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The identification of ornithine and lysine at high concentrations in the red cells of sheep with an inherited deficiency of glutathione
481
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 26954
T H E IDENTIFICATION OF O R N I T H I N E AND LYSINE AT H I G H CONCENTRATIONS IN T H E RED CELLS OF S H E E P W I T H AN I N H E R I T E D DEFICIENCY OF GLUTATHIONE
J. c. ELLORY, ELIZABETH M. T U C K E R A~B E. V. DEVERSON
A .R.C. Institute of Animal Physiology, Babraham, Cambridge CB2 4 A T (Great Britain) (Received June Igth, 1972)
SUMMARY
Red cells from Finnish Landrace sheep with an inherited deficiency of reduced glutathione were found to contain high concentrations of ornithine (5.3 • 0.8 mmoles/1 packed cells) and lysine (7.8 ± I.O mmoles/1 packed cells), in contrast to red cells from normal individuals where the concentrations were 0.20 ± 0.08, and 0.28 ~ o.II mmole/1 packed cells, respectively. Red cells from Merino sheep with low glutathione levels showed no significant concentrations of these amino acids.
Analysis of red cells from both high potassium (HK) and low potassium (LK) type Finnish Landrace ("Finn") sheep with an inherited deficiency of reduced glutathione (GSH-Iow type) showed significantly lower intracellular concentrations of Na ÷ and K + than cells from normal (GSH-high type) sheep amounting to a total of some 20 mmoles/1 packed cells1, *. Since the mean corpuscular haemoglobin concentration, dry weight and intracellular C1- concentration of the GSH-low cells were not significantly different from those of normal cells, it was postulated that another cation or cations must be present in these GSH-low type red cells. In contrast, we have recently observed that in the Merino breed, sheep with GSH-deficient red cells have normal Na ÷ and K ÷ concentrations. The present report is directed to identifying the extra components present in GSH-low type red cells from "Finn" sheep and comparing these results with those obtained for Merino sheep. Blood was drawn from sheep into heparinized containers. The cells were packed by centrifugation of whole blood and were either analysed unwashed or were washed three times by centrifugation in IO vol. of an isotonic saline. They were lysed by freezing and thawing, and samples (0.3/~1) were run on Millipore Phoroslide electrophoresis strips in 0.o35 M phosphate buffer, pH 6.5 (IOO V, 6 rain). On treatment of the strips with ninhydrin (0.3% in ether) a strongly staining band running towards the cathode was observed in lysates from both unwashed and washed GSH-low type " F i n n " red cells. In contrast, GSH-high type cells gave only a very weakly positive reaction. A fainter band running towards the anode was also observed only in GSH-low samples. Biochim. Biophys. Acta, 279 (1973) 481-483
482
J . c . ELLORY et al.
In both high and low types other faintly staining bands were present, especially in the unwashed samples. Samples of washed, lysed red cells still gave consistent results after storage for long periods at --20 °C. The summarised results of analyses for 300 "Finn" and 135 Merino sheep are presented in Table I. All except 6 of the 86 GSH-low type "Finn" samples gave the strong ninhydrin-positive reaction, whilst none of the GSH-high type "Finn" samples, or GSH-low or high Merino samples gave this positive reaction. TABLE I NINHYDRIN STAINING REACTIONS OF GSH HIGH AND LO'VVTYPE RED CELLS R e d cell l y s a t e s were classified as n i n h y d r i n p o s i t i v e or n e g a t i v e a f t e r e l e c t r o p h o r e t i c s e p a r a t i o n on P h o r o s l i d e s t r i p s a n d t r e a t m e n t w i t h n i n h y d r i n .
