Aiiiin. Blood Grps biochem. Genet. 7 (1976) 207-215
Some physiological aspects of genetic variation in the blood of sheep' Elizabeth M. Tucker ARC Institute of Animal Physiology, Babraham, Cambridge CB2 4AT, EngIand Received: 16 September 1976
Summary The principal genetic variants in sheep red cells and plasma are listed. Current hypotheses as to how the L blood group antigen affects active potassium transpait across the red cell membrane are summarized. Recent work on an inherited defect in amino acid transport which results in a red cell GSH deficiency is also described.
Introduction Tables 1 , 2 and 3 summarize the principal genetic systems which have been described for sheep plasma and red blood cells. For further details see review papers by Tucker, 1971; Agar et a]., 1972; Hall, 1975; Tucker, 1975; McDermid el al., 1975. The sheep can claim something of a unique position in that it provides several impressive example; of how the discovery of an apparently simple genetic variation can lead to or stimulate research in a wide area of inte-e;t. In this paper I shall give two Table 1. Genetic markers in the plasmn. Locus symbol Alleles Albumin Pre-albumin
A1 Pr
X Transferrin Esterase j?-Lipoprotein upMacroglobulin Immunoglobulin
Tf
Es Lp Ap
Irn (1) Zin (2)
D,F, S, T , V, H7 F , S, 0 E M, S I , A , G, H , B, K, C, M , D,Q,E, P 0,b, c 1, 1, 2 Pa,Y l b
-
p=
1 Review paper presented at the 15th International Conference on Animal Blood Groups agd Biochemical Polymorphism (Dublin, Ireland, 1976).
207
ELIZABETH hl. TUCKER
Table 2. Blood group antigens.
Locus symbol
Alleles
R I .A C M
R, r c I, i O, 6, n, ab, b, a, 6, nc, c ( M , L , MM', M ' )
D
fl,
B .Y- Z He1 C O I1
-
n, nb, nbc,
x,x=
-,err
Hel, he1 Cot+, con"
examples of how genetic variants in sheep red cells have proved to be useful tools for investigating basic physiological processes. Both examples are concerned with transport processes across the redcell membrane. The first deals with active transport ofpotassium ( K e , M locus) and the second with transport of amino acids (7" locus).
A4ctivepotassium transport Sheep show 2 distinct phenotypes with respect to their internal red cell sodium (Na') and potassium (K+) concentrations. Some sheep have red cells with high K' and low Na+ and some with low K- and high Na+. This phenomenon has been known for many years and the HK and LK type cells, as they are called, have provided useful model systems for studying the mechanism whereby Na+ and K+ are transported across the membrane (Tostejon & Hoffman, 1960). A most significant discovery was made by Rasmusen & Hall (1 966) when they found that the M blood group system is associated with potassium types. This led to further investigations (Tucker & Ellory, 1970), and the relationship is now clearly established (Table 4). H K cells are always homozygous
Table 3 . Genetic markers in the red cells, ___ Locus symbol Alleles Efaemoglobin
Hb
Carbonic anhqdrase
CA
'X'Protein
'X' Di Ke
Diaphorase Potassium Lysine i Tr Glutathione \ Glutathione GSH Nucleoside phosphorylase NP
208
I
Anim. Blood Grps biochem. Genet. 7 (1976)
GENETIC VARIATION IN SHEEP BLOOD
Table 4. The relationship between potassium types and the M blood group system. Potassium type
Blood group
Homozygous LK Heterozygous LK Homozygous HK
LIL ( M b J M b *) MIL (M"lMb)* M / M ( M a / M a *)
*
New nomenclature.
for the gene controlling blood group factor M. LK cells are heterozygous or homozygous for the allelomorph controlling factor L. Since all LKcells have L antigen present it has been postutated that this antigen is acting as an inhibitor ot active K+ transport (Tucker & Ellory, 1970). In order to interpret these findings it is necessary to know something about the Na-I< pump in red cells. The red cell can be considered to be in a steady state because it maintains its internal ionic concentrations at a constant level. This steady state is achieved by a mechanism which involves a balanced process of passive diffusion or leak and active transport or pump. A pump is required to pump Kf in and Na+ out because the transport of ions has to take place against a concentration gradient. This mechanism requires a supply of energy which is obtained by the hydrolysis of ATP by a ?la+-K+ dependent ATP-ase activity. Now the question arises : how do HK and LK cells achieve their different internal Na+ and K+ concentrations? Table 5 shows that these two cell types differ in a number of properties (for references see review by Lauf, 1975a). Perhaps the two most important points to note are (1) that LK cells have antigen L and (2) that the pump of LK cells is more sensitive than that of HK cells to inhibition by intracellular K+ (Hoffman & Tosteson, 1971; Ellory et a1 1972). In other words, LK cells are LK because they have a special inner Na site which has a low affinity for Na+ and which is inhibited by K + (Glynn & Ellory, 1972). It must be remembered that all the major differences listed in Table 5 are apparently the result of a single gene mutation at one locus, and the most likely 'candidate' to bring about all these conse-
Table 5 . Comparison of high and low potassium type sheep red cells.
