Isolation of O-Phosphorylethanolamine from the Erythrocytes of an Elasmobranch (the Southern Fiddler Rav - Trygonorhina fascia ta guanerius)

MICHAEL COATES,' JUDITH THOMPSON ' AND M. E. TATE 'Department of Zoology, University of Adelaide, Adelaide, South Australia, 5000 and 'Department of Agricultural Biochemistry, University of Adelaide, Waite Agricultural Research Institute, Glen Osmond, South Australia, 5064

ABSTRACT An amphoteric organic phosphate, which represents ZOY, of erythrocyte acid soluble phosphate components from an elasmobranch (the Southern Fiddler Ray - Trygonorhina fasciata guanerius) has been isolated as a colourless crystalline solid. Its phosphorus content, melting point behaviour, chromatographic, electrophoretic and ion-exchange properties, together with its infrared and electron impact mass spectra are consistent with its formulation as 0- phosphorylethanolamine.

Assuming minimal degradation in the isolation procedure, the calculated erythrocyte intracellular concentrations of characterised phosphates from the Southern Fiddler Ray are as follows: O-phosphorylethanolamine (1.89 mM), adenosine triphosphate (1.61 mM), inorganic phosphate (0.26 mM), inosine monophosphate (0.47 mM), guanosine triphosphate (0.34 mM) and adenosine di- phosphate (0.11 mM). On a molar basis O-phosphorylethanolamine is the major acid soluble phosphate of the Southern Fiddler Ray erythrocyte, but examina- tion of the literature suggests its concentration may vary considerably among other elasmobranchs and possible reasons for such variance are considered.

The erythrocytes of representatives from most of the classes of vertebrates have been shown to contain high levels of strongly anionic organic phosphate molecules (e.g., Bartlett, '76; Coates, '75a; Coates et al., '78; Isaacks et al., '77). These anions react with cationic groups of hemoglobin to reduce its oxygen affinity (Benesch and Benesch, '67; Chanutin and Curnich, '67: see Coates, '75a, for review). In contrast, high concentrations of the considerably weaker anionic organic phosphate, inosine monophosphate (IMP) were reported in the erythrocytes of three elasmobranchs (Coates e t al., '78). IMP showed no significant effect upon the hemo- globin-oxygen dissociation curves using shark haemoglobin. Furthermore, the presence in all three elasmobranchs (Galeorhinus australis, Notorynchus cepedianus and Trygonorhina fasciata guanerius) of a large amount of an unidentified neutral or cationic organic phos- phate, emerging a t the front in Dowex 1 col- umn chromatography, was noted. This com- pound has now been identified in one of the

J. EXP. ZOOL. (1979) 210: 489-496.

species (the Southern Fiddler Ray - T.f. gua- nerius) as O-phosphorylethanolamine (PE).

MATERIALS AND METHODS

Dowex 1 chromatography Blood samples were collected from Southern

Fiddler Rays and trichloracetic acid extracts of erythrocytes were prepared as previously described (Coates, '75b; Coates e t al., '78). Dowex 1 column chromatography of the ex- tracts was carried out by the method of Bartlett ('70) with the following modifica- tions: A 2.7 x 10.5 cm column of anion ex- change resin AG 1-X8 200- to 400-mesh for- mate form supplied by Bio Rad was utilized. The neutralized extract from 5-10 ml of erythrocytes was applied to the column and eluted with water (200 ml). A concave gra- dient of water (1400 ml) and 5 M ammonium formate (750 ml) was then applied. The gra- dient was followed by 1 M HC1 (200 ml) (Isaacks e t al., '77). Six-milliliter fractions were collected from the time of sample ap- plication, the ultraviolet absorbance (260 nm)

489

490 M. COATES, J. THOMPSON AND M. E. TATE

was measured, and aliquots were analysed for phosphate (Bartlett, '59). Fractions under peaks were pooled, concentrat,ed by freeze dry- ing, the ammonium formate removed by passage through a G-10 sephadex column equilibrated with water and then again lyo- philised.

