RAPID COMMUNICATION
ZIPGRAM
INOSITOL PENTAPHOSPHATE IN FISH R E D BLOOD CELLS THOMAS A. BORGESE AND RONALD L . NAGEL Herbert H . Lehman College, CUNY, Bronx, New York 10468, Albert Einstein College of Medicine, Bronx, New York 10461, Marine Biological Laboratory, Woods Hole, Massachusetts 02543 ABSTRACT Inositol pentaphosphate (IP5) has long been c h a r a c t e r i s t i c of avian erythrocytes. We now report t h a t t h i s compound i s a l s o present i n the red c e l l s o f two species of elasmobranch f i s h e s , Squalus acanthias (spiny dogfish) and Narcacion nobiliana (torpedo r a y ) . The mean concentration of IP5 i s 0.36 and 0.24 micromoles per ml o f red c e l l s respectively. ATP i s the major organic phosphate i n both f i s h e s . 2,3-diphosphoglycerate i s absent and GTP levels correspond approximately t o t h e l e v e l s of IP5. I t was believed f o r some time t h a t t h e erythrocytes of birds were unique among nucleated and non-nucleated red blood c e l l s in t h a t they contained no 2,3-DPG and considerable amounts o f i n o s i t o l polyphosphate.
Previously be-
lieved t o be i n o s i t o l hexaphosphate (Rapoport and Guest, ' 4 1 ) , i t i s now evident t h a t t h i s compound i s a c t u a l l y the pentaphosphate (IP5) d e r i v a t i v e (Johnson and Tate, '69).
2,3-diphosphoglycerate present in most mammalian
red c e l l s i s a l s o found f o r a limited period d u r i n g embryonic development i n birds (Isaacks e t a l . , ' 7 7 ; Borgese and Lampert, ' 7 5 ) .
The importance of
2,3-DPG a s an a l l o s t e r i c modifier of oxygen binding among human hemoglobins (Benesch and Benesch, '67; Chanutin and Curnish, '67) focused a t t e n t i o n on other organic phosphates as possible regulators.
Inositol hexaphosphate
(IP6) has been subsequently shown t o decrease oxygen a f f i n i t y of avian and human hemoglobins i n v i t r o (Benesch and Benesch, '69; Vandercasserie e t a l . , '73; Isaacks and Harkness, ' 7 5 ) .
We have recently established t h a t IP5, t h e
133
n a t u r a l l y occurring major phosphorylated intermediate i n avian red c e l l s , i s more e f f e c t i v e than ATP o r 2,3-DPG i n causing a r i g h t s h i f t of the oxygen equilibrium of f e t a l and a d u l t type duck hemoglobins (Borgese and Nagel , ' 7 7 ) . IP5 i s present in green sea t u r t l e (Rapoport and Guest, '41)
and changes
i n concentration d u r i n g development have been observed ( B a r t l e t t , ' 7 6 ) .
B a r t l e t t ( ' 7 6 ) has a l s o found small amounts of IP5
in the frog.
In a recent
publication (Isaacks e t a l . , ' 7 7 ) reported appreciable amounts o f IP5 in the red c e l l s of a f r e s h water (Amazon) t e l e o s t Piraracu.
The purpose of t h i s
paper i s t o report, f o r the f i r s t time, t h a t IP5 i s a l s o present i n two species of elasmobranch f i s h e s , Squalus acanthias (spiny dogfish) and Narcacion nobiliana (torpedo r a y ) . MATERIALS AND METHODS Spiny dogfish and torpedo ray were obtained from the supply department of t h e Marine Biological Laboratory i n Woods Hole, Massachusetts.
The animals were stunned by a blow on t h e head and the Blood was removed from the heart with
pericardial cavity exposed (torpedo). a heparinized syringe.
Alternatively, t h e major blood vessels were severed
and blood removed w i t h syringes o r Pasteur pipets and transferred t o a f l a s k . Dogfish were bled from the caudal vein. centrifuged and t h e plasma removed.
The blood was f i l t e r e d through gauze,
The packed c e l l s were washed t h r e e times
with 0.33 M NaCl and quadruplicate microhematocrits obtained on t h e recons t i t u t e d suspension.