Breed : GSH type: No. of n i n h y d r i n - p o s i t i v e sheep No. of n i n h y d r i n - n e g a t i v e sheep Total
Finnish landrace Low type High type
Merino Low type
High type
80 6 86
o 54 54
o 81 8i
o 214 2 i4
In an attempt to identify these components, washed red cells were lysed in io vol. of lO% (w/v) trichloroacetic acid, and the trichloroacetic acid removed by ether extraction. Samples (50 ffl) were run on high-voltage paper electrophoresis 3-5 at pH 2.1 (6 kV, 15 rain) and p H 6.5 (3 kV, 7 ° rain), and sprayed with 0.5% ninhydrin, 0.5% collidine in acetone. Two major spots migrating towards the cathode were obtained, which co-chromatographed with lysine and ornithine. Finally, aliquots of the trichloroacetic acid supernatant, ether extracted, were diluted 1:5 with the pH 2.2 starting buffer and analysed in a Locarte amino acid analyser, either with a 50 cm × I cm column for physiological fluids, or a 24 c m x i cm column. The results for determinations on both GSH-low and high type "Finn" and Merino red cells are given in Table II. The only detectable amino acid in washed GSH-high or low Merino red cells was glycine. In contrast very high concentrations of ornithine and lysine were found in GSH-low "Finn" sheep red cells. Significant amounts of several other amino acids were also found in the latter and the total contribution was some 20 mmoles amino acid/1 red cells, of which positively charged amino acids contributed 60-700/0 . Normal GSH-high "Finn" red cells contained small concentrations of lysine, ornithine and glycine amounting to some 0. 7 mmole/1 packed cells. Plasma levels of lysine and ornithine in sheep are about 0.08 and 0.05 mmole/1, respectively (Mangan, J. L. and Wright, P. C., personal communication). These high concentrations persisted in GSHlow type red cells after washing the cells 3 times, and experiments with [i4C~lysine (0.5 mM) revealed no significant uptake over 4 h indicating that the permeability of these cells to the amino acids must be low. At the present moment it is difficult to explain how the presence of the amino acids, the lowered Na + and K + concentrations, and the lowered GSH levels in these "Finn" cells are related. The results with the Merino sheep show that a lowered red cell Na + and K + concentration is not a necessary accompaniment to a lowered GSH concentration. It is possible however that the lesion resulting in lowered GSH levels in the Merino is different from that in the "Finn". The correlation between low GSH and high lysine and ornithine concentrations in the "Finn" is very good, but the fact Biochim. Biophys. Acta, 279 (1972) 481-483
AMINO
ACIDS
IN SHEEP
RED
483
CELLS
TABLE II AMINO ACID CONCENTRATIONS IN G S H - H I G H
AND GSH-LOW
T Y P E S H E E P RED CELLS
Amino acid concentrations in mmoles/1 packed cells. Significant amounts of histidine, sarcosine, aspartic acid and an unknown were also found in some GSH-low Finn red cells, at concentrations of about o.i raM. Values are for washed red cells. Breed: GSH type:
No. of samples Lysine Ornithine Threonine Glutamic acid Glutamine Serine Glycine Alanine Tyrosine
Finnish landrace Low type High type
Merino Low+high type
6 7.8 -E I.O 5.3 =E 0.8 1.13 -4- o.14 o.81 ± 0.20 0.38 ± o.14 o.58 -t- o.32 0.45 -t- 0.09 o.90 i o.16 o.51 ± 0.09
8 o o o o o o 0.66 :~ o.o2 o o
6 0.28 -4- o.II 0.20 -1- o.o8 o o o o 0.28 ± 0.o9 o o
t h a t 6 e x c e p t i o n s w e r e f o u n d w o u l d s u g g e s t t h a t t h e a s s o c i a t i o n m a y be d u e to close g e n e t i c linkage. O n t h e o t h e r h a n d , if this w e r e so, a n d c r o s s i n g - o v e r w e r e t a k i n g p l a c e o c c a s i o n a l l y , one w o u l d e x p e c t to find s o m e G S H - h i g h t y p e i n d i v i d u a l s w i t h h i g h lysine a n d o r n i t h i n e levels. S u c h s h e e p h a v e n o t y e t b e e n f o u n d . M o r e o v e r , s t u d i e s in t h i s l a b o r a t o r y h a v e s h o w n t h a t G S H - h i g h t y p e s h e e p m a y h a v e low G S H levels if t h e y are a n a e m i c . T h e e x c e p t i o n s n o t e d , c o u l d t h e r e f o r e be d u e to m i s c l a s s i f i c a t i o n as to G S H t y p e . F u r t h e r g e n e t i c a n d b i o c h e m i c a l s t u d i e s will h a v e to be c a r r i e d o u t b e f o r e t h e e x a c t n a t u r e of t h e a s s o c i a t i o n can be d e t e r m i n e d . ACKNOWLEDGEMENTS W e w o u l d like to t h a n k M r P e t e r W r i g h t for p e r f o r m i n g t h e a m i n o a c i d a n a l y s e s on t h e 50 c m × I c m L o c a r t e a n a l y s e r , a n d M r T. H. M c C l e l l a n d of t h e A n i m a l B r e e d i n g R e s e a r c h O r g a n i s a t i o n , E d i n b u r g h , for k i n d l y p r o v i d i n g b l o o d s a m p l e s f r o m T a s m a n i a n Merinos. REFERENCES i 2 3 4 5
Tucker, E. M. and Kilgour, L. (197 o) Experientia, 26, 2o 3 Tucker, E. M. and Ellory, J. C. (1971) Res. Vet. Sci. 12, 6o0 Michl, H. (1951) Monatsh. Chem. 82, 489 Ambler, R. P. (1963) Biochem. J. 89, 349 Milstein, C. P. and Feinstein, A. (1968) Biochem. J. lO7, 559 Biochim. Biophys. Acta, 279 (1972) 481-483
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 26954
T H E IDENTIFICATION OF O R N I T H I N E AND LYSINE AT H I G H CONCENTRATIONS IN T H E RED CELLS OF S H E E P W I T H AN I N H E R I T E D DEFICIENCY OF GLUTATHIONE
J. c. ELLORY, ELIZABETH M. T U C K E R A~B E. V. DEVERSON
A .R.C. Institute of Animal Physiology, Babraham, Cambridge CB2 4 A T (Great Britain) (Received June Igth, 1972)
SUMMARY
Red cells from Finnish Landrace sheep with an inherited deficiency of reduced glutathione were found to contain high concentrations of ornithine (5.3 • 0.8 mmoles/1 packed cells) and lysine (7.8 ± I.O mmoles/1 packed cells), in contrast to red cells from normal individuals where the concentrations were 0.20 ± 0.08, and 0.28 ~ o.II mmole/1 packed cells, respectively. Red cells from Merino sheep with low glutathione levels showed no significant concentrations of these amino acids.