Potassium concentration Sodium concentration Na+-K+ activated ATP-ase activity Active transport rate Sensitivity of pump to intracellular Kr Pump sites Potassium permeability M blood group factors
HK
LK
High Low High High Low More Low M
Low High Low Low High Less High ML or LL
Anim. Blood Grps biochem. Genet. 7 (1976)
209
ELIZABETH M. TUCKER
inside
outs&
ant!.
I
I
I
I
L
b
@
Fis. 1. Diagram to show the possible location of L antigen - in the sheeu rsd cell membrane and the effect of sensitization with anti-L (J. C. Ellory, unpublished). a) section of an Linsensitized red cell membrane; L antigen is designated by the black area; b) section of same membrane after sensitization with anti-L.
yuential effccts is the L antigen. Where then is L situated and how could it be acting 10 have this striking effect? An important step forward was made when it was shown [hat both the active uptake of K+ and the activity of Na-K activated ATP-ase can be dramatically stimulated (5-8 fold) in LK cells when they are sensitized with the blood group reagent anti-L (Ellory & Tucker, 1969; Lauf et al., 1970). It would seein from rhis re;ult that the effect of anti-L is to remove or reduce the inhibitory action of the L antigea. Fig. 1 shows the possible location of L in the membrane (Eilory, unpublished) Prejumably it is accessible from the outside of the membrane because anti-L binds to intact red cell;. It could also be an integral part of a carrier protein linking outside t 3 inside and ii wuld also be acting at the in'ierior surface of the membrane. Kinetic studies have shown that anti-L doe; indeed have an effect here, at the inner surface of the membraneand current belief is that L has its effeci by altering the afinity for Na+ and K+ at this inner Na site. Thus the affinity for Na+/K+,which is 1/6 in L K cells, is I :2 after treitment with the a n t i b d y (Caviere; & Ellory, 1975). Anti-L stimulates the pump because, by a mechanism as yet not fully understood, it reduces the sensitivity of the internal Na sites to inhibition by intracellular K+ (Lauf et al. 1970; Ellory et al. 1972a). 210
Anirn. Blood Grps biochem. Genet. 7 (1976)
GENETIC VARIATION IN SHEEP BLOOD
Since L is intimately involved with the pump it was of interest to know if the number of L antigen sites correlate with the number of pump sites on the membrane. Estimate; of the latter have been made by using radioxtively-labelled ouabain which binds spezifically to the KT sites on the exterior surface of the membrane. Two approaches have been made to determine the number of L antigen site;. By estimating the number of molecule; of 12'I-labeIled antibody bound to homozygous LK ceils, Lauf (1975b) estimated that there were l o 3 L antigen sites. Our approach was different: we employed the technique of visualizing L antigen site; by electron microscopy, using haemocyanin coupled to antiglobulin as a marker according to the method of Ostrand-Rosenberg (1975). Our estimate (-300) was lower (Tucker et al., 1976) but was still considerably higher than the figure of 50-70 pump site; by ouabain-binding published by Joiner & Lauf (1975). A complication in studies such as this is that all batches of anti-L d o not behave in a similar way, and an anti-L which has a seroiogically high titre may be less efficient at stimulating the pump than an appsrently serologically much weaker antiserum. Both specificity and affinity of antibody probably play a part here and until more experiments have been done any estimate; of antigen sites must be considered tentative. Current hypotheses in fact suggest that there are probably two specificities of antigen L (Ellory &Tucker, 1970; Lauf et al., 1971; Tucker et al., 1976). This is where the inve;tigation rests at the moment. There are still many experiments to be done and not the lesst will be to isolate and analyse the L antigen itself.