Osmotic f r a g i l i t y s t u d i e s indicated 7-5% hemolysis with
NaCl solutions o f 0.2 t o 0.5 M with no apparent change between 0.3 and 0.5 M. Trichloracetic acid e x t r a c t s were prepared and t h e neutral ized e x t r a c t s adsorbed onto anion exchange columns (AG 1 x 8 r e s i n and 1 cm x 18.5 cm) a f t e r conversion from the chloride t o t h e formate form.
The adsorbed phosphorylated intermedi-
a t e s were eluted with a l i n e a r gradient of 2.5 l i t e r s of 0-5 M formate buffer, pH 3.0.
IP5 was eluted, a t the end of t h e r u n , by t h e addition of 200 ml of
134
Range (n=3)
Torpedo
Range (n=6)
Spiny dogfish 1.47
Pi 0.35
ADP
3.01
ATP
0.56
GTP
0.36
IP5
1.76
0.09
1.19
0.30
0.24
(0.24-0.34) (1.69-1.85) (0.05-0.15) (1.06-1.29) (0.11-0.57) (0.19-0.31)
0.27
(0.19-0.74) (1.04-2.94) (0.17-0.57) (2.59-3.59) (0.26-1.22) (0.29-0.45)
0.34
AMP
u moles / ml cells
Mean concentration of some major elasmobranch red cell phosphorylated intermediates
TABLE 1
1.0 N HCl.
The i s o l a t e d intermediates were i d e n t i f i e d on the b a s i s of their
e l u t i o n position and confirmed by a p p r o p r i a t e assay procedures f o r inorganic and t o t a l phosphorus, ADP, ATP and GTP a s described by B a r t l e t t ( ' 5 9 ) .
The
i n o s i t o l phosphate e l u t e d from t h e f i r s t column w i t h 1 . 0 N HC1 was evaporated t o dryness a t 60' C , r e c o n s t i t u t e d w i t h water n e u t r a l i z e d and readsorbed onto a second column of AG 1 x 8 resin. 1 . 5 l i t e r s of a 0-1 N HC1 g r a d i e n t .
Elution was accomplished t h i s time w i t h
The e l u t i o n p o s i t i o n s of duck IP5 and IP6
(Sigma Chemical) were used as references. and i n combination.
The two compounds were run alone
Fish i n o s i t o l phosphate was a l s o r u n alone and i n combina
t i o n w i t h standard IP6.
Samples of t h e phosphate compound e l u t e d w i t h 1.0 N
HC1 gave a p o s i t i v e r e a c t i o n w i t h the Lornitzo method f o r i n o s i t o l ( ' 6 8 ) . RESULTS AND DISCUSSION
Figure 1A shows t h e t y p i c a l e l u t i o n p r o f i l e of
red c e l l e x t r a c t s from the a d u l t white Peking duck.
The formate e l u t i o n posi-
t i o n s of t h e principal phosphate compounds coincide w i t h t h e corresponding compounds i n t h e spiny dogfish ( f i g . 1 B ) and the torpedo ray ( f i g . 1 C ) .
In-
organic phosphorus, AMP, ADP and GTP a r e among the principal intermediates FIGURE LEGENDS 1
Ion-exchange chromatography (anion exchange r e s i n AGlx8, lx18.5 cm) of a d u l t duck ( A ) , spinydogfish ( B ) and torpedo ray ( C ) red blood c e l l e x t r a c t s . 14.8 ml f r a c t i o n s were c o l l e c t e d and a l l samples were analyzed f o r t o t a l phosphate ( 0 - 0 ) and absorbance a t 260 nm (a-0). Abbreviations: Inorganic phosphorous ( P i ) , adenosine monophosphate (AMP) , adenosine diphosphate (ADP) adenosine triphosphate (ATP) , c y t i d i n e triphosphate ( C T P ) , u r i d i n e t r i p h o s p h a t e ( U T P ) , guanosine triphosphate ( G T P ) , i n o s i t o l polyphosphate (INOS-P), i n o s i t o l pentaphosphate ( I P 5 ) . 2 Ion-exchange chromatography of a mixture o f i s o l a t e d duck IP5 p l u s standard IP6 on Bio Rad AG1 x 8 r e s i n , converted from c h l o r i d e t o formate form. I n o s i t o l phosphate compounds from lA, B , and C were evaporated t o dryness a t 60' C , r e c o n s t i t u t e d w i t h d i s t i l l e d water and t i t r a t e d t o pH 7.0 w i t h 0.05 N NaOH. Standard IP6 was added t o t h e s o l u t i o n and the mixture adsorbed onto the resin. Elution was accomplished w i t h a l i n e a r g r a d i e n t of 0- IN HC1. All samples were analyzed f o r t o t a l phosphorus. The mixture of duck IP5 and standard IP6 i s shown resolved i n t o two components ( A ) . The phosphate p r o f i l e s o f mixtures of standard IP6 and i n o s i t o l polyphosphate from th e dogf i s h ( B ) and the torpedo ray ( C ) show i d e n t i c a l e l u t i o n p o s i t i o n s f o r the penta and hexaphosphate d e r i v a t i v e s of i n o s i t o l .
