BIOINORGANIC

CHEMISTR Y 5,189-l

Inorganic Polyphosphate

of Alkaline Phosphatase

189

97 (1976)

as an Idegral Preparations

Part

NORMAN W. GABEL Laboratory of Chemical Evolution. Department of Chemirtry. University of Maryland, College Park, Maryland 20742 and VIRGINIA THOMAS Research Department, Illinois State Psychiatric Institute, 1601 W_ Taylor Street, Chicago. Illinois 60612 ABSTRACT Alkaline phosphatase (from chicken intestinal sources) was shown to contain a considerable amount of polyanionic phosphorus which was released by basic digestion. The polyanionic phosphorus of alkaline phosphatase is not associated with protein or polyalcohols and does not exhibit a visible or ultraviolet absorption spectrum_ Alkaline phosphatase and abiogenic inorganic polyphosphate were found to incorporate 3 * P-orthophosphate under similar experimental conditions. It has been previously reported that this enzyme will incorporate “P-orthophosphate into its protein phosphoserine without the apparent concomitant utilization of an energy source. This reported phosphorylation was immediately reversible upon dilution of the phosphorylated enzyme with unlabelled orthophosphate, which indicates that the initial phosphorylation was an exchange reaction. These observations suggest that this polyanionic phosphorus from alkaline phosphatase may be inorganic polyphosphate.

INTRODUCTION Alkaline phosphatase is the most overt example of a phosphoprotein that directly incorporates orthophosphate without the apparent concomitant utilization of an energy source. It was reported in 1958 that the enzyme (from calf-intestinal sources) incorporates Pi into phosphoserine under acidic conditions [ I] _ The amino acid sequence around this phosphorylated serine subsequently was shown to be asp-serP-ala from both E. coli and calf-intestinal sources [2,3] _ Schwartz [4] demonstrated that this phosphorylation was immediateIy reversibIe (IS seconds) upon dilution of the phosphorylated enzyme with unlabelIed Pi, which suggested that the initial phosphorylation was an exchange reaction_ In spite of the disposition of “P as phosphoserine, it 0 American Elsevier Publishing Company, Inc., 1976

190

NORMAN

W. GABEL

AND VIRGINIA

THOMAS

seemed unreasonable to us that a phosphate-ester bond, the hydrolysis of which has a smalI free energy and is coupled with a large free energy of activation, could participate in any exchange reactions under these conditions. Since it is our contention that rapidly exchangeable phosphoproteins contain, as part of their structural components, inorganic polyphosphate chains that would readily exchange with Pi [5,6,], we decided to test the possible existence of polyphosphates as part of the alkaline phosphatase preparation. Inorganic polyphosphates have been shown to occur throughout the biosphere [6,7] and have been proposed to have played a role in the structuraiization [S,9] and energetics of prebiological systems [ 10,ll I _

MATERIALS

AND METHODS

E.C.3.1.3.1., (Worthington Biochemical In general, alkaline phosphatase, Corp., Freehold, New Jersey) or a synthetic polyphosphate in 10 ml. of a 0.002 M acetate buffer (sodium or potassium) at pH 5 was treated at 4OC with 0.6-I .2 mC of Naa H32P04 (New England Nuclear Corp., Boston, Mass.) for 30 min. [12,13]_ Sufficient KOH pellets then were added to make the solution 0.33 M KOH. The resultant solution was heated at 40° for 18 hrs [5] and was then applied to an anion-exchange chromatographic column of Dowen l-X8 (formate form) as described by Tanzer and Krichevsky [ 14]_ The Nap H32 PO4 itself contained less than 0.01% of polyanionic phosphorus, which was removed prior to use by ion-exchange chromatography [IS] _ The water washings and the eluted fractions that contained phosphorus were analyzed for protein by the method of Lowry, Rosebrough, Far-r, and Randall [ 16]_ Phosphorus determinations were obtained by the method of Bartlett [ 17]_ The eerenkov radiation of the 32P was measured directly in aqueous portions of eluate from the anion-exchange column using a Packard Model 4322 Spectrometer [ 18]_ Abiogenic inorganic polyphosphate was obtained from Dr. E. J. Griffith (inorganic Research Department, Monsanto Industrial Chemicals Company, St Louis, MO.).