Amino acid transport My second example is concerned with glutathione deficiency and amino acid transport. Approximately 25 7; of Finnish Landrace sheep have red cells which are deficient in reduced glutathione (GSH), that is they have less than 1.6 mmol compared with the normal 2-4 mmol per Iitre red cells (Tucker & Kilgour, 1970). A concomitant feature of such red cells is that they have high concentrations (up to 20 mmol/litre) of the basic amino acids ornithine and lysine and correspondingly lower than normal total Na+
+
+
Fig. 2. Red cell lysates stained with ninhydrin to show lysine and ornithine (2) in GSH-deficient sheep. Note the absence of these amino acids from normal sheep red cells. Band 1 is haemoglobin. Electrophoretic separation at pH 6.5 on Millipore Phoroslide strips, a and c: normal red cells; b: GSHdeficient red cells.
Anim. BIood Grps biochem. Genet. 7 (1976)
21 1
ELIZABETH M. TUCKER
v)
u
a
n
4
a
E
a!
C
m
I
212
Anim. Blood Grps biochem. Genet. 7 (1976)
GENETIC VARIATION IN SHEEP BLOOD
K+ concentrations (i.e. 80-90 mmol instead of 110 mmol per litre). The ornithne and lysine concentrations are so high that these cells can be identified by a simple electrophoretic test with ninhydrin' to stain for these amino acids in red cell lysates (Fig. 2; Ellory et al., 1972b). GSH is a tripeptide of glycine, cysteine and glutamic acid and it is synthesized within the red cells from these 3 constituent amino acids. Its chief function is apparently to protect the cell against oxidative damage. Fig. 3 shows the 3 major stages which are involved in GSH synthesis: 1) the supply of amino acids; 2) the first step of synthesis - the production of y-glutamylcysteine by the action of y-glutamyl cysteine synthetase (GC-S) ; 3) the second step of synthesis -the production of GSH by the enzyme glutathione synthetase (GSH-S). The mutation causing the GSH deficiency could therefore be exerting its effect at any of these 3 stages. Biochemical analyses have shown that stages 2 and 3 are apparently normal in the GSH-deficient cells of the Finnish Landrace breed (Young & Nimmo, 1975) so that a likely point of lesion is at stage one, i.e. the supply of amino acids. Tests using 1aC-labelled amino acids have indeed shown this to be the case, and it has been demonstrated that the GSH-deficient cells take up cysteine about 10 times less rapidly than normal cells (Young et al., 1975). It would seem, therefore, that a diminished availability of cysteine is responsible for the low concentration of red cell GSH and that the lesion represents a specific defect in the red cell membrane transport system for this amino acid. Like the HK and LK types these high and low GSH types therefore provide a useful model system for studying transport across the membrane, and by comparing the relative uptake rates of different amino acids it has been possible to obtain basic information about amino acid transport in sheep red cells. It seems that normal red cells have a transport system which is rather specific and shows a relatively large capacity to transport amino acids such as L-alanine, cysteine, ornithine and lysine. A second quantitatively less important component possibly equivalent to passive diffusion accounts for the movement of other amino acids such as leucine, arginine and glutamine (Young et al., 1976). In the GSH-deficient sheep it apparently is the first system which is affected, the second system being identical to that of normal sheep. We still do not know why the cells accumulate lysing and ornithine. The simplest explanation is that these amino acids are breakdown products of reticulocyte maturation which are normally transported out of the cell. In the GSH-deficient cell they presumably accumulate because the membrane is I elatively impermeable t o them. A corollary to this study is that recently we have found that certain sheep have a genetically determined red cell arginase deficiency. Arginase is an important enzyme of the urea cycle and it acts on arginine to produce ornithine. The question therefore arises as to whether or not those GSH-deficient sheep which also are arginase deficient accumuiate arginine instead of ornithine. Experiments are in progress to test this, and Anim. Blood Grps biochem. Genet. 7 (1976)
21 3
ELIZABETH M. TUCKER
it seems likely thst this will provide an example of how a genetic defect in one system
can affect the phenoiypic expression of another system. There is no doubt that the m3re we loDk into the physiology and bio2hemi;try of red cells the more interejting findings emerge and I am sure thzt genetic markers will continue to be invaluable tools for elucidating some of these diverse mechanisms in the sheep.