DUCK
II
1 80
IPS(DUCK)
IP6 (STANDARD)
3
LITERS
LITERS TORPEDO RAY
YI
80
3 -
60-
z
Ly
.fP ADP
~0.01
CTP
GTP UTP
IWS-P
n
-
IP5 (TORPEDO RAY)
I a.
ow LITERS
IN HCI
LITERS
IP6 (STANDARD)
seen in these e x t r a c t s .
The nucleotides CTP and UTP were i d e n t i f i e d on the
basis of t h e i r elution positions.
No 2,3-DPG was detected in these e x t r a c t s ,
Following the formate gradient, duck IP5 was rapidly eluted from the column by the addition of 1 . 0 N H C 1 .
Similar phosphate peaks, which tested
positively f o r i n o s i t o l , were eluted from the spiny dogfish and torpedo extracts.
In a l l t h r e e cases the inositol polyphosphate was eluted within
15-45 ml a f t e r HC1 addition. One normal HC1 will normally c l e a r the columns of a l l phosphorylated intermediates.
Since the penta and hexaphosphates would a l s o be eluted as
a n inseparable peak under these conditions, t h e exact nature of the dogfish and torpedo i n o s i t o l polyphosphate was uncertain.
Therefore, duck IP5 f r a c t i o n s
were removed from peak tubes a f t e r addition of HC1 and used as standards. The samples were evaporated t o dryness in a forced d r a f t oven a t 60' C. D i s t i l l e d water was then added and the sample neutralized w i t h 0.05 N NaOH. Inositol hexaphosphate was added and the mixture adsorbed onto a second formate column.
A l i n e a r gradient of 1.5 l i t e r s of 0.1 N HC1 was used t o separate
and e s t a b l i s h the e l u t i o n positions of each compound. in t h e mixture duck IP5 i s eluted between 0.814 IP6 e l u t e s between 1.036 - 1.317 l i t e r s .
-
Figure 2A shows t h a t
1.011 l i t e r s and standard
Preliminary experiments established
t h a t these were the identical elution positions f o r t h e penta and hexaphosphates respectively when chromatographed separately.
Mixtures of i n o s i t o l polyphos-
phate from the dogfish o r torpedo plus standard IP6 show identical rechromatography p r o f i l e s when compared w i t h each other ( f i g s . 2B, 2C) or w i t h duck IP5
t
IP6 ( f i g . 2A).
The q u a n t i t a t i v e data of t h e red c e l l phosphate intermediates i s shown in t a b l e 1 .
The r e s u l t s i n d i c a t e t h a t ATP i s the major organic phosphate in
both species of elasmobranchs.
