Purine nucleoside phosphorylase heterogeneity in the mouse as analyzed by isoelectric focusing and specific activity

Comp. Biochem. Physiol. Vol. 8911,No. 2, pp. 427-431, 1988 Printed in Great Britain

0305-0491/88 $3.00+0.00 © 1988 Pergamon Journals Ltd

PURINE NUCLEOSIDE PHOSPHORYLASE HETEROGENEITY IN THE MOUSE AS A N A L Y Z E D BY ISOELECTRIC FOCUSING A N D SPECIFIC ACTIVITY* ELLEN R. MABLY, THERESE CARTER-EDWARDS, FRED G. BIDDLE and FLOYO F. SNYDERt Departments of Medical Biochemistry and Pediatrics, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4NI, Canada (Tel: 403 284-6876) (Received 22 April 1987)

Abstract--l. Isoelectric focusing in polyacrylamide gels identified three erythrocytic electrophoretic patterns of purine nucleoside phosphorylase in a survey of 16 mouse strains. 2. Three strain-specific electrophoretic types were also evident in liver, kidney and spleen leukocytes. 3. There are 3-fold differences in purine nucleoside phosphorylase activities between strains for several tissues; C57BL/6J and Mus spretus having the greatest and the least activity, respectively. 4. Within strains there were up to 8-fold tissue-specific differences in activity with the order from greatest to least being: liver, kidney, spleen leukocyte, erythrocyte, heart. INTRODUCTION

Purine nucleoside phosphorylase (NP; EC 2.4.2.1) catalyzes the reversible phosphorolysis of inosine, guanosine, deoxyinosine and deoxyguanosine. The inherited human deficiency of this activity results in cellular immune dysfunction and normal B cell function (Giblett et al., 1975). In the mouse purine nucleoside phosphorylase (Np-1) has been mapped to chromosome 14 and is linked to esterase-10 (Es-10) (Womack et al., 1977). Most inbred mouse strains exhibit a common erythrocytic pattern for purine nucleoside phosphorylase on cellulose acetate eleetrophoresis designated NP-1A. An additional erythroeytic electrophoretic band observed only by starch gel eleetrophoresis, designated NP-2, is coincident with high specific activity and was found only in strains of the C57 or C58 background (Bremner et al., 1978; Snyder et aL, 1983). Np-2 has also been shown to be linked to Es-10 (Taylor, 1981; Lukey et aL, 1985) and no reeombinants were found between Np-1 and Np-2 in a limited study (Lukey et al., 1985) indicating that NP-1 and NP-2 are determined either by alleles at a single locus or by very closely linked loci. The biochemical heterogeneity of mammalian purine nueleoside phosphorylase has been examined by quantitative assay or electrophoresis for man (Edwards et aL, 1971), cattle (Ansay and Hanset, 1972; Mahin and Mammad, 1982), sheep (Board and Smith, 1977) and mouse (Womack et al., 1977; Bremner et al., 1978; Snyder et al., 1983; Lukey et al., 1985; Soodeen and Bremner, 1985). The electrophoretic analyses have employed starch gels or cellulose acetate. We describe here the use of vertical isoelectric focusing in polyacrylamide gels to study heterogeneity in the erythrocytes of 16 mouse strains. Tissue purine nucleoside phosphorylase from five of *Supported by the Medical Research Council of Canada grants MT-6376 and MT-6736. i'To whom correspondence may be addressed.

these strains was also compared electrophoretically. The quantitative activity of purine nucleoside phosphorylase was examined in erythrocytes, liver, kidney, heart and spleen leukocytes for the same five strains. MATERIALSAND METHODS Mouse strains The strains of mice used in this study and their source are indicated in Table 1. Lysate preparation and enzyme assay Blood was obtained from the orbital sinus and erythrocyte lysates were prepared as previously described (Lukey et al., 1985). Mice were killed by cervical dislocation and liver, kidney and heart were removed and homogenized at 4°C in 50raM phosphate, pH 7.4, with a Polytron (Brinkmann Instruments) and the 10,000g supernatants were recovered for enzyme assay. Spleens were removed, minced and passed through a stainless steel mesh and spleen leukocytes were prepared by lysis of contaminating erythrocytes with Trisbuffered ammonium chloride (Lukey and Snyder, 1983). Purine nucieoside phosphorylase activity was assayed at 37°C by following the conversion of [8-14C] inosine (52 Ci/mol; Amersham Corp., OakviUe, Ontario, Canada) to hypoxanthine as previously described (Snyder et aL, 1976). Isoelectric focusing in polyacrylamide gels Non-denaturing isoelectric foscusing slab gels were prepared by mixing 30 mg each of the amino acids, nL-aspartic acid, L-arginlne HCI and L-lysine HC1 in 18.31 ml of water, 11.66 ml 50% glycerol, 8.0 ml acrylamide solution (24.25% acrylamide, 0.75% bis-acrylamide), and stock ampholytes (Pharmacia), 1.27 ml each of pH 5-8 and pH 4.5-5.4. The solution was degassed and 0.0156ml of TEMED and then 0.320 ml of 10% ammonium persulfate were added prior to pouring between glass plates (180 x 160 x 3 mm) in a Protean I cassette (Biorad Laboratories) using 0.75 mm spacers. After 1 hr, sample wells were washed three times with water then the gel was pre-focused at 200V for 15 min, 300V for 30 min and 400 V for 30 min using tap water to cool the system. Degassed L-glutamic acid, 0.01 M, was in the lower