.References Agar, N.S., J. V. Evans & J. Roberts, 1972. Red bloodcell potassium and haemoglobin polymorphism in sheep. Anim Breed. Abstr. 40: 407-436. Cavieres, J. D. & J. C. Ellory, 1975. The change induced by anti-L in the sodium and potassium affinities in the sodium pump in LK erythrocytes. J. Physisl., Lond. 245: 93P. E,llory, 3. C., & E. M. Tucker, 1969. Stimulation of the potassium transport system in low potassium type sheep red cells by a specific antigen antibody reaction. Nature, Lond. 222: 477-478. Ellory, J. C . & E. M. Tucker, 1970. Active potassium transport and the L and M antigens of sheep and goat red cells. Bioclzinz. biophys. Acta 219: 160-168. Ellory, J. C., J. R. Sachs, P. B. Dunham & J. F. Hoffman, 1972a. The Lantibodyand potassium fluxes in LK red cells of sheep and goats. In: F. Kreuzer & J. F. G. Slegers (Ed.), Biomembranes. 3. Passive permeability of cell membranes, Plenum, New York, pp. 237-245. E:llory, J. C., E. M. Tucker & E. V. Deverson, 1972b. The identification of ornithine and lysine at high concentrations in the red cells of sheep with an inherited deficiency of glutathione. BiochDn. biophys. Act0 279: 481-483. Glynn, I. M. & J. C. Ellory, 1972. Stimulation of a sodium pump by an antibody that increases the apparent affinity for sodium ions of the sodium-loading sites. In: Role of membranes in secretory processes, pp. 224-237. North-Holland, Amsterdam. Hall, J. G., 1975. Blood groups in sheep. Vet. Rec. 96: 400-401. Hoffman, P. G. & D. C. Tosteson, 1971. Active sodium and potassium transport in high potassium and low potassium sheep red cells. J . gen. Pliysiol. 58 : 438466. Joiner, C. H. & P. K. Lauf, 1975. The effect of anti-L on ouabain binding to sheep erythrocytes. J. Membrane Biof. 21 : 99-1 12. Lauf, P. K., 1975a. Antigen-antibody reactions and cation transport in biomembranes: irnmunophysiological aspects. Biochim. biophys. Acta 415: 173-229. Lauf, P. K., 1975b. Abstract presented at Society of General Physiologists Meeting (Sept. 1975). Lauf, P. K., M. L. Parmelee, J. J. Snyder & D. C. Tosteson, 1971. Enzymatic modification of the L and M antigens in LK and H K sheep erythrocytes and their membranes: the action of neuraminidase and trypsin. J. Membrane Biol. 4: 52-67. Lauf, P. K., B. A. Rasmusen, P. G. Hoffman, P. B. Dunham, P. Cook, M. L. Parmelee & D. C. Tosteson 1970. Stimulation of active potassium transport in LK sheep red cells by blood group - L antiserum. J . Membrane Biol. 3: 1-13. McDermid, E. M., N. S. Agar & C. K. Chai, 1975. Electrophoretic variation of red cell enzyme systems in farm animals (Review). Aninz. Blood Grps biochem. Genet. 6 : 127-174. Ostrand-Rosenberg, S., 1975. Gene dosage and antigenic expression on the cell surface of bovine red cells. Aizim. Blood Grps biochem. Genet. 6 : 81-99. Rasmusen, B. A. & J. G. Hall, 1966. Association between potassium concentration and serological type of sheep red blood cells. Science, N . Y. 151: 1551-1552. Tosteson, D. C. & J. F. Hoffman, 1960. Regulation of cell volume by active cation transport in high and low potassium sheep red cells. J. gem Physiol. 44: 169-194. Tucker, E. M., 1971. Genetic variation in the sheep red blood cell. Biol. Rev. 46: 341-386. Tucker, E. M., 1975. Genetic markers in the plasma and red blood cells. In: M. H. Blunt (Ed.), The blood of sheep. Springer-Verlag, Berlin. Tucker, E. M. & J. C. Ellory, 1970. The M-L blood group system and its influence on red cell potassium levels in sheep. Anim. Blood Grps biochem. Genet. 1: 101-112. Tucker, E. M. & L. Kilgour, 1970. An inherited glutathione deficiency and a ccncomitant reduction in potassium concentration in sheep red cells. Experientia 26: 203-204.
214
Anim. Blood Grps biochem. Genet. 7 (1976)
GENETIC VARIATION IN SHEEP BLOOD
Tucker, E. M., J. C. Ellory, F. B. P. Wooding, G. h4organ & J. Herbert, 1976. The number and specificity ofL antigen sites on low potassium type sheep redcells. Proc. R. Soc. B (in press). Young, J. D. &I. A. Nimrno, 1975. GSH biosynthesis in glutarhione deficient erythrocytes from Finnish Landrace and Tasmanian Merino sheep. Biochim. biophys. Actn 404: 132-141. Young, J. D., J. C . Ellory, & E. M. Tucker, 1975. An amino acid transport defect in glutathionedeficient sheep erythrocytes. Nntiire, Lond, 254: 156-157. Young, J. D., J. C. Ellory & E. M. Tucker, 1976. Amino acid transport in normal and glutathionedeficient sheep erythrocytes. Biochenr. J . 154: 43-48.