This a l s o c o n t r a s t s with thesmooth dogfish
138
(Mustelus canis) red cells in which the major organic phosphate is GTP (Borgese et al., '78). ATP levels vary markedly between the spiny dogfish and the torpedo. The former contains approximately 3.0 micromoles ATP per ml cells compared to 1.2 micromoles for the torpedo, the mean red cell levels of IP5 are 0.36 for squalus and about 0.24 micromoles per ml of cells for the torpedo. The presence of IP5 in elasmobranchs suggests that it may play a role in regulating the oxygen affinity of dogfish and torpedo hemoglobins. Although recent evidence indicates that some invertebrate hemoglobins are insensitive to IP6, 2,3-DPG and ATP (Weber et al. , '77), IP5 and IP6 are much more effective than 2,3-DPG and ATP in decreasing the oxygen affinity of vertebrate (human and avian) hemoglobins (Benesch and Benesch, '69; Borgese and Nagel, '77). The fact that IP5, in these experiments, is present at levels under 0.5 micromoles per ml of cells does not preclude an effect on the oxygen equilibria of dogfish and torpedo hemoglobins. Our finding that IP5 is present in elasmobranch red cells also has evolutionary significance since it is now clearthatIP5, once thought to be unique among birds, is more ubiquitous than previously believed. ACKNOWLEDGMENTS Research was supported by grants from the Research Foundation
-
CUNY #11395, 11620, NIH-2P50-GM 19100 and an American Heart
Grant-in-Aid. We gratefully acknowledge the technical assistance of Ms. Barbara Colucci and Mr. Robert Goldschmidt. LITERATURE CITED Bartlett, G. R. 1959 Methods o isolation of glycolytic intermediates by column chromatography with ion-exchange resins. J . Biol. Chem., 234: 459-465. Bartlett, G. R. 1976 Phosphate compounds in red cells of reptiles, amphibians and fish. Comp. B ochem. Physiol . , 55A: 211-214. Benesch, R., and R. E. Benesch 967 The effect of organic phosphates from the human ervthrocvte on the allosteric properties of hemoglobin. Biochem. Biophys." Res. iommun. , 26: 162-166. Benesch, R., and R. E. Benesch 1969 Intracellular organic phosphate as regulators of oxygen release by hemoglobin. Nature, 221: 618-622.
139
Borgese, T. A . , and L . M. Lampert 1975 Duck red c e l l 2,3-diphosphoglycerate: I t s presence i n the embryo and i t s disappearance i n the a d u l t . BBRC. 65: 822-827. Borgese, T. A . , and R . L . Nagel 1977 D i f f e r e n t i a l e f f e c t s of 2,3-DPGY ATP and i n o s i t o l pentaphosphate (IP5) on the oxygen e q u i l i b r i a of duck embryonic f e t a l and a d u l t hemoglobins. Comp. Biochem. Physiol., 56A: 539-543. Borgese, T. A . , R. L . Nagel, E. Roth, D. Murphy and J . Harrington 1978 Guanosine triphosphate (GTP): The major organic phosphate i n the erythrocytes of t h e elasmobranch mustelus c a n i s (smooth d o g f i s h ) . Comp. Biochem. Physiol., i n press. Chanutin, A . , and R. R. Curnish 1967 Effect of organic and inorganic phosphates on t h e oxygen equilibrium of human erythrocytes. Arch. Biochem. Biophys. , 121: 96-102. Isaacks, R. E . , and D. R. Harkness 1975 2,3-diphosphoglycerate i n erythrocytes of chick embryos. Science, 189: 393-394. Isaacks, R. E . , H. D. K i m , G . R. B a r t l e t t and D. R. Harkness 1977 I n o s i t o l pentaphosphate in erythrocytes of a freshwater f i s h , Piraracu (Arapaima G i Gas) Life Sciences, 20: 987-990. Johnson, L . F., and M. E . Tate 1969 S t r u c t u r e of "phytic a c i d s " . Can. J . Chem., 47: 63-73. Lornitzo, F. A. 1968 A method f o r c a l o r i m e t r i c assay of i n o s i t o l and some of i t s phosphate d e r i v a t i v e s . Anal. Biochem., 25: 396-405. Rapoport, S . , and A. M. Guest 1941 D i s t r i b u t i o n of a c i d s o l u b l e phosphorus i n the blood c e l l s of various v e r t e b r a t e s . J . Biol. Chem., 138: 269-282. Vandercasserie,C., C . Paul, A. C . Schnek and J . Leonis 1973 Oxygen a f f i n i t y o f avian hemoglobins. Comp. Biochem. Physiol., 44A; 71 1-71 8. Weber, R. E., B. S u l l i v a n , J . Bonaventura and C. Bonaventura 1977 The hemoglobin systems of the blood worms Glycera dibranchiata and G . Americana: Oxygen binding p r o p e r t i e s of the hemolysates and component hemoglobins. Comp. Biochem. Physiol., 58B: 183-187.