Analysis of red cells from both high potassium (HK) and low potassium (LK) type Finnish Landrace ("Finn") sheep with an inherited deficiency of reduced glutathione (GSH-Iow type) showed significantly lower intracellular concentrations of Na ÷ and K + than cells from normal (GSH-high type) sheep amounting to a total of some 20 mmoles/1 packed cells1, *. Since the mean corpuscular haemoglobin concentration, dry weight and intracellular C1- concentration of the GSH-low cells were not significantly different from those of normal cells, it was postulated that another cation or cations must be present in these GSH-low type red cells. In contrast, we have recently observed that in the Merino breed, sheep with GSH-deficient red cells have normal Na ÷ and K ÷ concentrations. The present report is directed to identifying the extra components present in GSH-low type red cells from "Finn" sheep and comparing these results with those obtained for Merino sheep. Blood was drawn from sheep into heparinized containers. The cells were packed by centrifugation of whole blood and were either analysed unwashed or were washed three times by centrifugation in IO vol. of an isotonic saline. They were lysed by freezing and thawing, and samples (0.3/~1) were run on Millipore Phoroslide electrophoresis strips in 0.o35 M phosphate buffer, pH 6.5 (IOO V, 6 rain). On treatment of the strips with ninhydrin (0.3% in ether) a strongly staining band running towards the cathode was observed in lysates from both unwashed and washed GSH-low type " F i n n " red cells. In contrast, GSH-high type cells gave only a very weakly positive reaction. A fainter band running towards the anode was also observed only in GSH-low samples. Biochim. Biophys. Acta, 279 (1973) 481-483
482
J . c . ELLORY et al.
In both high and low types other faintly staining bands were present, especially in the unwashed samples. Samples of washed, lysed red cells still gave consistent results after storage for long periods at --20 °C. The summarised results of analyses for 300 "Finn" and 135 Merino sheep are presented in Table I. All except 6 of the 86 GSH-low type "Finn" samples gave the strong ninhydrin-positive reaction, whilst none of the GSH-high type "Finn" samples, or GSH-low or high Merino samples gave this positive reaction. TABLE I NINHYDRIN STAINING REACTIONS OF GSH HIGH AND LO'VVTYPE RED CELLS R e d cell l y s a t e s were classified as n i n h y d r i n p o s i t i v e or n e g a t i v e a f t e r e l e c t r o p h o r e t i c s e p a r a t i o n on P h o r o s l i d e s t r i p s a n d t r e a t m e n t w i t h n i n h y d r i n .