RESULTS The experiment illustrated by Fig. 1 was carried out to demonstrate the ease with which abiogenic inorganic polyphosphate incorporates orthophosphate. The exchange-reaction appears to be somewhat more extensive with pyrophosphate than with the phosphate material eluted by 5.0 M ammonium formate. The pyrophosphate was probably part of the original polyphosphate preparation while the major portion of eluted orthophosphate presumably arose from the “P-orthophosphate introduced during the experiment. The chicken intestinal alkaline pbosphatase (Fig. 2) was shown to contain a considerable amount of polyanionic phosphorus that could be released by basic digestion_ These polyanionic fractions, eluted by 5.0 M ammonium formate, did not contain

PIG. I. Anion-cxchnngc column cliromatogr;lpby of 5.0 mg of sodium polyphosphtc with nn avcmgc chin-Jcngth of 5.5 (dctcrmincd by ia 10 ml of 0.002 M sodim ;mlnk btrffer (pH 5.0) at 5’ C for 30 min. end-group titration) which had been trcatcd with Na,ll “PO,, Sufficient ROll pellets WC added to make the solution 0.33 M KOH. This mixture ww hted at 40” C for 18 brs. and was then poured directly onto ;I Dowcx I-X8 (formatc form) CO~UIIIII, 12.5 cm X 1,O cm, followctl by 20 mlof wntcr washings. The N,O -) 4-O M formic acid grndicnt (ortlrophosphatc) was producctl by ;I constnnl lcvcl clution apparatus consisting of two cylindrical bottjcs of cqual diameter in tnlldcm with 225 ml of I-I,0 nnd 225 ml of 4.0 M formic acid in ench, rcspcctivcly, This wns followed by 300 ml of a solution which was 0.5 M anmonium formntc and 3,s M formic acid (pyrophosphte). Finally, 150 ml of 5.0 M ammonium forma wns pnsscd through the column (lripolyphosphntc nnd higher oligomcrs.) Fractions of 9.0 ml were collected nt approximately 0.75 ml/min.

192

NORMAN

W. GABEL

AND VIRGINIA

THOMAS

POLYPHOSPHATES

AND ALKALINE

PHOSPHATASE

193

protein [ 161 or polyalcohols [ 191 and did not exhibit an ultraviolet or visible absorption spectrum_ Approximately 90% of the enzymatic protein was recovered from the aqueous washings during application of the sample to the anion-exchange column. In addition to the 32Pi incorporated into the polyanionic phosphorus, some 3 * Pi was incorporated into other fractions eluted by the solution which was 0.5 M ammonium formate and 3.5 M formic acid. From its elution-pattern, the material was apparently not pyrophosphate. No further identification was attempted, but the distribution of incorporated 32Pi would seem to indicate that this material contained more than one component. The time-course for- the incorporation of 32P-orthophosphate into alkaline phosphatase and abiogenic inorganic polyphosphate was followed for 15 min. (I:ig. 3). The rates of incorporation were virtually identical under the conditions of the experiment_ The rate-limiting parameter was apparently the concentration of radioactive phosphate_ In order to determine whether the incorporation of 32Pi into the polyanionic phosphorus was dependent on the viability of the enzymatic activity of alkaline phosphatase, the enzyme was first boiled for 30 min. in 0.002 M sodium acetate buffer (pH 5.0) and then cooled to 4O C before the addition of the 32Pi to the incubation mixture_ After basic digestion and application to the anionexchange column, radioactive phosphorus was again found in the material

1

IO TIME (minuted 20 3. In triplicate, 5.0 mg of sodium polyphosphste (average chain-length of 5.5) dissolvedin 10 ml of 0.002 M sodium acetate buffer (pH 5.0) was treated with Na, H “PO, at 5” C. At intervals of 5, 10, and 15 min sufficie& KOH pellets were added to one of the samples to make the solution 0.33 M KOH. These.miutures were then heated at 40” C for FIG.