140
ZIPGRAM
INOSITOL PENTAPHOSPHATE IN FISH R E D BLOOD CELLS THOMAS A. BORGESE AND RONALD L . NAGEL Herbert H . Lehman College, CUNY, Bronx, New York 10468, Albert Einstein College of Medicine, Bronx, New York 10461, Marine Biological Laboratory, Woods Hole, Massachusetts 02543 ABSTRACT Inositol pentaphosphate (IP5) has long been c h a r a c t e r i s t i c of avian erythrocytes. We now report t h a t t h i s compound i s a l s o present i n the red c e l l s o f two species of elasmobranch f i s h e s , Squalus acanthias (spiny dogfish) and Narcacion nobiliana (torpedo r a y ) . The mean concentration of IP5 i s 0.36 and 0.24 micromoles per ml o f red c e l l s respectively. ATP i s the major organic phosphate i n both f i s h e s . 2,3-diphosphoglycerate i s absent and GTP levels correspond approximately t o t h e l e v e l s of IP5. I t was believed f o r some time t h a t t h e erythrocytes of birds were unique among nucleated and non-nucleated red blood c e l l s in t h a t they contained no 2,3-DPG and considerable amounts o f i n o s i t o l polyphosphate.
Previously be-
lieved t o be i n o s i t o l hexaphosphate (Rapoport and Guest, ' 4 1 ) , i t i s now evident t h a t t h i s compound i s a c t u a l l y the pentaphosphate (IP5) d e r i v a t i v e (Johnson and Tate, '69).
2,3-diphosphoglycerate present in most mammalian
red c e l l s i s a l s o found f o r a limited period d u r i n g embryonic development i n birds (Isaacks e t a l . , ' 7 7 ; Borgese and Lampert, ' 7 5 ) .
The importance of
2,3-DPG a s an a l l o s t e r i c modifier of oxygen binding among human hemoglobins (Benesch and Benesch, '67; Chanutin and Curnish, '67) focused a t t e n t i o n on other organic phosphates as possible regulators.
Inositol hexaphosphate
(IP6) has been subsequently shown t o decrease oxygen a f f i n i t y of avian and human hemoglobins i n v i t r o (Benesch and Benesch, '69; Vandercasserie e t a l . , '73; Isaacks and Harkness, ' 7 5 ) .
We have recently established t h a t IP5, t h e
133
n a t u r a l l y occurring major phosphorylated intermediate i n avian red c e l l s , i s more e f f e c t i v e than ATP o r 2,3-DPG i n causing a r i g h t s h i f t of the oxygen equilibrium of f e t a l and a d u l t type duck hemoglobins (Borgese and Nagel , ' 7 7 ) . IP5 i s present in green sea t u r t l e (Rapoport and Guest, '41)
and changes
i n concentration d u r i n g development have been observed ( B a r t l e t t , ' 7 6 ) .
B a r t l e t t ( ' 7 6 ) has a l s o found small amounts of IP5
in the frog.
In a recent
publication (Isaacks e t a l . , ' 7 7 ) reported appreciable amounts o f IP5 in the red c e l l s of a f r e s h water (Amazon) t e l e o s t Piraracu.
The purpose of t h i s
paper i s t o report, f o r the f i r s t time, t h a t IP5 i s a l s o present i n two species of elasmobranch f i s h e s , Squalus acanthias (spiny dogfish) and Narcacion nobiliana (torpedo r a y ) . MATERIALS AND METHODS Spiny dogfish and torpedo ray were obtained from the supply department of t h e Marine Biological Laboratory i n Woods Hole, Massachusetts.
The animals were stunned by a blow on t h e head and the Blood was removed from the heart with
pericardial cavity exposed (torpedo). a heparinized syringe.
Alternatively, t h e major blood vessels were severed
and blood removed w i t h syringes o r Pasteur pipets and transferred t o a f l a s k . Dogfish were bled from the caudal vein. centrifuged and t h e plasma removed.
The blood was f i l t e r e d through gauze,
The packed c e l l s were washed t h r e e times
with 0.33 M NaCl and quadruplicate microhematocrits obtained on t h e recons t i t u t e d suspension.
