VOL. 39, 1953 ZOOLOGY: T. F. GOREA U 1291

PHOSPHOMONOESTERASES IN REEF-B UILDING By THOMAS F. GOREAU UNIVERSITY COLLEGE OF THE WEST INDIES, MONA, JAMAICA, B. W. I. Communicated by G. E. Hutchinson, October 16, 1953 Alkaline and acid phosphomonoesterases have now been found in a num- ber of West Indian reef corals. This is believed to be the first demonstra- tion of these enzymes in the coelenterata. Although this report is con- fined to a description of the phosphomonoesterases found in 26 of corals belonging to eight different families,' our evidence also shows that such enzymes exist in at least one zoanthidean, Palithoa caribaea, and a hydrocoralline, Millepora alcicornis. Materials and Methods.-With one exception, all the species used for this study were collected from shallow fringing reefs at Lime Cay, near Kings- ton, and on the North Coast near the town of Ocho Rios. The freshly collected was promptly drained and kept in jars on ice for not more than two hours, then stored in deep freeze until ready to use. The coral tissue homogenate was prepared by carefully cleaning 50 g. of the freshly frozen coral of all the encrusting growths and dead corallum. The remainder was pounded to fine bits in a mortar, triturated in a blen- dor with 200 ml. distilled water and centrifuged at 2000 r. p. m. for ten minutes. The light brown supernate was then decanted away from the precipitate and stored in a refrigerator with a few milliliters toluene as pre- servative. The precipitate was seen to be composed of zooxanthellae, nematocysts and finely ground corallum. Phosphatase analyses of the washed precipitate showed low orders of activity which is attributable to remaining bits of coral tissue rather than to intrinsic activity of the zooxan- thellae. The plI optima of the phosphomonoesterases were determined as fol- lows: 5 nml. aliquots of an ice cold suspension containing 25 ml. of the fresh homogenate, 50 ml. 10% beta-sodium glycerophosphate and 100 ml. of a 1:1 0.2 N glycine-NaOH buffer were pipetted into volumetric test tubes. Fifteen points were chosen in the pH range 11.50 to 9.60 and an identical number tetween pH 3.50 to 6.00. The pH in each tube was then carefully adjusted to one of these points with 0.2 N NaOH or 0.2 N HAc using glass electrodes. The tubes were allowed to incubate in a thermostat at 370C. and the reaction stopped with TCA at the end of one hour. Final pH was measured at first owing to the possibility of drift, but as this was seldom more than 0.1 unit in the course of an experiment, this step was omitted in most of the final determinations. After stopping the runs, the volumes were made up to 10 ml., and 2 ml. aliquots were taken for colorimetric phos- phate analysis according to the method of Taussky and Shorr.2 There was Downloaded by guest on September 26, 2021 1292 ZOOLOGY: T. F. GOREA U PROC. N. A. S.

usually a slight turbidity in these samples owing to the presence of small amounts of mucopolysaccharide-like substances which were not precipita- ted by 'fCA. Controls run under identical conditions but without the phosphate determinations showed that the optical density of the turbid material remained constant over a pH range of 3.40 to 11.80 and that the effect could be corrected for by running the experimental TCA control as the color blank.

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3 4 5 6 7 e 9 10 11 12 pH FIGURE 1 Sample plot showing pH optima of P-ases from Mussa angulosa Results.-The activity versus hydrogen ion concentration curves for scleractinian alkaline phosphomonoesterases are markedly different from previously published data which are derived exclusively from mammalian material.3-5 In all except one of the species examined, the optimum ac- tivity is between pH 10.50 and 11.10 (data corrected for sodium error of the glass electrodes). The activity falls off with very great rapidity on either side of the optimum pH, producing in most cases a needle sharp peak (Fig. 1). The mean optimum pH of 25 species, not including Porites divaricata, is in the range of 10.85 to 10.90 with relatively small degree of scatter on either side of the mean (Fig. 2). Downloaded by guest on September 26, 2021 VOL. 39, 1953 ZOOLOG Y: T. F. GOREA U 1293

The acid phosphomonoesterase optima have a much wider scatter in the pH range of 3.60 to 5.50. The peaks are blunt and rounded and the en- zyme activity is very variable in intensity relative to the degree of alkaline phosphomonoesterase activity. In four cases the acid phosphomonoesterase activity was not measurable, but it remains uncertain whether this is due to actual absence, excessive dilution, inhibition or deterioration of the enzyme. The pH optima of the phosphomonoesterases of 26 species of corals are listed in table 1. It will be noted that local representatives of the families Astrangiidae, Trochosmiliidae, Astrocoeniidae, and Seriatoporidae, eight species in all, were not available and had to be omitted from this survey.