Anim. Blood Grps biochem. Genet. 7 (1976)
Some physiological aspects of genetic variation in the blood of sheep' Elizabeth M. Tucker ARC Institute of Animal Physiology, Babraham, Cambridge CB2 4AT, EngIand Received: 16 September 1976
Summary The principal genetic variants in sheep red cells and plasma are listed. Current hypotheses as to how the L blood group antigen affects active potassium transpait across the red cell membrane are summarized. Recent work on an inherited defect in amino acid transport which results in a red cell GSH deficiency is also described.
Introduction Tables 1 , 2 and 3 summarize the principal genetic systems which have been described for sheep plasma and red blood cells. For further details see review papers by Tucker, 1971; Agar et a]., 1972; Hall, 1975; Tucker, 1975; McDermid el al., 1975. The sheep can claim something of a unique position in that it provides several impressive example; of how the discovery of an apparently simple genetic variation can lead to or stimulate research in a wide area of inte-e;t. In this paper I shall give two Table 1. Genetic markers in the plasmn. Locus symbol Alleles Albumin Pre-albumin
A1 Pr
X Transferrin Esterase j?-Lipoprotein upMacroglobulin Immunoglobulin
Tf
Es Lp Ap
Irn (1) Zin (2)
D,F, S, T , V, H7 F , S, 0 E M, S I , A , G, H , B, K, C, M , D,Q,E, P 0,b, c 1, 1, 2 Pa,Y l b
-
p=
1 Review paper presented at the 15th International Conference on Animal Blood Groups agd Biochemical Polymorphism (Dublin, Ireland, 1976).
207
ELIZABETH hl. TUCKER
Table 2. Blood group antigens.
Locus symbol
Alleles
R I .A C M
R, r c I, i O, 6, n, ab, b, a, 6, nc, c ( M , L , MM', M ' )
D
fl,
B .Y- Z He1 C O I1
-
n, nb, nbc,
x,x=
-,err
Hel, he1 Cot+, con"
examples of how genetic variants in sheep red cells have proved to be useful tools for investigating basic physiological processes. Both examples are concerned with transport processes across the redcell membrane. The first deals with active transport ofpotassium ( K e , M locus) and the second with transport of amino acids (7" locus).
A4ctivepotassium transport Sheep show 2 distinct phenotypes with respect to their internal red cell sodium (Na') and potassium (K+) concentrations. Some sheep have red cells with high K' and low Na+ and some with low K- and high Na+. This phenomenon has been known for many years and the HK and LK type cells, as they are called, have provided useful model systems for studying the mechanism whereby Na+ and K+ are transported across the membrane (Tostejon & Hoffman, 1960). A most significant discovery was made by Rasmusen & Hall (1 966) when they found that the M blood group system is associated with potassium types. This led to further investigations (Tucker & Ellory, 1970), and the relationship is now clearly established (Table 4). H K cells are always homozygous
Table 3 . Genetic markers in the red cells, ___ Locus symbol Alleles Efaemoglobin
Hb
Carbonic anhqdrase
CA
'X'Protein
'X' Di Ke
Diaphorase Potassium Lysine i Tr Glutathione \ Glutathione GSH Nucleoside phosphorylase NP
208
I
Anim. Blood Grps biochem. Genet. 7 (1976)
GENETIC VARIATION IN SHEEP BLOOD
Table 4. The relationship between potassium types and the M blood group system. Potassium type
Blood group
Homozygous LK Heterozygous LK Homozygous HK
LIL ( M b J M b *) MIL (M"lMb)* M / M ( M a / M a *)
*
New nomenclature.
for the gene controlling blood group factor M. LK cells are heterozygous or homozygous for the allelomorph controlling factor L. Since all LKcells have L antigen present it has been postutated that this antigen is acting as an inhibitor ot active K+ transport (Tucker & Ellory, 1970). In order to interpret these findings it is necessary to know something about the Na-I< pump in red cells. The red cell can be considered to be in a steady state because it maintains its internal ionic concentrations at a constant level. This steady state is achieved by a mechanism which involves a balanced process of passive diffusion or leak and active transport or pump. A pump is required to pump Kf in and Na+ out because the transport of ions has to take place against a concentration gradient. This mechanism requires a supply of energy which is obtained by the hydrolysis of ATP by a ?la+-K+ dependent ATP-ase activity. Now the question arises : how do HK and LK cells achieve their different internal Na+ and K+ concentrations? Table 5 shows that these two cell types differ in a number of properties (for references see review by Lauf, 1975a). Perhaps the two most important points to note are (1) that LK cells have antigen L and (2) that the pump of LK cells is more sensitive than that of HK cells to inhibition by intracellular K+ (Hoffman & Tosteson, 1971; Ellory et a1 1972). In other words, LK cells are LK because they have a special inner Na site which has a low affinity for Na+ and which is inhibited by K + (Glynn & Ellory, 1972). It must be remembered that all the major differences listed in Table 5 are apparently the result of a single gene mutation at one locus, and the most likely 'candidate' to bring about all these conse-
Table 5 . Comparison of high and low potassium type sheep red cells.
