404 TIBS - October 1985

Phosphodiesters and NMR: a tale of rabbits and chickens

C. Tyler Burr

Comparative biochemistry can often encourage a wider view of a biochemical system by showing how different species have solved similar problems using different bio- chemicals (enzymes). Further, by comparing how similar the two solutions are, it can

be seen to what extent universal principles are involved.

A good example is the high-energy phosphate buffer phosphocreatine (PCr). It is the only high-energy buffer in vertebrates l: the highest con- centrations are found in muscle but sig- nificant amounts are also present in heart and brain. In these tissues essen- tially one enzyme, creatinephospho- kinase (EC 2.7.3.2), allows ADP to be phosphorylated to ATP using PCr as the phosphate donor. By contrast, several types of invertebrate possess phospho- arginine (PArg) instead of PCr 2. In earthworms yet another compound, phospholumbricine, contains a phos- phodiester bond besides a transferable phosphate 3. PArg matches most of the characteristics of PCr: it decreases in concentration during high-energy chal- lenge to a tissue; it occurs in approxi- mately the same concentration; it shows a similar distribution among various tissue; and there is an enzyme that cata- lyses the transfer of the compounds' phosphate to ADP to make ATP.

From this comparison comes the con- cept of the phosphorylated guanido nitrogen moiety being an appropriate substrate for energy storage in tissues subject to intense physiological strain. Even synthetic analogs containing phos- phorylatable nitrogens such as homo- cyclocreatine can be taken up in the unphosphorylated form and phosphoryl- ated like creatine 4.

Interestingly, NMR is a sensitive tool for indexing these compounds since 3~p spectra of PArg and PCr have chemical shifts that differ by 0.5 ppm 5. Similar spectra of various mammalian tissues revealed a series of compounds with resonances between those of PCr and inorganic phosphate (see Fig. 1) 6. Ini- tially listed as unknowns, they were later (after chemical isolation and a literature search) identified as phosphodiesters such as glycerolphosphorylcholine

(GPC) and glycerolphosphorylethanol- amine (GPE). A survey showed that their distribution varied between tissue types (e.g. higher in red muscle than white) and between species (e.g. gener- ally lower in rat tissue than in rabbit or man) 7. Table I shows the relative dis- tribution of phosphodiesters in several tissues and species.

From the mammal results, it is tempt- ing to conclude that the appearance of GPC or GPE is a measure of the break- down of phospholipids. This is sup- ported by the finding that rabbit red

C. T. Burr is at the Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, PO Box 12233, Research Triangle Park, NC 27709, USA.

muscle (soleus) which contains milli- molar concentrations of GPC has three times the activity of lysophosphohpase found in white muscle (extensor digi- torum longus) which contains little GPC 8. Also, isolated mouse livers per- fused with glucagon in saline have increased phosphodiester concentrations 9 and, according to the literature, glucagon also stimulates phospholipases. However, these conclusions seem pre- mature if comparative data are taken into account.

A prominent 31p-NMR resonance in the phosphodiester region was also seen in samples from chickens and turkeys of tissues corresponding to those in which phosphodiesters occur in mammals (see Table I). However, the resonance pos- ition was significantly upfield from known phosphodiesters (about 0.3 ppm from GPC at pH 7). The compound proved to be serine ethanolamine phos- phodiester (SEP) ~°. It is a compound which must be specifically synthesized and is not directly related to lipid break- down. The question is then: what else

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Fig. 1.3tp NMR spectra of isolated human chest muscle undergoing isehemia in an NMR tube. The two spectra are for the average over the first and second half-hour of NMR examination. Phosphocreatine, PCr, (the peak to the right o f the phosphodiester) decreases in area with time, while inorganic phosphate, P~ (to the left) increases. The area of the phosphodiester peak does not change appreciably. Between the two resonances is the peak for glycerolphosphorylcholinc, labeled phosphodiesters.

~) 1985, Elsevier Science Publislcrs B.V., Amsterdam 0376 - 5067/85/$02.00

T 1 B S - O c t o b e r 1985

Table 1. Phosphodiester concentrations and distribution in some representative tissue

405

Tissue Species Type Compound(s) Concentration Ref.

muscle rabbit red GPC >1 mM 8 white trace

human biceps GPC trace 17 gastrocnemius ~1 mM

toad gastroenemius GPC, SEP >1 mm 5 chicken red SEP >1 mM 17

white trace erythrocytes rabbit reticulocytes GPC, GPE >1 mM 18

red cells trace turtle red cells SEP -1 mM 19

semen ram whole GPC -100 mM 20 human whole GPC >1 mM 21 chicken whole SEP >1 mM 11

brain calf grey GPC, GPE <1 mM 22 white >1 mM

human whole GPC, GPE >1 mM 23 fish/reptile whole SEP, TEP" >1 mM 24

kidney dog cortex trace medulla GPC >1 mM 25 papillary ~100 mM

chicken total SEP >1 mM 26

• Threonine ethanolamine phosphate is present in teleosts.

besides a similar N M R resonance pos- ition could SEP and GPC have in com- mon? Remember that a similar N MR resonance position for PCr and PArg represents the tip-of-an-iceberg of many other biochemical similarities between them. Perhaps GPC and SEP behave similarly, even though PCr and PArg function in energetics while GPC and SEP are involved in lipid metabolism. Table I shows that, as for PCr and PArg, the concentrations and distributions of GPC and SEP are strikingly similar. Burt and Chalovich u used the analogy to predict and find SEP in chicken semen: this eluded previous investiga- tors who looked predominantly for GPC.