Dowex 50H + chromatography A 2.7 x 27.0 cm column of a cation ex-

change resin (Dowex-50W hydrogen form, 8% cross linked, 50-100 dry mesh) was utilized. The column was first washed with 5 M HC1 1500 ml) followed by double distilled water until the pH of the column eluate was the same as that of the water as measured with litmus paper. Erythrocyte extract, or t,he water eluate from the Dowex 1 column, adjusted to pH 2.0, was applied to the column and eluted with water (200 ml), followed by a linear gradient of water (500 ml) and 5 M IlCl (500 ml). Six-milliliter fractions were col- lected, absorbance a t 260 nm was measured and a small aliquot removed from each frac- tion for phosphate analysis. Fractions under peaks were pooled and vaccum desiccated.

Paper chromatography and electrophore.sis Descending paper chromatography was car-

ried out on Whatman No. 1 paper with the fol- lowing solvent systems: Solvent 1, water ad- justed to pH 10.0 with NH,OH; Solvent 2, pheno1:water (4: l ) 100, glacial acetic acid 10, anhydrous ethanol 12; Solvent 3, ethylacetate 10, pyridine 4, water 4.

High voltage paper electrophoresis was car- ried out using a solvent cooled system at 35 voltsicm on Whatman No. 1 paper with the following buffers: Buffer 1, 0.05 M Na2B,07, pH 9.4; Buffer 2, 0.1 M citrate, pH 5.5; Buffer 3, 0.76 M formic acid containing 1.0 M acetic acid, pH 1.70. Paper chromatograms and elec- trophoretograms were first examined with ul- traviolet light (250 nm) for absorbing spots and then dipped in an acetone solution of ninhydrin (0.25% w/v) and heated a t 60-70" to detect primary amines. Finally, phosphory- lated components were detected by the molyb- date procedure of Harrap ('60). Mobilities (Mot values) are measured relative to the anionic dyestuff Orange G ( M O G = 1.0) using a suitable non-migrating marker, e.g., 2-deox- yadenosine for t he borate buffer No. 1, and fructose for buffers No. 2 and No. 3.

Enzymic hydrolysis An enzyme suspension of Escherichia coli

alkaline phosphatase, Sigma product No. 4252, type 111, in 2.5 M ammonium sulphate containing 8.8 mg protein/ml was used. The enzyme suspension contained 256 unitdm1 where one unit will hydrolyse 1.0 +mole of p- nitrophenyl phosphate per minute a t pH 8.0 a t 25". In a total volume of 15 ~ 1 , the enzyme suspension (5 pl) , 1.5 M ammonium hydroxide 5 +1 and a 0.16 M solution (5 p l ) of the crystalline P.E. sample were incubated at 25" for 2 hours. Appropriate control hydroly- ses including the authentic phosphorylethanol- amine were carried out simultaneously. The progress of each hydrolysis was followed by electrophoresis at pH 1.7 using aliquots (2 p1). Hydrolysis to yield inorganic phosphate (MOC 0.49) and ethanolamine (MOC; - 2.16) was vir- tually complete within 2 hours.

Spectroscopy Ultraviolet spectra were recorded using a

Varian Superscan 3 spectrophotometer. In- frared spectra were determined using 0.5% samples in potassium chloride discs with a Model 237 Perkin Elmer Grating Spectropho- tometer. Electron impact low resolution mass spectra were obtained with a n A.E.I. MS3D74 double beam mass spectrometer operating at 70 eV with direct probe insertion a t T = 80".

Crystallisation of O-phosphoryl- ethanolamine

The combined Dowex 1 water eluate (fig. 1: peak 1) obtained from the separation of the acid soluble phosphates of a pellet (48.5 ml) of packed Southern Fiddler Ray erythrocytes, were further separated by Dowex 50 cation ex- change chromatography (fig. 2). Browning ('78) noted tha t for calculation of actual erythrocyte volumes from pellet volumes (packed at 2,500 rpm for 5 minutes) a factor of x 0.94 should be applied to account for the plasma content of the pellet.