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Q.6 9.7 9.8 Q9 M0 IQI 1O2 IQ3 14 K)S Q6 10.7 Q8 lQ9 11.0 11.1 -H FIGURE 2 Frequency distribution of alkaline P-ase optima from 26 species of Jamaican reef corals. Discussion.-The exceedingly high pH optima of the alkaline phospho- monoesterases of reef corals are very interesting when considered in the light of possible adaptive advantages they may have for rapid and efficient deposition of CaCO3 into the corallum. Due to the fact that enzyme activ- ity falls off so very rapidly with even small pH changes away from the op- timum, and assuming these enzymes to have specific internal functions, it becomes necessary to postulate that very high physiological pH values can occur in tlhe coral . Several mechanisms for achieving this are pos- sible. CO2 can be lost from the body fluids either through photosynthetic fixation by zooxanthellae or by precipitation into the corallum as carbonate. The same result would also be produced through massive localized produc- Downloaded by guest on September 26, 2021 1294 ZOOLOG Y: T. F. GOREA U PROC. N. A. S.

tion of ammonia at night. It is naturally possible to look at the probleni from the other side and postulate that carbonate deposition is the mech- anism whereby the high pH necessary for the function of these particular TABLE 1 ALKALINE ACID FAMILY AND SPECIES* OPTIMUM PH OPTIMUM PH Faviidae natans 10.78 4.50 Diploria clivosa 10.86 4.50 Diploria strigosa 10.86 4.50 Diploria labyrinthiformis 10.86 4.50 areolata 10.86 4.20 Cladocora arbusculata 10.75 Montastrea annularis 10.53 4.50 Montastrea cavernosa 10.64 5.50 Favia fragum 10.86 4.50 Oculiniidae diffusa 10.53 5.00 Mussa angulosa 10.86 5.00 Isophyllastrea rigida 10.75 Mycetophyllia lamarkana 10.86 4.50 Isophyllia sinuosa 10.86 4.50 Isophyllia multiflora 10.96 4.90 Caryophylliidae Eusmilia fastigiata 10.86 5.50 Acroporiidae Acropora cervicornis 10.96 4.00 Acropora palmata 10.96 4.00 Acropora prolifera 10.96 4.00 agaricites 10.75 Agaricia fragilis 10.86 Siderastreidae Siderastrea siderea 10.86 3.60 Siderastrea radians 10.75 3.80 Poritidae Porites astreoides 11.07 4.80 Porites porites 10.96 5.00 Porites furcata 10.86 4.50 Porites divaricata 9.70 3.80 * Generic and specific nomenclature was taken from Smith's monograph on Atlantic reef corals.6 Familial nomenclature for the species cited is according to Vaughan and Wells.' phosphomonoesterases is produced. This may well be the case if these enzymes are not concerned with calcification at all. It is hoped that work now in progress will help to clarify this problem. The alkaline phosphomonoesteraseof Porites divaricata appears tobe a spe- Downloaded by guest on September 26, 2021 VOL. 39, 1953 NATIONAL ACADEMY OF SCIENCES: E. B. WILSON 1295

cial case that deserves some discussion. The optimum pH in this species is 9.70 whereas the mean value for all the other species is 10.86. The peak is also not so sharp and much wider at the base than the others. There is strong suspicion that this difference may be the result of environmental changes. The specimen of Porites divaricata on which these analyses were performed was the only coral specimen in this series which was not collected in shallow, clear and sunny reef waters that are the normal environment of reef building . This particular specimen was collected from the shady side of a submerged sea wall, growing at a depth of more than fifteen feet in extremely turbid water at Port Royal. It is hoped that additional specimens will be collected from other localities to investigate the possibility that environmental changes might produce adaptive changes in the func- tional characteristics of alkaline phosphomonoesterases in corals. Investigations to determine the specific localizations of these enzymes in coral tissue are now in progress. This work, together with that described above constitutes part of a more generalized research program being carried out on this laboratory on problems of growth and calcium metabolism in reef corals. I Vaughan, T. W., and Wells, J. W., Geol. Soc. of America, Special Paper No. 44 (1943). 2 Taussky, H. H., and Shorr, E., J. Biol. Chem., 202, 675 '1953). 3 Belfanti, S., Contardi, A., and Ercoli, A., Biochem. J., 29, 517 (1935). 4 Kroon, D. B., Neumann, H., and Krayenhoff-Sloot, W. J. A. T., Enzymologia, 11, 186 (1944). 5 Roche, J., and Bullinger, E., Ibid., 7, 278 (1939). 6 Smith, F. G. W., Atlantic Reef Corals, University of Miami Press (1948).

VITAL STA TISTICS OF OUR FOREIGN ASSOCIA TES By EDWIN B. WILSON OFFICE OF NAVAL RESEARCH, BOSTON Read before the Academy, November 9, 1953 Many years ago incident to committee considerations about elections to the Academy, Raymond Pearl' prepared reports on "Vital Statistics of the National Academy of Sciences." More recently Fred E. Wright,2 who served us long as Home Secretary, prepared another analysis. It seems now desirable to prepare a brief analysis about our foreign associates; their numbers are too few to justify either great length or much technique in the treatment. From such records as appear in our Annual Report supplemented by data available in the office of the Home Secretary and supplied by his staff,3 Downloaded by guest on September 26, 2021