Having established this correlation, one can look for similarity in structure. An allowed configuration (using Carey- Paulin-Koltun (CPK) models) for GPC and SEP (see Fig. 2) reveals quite simi- lar configurations, with a hydrophilic and hydrophobic side and also a chain of oxygens on each, suggesting that both could serve a similar role in a lock-and- key enzyme mechanism.

It is at this point, where N M R and model-building have provided as much information as possible, that the argu- ment becomes more speculative. Never- theless, the inferential evidence, once a common role is assumed for the two types of phosphodiester, points in the same direction.

Preliminary results with GPC and lysophospholipase show that GPC at millimolar concentration can inhibit lysophospholipase by up to 50% 12,13. Such inhibition could lead to the

Hydroohilic Surface

Fig. 2. CPK (Corey-Paulin--Kolam) model o f serine ethanolamine phosphodiester (top) and glycerophosphorylcholine (bottom). Left: a view of the model with most of the oxygens showing. Right: the models viewed from the other side. The models have been aligned so that the amine ends (ethanolamine and choline) face each other at the midline. The atoms are marked as follows: plain - hydrogen, black - carbon, hatched - oxygen, striped - nitrogen and stipled - phosphorus. Note the hydrophilic surface of oxygens, the bent shape of the molecules, and the presence of permanent dipole at p H Z

build-up of lysolecithin as speculated by Burt and Ribolow 7. However, the litera- ture also reveals that the phospholipid content as a percentage of total lipid is appreciably lower in tissue containing phosphodiesters (e.g. brain white matter and red muscle) than in corresponding tissue of the same species which lacks phosphodiesters (i.e. brain grey matter and white muscle) 14,15. Thus, GPC and SEP may serve as endogenous lysophospholipase inhibitors to preserve the phospholipid composition in tissue in which the percentage phospholipid has fallen. This would be accomplished by promoting the futile cycle of phos- pholipid breakdown via phospholipase

Fig. 3. The distn'bution of glycerophosphoryl- choline (GPC) and serine ethanolamine phospho- diesterase (SEP) among vertebrates. Amphibians can have both GPC and SEP while reptiles and birds largely have SEP and mammals have the glycerophospholipid derivatives, GPC and GPE.

406

and reconstruction by transacylase, rather than complete phospholipid loss through breakdown by phospholipase and then lysophospholipase. Although this is an explanation for long-term cel- lular adaption, short-term effects may need to take into account inhibition of other phospholipid-processing enzymes, including the transacylases t6. However, in all long-term adaptions including many chronic disease processes, the interpretation of the appearance or dis- appearance of phosphodlesters pres- ented above holds.

Thus, a phenomenon which on the surface appears simple and easily explained becomes much more complex when a wider range of species is ex- amined. Indeed, evolutionary diver- gence is apparent in that mammals use GPC and GPE while reptiles and birds use SEP (see Fig. 3). However, there is experimental evidence for another scheme which proposes that phos- phodiesters are lysophospholipase inhibitors. This hypothesis presents a new mechanism by which some cells may control membrane composition.

Rlff~'ellom 1 Hill, A. V. (1965) Trails and Trials in Muscle

Physiology, pp. 164--167, Williams and Wilkins CO.