Figure 2 fractions (48-60, "PE") were con- centrated to dryness in vacuum and redis- solved in water (0.5 ml) at 24", acetone ( - 3 ml) was slowly added to produce a faint tur- bidity. The mixture was maintained a t 4" and gradually deposited crystals which were washed with 75% v/v acetone (2 x 1 ml) and dried (17 h/25"/0.1 mm HgiSiO,) to yield col- ourless rods (3.41 mg), mp 240-241 (Pyrex cap-

PHOSPHOETHANOLAMINE IN FIDDLER RAY ERYTHROCYTES 491

3

I 7

t m I I I I I m

0 start 150 300 gradient Fraction No.

Fig. 1 Dowex 1, anion exchange chromatography of organic phosphates from a trichloroacetic acid extract of Southern Fiddler Ray erythrocytes. Phosphorus-containing peaks are assigned as follows: 1, neutral and/or cationic ma- terial eluting with the water eluant; 2, inorganic phosphate; 3, inosine monophosphate; 4, unknown phosphate; 5, ade- nosine diphosphate; 6, adenosine triphosphate; 7, guanosine triphosphate.

illary), P, 21.7%. Recrystallisation from 80% methanol did not alter the mp. The infrared spectrum (0.5% KCl Disc) showed the follow- ing absorption maxima: 760s, 900sh, 935s, 1,004s, 1,023s, 1,075s, 1,155s, 1,185sh, 1,24Om, 1,44Ow, 1,54Om, 1,635m, 2,04Om, 2,300-3,400 S.br. cm-'. The general pattern of absorption intensities agreed well with published spectra for 0-phosphorylethanolamine (Baer and Stancer, '56; Fraser and Lucas, '56; Grollman and Osborn, '64; Akutsu and Kyoguku, '75) and also with the spectrum of an authentic sample of 0- phosphorylethanolamine.

RESULTS

Figure 1 shows the anion exchange (Dowex 1) chromatography of acid soluble organic phosphates from Southern Fiddler Ray erythrocytes. Peak 1 is the main subject of this investigation; the remaining numbered peaks have been tentatively assigned on the basis of their retention times, chromato-

graphic properties and spectral behaviour a t pH 1.0, 7.0 and 11.0 before and after acid hydrolysis (6 N HC1/100"/1.5 h). The num- bered peak assignments and percentages of the total acid soluble phosphate are as follows: 1. unknownk) 21%; 2. inorganic phosphate 3%; 3, inosine monophosphate 5%; 4, unknown 3%; 5, adenosine diphosphate 2%; 6, adenosine triphosphate 51%; 7, guanosine triphosphate 11%. Subsequent elution of the column with 1 M hydrochloric acid yielded another peak of strongly bound phosphate, 3%, which has not been studied in detail. Borgese and Nagel ('78) provided evidence for an inositol pentaphos- phate in a similar strongly bound fraction from other elasmobranchs, but the data did not exclude the possibility that the inositol may not have been myo-inositol.

Figure 1 also shows tha t peak 1 which elutes with water from the Dowex 1 column is strongly U.V. absorbing. This peak was not ini- tially analysed for phosphate, but subsequent

492 M. COATES, J. THOMPSON AND M. E. TATE

1

0

0 100

Fraction No. Fig. 2 Dowex 50 H’ chromatography of organic phosphates present in peak 1 of figure 1. In this separation, the ap-

plied sample contained neutral and/or cationic phosphates equivalent to a pellet (48.6 ml) of packed erythrocytes. Absorb- ance a t 660 nm represents a measure of the phosphate content in aliquots used for the Bartlett (’59) phosphate assay.

analysis showed the bulked fraction contained 21% of the total acid soluble phosphate. Figure 2 shows that cation exchange chromatography using a linear gradient of 0-5 M HCl, partially resolves the ultraviolet absorbing ( A max; 239.9 nm, pH 1; 235.8 nm, pH 7; 238.8 nm, pH 10.0) component from the major organic phos- phate component “PE” in fractions 48-60. After removal of the hydrochloric acid and dis- solving the residue in a small volume of water, electrophoretically and chromatographically homogeneous crystals were obtained from the combined fractions labelled “PE” in figure 2, by the addition of acetone.