Potassium concentration Sodium concentration Na+-K+ activated ATP-ase activity Active transport rate Sensitivity of pump to intracellular Kr Pump sites Potassium permeability M blood group factors
HK
LK
High Low High High Low More Low M
Low High Low Low High Less High ML or LL
Anim. Blood Grps biochem. Genet. 7 (1976)
209
ELIZABETH M. TUCKER
inside
outs&
ant!.
I
I
I
I
L
b
@
Fis. 1. Diagram to show the possible location of L antigen - in the sheeu rsd cell membrane and the effect of sensitization with anti-L (J. C. Ellory, unpublished). a) section of an Linsensitized red cell membrane; L antigen is designated by the black area; b) section of same membrane after sensitization with anti-L.
yuential effccts is the L antigen. Where then is L situated and how could it be acting 10 have this striking effect? An important step forward was made when it was shown [hat both the active uptake of K+ and the activity of Na-K activated ATP-ase can be dramatically stimulated (5-8 fold) in LK cells when they are sensitized with the blood group reagent anti-L (Ellory & Tucker, 1969; Lauf et al., 1970). It would seein from rhis re;ult that the effect of anti-L is to remove or reduce the inhibitory action of the L antigea. Fig. 1 shows the possible location of L in the membrane (Eilory, unpublished) Prejumably it is accessible from the outside of the membrane because anti-L binds to intact red cell;. It could also be an integral part of a carrier protein linking outside t 3 inside and ii wuld also be acting at the in'ierior surface of the membrane. Kinetic studies have shown that anti-L doe; indeed have an effect here, at the inner surface of the membraneand current belief is that L has its effeci by altering the afinity for Na+ and K+ at this inner Na site. Thus the affinity for Na+/K+,which is 1/6 in L K cells, is I :2 after treitment with the a n t i b d y (Caviere; & Ellory, 1975). Anti-L stimulates the pump because, by a mechanism as yet not fully understood, it reduces the sensitivity of the internal Na sites to inhibition by intracellular K+ (Lauf et al. 1970; Ellory et al. 1972a). 210
Anirn. Blood Grps biochem. Genet. 7 (1976)
GENETIC VARIATION IN SHEEP BLOOD
Since L is intimately involved with the pump it was of interest to know if the number of L antigen sites correlate with the number of pump sites on the membrane. Estimate; of the latter have been made by using radioxtively-labelled ouabain which binds spezifically to the KT sites on the exterior surface of the membrane. Two approaches have been made to determine the number of L antigen site;. By estimating the number of molecule; of 12'I-labeIled antibody bound to homozygous LK ceils, Lauf (1975b) estimated that there were l o 3 L antigen sites. Our approach was different: we employed the technique of visualizing L antigen site; by electron microscopy, using haemocyanin coupled to antiglobulin as a marker according to the method of Ostrand-Rosenberg (1975). Our estimate (-300) was lower (Tucker et al., 1976) but was still considerably higher than the figure of 50-70 pump site; by ouabain-binding published by Joiner & Lauf (1975). A complication in studies such as this is that all batches of anti-L d o not behave in a similar way, and an anti-L which has a seroiogically high titre may be less efficient at stimulating the pump than an appsrently serologically much weaker antiserum. Both specificity and affinity of antibody probably play a part here and until more experiments have been done any estimate; of antigen sites must be considered tentative. Current hypotheses in fact suggest that there are probably two specificities of antigen L (Ellory &Tucker, 1970; Lauf et al., 1971; Tucker et al., 1976). This is where the inve;tigation rests at the moment. There are still many experiments to be done and not the lesst will be to isolate and analyse the L antigen itself.