2 Roche, J. (1967) Biochim. Biophys. Acta 24, 514--519

3 Prosser, C. L. and Brown, F. A., Jr (1961) Comparative Animal Physiology, pp. 430-431, W. B. Saunders Co.

4 Roberts, J. J. and Walter, J. B. (1983) Arch. Biochem. Biophys. 220, 563-569

5 Bun, C. T., Glonek, T. and Barany, M. (1976) J. Biol. Chem. 251, 2584--2591

6 Glonek, T., Burt, C. T. and Barany, M. (1981) In NMR in Medicine (Damadian, R., ed.) pp. 121-159, Springer-Verlag

7 Butt, C. T. and Ribolow, H. J. (1984) BIO- chem. Med. 31, 21-30

8 Butt, C. T., Kleps, R. A., Kapin, M., Danon, M. J. and Perurena, O. (1982) Life Sci. 30, 39--44

9 Cohen, S. M. (1983) Z Biol. Chem. 258, 14294-14308

10 Chalovich, J. M., Burt, C. T., Cohen, S. M., Glonek, T. and Barany, M. (1977) Arch. BIO- chem. Biophys. 182, 683--689

11 Burt, C. T. and Chalovieh, J. M. (1978) BIO- chin*. Biophys. Acta 529, 186-188

12 Ribolow, H. J., Butt, C. T. and Martin, M. (1981) Biophys. Z 33, 251a

13 Ribolow, H. J. and But't, C. T. (1981) Fed. Proc. 40, 1632

T I B S - October 1985

14 Sarzala, M. G., Szymanska, G., Weihrer, W. and Pette D. (1982) Eur. J. Biochem. 123, 241-245

15 Spector, W. S., ed. (1956) Handbook of Bio- logical Data, p. 70, W. B. Saunders and Co.

16 Gross, R. W. and Sobel, B. E. (1981) Clin. Res. 29, 201a

17 Chalovich, J. M., Burt, C. T., Danon, M. O., Glonek, T. and Barany, M. (1979) Ann. N. Y. Acad. Sci. 317, 649-669

18 Labotka, R. J., Glonek, T., Hruby, M. A. and Honig, G. R. (1976) Biochem. Med. 35, 311- 329

19 Armda, J. A. L., Lubansky, H., Dytko, G., Mola, R., Kleps, R. A. and Burr, C. T. (1984) Min. Elec. Metab. 10, 233-238

20 Scott, T. W., Wales, R. G., Wallace, J. C. and White, I. G. (1963) J. Reprod. Fertil. 6, 49-59

21 Arrata, W. S. M., Butt, C. T. and Corder, S. (1978) Fertil. Steril. 30, 329-333

22 Butt, C. T., Pluskal, M. and Sreter, F. (1983) Biophys. J. 41, 141a

23 Hope, P. L., Costello, A. M. de L., Cady, E. B., Delby, D. T., Torts, P. S., Chu, A., Hamilton, P. A., Reynolds, F. D. R. and Wilki¢, D. R. (1984) Lancet ~, 366-370

24 Porcellati, G., Floridi, A. and Ciammaruglti, A. (1965) Comp. Biochem. Physiol. 14, 413-418

25 Schimassek, H., Kohl, D. and Bucher, Th. (1959) Biochem. Z. 331, 87-97

26 Chalovich, J. M. (1978) PhD Thesis, Univer- sity of Illinois Medical Center, Chicago, p. 58

Biochemical Explanations for Folk Tales

Auslralian madness and mad hatters: a unifying hypothesis

Responding to the TIBS Editor's request for biochemical explanations of folk-tales and literary references, R. K. Poole wrote in the June issue that the origin of the term 'mad as a hatter' popularized in Louis Carroll's Alice in Wonderland was subject to two inter- pretations t. He indicated that the term 'hatter' in Australia refers to lonesome prospectors overtaken by madness pre- sumably because of their solitary life- style. An alternative, more widely held, view of the origin of the term is that it refers to the psychiatric symptoms of felt-hat makers suffering from inorganic mercury intoxication.

At the time Alice in Wonderland was penned, the great American and Euro- pean hatteries produced many cases of inorganic mercury intoxication by the use of a solution of mercury and nitric acid as a stiffening agent or 'carrot' for felt hats. The psychiatric symptoms of intoxication were referred to by the great physician Kussmaul as 'erythismus mercurialis' characterized by difficulty in concentrating, insomnia, impaired judgement, easily aroused anger, and maniacal excitability 2. Another very common neurological symptom was

tremor which often preceded the psychi- atric manifestations, and which, on occasion, was sufficiently severe that workers had to be helped to their benches lest they lurch into the factory machinery. Mercury intoxication was extraordinarily common among hatters, affecting 37% of workers in 'good' fac- tories, and up to 89% in 'bad' factories according to industrial hygienist Alice Hamilton in her definitive work 'Indus- trial diseases of fur cutters and hatters '3. This occupational hazard was well known to government officials and edu- cated persons alike, and it appears prob- able that the author of Alice in Wonderland knew of this condition, and incorporated it into his story.

The origin of the use of the term in Australia is problematical, but it should be noted that workers other than hatters suffered from mercury intoxication. Among these were mirror silverers, gilders, scientific instrument manufac- turers, and especially relevant to Poole's observations, miners and gold refiners. Is it possible that the lonely mad Aus- tralian workers suffered from inorganic mercury poisoning and that the origina- tors of the Australian colloquialism 'hat-

ter' were familiar with the European industrial affliction?

Regardless of the validity of these lin- guistic speculations, it is clear that inorganic mercury intoxication was a very common occupational hazard in early industrial Europe and America, whereas more recent industrial disasters have involved organic mercurials. The precise biochemical basis of mercury intoxication remains obscure, largely because of the remarkable diversity of metabolic sites adversely impacted by mercur#, 5.

References 1 Poole, R. K. (1985) Trends Biochem. Sci. 2 Cowers, W. R. (1888) 10, V A Mammal of Dis-

eases of the Nervous System, pp. 1275-1277, P. Blakiston, Son, & Co., Philadelphia

3 Hamilton, A. (1922) J. Ind. Hyg. 4, 219-234 4 Chang, L.W. (1980) In Experimental and

Clinical Neurotoxicology (Spencer, P.S. and Schwaumburg, H. H., eds), pp. 508-526, Williams and Wilkins

5 Kark, R. A. P. (1979) In Handbook of Clinical Neurology, Vol. 36 (Winkan, P. J., and Bruyn, G. W. eds), pp. 147-197, Elsevier North-Hol- land

J. CLAY GOODMAN

Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA.