pp. 237-280. Kalckar, H. M. (1947). Differential spectrophotometry of purine compounds by means of specific enzymes. III. Studies of the enzymes of purine metabolism. J. Biol. Chem. 167:461. Strickberger, M. W. (1968). In Genetics, Macmillan, New York, p. 132.
Purine Nucleoside Phosphorylase Polymorphism in Sheep Erythrocytes P. G. Board 1 and J. E. Smith ~
Received 30 July 1976--Final 11 Nov. 1976
A polymorphism of purine nucleoside phosphorylase is described in sheep erythrocytes. Two isozymes were distinguished eleetrophoretically, one with high activity (NP-!) and one with low activity (NP-2). Breeding data suggest that the two isozymes are the product of two eodominant alleles, N P 1 and N P z. The Km's for inosine did not differ between NP-1 and NP-2; however, NP-2 had a lower p H optimum and was relatively unstable when incubated at 48 C. KEY WORDS: purine nucleoside phosphorylase; polymorphism; erythrocyte; sheep.
INTRODUCTION Purine nucleoside phosphorylase (NP; E.C. 2.4.2.1) is found in many tissues and may catalyze both the synthesis and degradation of purine nucleosides (Friedkin and Kalckar, 1961). Inherited variants of N P from erythrocytes have been reported in humans (Edwards et al., 1971) and cattle (Ansay and Hanset, 1972; Ansay, 1975). Previous studies have demonstrated many biochemical polymorphisms in sheep erythrocytes (Blunt, 1975). This article reports a biochemical and genetic study of a new NP polymorphism in sheep erythrocytes. Contribution No. 421-J, from the Department of Pathology, Kansas Agricultural Experiment Station, Manhattan, Kansas. Supported in part by USPHS Grants HL-70119 and HL 12072. 1 Department of Pathology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas. 439 © 1977 Plenum Publishing Corp., 227 West 17th Street, N e w York, N.Y. 10011. To promote freer access to published material in the spirit o f the 1976 Copyright Law, Plenum sells reprint articles f r o m all its journals. This availability underlines the fact that no part o f this publication m a y be reproduced, stored in a retrieval system, o r transmitted, in any f o r m or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission o f the publisher. Shipment is p r o m p t ; rate per article is $7.50.
440
Board and Smith METHODS
Red cell hemolysates were prepared by the method described by Ansay and Hanset (1972). NP activity was determined using inosine as a substrate and by recording the formation of uric acid at 293 nm by the method of Kalckar (1947). The activities were expressed as enzyme units (/~Muric acid formed per gram of hemoglobin per minute at 37 C). Electrophoresis was carried out on 12~ starch gels with the following buffers: tris, 200 mM; EDTA, 20 mM; boric acid, 162 mM, pH 8.3. The gel buffer was made by diluting the bridge buffer ten-fold. Enzyme activity was localized on sliced gels by the method of Edwards et al. (1971). NP was partially purified and the optimum pH, heat stability and Michaelis constant (Kin) for inosine were determined by the methods used to compare erythrocyte glucose-6-phosphate dehydrogenase variants (Beutler et al., 1968).
RESULTS Electrophoresis of sheep hemolysates and subsequent localization of NP activity indicated two phenotypes. In most sheep, NP migrates as a large band immediately in front of the hemoglobin zone. In some, NP activity was much weaker and was localized as a very faint zone (Fig. 1). We called the two phenotypes NP-1 and NP-2, respectively. Quantitative determination
4"
Fig. 1. Starch gel electrophoretic pattern of the two NP phenotypes: NP-1 (left)and NP-2 (right).
Polymorphism in Sheep Erythroeytes
441
15
c
LL
0
1
2
t 3
I 4
i
I 5
]
I 6
L 7
8
9
r-"] 1 10 11 12
pMlgHb/min Fig. 2. Distribution o f erythrocyle N P Suffolk ewes.
activities in
of NP activity in a group of Suffolk ewes demonstrated three subpopulations (Fig. 2). The animals with lower NP activities ( < 1 unit) correspond to animals with the weakly staining phenotype (NP-2). The animals from the two populations with the higher activities ( > 1 unit), correspond to animals with the strongly staining electrophoretic phenotype (NP-1). The data suggested the existence of two alleles, N P 1 and N P z, animals with NP activities of 7 units or greater being homozygous ( N P 1-1) and those with less than 1 unit of activity being homozygous for the alternate allele ( N P Z - 2 ) . With this scheme, sheep with intermediate values (1-6 units) may be considered as heterozygotes ( N P I - 2 ) . To test that possibility, we examined breeding data from small flocks of Suffolk, Hampshire, and Southdown sheep. Table I shows the available breeding data and the expected occurrence of each genotype, assuming a codominant relationship between the two alleles. There were more N P ~-~ progeny than expected on the basis of a two coTable I. T h e Inheritance o f N P G e n o t y p e s Progeny" Matings
1-1
1-2
1-1 x 1-1
8 (8) 31 (22.5) 7 (4.75) 2 (0) o (o) 0 (0)
0 (0) 14 (22.5) 8 (9.5) 5 (7) 6 (3) 0 (0)
1-2 x 1-1 1-2 x 1-2 2-2 x 1-1 2-2 x 1-2 2-2 × 2-2
" E x p e c t e d n u m b e r in parentheses. C
2-2
0 (0) 0 (0) 4 (4.75) 0 (0) o (3) 1 (1)
442
Board and Smith
.15
1 V (u'l)
.1
.05
J
-I
I
.05
I
.1
.15
I
.2
! (pM"1) S Fig. 3. Lineweaver-Burk NP-1 ; - - , NP-2.