Amino acid transport My second example is concerned with glutathione deficiency and amino acid transport. Approximately 25 7; of Finnish Landrace sheep have red cells which are deficient in reduced glutathione (GSH), that is they have less than 1.6 mmol compared with the normal 2-4 mmol per Iitre red cells (Tucker & Kilgour, 1970). A concomitant feature of such red cells is that they have high concentrations (up to 20 mmol/litre) of the basic amino acids ornithine and lysine and correspondingly lower than normal total Na+
+
+
Fig. 2. Red cell lysates stained with ninhydrin to show lysine and ornithine (2) in GSH-deficient sheep. Note the absence of these amino acids from normal sheep red cells. Band 1 is haemoglobin. Electrophoretic separation at pH 6.5 on Millipore Phoroslide strips, a and c: normal red cells; b: GSHdeficient red cells.
Anim. BIood Grps biochem. Genet. 7 (1976)
21 1
ELIZABETH M. TUCKER
v)
u
a
n
4
a
E
a!
C
m
I
212
Anim. Blood Grps biochem. Genet. 7 (1976)
GENETIC VARIATION IN SHEEP BLOOD
K+ concentrations (i.e. 80-90 mmol instead of 110 mmol per litre). The ornithne and lysine concentrations are so high that these cells can be identified by a simple electrophoretic test with ninhydrin' to stain for these amino acids in red cell lysates (Fig. 2; Ellory et al., 1972b). GSH is a tripeptide of glycine, cysteine and glutamic acid and it is synthesized within the red cells from these 3 constituent amino acids. Its chief function is apparently to protect the cell against oxidative damage. Fig. 3 shows the 3 major stages which are involved in GSH synthesis: 1) the supply of amino acids; 2) the first step of synthesis - the production of y-glutamylcysteine by the action of y-glutamyl cysteine synthetase (GC-S) ; 3) the second step of synthesis -the production of GSH by the enzyme glutathione synthetase (GSH-S). The mutation causing the GSH deficiency could therefore be exerting its effect at any of these 3 stages. Biochemical analyses have shown that stages 2 and 3 are apparently normal in the GSH-deficient cells of the Finnish Landrace breed (Young & Nimmo, 1975) so that a likely point of lesion is at stage one, i.e. the supply of amino acids. Tests using 1aC-labelled amino acids have indeed shown this to be the case, and it has been demonstrated that the GSH-deficient cells take up cysteine about 10 times less rapidly than normal cells (Young et al., 1975). It would seem, therefore, that a diminished availability of cysteine is responsible for the low concentration of red cell GSH and that the lesion represents a specific defect in the red cell membrane transport system for this amino acid. Like the HK and LK types these high and low GSH types therefore provide a useful model system for studying transport across the membrane, and by comparing the relative uptake rates of different amino acids it has been possible to obtain basic information about amino acid transport in sheep red cells. It seems that normal red cells have a transport system which is rather specific and shows a relatively large capacity to transport amino acids such as L-alanine, cysteine, ornithine and lysine. A second quantitatively less important component possibly equivalent to passive diffusion accounts for the movement of other amino acids such as leucine, arginine and glutamine (Young et al., 1976). In the GSH-deficient sheep it apparently is the first system which is affected, the second system being identical to that of normal sheep. We still do not know why the cells accumulate lysing and ornithine. The simplest explanation is that these amino acids are breakdown products of reticulocyte maturation which are normally transported out of the cell. In the GSH-deficient cell they presumably accumulate because the membrane is I elatively impermeable t o them. A corollary to this study is that recently we have found that certain sheep have a genetically determined red cell arginase deficiency. Arginase is an important enzyme of the urea cycle and it acts on arginine to produce ornithine. The question therefore arises as to whether or not those GSH-deficient sheep which also are arginase deficient accumuiate arginine instead of ornithine. Experiments are in progress to test this, and Anim. Blood Grps biochem. Genet. 7 (1976)
21 3
ELIZABETH M. TUCKER
it seems likely thst this will provide an example of how a genetic defect in one system
can affect the phenoiypic expression of another system. There is no doubt that the m3re we loDk into the physiology and bio2hemi;try of red cells the more interejting findings emerge and I am sure thzt genetic markers will continue to be invaluable tools for elucidating some of these diverse mechanisms in the sheep.