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ELLEN R. MABLY et al. Table 1. Inbred and incipient inbred strains of laboratory mice maintained by sib mating Name

Source and year imported

Reference

Mus musculus complex

ABP/Le C57BL/6J DBA/2J WB/ReJ C3H/HeHa.Pgk-I a NPG SWV

Jackson Laboratory Jackson Laboratory Jackson Laboratory Jackson Laboratory V. Chapman G. Bulfield J. Miller

1984 1978 1980 1983 1982 1983 1975

Staats (1980) Staats (1980) Staats (1980) Staats (1980) Chapman et al. (1983) Bulfield et al. (1984) Staats (1980)

M. Potter M. Potter

1985

Potter (1986) Potter (1986)

M. M. M. M.

1984

Wallace(1985) Wallace (1985) Wallace (1985) Wallace (1985)

Jackson Laboratory M. Potter

1984 1985

Potter (1986) Potter (1986)

M. Potter

1982

Potter (1986)

Mus domesticus

CLA (Centreville Light; North America) Posch-2 (Zalende, Switzerland) Peru W-I (Peru-Coppock; Rimac Valley, Peru) Peru W-2 Peru W-9 Peru W-10

Wallace Wallace Wallace Wallace

Mus musculus molossinus

MOLD/Rk MOLO Mus spretus

SPRET-1 (Puerto Real, Spain)

cathode reservoir and L-histidine, 0.01 M, was in the upper anode reservoir. Lysates appropriately diluted with water were mixed 1: 3 with sample buffer comprised of 3.086 rnl H20, 0.709 ml 50% glycerol, 0.168 ml of each ampholyte, pH 5--8 and pH 4.5-5.4 and 0.037 ml 1% mercaptoethanol. A loading of approx. 400 #g of protein was adequate for erythrocyte lysates. Wells were again water-washed and equilibrated with sample buffer for 10 min before loading samples. The anode solution was replaced and the samples were electrophoresed overnight at 300 V for 4800V-hr, followed by 45 min at 800 V. The pls were determined at room temperature with a surface pH electrode placed on the moistened gel after electrophoresis and should be considered relative rather than absolute pI values. The gels were stained for purine nucleoside phosphorylase with an agarose overlay as previously described (Womack et al., 1977).

RESULTS Isoelectric focusing of erythrocyte lysates revealed three distinct m u l t i b a n d e d p a t t e r n s (Fig. 1). T h e M u s musculus a n d M u s domesticus strains showed a pred o m i n a n t three b a n d e d p a t t e r n with pls o f 4.80--4.92 a n d evidence o f a m o r e faint f o u r t h b a n d (Fig. 1). M u s musculus molossinus b o t h M O L D / R k and M O L O , gave a four b a n d e d p a t t e r n with pIs between 4.90 a n d 5.05, distinctly less acidic t h a n the o t h e r i n b r e d strains. M u s spretus ( S P R E T - I ) exhibited a four b a n d e d p a t t e r n h a v i n g the m o s t neutral pI species between 5.24 a n d 5.44. Thus, in this survey, three distinct alleles were evident by isoelectric focusing in polyacrylamide gels.

Fig. 1. Isoelectric focusing of erythrocytic purine nucleoside phosphorylase on polyacrylamide gels: survey of 16 inbred mouse strains.

Mouse purine nucleoside phosphorylase

429

Fig. 2. Isoelectric focusing of mouse liver and kidney purine nucleoside phosphorylase. Isoelectric focusing of liver and kidney lysates from five strains demonstrated the three major patterns of purine nueleoside phosphorylase which were observed in erythrocytes but the patterns were much simpler in composition (Fig. 2). There were two

bands present with one of these being predominant. The major kidney and liver bands appeared to have the same pI as the most neutral erythrocyte band. In addition a distinction appeared to be evident between DBA/2J and C57BL/6J or N P G in these tissues

Fig. 3. Isoelectric focusing of mouse spleen leukocyte purine nucleoside phosphorylase.