The electrophoretic mobilities (MOG values) of the crystalline phosphate “PE,” relative to the anionic dyestuff standard Orange G fMoc = 1.0) using fructose as a non-migrating marker (MOc = 0.01, were found to be MOG -0.05 a t pH 1.7 and MOG 0.28 a t pH 5.5. For the borate buffer a t pH 9.4, 2-deoxyadenosine was used to replace fructose as the non-mi- grating marker and the relative mobility of “PE” was MOG 0.77. The weakly cationic nature denoted by the negative MOG value a t

pH 1.7 indicated the presence of a basic group; the presence of a primary amino group was then confirmed by a positive ninhydrin re- sponse.

A sealed tube acid hydrolysis (0.5 M HC1/2 h/ 110”) of an 0.08 M solution of crystalline “PE” and a sample of authentic O-phosphoryl- ethanolamine yielded inorganic phosphate (MOG 0.491, a ninhydrin positive base fMOG -2.16) and unreacted PE fMoc -0.05) when examined by electrophoresis a t pH 1.7. The ninhydrin positive base was found to be elec- trophoretically and chromatographically indistinguishable from ethanolamine in all systems.

In contrast to the slowness of the acid hydrolysis, a rapid ( < 2 h) enzymic hydrolysis ensued with commercial Escherichia coli alkaline phosphatase (E.C. 3.1.3.1). In both acid and enzymic hydrolyses, the behaviour of crystalline P.E. and authentic O-phos- phorylethanolamine were indistinguishable.

The simplest interpretation of these data is that the crystalline component “PE” of figure 2 is 0- phosphorylethanolamine, a conclusion

PHOSPHOETHANOLAMINE IN FIDDLER RAY ERYTHROCYTES 493

Q, 0 E m '0

f 0

c o

4 100

Q, > m

p!

.- +

50

0

4 0 8 0 120

M a s s - t o - C h a r g e R a t i o Fig. 3a Low resolution 70 e.v. electron impact mass spectrum of crystalline material from peak "PE" of figure 2.

b Corresponding mass spectrum of authentic O-phosphorylethanolamine. The molecular ion M' of mass to charge lm/e 141) was not observed, but the fragment (m/e 123) corresponding to a loss of water (m/e 18) is apparent. Cleavage a t carbon-carbon, carbon-oxygen and phosphorus-oxygen bonds in the molecular ion M' predicts the following ions: C,H,N+ Im/e 30). C,H&" (m/e 44) and H,PO,' (m/e 81). The fragment (m/e 98) probably corresponds to H ,PO,+ de- rived by a McLafferty type rearrangement with elimination of CH,:CHNH,.

supported by direct comparison with authen- tic O-phosphorylethanolamine using chroma- tography in solvents 1, Rf = 0.92; 2, Rf = 0.47; 3, Rf = 0.04, by electrophoresis a t pH 1.7, pH 5.5 and pH 9.4, and by cation exchange chromatography as in figure 2. Further evi- dence was obtained from the phosphorus con- tent P 21.7%, C,H,NO,P, MWt 141.1 required P 22.0%, the correspondence of the infrared and low resolution mass spectra (fig. 3) and the melting point and mixed melting point be- haviour. The latter is worthy of further com- ment: Baer and Stancer ('56) noted the wide range (228-245") of recorded melting points for both synthetic and natural product sam- ples of O-phosphorylethanolamine. These workers ascribed the problem to difficulties in purification of the samples, however we have observed that widely differing melting points can be obtained using a single sample de- pending on the exact melting point procedure