18 hrs. and poured onto individual Dowex 1-X8 (formate form) columns. Following 20 ml of water washing, each column was eluted wiith 300 ml of a solution which was 0.5 M ammonium formate and 3.5 M formic acid. Elution was c6ntinued with 5.0 M ammonium formate during which the first 50 ml was collected for radioactive counting. In a second experiment the above procedure was followed substituting 40.0 mg of chicken intestinal alkaline phosphatase for the 5.0 mg of sodium polyphosphate.

194

NORMAN WV.GABEL AND ViRGINIA THOMAS

eluted by 5.0 M ammonium formate (Fig. 4). The most noteworthy change brought about by boiiing the enzyme was that more than half of the phosphorus, which had been previously eluted as in Fig. 2, was now found in the aqueous washings with the protein when the basic digestion mixture was applied to the column. Pyrophosphate with a large incorporation of 32Pi appeared in -the boiled enzyme preparition and the amount of unknown phosphate material previoudy eluted by 0.5 M ammonium formate and 3.5 M formic acid was greatIy diminished. The effect of the cation of the buffer on the incorporation of 32Pi into the polyanionic phosphorus was determined by substituting polassium acetate buffer for the sodium acetate buffer. The incorporation of 32Pi into the material eluted by 5.0 M ammonium formate decreased and pyrophosphate with a relatively high specific radioactivity appeared in the eluted fractions. A further comparison of the effect of substituting potassium for sodium as the buffer cation was made with the abiogenic inorganic polyphosphate having an average chain-length of 5.5. A very sharp decrease in the incorporation of 32Pi was the result_ The differences of incorporation of 32Pi, due to changing the buffer cation, are summarized in Table 1. The relative specific radioactivities of the poiyanionic phosphorus materials were caIculated by dividing their specific radioactivities by the specific radioactivity of the orthophosphate introduced in each experiment. When an alkaline phosphatase preparation from E. coli was subjected to the same treatment as the chicken-intestinal enzyme in sodium acetate buffer, the amount of 32Pi incorporated into the polyaniouic phosphorus was of the same order of magnitude_

DISCUSSION The occurrence of inorganic polyphosphates in microorganisms has been we&documented [ 71 and they are apparently distributed throughout vertebrate tissrres as well [51Several investigators have proposed that these inorganic polyphosphates are metabohc fossils of primitive bioenergetic systems [7,1 I], A contemporary, regulatory function in biological transport has been proposed [6,20 1, and a role in nucIeic acid metabolism has been indicated [2 I] _ The conditions for incorporation of 32Pi into the alkaline phosphatase preparations were chosen on the basis of what has been shown to result in the maximum amount of incorporation of orthophosphate into the enzyme [ 12,13]_ ProIonged alkaline digestion of proteins has been and still is the standard method for removing metabolically active phosphorus from mammalian phosphoproteins [22,23] _ This same method also is used for separating microbiological poiyphosphate from proteins and nucleic acids [ 7,14]_ Although the polyphosphates can be separated from other biopolymers under alkaline conditions, they themselves are not as readily degraded in base because their high density of negative charge impedes the nucleophilic attack of water and hydroxyi ion at the P-O bond [24] _ Acidic digestion or the boiling of

POLYPHOSPHATES

AND ALKALINE

PHOSPHATASE

196

NORMAN

W. GABEL

AND VIRGINIA

TABLE

THOMAS

1

Relative incorporation of 32P-orthophosphate into polyanionic phosphorus material in the presence sodium acetate or potassium acetate buffers_ Relative Sodium Alkaline phosphatase Polyphosphate (5.5)

Specific

acetate

4.0x 1cr3 3.5 x I@

of

Radioactivities= Potassium

acetate

8.2 x I@ 8.0 x lo-6

?he relative specific radioactivities of the polyanionic phosphorus materials were calculated by dividing their specific radioactivities by the specific radioactivity of the orthophosphate for each experiment.