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ELLEN R. MABLY et al. Table 2. Strain variation o f purin¢ nuclvoside phosphorylas¢ activity in several mouse tissues and cells

Erythrocyte Strain DBA/2J C57BL/6J NPG MOLD/Rk SPRET-I

16.2 ± 43.9 ± 13.2 ± 22.2 ± 13. ! ±

2.0 a'l 0 . 2 c'2

0.3 *'1 2.6 b'l 1.0~2

Tissue or cell type Spleen leukocyte Kidney nmol/min per mg protein 37.9 ± 53.3 ± 31.9 ± 64.4 ± 25.4 ±

2.4 "~ 5.4 b'23

1.4i'2 9.7 b'3

2.5 s'3

62.3 ± 60.9 ± 59.2 ± 40.0 ± 32.4 ±

1.2~3 1.1~3 4.8 ¢'3 0.9 b'2

2.5 "(

Liver 68.7 ± 100 ± 74 ± 45.5 ± 36.4 ±

3.8 b'( 5.8c'( 15b'3 9.3 *~ 1.0*'4

Heart 16.7 ± 19.1 ± 6.97 ± 14.8 ± 6.85 ±

0.5 ~'1 2.6 c'l 0.28 R'l 1.5b'l 0.33 "~

Results are the mean + SD for three animals. Activity comparisons were made by the Student-Newman-Keuls test at P = 0.01 (Sokal and Rohlf, 1969). Supcrscript letters indicate significant differences between strains for each tissue. Superscript numbers indicate significant differences between tissues for each strain. Double letters or numbers indicate overlap between two groups.

which was not evident in the erythrocytes. The three electrophoretic patterns were also found for spleen leukocyte purine nucleoside phosphorylase (Fig. 3). We have previously shown that there are significant differences in erythrocyte purine nucleoside phosphorylase activity between strains of the C57 and C58 background and other inbred strains (Snyder et al., 1983). Thus, a comparison of specific activity between these strains and those showing electrophoretic differences, namely M. molossinus and M. spretus was conducted for several tissues (Table 2). For erythrocytes, C57BL/6J has the greatest activity with MOLD/Rk being intermediate and DBA/2J, NPG and SPRET-1 having lower activity. There was a 2.5-fold difference between strains for spleen leukocyte activity with MOLD/Rk and C57BL/6J having the greatest activity. The differences in activity between strains were for kidney, 2-fold, liver, 3-fold and heart, 3-fold. Tissues from C57BL/6J were consistently among those having greatest activity whereas those from M. spretus were the least (Table 2). DISCUSSION

Isoelectric focusing in polyacrylamide gels has shown three distinct patterns amongst the mice surveyed (Fig. 1). DBA/2J, C57BL/6J and eleven other inbred strains all showed the same profile for erythrocytes, the NP-1A pattern. These include wild caught mice from diverse geographical locations, the PeruCoppock strains from Peru, NPG from Greece, CLA from North America, and the Posch-2 from Switzerland (Table 1). The M. molossinus-derived MOLD/ Rk and MOLO strains gave a more neutral species, characteristic of the NP-1B phenotype. SPRET-1 gave a third pattern which was clearly distinct from the others and further reflects the utility of Mus spretus as a source of polymorphic loci (Guenet, 1986). Interestingly, the erythrocyte C57BL/6J pattern was identical to that of DBA/2J by isoelectric focusing whereas the much cruder electrophoresis on starch gels shows a unique second band for C57BL/6J mice (Bremner et al., 1978; Snyder et al., 1983). Multibanded purine nucleoside phosphorylase patterns for human erythrocytes are presumed to arise from post-translational modification of the protein; the native structure being comprised of three identical subunits which can associate in various charge configurations (Zannis et al., 1978). The subunit composition of the mouse enzyme is not known.

Starch gel electrophoresis of spleen has been interpreted to show the same NP-IA electromorph for M. molossinus, DBA/2J and C57BL/6J (Soodeen and Bremner, 1985). Our studies show that isoelectric focusing of spleen leukocytes, liver or kidney (Figs 2 and 3) gave three distinct patterns characteristic of M. spretus, M. molossinus and the other inbred strains as typified by DBA/2J or C57BL/6J. Although an identical pattern is present for DBA/2J, C57BL/6J and NPG in erythrocytes (Fig. 1), DBA/2J could be distinguished from C57BL/6J and NPG in liver and kidney (Fig. 2). The distinction, however, was subtle compared to that observed between DBA/2J and M. molossinus.

Quantitative assay of purine nucleoside phosphorylase activity in five inbred strains revealed 2.5-3 fold strain variation for all tissues examined. There were in general three activity groups with respect to strain (see legend to Table 2) with C57BL/6J being amongst the greatest activity group and SPRET-1 consistently having the lowest activity. For all strains purine nucleoside phosphorylase was, in general, lowest in heart and greatest in liver or kidney although spleen leukocytes had nearly similar activity. The NPG strain was previously reported to have approximately half the erythrocyte activity of DBA/ZI (Bulfield et al., 1984). In the present study NPG activity was only fractionally less than that of DBA/2J (Table 2). The NPG strain was inbred for eight generations in Calgary without typing and it is possible that the stock was segregating for alleles at the purine nucleoside phosphorylase locus which resulted in a loss of the low activity variant. These studies have further characterized both the quantitative and elcctrophoretic heterogeneity of purine nucleoside phosphorylase in the mouse. We believe the isoeleetric focusing procedure described here provides a greatly improved method for the typing of purine nucleoside phosphorylase. Acknowledgements--We t h a n k G . A n t o u a n d G . L a u z o n

for guidance with the isoelectric focusing. REFERENCF~

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Board P. G. and Smith J. E. (1977) Purine nucleoside phosphorylase polymorphism in sheep erythrocytes. Biochem. Genet. 15, 347-355.

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