which is employed. In our hands, dry, recrystallised, electrophoretically and chro- matographically homogeneous authent ic O-phosphorylethanolamine gave a sharp mp 241-242" (corr.) in pyrex capillaries; Baer and Stancer ('56) recorded a melting point 244- 245" for analytically pure material using a capillary procedure. Using soda glass capil- laries a melting point of 238-240" (corr.) was observed by us and by employing the Kofler hot stage microscope with a heating rate of 2"/ min, an extremely broad melting point 226- 234" (corr.) with decomposition was observed. Using the pyrex capillary procedure, the crystalline "PE" sample from erythrocytes showed a mp 240-241" (corr.) and a mixed mp 240-242" (corr.) with the authentic O-phos- phorylethanolamine mp 241-242" (corr.1.

The isolated O-phosphorylethanolamine represents 20% of the total acid soluble phos- phate of the Southern Fiddler Ray's erythro-

494 M. COATES, J. THOMPSON AND M. E. TATE

cytes and providing i t is not the product of some acid labile precursor the intracellular erythrocyte concentration of 0 -phosphoryle t h.- anolamine was estimated to be 1.89 mM (mean of 3 determinations).

DISCUSSION

This project was commenced in order to identify a phosphorus component previously observed (Coates e t al., '78) a t the eluate front during anion exchange separations of t h e organic phosphorus consti tuents of elasmobranch erythrocytes. This phosphate was detected as a major erythrocyte compo- nent in the Seven Gilled Shark (Notorynchus cepedianus), the School Shark (Galeorhinus australis) and the Southern Fiddler Ray (Tri- gonorhina fasciata guaneriusi. The evidence for its identity with O-phosphorylethanol- amine in the case of the Southern Fiddler Ray and possible explanations for i ts apparent var- iation amongst other elasmobranchs will be discussed.

Free 0- phosphorylethanolamine was first isolated in crystalline form from bovine, tumour tissue (Outhouse, '36). I t has also been isolated from the intestines of rabbits and pigs (Colowick and Cori, '39) and the urine of patients suffering from the hereditary bone disease Hypophosphatasia (Fraser and Lucas, '57; Cusworth, '58). In addition, crystalline material has been isolated from hydrolysates of E. coli rest protein (de Verdier and Agren, '58) and a lymphpolysaccharide from Sal - monella typhimurium (Grollman and Os- born, '64).

The best characterised synthetic O-phos- phorylethanolamine appears to be the report of Baer and Stancer ('56). The wide range of reported melting points for both synthetic and natural produce 0-phosphorylethanolamine was noted by Baer and Stancer ('561, who a t - tributed it to varying degrees of purification. In the present study i t has been found to be also dependent on the actual melting point technique.

The amount of crystalline material (3.4 mg) isolated in the present study precluded a com- plete elemental analysis. However, there was satisfactory agreement between the observed and calculated phosphorus content. The claim for identity with authentic O-phosphoryl- ethanolamine rests mainly upon the following independent criteria: (i) electrophoresis a t pH 1.7, 5.5, 9.4 to ensure tha t the mobilities were

compared with each ionisation reported by Folsch and Osterberg ('59); (ii) paper chroma- tography in three different systems; (iii) acid and enzymic hydrolysis behaviour; (iv) cation exchange ch romatography ; ( v ) e l ec t ron impact mass spectra; (vi) infrared spectra; (vii) melting point and mixed melting point behaviour. Within the bounds of experimental error, the authentic and isolated samples were found to be indistinguishable and it is con- cluded tha t the major phosphorus component which may be isolated from the erythrocytes of the Southern Fiddler Ray is O-phosphoryl- ethanolamine.