polyphosphate-protein complexes would be expected either to hyrolyze the inorganic polyphosphate or phosphorylate the protein_ The decrease of polyanionic phosphorus in the boiled enzyme preparation with the concomitant increase in phosphorylated protein may be due to thermal phosphorylation of the enzymatic protein with inorganic polyphosphate. Similarly, acidic digestion procedures may be the source, as suggested by Rodnight et al. [25], of the 32P-labelled phosphoserine isolated from some neural phosphoproteins which 32P from either ATP y”P or 32Pi (231. in any isolation can incorporate technique applied to biological samples, the isolated materials should be considered to be possible reaction products of the isolation technique that arise from the original tissue components and the reagents introduced during the extraction procedure_ into both abiogenic, The similarity of the decrease in 32Pi incorporation inorganic polyphosphate and the polyanionic phosphorus derived from alkaline phosphatase when potassium was substituted for sodium as the cation of the buffer gives corroborative evidence for the identity of the enzymatic, polyanionic phosphorus as inorganic polyphosphate. Hems and Rodnight [26] have shown that sodium ion is necessary for the formation of the unstable phosphorylated intermediate of neural, microsomal ATPase while the additional presence of potassium ion enhances its breakdown_ Albers, Fahn, and Koval [27] had previously given evidence that potassium ion was associated with the phosphohydrolase activity of ATPase. In addition, the initial rates of incorporation of 32Pi into alkaline phosphatase and into abiogenic, inorganic polyphosphates are virtually identical. The results of the present experiments with alkaline phosphatase and abiotic polyphosphate would appear to suggest that inorganic polyphosphate may be an integral part of some materials, which have been designated as rapidly metabolizing phosphoproteins.

POLYPHOSPHATES

AND ALKALINE

PHOSPHATASE

197

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12_ I3_ 14 15. 16. 17. 18. 19. 20_ 21. 22. 23. 24. 2526. 27_

L. Engstrom, and G. Agren, Acta Chem Scand 12,357 (1958). C. Mihtein, Biochim, Biophys. Acta 67,171-172 (1963). L. Engstrom, Biochim. Biophys. Acta 92,79-84 (1964). J-H. Schwartz, Proc. Nat. Acad. Sci. (USA) 49,871-878 (1963). N-W_ Gabel and V. Thomas, J. Neurochem. 18, 1229-1242 (1971). N.W. Gabel, Perspectives Biot Med. 15,640-643 (1972). FM_ Harold, BacteriaL Revs. 30,772-794 (1966). N-W. Gabel. Life Sci 4,2085-2096 (1965). N-W_ Gabel, in Biogenesis-Evolution-Homeostasis (A. Locker, Ed.), Springer-Verlas.. New York, 1973, p_ 85 F. Lipmann, in The Origins of PrebioIogicai Systems and of their Molecular Matrices (S-W. Fox, Ed.), Academic Press, New York, 1965. p_ 195. IS. Kulaev, in ~folecular Evolution. Vol. I. Chemical Evolution and the Origin of Life (R. Buvet and C. Ponnamperuma, Eds.), North-Holland, Amsterdam, 197 1, p_ 458. J-H_ Schwartz and F. Lipmann, Proc. Nat. Acad. Sci. (USA) 47. 1996-2005 (1961). H_M_ Sarau, Ph.D. Dissertation, University of IBinois at the Medical Center, Chicago, Illinois, 1968. J-M_Tanzet and M.I. Krichevsky, Biochim. Biophys. Acta. 215, 368-376 (1970). W-B. Chess and D-N. Bemhart, AnaL Cf?em 31,1116 (1959). 0-H. Lowry, N.J. Rosebrough, A.L. Farr, and R.J. Randall, J. Biol. Chem. 193, 265-275 (1951). G, R. Bartlett, J. f3ioL Chem 234.466468 (1959). R-T. Haviiand and L.L. Bieber, AnaL Biochem. 33,323-324 (1970). M-K. Gaitonde and M. Griffiths, AnaL Biochem. 15,532~535 (i966). F-A. Deierkauf and Hi.. Booij, Biochim Biophys. AC?Q 150,214-225