Osmotic f r a g i l i t y s t u d i e s indicated 7-5% hemolysis with
NaCl solutions o f 0.2 t o 0.5 M with no apparent change between 0.3 and 0.5 M. Trichloracetic acid e x t r a c t s were prepared and t h e neutral ized e x t r a c t s adsorbed onto anion exchange columns (AG 1 x 8 r e s i n and 1 cm x 18.5 cm) a f t e r conversion from the chloride t o t h e formate form.
The adsorbed phosphorylated intermedi-
a t e s were eluted with a l i n e a r gradient of 2.5 l i t e r s of 0-5 M formate buffer, pH 3.0.
IP5 was eluted, a t the end of t h e r u n , by t h e addition of 200 ml of
134
Range (n=3)
Torpedo
Range (n=6)
Spiny dogfish 1.47
Pi 0.35
ADP
3.01
ATP
0.56
GTP
0.36
IP5
1.76
0.09
1.19
0.30
0.24
(0.24-0.34) (1.69-1.85) (0.05-0.15) (1.06-1.29) (0.11-0.57) (0.19-0.31)
0.27
(0.19-0.74) (1.04-2.94) (0.17-0.57) (2.59-3.59) (0.26-1.22) (0.29-0.45)
0.34
AMP
u moles / ml cells
Mean concentration of some major elasmobranch red cell phosphorylated intermediates
TABLE 1
1.0 N HCl.
The i s o l a t e d intermediates were i d e n t i f i e d on the b a s i s of their
e l u t i o n position and confirmed by a p p r o p r i a t e assay procedures f o r inorganic and t o t a l phosphorus, ADP, ATP and GTP a s described by B a r t l e t t ( ' 5 9 ) .
The
i n o s i t o l phosphate e l u t e d from t h e f i r s t column w i t h 1 . 0 N HC1 was evaporated t o dryness a t 60' C , r e c o n s t i t u t e d w i t h water n e u t r a l i z e d and readsorbed onto a second column of AG 1 x 8 resin. 1 . 5 l i t e r s of a 0-1 N HC1 g r a d i e n t .
Elution was accomplished t h i s time w i t h
The e l u t i o n p o s i t i o n s of duck IP5 and IP6
(Sigma Chemical) were used as references. and i n combination.
The two compounds were run alone
Fish i n o s i t o l phosphate was a l s o r u n alone and i n combina
t i o n w i t h standard IP6.
Samples of t h e phosphate compound e l u t e d w i t h 1.0 N
HC1 gave a p o s i t i v e r e a c t i o n w i t h the Lornitzo method f o r i n o s i t o l ( ' 6 8 ) . RESULTS AND DISCUSSION
Figure 1A shows t h e t y p i c a l e l u t i o n p r o f i l e of
red c e l l e x t r a c t s from the a d u l t white Peking duck.
The formate e l u t i o n posi-
t i o n s of t h e principal phosphate compounds coincide w i t h t h e corresponding compounds i n t h e spiny dogfish ( f i g . 1 B ) and the torpedo ray ( f i g . 1 C ) .
In-
organic phosphorus, AMP, ADP and GTP a r e among the principal intermediates FIGURE LEGENDS 1
Ion-exchange chromatography (anion exchange r e s i n AGlx8, lx18.5 cm) of a d u l t duck ( A ) , spinydogfish ( B ) and torpedo ray ( C ) red blood c e l l e x t r a c t s . 14.8 ml f r a c t i o n s were c o l l e c t e d and a l l samples were analyzed f o r t o t a l phosphate ( 0 - 0 ) and absorbance a t 260 nm (a-0). Abbreviations: Inorganic phosphorous ( P i ) , adenosine monophosphate (AMP) , adenosine diphosphate (ADP) adenosine triphosphate (ATP) , c y t i d i n e triphosphate ( C T P ) , u r i d i n e t r i p h o s p h a t e ( U T P ) , guanosine triphosphate ( G T P ) , i n o s i t o l polyphosphate (INOS-P), i n o s i t o l pentaphosphate ( I P 5 ) . 2 Ion-exchange chromatography of a mixture o f i s o l a t e d duck IP5 p l u s standard IP6 on Bio Rad AG1 x 8 r e s i n , converted from c h l o r i d e t o formate form. I n o s i t o l phosphate compounds from lA, B , and C were evaporated t o dryness a t 60' C , r e c o n s t i t u t e d w i t h d i s t i l l e d water and t i t r a t e d t o pH 7.0 w i t h 0.05 N NaOH. Standard IP6 was added t o t h e s o l u t i o n and the mixture adsorbed onto the resin. Elution was accomplished w i t h a l i n e a r g r a d i e n t of 0- IN HC1. All samples were analyzed f o r t o t a l phosphorus. The mixture of duck IP5 and standard IP6 i s shown resolved i n t o two components ( A ) . The phosphate p r o f i l e s o f mixtures of standard IP6 and i n o s i t o l polyphosphate from th e dogf i s h ( B ) and the torpedo ray ( C ) show i d e n t i c a l e l u t i o n p o s i t i o n s f o r the penta and hexaphosphate d e r i v a t i v e s of i n o s i t o l .
