RAPID COMMUNICATION ZIPGRAM

H I G H LEVELS O F INOSINE MONOPHOSPHATE IN THE

ERYTHROCYTES OF ELASMOBRANCHS (1)

MICHAEL COATES, BARBARA C . PATON ( 2 ) AND JUDITH THOMPSON Department of Zoology, University of Adelaide, Adelaide, South Australia 5001

ABSTRACT The acid soluble organic phosphates of the erythrocytes of three species of elasmobranchs were assayed by chromatography on Dowex 1 anion exchange columns. Organic phosphates in the peaks eluted from these columns were identified by their ultraviolet absorption spectra and by further chromatography on paper. unusual amongst the vertebrates in that their erythrocytes contain h i g h levels of inosine monophosphate (IMP). oxygen aff ini ty o f the hemoglobins of the two species tested.

All three species are

IMP has l i t t l e effect on the

The acid soluble organic phosphates of the erythrocytes of a number of

vertebrate species have been assayed (see Coates, '75a for review).

more, i n those species w i t h h i g h levels of organic phosphates such as 2,3

diphosphoglyceric acid (DPG), adenosine triphosphate (ATP) or guanosine t r i -

phosphate (GTP) these molecules were found t o be present i n concentrations

nearly equimolar w i t h hemoglobin and t o modify the oxygen aff ini t ies of the

species' hemoglobins (Coates, '75a). However, no such studies had been carried

o u t for any species of the class Elasmobranchii. This study was undertaken

t o f i l l th i s gap i n our knowledge.

Further-

MATERIALS A N D METHODS Three species of Elasmobranchii were collected

from coastal waters off South Australia: the School Shark (Galeorhinus

austral i s ) , the Seven Gilled Shark (Notorynchus cepedianus) and the Southern

Fiddler Ray (Trygonorhina fasciata guanerius). The pelagic School and Seven

Gilled Sharks were caught i n g i l l nets. The t a i l was severed about 3-5 cm

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from i t s end and the blood allowed t o spurt from the caudal a r t e r y in to

heparinized p l a s t i c containers. The blood of these species was unavoidably

s tored a t 0-5' C f o r 6-8 hours. The bottom dwelling Southern Fiddler Rays

were caught by hand i n shallow water and transported a l ive , i n seawater aquaria,

t o t he laboratory where they were anaesthetized with MS222 Sandoz, and the

per icardial cav i ty opened. The blood was then taken in to heparinized syringes

by heart puncture and ch i l led t o 0-5O C.

For determination of hemoglobin-oxygen equilibrium curves hemolysates

were produced by suspending the erythrocytes i n 3.5% NaCl and lysing by

sonication.

hemolysate through a 6-25 Sephadex column equi l ibrated with 0.05M Bistris-HCl,

0.3M NaCl, pH 7.3. Hemoglobin-oxygen equilibrium curves were determined as

previously described (Coates, '75b). For tonometry a l l solut ions were 3lrtM

hemoglobin (assuming a molecular weight of 65,000 da l tons) , 0.05M Bistris-HC1,

0.3M NaCl, 0.36M urea adjusted t o pH 6.7 o r 7.3.

contained i n addition 1.36mM inosine monophosphate o r adenosine tr iphosphate.

Solutions of "stripped" hemoglobin were produced by passing the

Some of these solut ions

For analysis of acid soluble organic phosphates the erythrocytes were

extracted w i t h t r i ch lo race t i c acid as previously described (Coates, '75b).

Dowex 1 column chromatography was car r ied out by the method of Bar t l e t t ( ' 7 0 )

except t h a t a concave gradient of 1400 ml water and 750 ml 5N ammonium formate

was used. Total phosphate was determined fo r each f rac t ion by the method of

Bar t l e t t ( ' 5 9 ) . The E260 of each f rac t ion was a l so determined.

Fractions under the peaks eluted from the Dowex 1 column were pooled,

concentrated by freeze drying, the ammoni um formate removed by passage through

a G-10 Sephadex column equi l ibrated with water and then again freeze dr ied.

Nucleotides in these f rac t ions were t en ta t ive ly ident i f ied by t h e i r e lu t ion

posit ion from Dowex 1 and by their U.V. absorption spectra . Ident i f ica t ions

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TABLE 1

Est imated concent ra t ions (mMo1ar) o f hemoglobin and nuc leo t i des w i t h i n t h e e ry th rocy tes o f t h r e e species o f Elasmobranchi i .

A l l values a re averages o f t h r e e es t imates .

Species Hb I M P AMP GMP ATP GT P

Southern F i ddl e r 2.84 .47 .03 .05

School Shark 2.32 .71 - .55 .58

Ray

Seven G i l l e d Shark 4.01 5.33 3.33

TABLE 2

Resu l ts o f de termina t ions o f P50 values o f hemoglobin-oxygen e q u i l i b r i u m curves f rom two species o f Elasmobranchs.

w i t h o r w i t h o u t 1.86mM nuc leo t i de . Values a re averages o f two de terminat ions .