.References Agar, N.S., J. V. Evans & J. Roberts, 1972. Red bloodcell potassium and haemoglobin polymorphism in sheep. Anim Breed. Abstr. 40: 407-436. Cavieres, J. D. & J. C. Ellory, 1975. The change induced by anti-L in the sodium and potassium affinities in the sodium pump in LK erythrocytes. J. Physisl., Lond. 245: 93P. E,llory, 3. C., & E. M. Tucker, 1969. Stimulation of the potassium transport system in low potassium type sheep red cells by a specific antigen antibody reaction. Nature, Lond. 222: 477-478. Ellory, J. C . & E. M. Tucker, 1970. Active potassium transport and the L and M antigens of sheep and goat red cells. Bioclzinz. biophys. Acta 219: 160-168. Ellory, J. C., J. R. Sachs, P. B. Dunham & J. F. Hoffman, 1972a. The Lantibodyand potassium fluxes in LK red cells of sheep and goats. In: F. Kreuzer & J. F. G. Slegers (Ed.), Biomembranes. 3. Passive permeability of cell membranes, Plenum, New York, pp. 237-245. E:llory, J. C., E. M. Tucker & E. V. Deverson, 1972b. The identification of ornithine and lysine at high concentrations in the red cells of sheep with an inherited deficiency of glutathione. BiochDn. biophys. Act0 279: 481-483. Glynn, I. M. & J. C. Ellory, 1972. Stimulation of a sodium pump by an antibody that increases the apparent affinity for sodium ions of the sodium-loading sites. In: Role of membranes in secretory processes, pp. 224-237. North-Holland, Amsterdam. Hall, J. G., 1975. Blood groups in sheep. Vet. Rec. 96: 400-401. Hoffman, P. G. & D. C. Tosteson, 1971. Active sodium and potassium transport in high potassium and low potassium sheep red cells. J . gen. Pliysiol. 58 : 438466. Joiner, C. H. & P. K. Lauf, 1975. The effect of anti-L on ouabain binding to sheep erythrocytes. J. Membrane Biof. 21 : 99-1 12. Lauf, P. K., 1975a. Antigen-antibody reactions and cation transport in biomembranes: irnmunophysiological aspects. Biochim. biophys. Acta 415: 173-229. Lauf, P. K., 1975b. Abstract presented at Society of General Physiologists Meeting (Sept. 1975). Lauf, P. K., M. L. Parmelee, J. J. Snyder & D. C. Tosteson, 1971. Enzymatic modification of the L and M antigens in LK and H K sheep erythrocytes and their membranes: the action of neuraminidase and trypsin. J. Membrane Biol. 4: 52-67. Lauf, P. K., B. A. Rasmusen, P. G. Hoffman, P. B. Dunham, P. Cook, M. L. Parmelee & D. C. Tosteson 1970. Stimulation of active potassium transport in LK sheep red cells by blood group - L antiserum. J . Membrane Biol. 3: 1-13. McDermid, E. M., N. S. Agar & C. K. Chai, 1975. Electrophoretic variation of red cell enzyme systems in farm animals (Review). Aninz. Blood Grps biochem. Genet. 6 : 127-174. Ostrand-Rosenberg, S., 1975. Gene dosage and antigenic expression on the cell surface of bovine red cells. Aizim. Blood Grps biochem. Genet. 6 : 81-99. Rasmusen, B. A. & J. G. Hall, 1966. Association between potassium concentration and serological type of sheep red blood cells. Science, N . Y. 151: 1551-1552. Tosteson, D. C. & J. F. Hoffman, 1960. Regulation of cell volume by active cation transport in high and low potassium sheep red cells. J. gem Physiol. 44: 169-194. Tucker, E. M., 1971. Genetic variation in the sheep red blood cell. Biol. Rev. 46: 341-386. Tucker, E. M., 1975. Genetic markers in the plasma and red blood cells. In: M. H. Blunt (Ed.), The blood of sheep. Springer-Verlag, Berlin. Tucker, E. M. & J. C. Ellory, 1970. The M-L blood group system and its influence on red cell potassium levels in sheep. Anim. Blood Grps biochem. Genet. 1: 101-112. Tucker, E. M. & L. Kilgour, 1970. An inherited glutathione deficiency and a ccncomitant reduction in potassium concentration in sheep red cells. Experientia 26: 203-204.
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Tucker, E. M., J. C. Ellory, F. B. P. Wooding, G. h4organ & J. Herbert, 1976. The number and specificity ofL antigen sites on low potassium type sheep redcells. Proc. R. Soc. B (in press). Young, J. D. &I. A. Nimrno, 1975. GSH biosynthesis in glutarhione deficient erythrocytes from Finnish Landrace and Tasmanian Merino sheep. Biochim. biophys. Actn 404: 132-141. Young, J. D., J. C . Ellory, & E. M. Tucker, 1975. An amino acid transport defect in glutathionedeficient sheep erythrocytes. Nntiire, Lond, 254: 156-157. Young, J. D., J. C. Ellory & E. M. Tucker, 1976. Amino acid transport in normal and glutathionedeficient sheep erythrocytes. Biochenr. J . 154: 43-48.
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