Due to the acidic nature of the deproteina- tion procedure, the present data does not ex- clude the possibility tha t they may be some hypothetical highly acid labile precursor of the 0- phosphorylethanolamine. However, the high proportion of adenosine triphosphate (1.61 mM) relative to adenosine diphosphate (0.11 mM) indicates t ha t such a precursor would need to be considerably more acid labile than ATP. In connection with Southern Fid- dler Ray erythrocyte ATP concentrations, i t should be noted tha t a n error in the gradi- en t elution conditions employed previously (Coates et al., '78) was responsible for the failure to detect high levels of ATP and GTP in the Southern Fiddler Ray in that study.

The recorded proton dissociation constants (pK <1, pK, 5.57, P K , ~ 10.13, Folsch and Oster- berg, '59) provide information on the net charge characteristics of 0- phosphoryletha- nolamine. Below pH 2, it will be cationic, in t he range pH 2-3.6 it will carry a net charge of zero, which accounts for the observed behav- iour on anion and cation exchange columns as shown in figure 1 and 2. In the physiological range pH 6.5-8, the net negative charge of 0-phosphorylethanolamine will be close to minus one and as in the case of inosine mono- phosphate (Coates et al., '78) i t is to be ex- pected that 0-phosphorylethanolamine even at a concentration of 1.84 mM, will be unlikely to exhibit a significant effect upon haemoglo- bin-oxygen dissociation curves, when stronger anions such as adenosine triphosphate (1.61 mM) and guanosine triphosphate (0.34 mM) are present. I t therefore seems likely that the metabolic significance of the relatively high concentration (1.84 mM) of O-phosphoryletha- nolamine in Southern Fiddler Ray erythro- cytes lies elsewhere.

Three other studies of elasmobranch eryth-

PHOSPHOETHANOLAMINE IN FIDDLER RAY ERYTHROCYTES 495

rocyte components are relevant. Borgese and Nagel ('78) and Borgese et al. ('78) published anion exchange chromatograms of the phos- phorus components in the Torpedo Ray (Nar- cacion nobilianai, the Spiny Dogfish (Squalus acanthias) and the Smooth Dogfish (Mustelis canis). The Torpedo Ray chromatogram shows a major unidentified phosphate component at the eluant front, which could correspond to the 0-phosphorylethanolamine found in this study. However, the corresponding peak in the Smooth and Spiny Dogfish chromatograms is only a minor component. In another study (Boyd et al., '77) of free amino acids in Skate (Raja erinacea) erythrocytes, using cation ex- change chromatography, a peak location ascribed to 0-phosphorylethanolamine was found to be below the level of detection.

Such a large variation in free erythrocyte concentrations of 0-phosphorylethanolamine (0-1.8 mM) among elasmobranchs, warrants further investigation. Several possible expla- nations may be considered: firstly, the dif- ferences may merely reside in the differing ex- perimental procedures. Secondly, significant species differences may be involved, as in the case of guanosine triphosphate, which has been shown (Borgese and Nagel, '78; Borgese et al., '78) to be a major component of the Spiny Dogfish erythrocyte and a minor compo- nent of the Smooth Dogfish erythrocyte. Thirdly, the concentrations of O-phosphoryl- ethanolamine may depend upon age, dietary factors, or circadian rhythms (Lenartowicz and Niemerko, '64; Licata e t al., '78). Finally, the weak, but measurable calcium chelating properties of 0- phosphoethanolamine in the physiological pH range are indicated by the stability constant (log KMHL 1.11) of the monoprotonated calcium complex (Osterberg, '62; Mohan and Abbot, '78). These reports lead us to consider the intriguing possibility that , as in the case of the hereditary bone disease Hypophosphatasia (Fraser and Lucas, '57), there may be a relationship between the pres- ence of 0-phosphorylethanolamine and the de- gree of ossification (or de-ossification) of the animal, which is a matter of some conse- quence for elasmobranchs.

ACKNOWLEDGMENTS

The assistance of Mr. T. Blumenthal of the Department of Organic Chemistry with the low resolution mass spectrometer comparison is gratefully acknowledged.

This work was supported in part by an A.R.G.C. grant to Dr. M. Coates.

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