Condi t ions: 31pM Hb, 0.05M B is t r i s -HC1, 0.3M NaCl , 0.36M urea,

Species Nuc leo t ide

pH 6.7 pH 7.3

School Shark none 10.9 6.9

I1 27.5 19.5 AT P

If I MP 9.8 7.3

Seven G i l l e d Shark none 10.4 8.2

I I 12.1 10.9

I 1 10.0 8.9

AT P

IMP

were confirmed by hydrolyzing the nucleotides to t h e i r bases i n 6N HC1 a t

100' C f o r 1 hour followed by descending chromatography on Whatman #1 f i l t e r

paper with standards adenine, hypozanthine, guanine, cytocine, and u r i c i l . Buffer f o r paper chromatography was water adjusted t o pH 10 with NH40H.

were visual ized under U . V . l i g h t and then cut ou t , e luted w i t h water and

Spots

absorption spectra determined.

The to t a l nucleotide in each peak was determined from the E260 of each

f rac t ion compared w i t h standard curves f o r the d i f f e ren t nucleotides. Hemo-

globin concentrations were estimated as previously described (Coates, '75b).

The volumes of t he erythrocyte pe l l e t s extracted were corrected f o r 6.5% plasma

trapping as determined fo r the Southern Fiddler Ray (Browning, '77). W i t h

these f igures the molarity of the nucleotides, and of hemoglobin, w i t h i n the

erythrocytes was es t i mated . RESULTS AND DISCUSSION Typical r e su l t s of Dowex 1 chromatography f o r the

three species are shown i n f igures 1, 2 and 3 . The r e su l t s of paper chroma-

tography of the hydrolyzed School Shark erythrocyte nucleotides a re shown in

f igure 4.

a c t e r i s t i c U . V . absorption spectrum w i t h a maximum a t 248 nm.

resces blue in U . V . l i g h t under these conditions.

1 tha t only the School Shark has appreciable amounts of the polyphosphates

Ident i f ica t ion of hypozanthine i s readi ly confirmed by i t s char-

Guanine fluo-

I t can be seen from f igure

FIGURE LEGENDS

Dowex 1 chromatography of erythrocyte organic phosphates o f :

1 Seven Gilled Shark 2 Southern Fiddler Ray 3 School Shark 4 Paper chromatography of hydrolysed nucleotides from School Shark

erythrocytes. Spots visualized under U . V . l i g h t . S = standards; H = hypozanthine; G = guanine; A = adenine. 1, 2 and 3 a re hydrolysed material from peaks marked IMP, ATP and GTP, respec- t i ve ly , in Fig . 3.

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FIG 1 FIG 4 8

6

0

FIG 2

$ 50

- E

f 0

40

I

tubes

Fffi 3

ATP and GTP.

rocyte. All species have an unidentified phosphate posi t ive, U . V . absorbing

peak i n the material which did not bind t o the Dowex 1 column and washed off

All species have IMP as a major organic phosphate of the eryth-

w i t h water.

within the erythrocytes of the three species a re given i n t ab le 1.

Estimates of the molarity of the nucleotides and of hemoglobin

The P50 values of School and Seven Gilled Shark hemoglobin solut ions w i t h

and without IMP, and w i t h and without ATP a re given i n t ab l e 2.

oxygen equilibrium curves could not be determined f o r Southern Fiddler Ray

because t h i s species ' hemoglobin i s unstable under the conditions used. I t

can be seen from t ab le 2 t h a t although ATP causes a substant ia l increase in

the P50 value (decrease in oxygen a f f i n i t y ) in the School Shark, IMP has no

detectable e f f ec t under these conditions on e i t h e r species ' hemoglobin. The

conditions of hemoglobin-oxygen equilibrium determination were selected t o

approximate the in vivo concentrations of NaCl and urea (Campbell, '73).

Hemoglobin-

, Therefore, we conclude t h a t the biological ro le of IMP i n the erythrocytes of

Elasmobranchii i s not the modification of hemoglobin function. The occurrence

of substant ia l l eve ls of IMP in elasmobranch erythrocytes i s unique among the

ver tebrates so f a r examined (Coates, '75a). Since high concentrations of urea

a re a l so found in elasmobranch blood and not i n t h a t of the o ther ver tebrates

assayed f o r erythrocyte organic phosphates, our working hypothesis i s tha t the

occurrence of these two compounds i n the erythrocytes of elasmobranchs i s

re la ted.

LITERATURE CITED

B a r t l e t t , G. R. 1959 Phosphorus assay in column chromatography.

Ba r t l e t t , G . R . 1970 Patterns of phosphate compounds in red blood

Browning, J .

J . Biol. Chem., 234: 466-468.

c e l l s of man and animals. In: Red Cell Metabolism and Function. G. J . Brewer, ed. , Plenum Press, New York, pp. 245-256.

Southern Fiddler Skate, Trygonorhina fasc ia ta guanerius. J . Exp. 1978 Urea leve ls i n plasma and erythrocytes of the

Zoo1 . , 203: 325-330.

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Campbell , J . W . 1973 Nitrogen exc re t ion . In: Comparative Animal

Coates, M. L. 1975a Hemoglobin function i n the ve r t eb ra t e s : An

Coates, M. L . 1975b Studies on the i n t e r a c t i o n of organic phosphates

Physiology, 3rd e d i t i o n . C . L . Prosser , e d . , Saunders, Phi ladelphia , pp. 279-316.

evolut ionary model . J . Mol . Evol . , 6: 285-307.

w i t h haemoglobin i n an amphibian (Bufo marinus), a r e p t i l e (Trachydosaurus rugosus) and man. Aust. J . Biol. S c i . , 28: 367-378.

REFERENCES

1 T h i s work was supported i n p a r t by an ARGC g ran t t o Dr. M . Coates. 2 Present address: Department of Zoology, Austral ian National

University, Canberra, A.C.T. 2600.

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