DUCK
II
1 80
IPS(DUCK)
IP6 (STANDARD)
3
LITERS
LITERS TORPEDO RAY
YI
80
3 -
60-
z
Ly
.fP ADP
~0.01
CTP
GTP UTP
IWS-P
n
-
IP5 (TORPEDO RAY)
I a.
ow LITERS
IN HCI
LITERS
IP6 (STANDARD)
seen in these e x t r a c t s .
The nucleotides CTP and UTP were i d e n t i f i e d on the
basis of t h e i r elution positions.
No 2,3-DPG was detected in these e x t r a c t s ,
Following the formate gradient, duck IP5 was rapidly eluted from the column by the addition of 1 . 0 N H C 1 .
Similar phosphate peaks, which tested
positively f o r i n o s i t o l , were eluted from the spiny dogfish and torpedo extracts.
In a l l t h r e e cases the inositol polyphosphate was eluted within
15-45 ml a f t e r HC1 addition. One normal HC1 will normally c l e a r the columns of a l l phosphorylated intermediates.
Since the penta and hexaphosphates would a l s o be eluted as
a n inseparable peak under these conditions, t h e exact nature of the dogfish and torpedo i n o s i t o l polyphosphate was uncertain.
Therefore, duck IP5 f r a c t i o n s
were removed from peak tubes a f t e r addition of HC1 and used as standards. The samples were evaporated t o dryness in a forced d r a f t oven a t 60' C. D i s t i l l e d water was then added and the sample neutralized w i t h 0.05 N NaOH. Inositol hexaphosphate was added and the mixture adsorbed onto a second formate column.
A l i n e a r gradient of 1.5 l i t e r s of 0.1 N HC1 was used t o separate
and e s t a b l i s h the e l u t i o n positions of each compound. in t h e mixture duck IP5 i s eluted between 0.814 IP6 e l u t e s between 1.036 - 1.317 l i t e r s .
-
Figure 2A shows t h a t
1.011 l i t e r s and standard
Preliminary experiments established
t h a t these were the identical elution positions f o r t h e penta and hexaphosphates respectively when chromatographed separately.
Mixtures of i n o s i t o l polyphos-
phate from the dogfish o r torpedo plus standard IP6 show identical rechromatography p r o f i l e s when compared w i t h each other ( f i g s . 2B, 2C) or w i t h duck IP5
t
IP6 ( f i g . 2A).
The q u a n t i t a t i v e data of t h e red c e l l phosphate intermediates i s shown in t a b l e 1 .
The r e s u l t s i n d i c a t e t h a t ATP i s the major organic phosphate in
both species of elasmobranchs.