plots of N P
activity. - - -
dominant allele system. However, g z analysis using the Yates correction term for small classes (Strickberger, 1968) indicated that the difference from the expected was not statistically significant. Partial purification of NP from presumed N P 1-1 and N P z - 2 animals was carried out to increase the specific activity of NP-2 and to facilitate the characterization of both enzymes. The Km's for inosine were calculated from double reciprocal plots of velocity against inosine concentration. The Km's of the NP-1 and NP-2 enzymes did not differ. The data presented in Fig. 3, giving a Km of 1.8 x 100'
80
~ %
60
~
~ ",o\ \
._~
40
20-
00
I 10
I 20
I 30
[ 40
I 50
I 60
Minutes Fig. 4. Effect of incubation at 48 C on the activity o f NP-1 ( - - - ) and NP-2 ( ).
Polymorphismin Sheep Erythrocytes
443
140
120
loo I ~6
\
80
g >,
.>
60
< 40
20--
0
I 6
I 7
I 8
I 9
I 10
t 11
pH Fig. 5. Effect o f p H variation on the activity o f NP-1 ( - - -) and NP-2 ( ).
10 . 5 M for NP-1 and 1.9 x 10 -5 M for NP-2 are representative of data obtained on several occasions from different sheep. When the partially purified enzymes were incubated at 48 C (Fig. 4), NP-2 was relatively unstable. The pH optimum was lower for NP-2 (6.5-7.0) than for NP-1 (7.5-8.0) (Fig. 5). Electrophoresis of the partially purified NP-1 and NP-2 showed little difference in mobilities of the two enzymes.
DISCUSSION The erythrocyte NP polymorphism in sheep appears to be similar to the twoallele system described in cattle (Ansay and Hanset, 1972). A third erythrocyte NP allele reported recently in cattle results in extremely high NP activities (Ansay, 1975). A similar allele in sheep may explain the difference of our breeding data from the pattern expected for a two-allele system. We have observed two animals with NP activities exceeding" 18 units. Such animals cannot be distinguished electrophoretically from N P 1-1 animals with NP activities of 7-10 units. Further biochemical and
444
Board and Smith
genetic analyses will be necessary to determine if they represent a third (NP-3) variant. The differences in p H optima and the decreased heat stability of the NP-2 variant suggest that the lower activity, of this enzyme in erythrocytes may result from instability of the enzyme and a shortened activity span in erythrocytes. NP polymorphism was observed in all the sheep breeds examined in this investigation. Further study of the inheritance of the two enzymes may permit them to be used as easily identified genetic markers. ACKNOWLEDGMENTS The authors thank Mrs. Kateri Moore and Mrs. Margret Lee for technical assistance. REFERENCES
Ansay, M. (1975). Note on a third allele in the erythrocytic NP system of cattle. Anhn. Blood Grps. Biochem. Genet. 6:121. Ansay, M., and Hanset, R. (1972). Purine nucleoside phosphorylase (NP) of bovine erythrocytes : Genetic control of electrophoretic variants. Anim. Blood Grps. Biochem. Genet. 3:219. Beutler, E., Mathai, C. K., and Smith, J. E. (1968). Biochemical variants of glucose-6phosphate dehydrogenase giving rise to congenital nonspherocytic hemolytic disease. Blood 31:131. Blunt, M. H. (1975). The Blood of Sheep, Springer-Verlag, New York. Edwards, Y. H., Hopkinson, D. A., and Harris, H. (1971). Inherited variants of human nucleoside phosphorylase. Ann. Hum. Genet. 34:395. Friedkin, M., and Kalckar, H. (1961). Nucleoside phosphorylases. In Boyer, P. D., Lardy, H., and MyrNick, K. (eds.), The Enzymes, Vol. 5, Academic Press, New York, pp. 237-280. Kalckar, H. M. (1947). Differential spectrophotometry of purine compounds by means of specific enzymes. III. Studies of the enzymes of purine metabolism. J. Biol. Chem. 167:461. Strickberger, M. W. (1968). In Genetics, Macmillan, New York, p. 132.