Breed : GSH type: No. of n i n h y d r i n - p o s i t i v e sheep No. of n i n h y d r i n - n e g a t i v e sheep Total
Finnish landrace Low type High type
Merino Low type
High type
80 6 86
o 54 54
o 81 8i
o 214 2 i4
In an attempt to identify these components, washed red cells were lysed in io vol. of lO% (w/v) trichloroacetic acid, and the trichloroacetic acid removed by ether extraction. Samples (50 ffl) were run on high-voltage paper electrophoresis 3-5 at pH 2.1 (6 kV, 15 rain) and p H 6.5 (3 kV, 7 ° rain), and sprayed with 0.5% ninhydrin, 0.5% collidine in acetone. Two major spots migrating towards the cathode were obtained, which co-chromatographed with lysine and ornithine. Finally, aliquots of the trichloroacetic acid supernatant, ether extracted, were diluted 1:5 with the pH 2.2 starting buffer and analysed in a Locarte amino acid analyser, either with a 50 cm × I cm column for physiological fluids, or a 24 c m x i cm column. The results for determinations on both GSH-low and high type "Finn" and Merino red cells are given in Table II. The only detectable amino acid in washed GSH-high or low Merino red cells was glycine. In contrast very high concentrations of ornithine and lysine were found in GSH-low "Finn" sheep red cells. Significant amounts of several other amino acids were also found in the latter and the total contribution was some 20 mmoles amino acid/1 red cells, of which positively charged amino acids contributed 60-700/0 . Normal GSH-high "Finn" red cells contained small concentrations of lysine, ornithine and glycine amounting to some 0. 7 mmole/1 packed cells. Plasma levels of lysine and ornithine in sheep are about 0.08 and 0.05 mmole/1, respectively (Mangan, J. L. and Wright, P. C., personal communication). These high concentrations persisted in GSHlow type red cells after washing the cells 3 times, and experiments with [i4C~lysine (0.5 mM) revealed no significant uptake over 4 h indicating that the permeability of these cells to the amino acids must be low. At the present moment it is difficult to explain how the presence of the amino acids, the lowered Na + and K + concentrations, and the lowered GSH levels in these "Finn" cells are related. The results with the Merino sheep show that a lowered red cell Na + and K + concentration is not a necessary accompaniment to a lowered GSH concentration. It is possible however that the lesion resulting in lowered GSH levels in the Merino is different from that in the "Finn". The correlation between low GSH and high lysine and ornithine concentrations in the "Finn" is very good, but the fact Biochim. Biophys. Acta, 279 (1972) 481-483
AMINO
ACIDS
IN SHEEP
RED
483
CELLS
TABLE II AMINO ACID CONCENTRATIONS IN G S H - H I G H
AND GSH-LOW
T Y P E S H E E P RED CELLS
Amino acid concentrations in mmoles/1 packed cells. Significant amounts of histidine, sarcosine, aspartic acid and an unknown were also found in some GSH-low Finn red cells, at concentrations of about o.i raM. Values are for washed red cells. Breed: GSH type:
No. of samples Lysine Ornithine Threonine Glutamic acid Glutamine Serine Glycine Alanine Tyrosine
Finnish landrace Low type High type
Merino Low+high type
6 7.8 -E I.O 5.3 =E 0.8 1.13 -4- o.14 o.81 ± 0.20 0.38 ± o.14 o.58 -t- o.32 0.45 -t- 0.09 o.90 i o.16 o.51 ± 0.09
8 o o o o o o 0.66 :~ o.o2 o o
6 0.28 -4- o.II 0.20 -1- o.o8 o o o o 0.28 ± 0.o9 o o
t h a t 6 e x c e p t i o n s w e r e f o u n d w o u l d s u g g e s t t h a t t h e a s s o c i a t i o n m a y be d u e to close g e n e t i c linkage. O n t h e o t h e r h a n d , if this w e r e so, a n d c r o s s i n g - o v e r w e r e t a k i n g p l a c e o c c a s i o n a l l y , one w o u l d e x p e c t to find s o m e G S H - h i g h t y p e i n d i v i d u a l s w i t h h i g h lysine a n d o r n i t h i n e levels. S u c h s h e e p h a v e n o t y e t b e e n f o u n d . M o r e o v e r , s t u d i e s in t h i s l a b o r a t o r y h a v e s h o w n t h a t G S H - h i g h t y p e s h e e p m a y h a v e low G S H levels if t h e y are a n a e m i c . T h e e x c e p t i o n s n o t e d , c o u l d t h e r e f o r e be d u e to m i s c l a s s i f i c a t i o n as to G S H t y p e . F u r t h e r g e n e t i c a n d b i o c h e m i c a l s t u d i e s will h a v e to be c a r r i e d o u t b e f o r e t h e e x a c t n a t u r e of t h e a s s o c i a t i o n can be d e t e r m i n e d . ACKNOWLEDGEMENTS W e w o u l d like to t h a n k M r P e t e r W r i g h t for p e r f o r m i n g t h e a m i n o a c i d a n a l y s e s on t h e 50 c m × I c m L o c a r t e a n a l y s e r , a n d M r T. H. M c C l e l l a n d of t h e A n i m a l B r e e d i n g R e s e a r c h O r g a n i s a t i o n , E d i n b u r g h , for k i n d l y p r o v i d i n g b l o o d s a m p l e s f r o m T a s m a n i a n Merinos. REFERENCES i 2 3 4 5
Tucker, E. M. and Kilgour, L. (197 o) Experientia, 26, 2o 3 Tucker, E. M. and Ellory, J. C. (1971) Res. Vet. Sci. 12, 6o0 Michl, H. (1951) Monatsh. Chem. 82, 489 Ambler, R. P. (1963) Biochem. J. 89, 349 Milstein, C. P. and Feinstein, A. (1968) Biochem. J. lO7, 559 Biochim. Biophys. Acta, 279 (1972) 481-483