This a l s o c o n t r a s t s with thesmooth dogfish
138
(Mustelus canis) red cells in which the major organic phosphate is GTP (Borgese et al., '78). ATP levels vary markedly between the spiny dogfish and the torpedo. The former contains approximately 3.0 micromoles ATP per ml cells compared to 1.2 micromoles for the torpedo, the mean red cell levels of IP5 are 0.36 for squalus and about 0.24 micromoles per ml of cells for the torpedo. The presence of IP5 in elasmobranchs suggests that it may play a role in regulating the oxygen affinity of dogfish and torpedo hemoglobins. Although recent evidence indicates that some invertebrate hemoglobins are insensitive to IP6, 2,3-DPG and ATP (Weber et al. , '77), IP5 and IP6 are much more effective than 2,3-DPG and ATP in decreasing the oxygen affinity of vertebrate (human and avian) hemoglobins (Benesch and Benesch, '69; Borgese and Nagel, '77). The fact that IP5, in these experiments, is present at levels under 0.5 micromoles per ml of cells does not preclude an effect on the oxygen equilibria of dogfish and torpedo hemoglobins. Our finding that IP5 is present in elasmobranch red cells also has evolutionary significance since it is now clearthatIP5, once thought to be unique among birds, is more ubiquitous than previously believed. ACKNOWLEDGMENTS Research was supported by grants from the Research Foundation
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CUNY #11395, 11620, NIH-2P50-GM 19100 and an American Heart
Grant-in-Aid. We gratefully acknowledge the technical assistance of Ms. Barbara Colucci and Mr. Robert Goldschmidt. LITERATURE CITED Bartlett, G. R. 1959 Methods o isolation of glycolytic intermediates by column chromatography with ion-exchange resins. J . Biol. Chem., 234: 459-465. Bartlett, G. R. 1976 Phosphate compounds in red cells of reptiles, amphibians and fish. Comp. B ochem. Physiol . , 55A: 211-214. Benesch, R., and R. E. Benesch 967 The effect of organic phosphates from the human ervthrocvte on the allosteric properties of hemoglobin. Biochem. Biophys." Res. iommun. , 26: 162-166. Benesch, R., and R. E. Benesch 1969 Intracellular organic phosphate as regulators of oxygen release by hemoglobin. Nature, 221: 618-622.
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Borgese, T. A . , and L . M. Lampert 1975 Duck red c e l l 2,3-diphosphoglycerate: I t s presence i n the embryo and i t s disappearance i n the a d u l t . BBRC. 65: 822-827. Borgese, T. A . , and R . L . Nagel 1977 D i f f e r e n t i a l e f f e c t s of 2,3-DPGY ATP and i n o s i t o l pentaphosphate (IP5) on the oxygen e q u i l i b r i a of duck embryonic f e t a l and a d u l t hemoglobins. Comp. Biochem. Physiol., 56A: 539-543. Borgese, T. A . , R. L . Nagel, E. Roth, D. Murphy and J . Harrington 1978 Guanosine triphosphate (GTP): The major organic phosphate i n the erythrocytes of t h e elasmobranch mustelus c a n i s (smooth d o g f i s h ) . Comp. Biochem. Physiol., i n press. Chanutin, A . , and R. R. Curnish 1967 Effect of organic and inorganic phosphates on t h e oxygen equilibrium of human erythrocytes. Arch. Biochem. Biophys. , 121: 96-102. Isaacks, R. E . , and D. R. Harkness 1975 2,3-diphosphoglycerate i n erythrocytes of chick embryos. Science, 189: 393-394. Isaacks, R. E . , H. D. K i m , G . R. B a r t l e t t and D. R. Harkness 1977 I n o s i t o l pentaphosphate in erythrocytes of a freshwater f i s h , Piraracu (Arapaima G i Gas) Life Sciences, 20: 987-990. Johnson, L . F., and M. E . Tate 1969 S t r u c t u r e of "phytic a c i d s " . Can. J . Chem., 47: 63-73. Lornitzo, F. A. 1968 A method f o r c a l o r i m e t r i c assay of i n o s i t o l and some of i t s phosphate d e r i v a t i v e s . Anal. Biochem., 25: 396-405. Rapoport, S . , and A. M. Guest 1941 D i s t r i b u t i o n of a c i d s o l u b l e phosphorus i n the blood c e l l s of various v e r t e b r a t e s . J . Biol. Chem., 138: 269-282. Vandercasserie,C., C . Paul, A. C . Schnek and J . Leonis 1973 Oxygen a f f i n i t y o f avian hemoglobins. Comp. Biochem. Physiol., 44A; 71 1-71 8. Weber, R. E., B. S u l l i v a n , J . Bonaventura and C. Bonaventura 1977 The hemoglobin systems of the blood worms Glycera dibranchiata and G . Americana: Oxygen binding p r o p e r t i e s of the hemolysates and component hemoglobins. Comp. Biochem. Physiol., 58B: 183-187.
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