- Email: [email protected]
The enthalpy change in the hydrolysis of the phosphate esters of myo-inositol
448
SHORT COMMUNICATIONS
two lines of evidence. First, when the sialic acids isolated from HeLa S 3 cells grown in presence of radioactive glucose were subjected to paper chromatography, a zone of radioactivity corresponding to the position of authentic NGNA was detected on the chromatogram (Fig. I). Second, hydrolysis of the material eluted from the NGNA zone of the paper chromatogram yielded radioactive glycolic acid as shown by recrystallizing a derivative to constant specific activity (Table I). HeLa Sa cells, a tissueculture line derived from a cervical cancer, represents the first material of human origin to be reported to contain NGNA. This investigation was supported in part by National Science Foundation Grant GB 6o43 and National Institutes of Health Grants AI o4954 and CA lO614. R.C. is recipient of a Research Career Development Award (I-K3-AM-38649) from the U.S. Public Health Service.
Oklahoma Medical Research Foundation and Department of Biochemistry, School of Medicine, University of Oklahoma, Oklahoma City, Okla. (U.S.A.)
R. CARUBELLI
M. J. GRIFFIN
I R. CARUBELLI A N D iV[. J. GRIFFIN, Science, 157 (1967) 693. 2 A. GOTTSCHALK,The Chemistry and Biology of Sialic Acids and Related S~tbstances, C a m b r i d g e U n i v e r s i t y Press, L o n d o n , 196o, p. 31. 3 L. SVENNERHOLM, Acta Chem. Scan&, 12 (1958) 547. 4 F~. SVENNERHOLM AND L. SVENNERHOLM, Nature, 181 (1958) 1154. 5 L. %VARREN, Nature, 186 (196o) 237. 6 E. I~LENK AND G. UHLENBRUCK,Z. Physiol. Chem., 307 (1957) 266. 7 G. TETTAMANTI, L. BERTONA, g . BERRA AND V. ZAMBOTTI, Italian J. Biochem., 13 (1964) 315 . 8 R. L. SHRINER AND R. C. FUSON, Identification of Organic Compounds, J o h n W i l e y a n d Sons, N e w York, 3rd ed., 1948, p. 157.
Received July 29th, I968 Biochim. Biophys. Acta, 17o (1968) 4 4 6 - 4 4 8
BBA 23472
The enthalpy change in the hydrolysis of the phosphate esters of myo-inositol Most of the phosphate in plant seeds is stored in the form of the hexaphosphate ester of myo-inositol (phytate) 1. The phytate is hydrolysed during germination to provide Pl for the developing embryo 1. Recent data indicate that in addition to its role as a reservoir of phosphate, phytate might also function as a phosphoryl donor in reactions with nueleoside diphosphates forming nucleoside triphosphates~, a. These recent findings indicate that at least one of the phosphate groups of phytate has a high phosphoryl transfer potential (so-called "high-energy" phosphate) and that phytate might function as a plant phosphagen 2. In an attempt to verify this proposal, measurements have been made of the enthalpy change during hydrolysis of inositol polyphosphates by phytase (myo-inositol hexaphosphate phosphohydrolase, EC 3.1.3.8). Biochim. Biophys. Acta, I7O (1968) 448-451
449
SHORT COMMUNICATIONS TABLE
I
THE HEAT OF HYDROLYSIS ~JHapp FOR THE PHOSPHATE ESTERS OF INOSITOL H e a t e v o l u t i o n w a s m e a s u r e d w i t h a m i c r o - c a l o r i m e t e r of t h e t y p e d e s c r i b e d b y EVANS AND CARNEY 7. O n e of t h e t w i n t a n t a l u m r e a c t i o n c e l l s c o n t a i n e d I m l of p h y t a s e s o l u t i o n i n o.2 M a c e t a t e b u f f e r ( p H 5.2), t h e o t h e r c o n t a i n e d b u f f e r o n l y . T h e s u b s t r a t e f o r e a c h r e a c t i o n (o.I m l o f t h e i n o s i t o l p h o s p h a t e e s t e r , e q u i v a l e n t t o 6 - 2 0 / * m o l e s of P t i n 0.2 M a c e t a t e b u f f e r ( p H 5.2), w a s p l a c e d i n t w o s y r i n g e s a n d d i s c h a r g e d s i m u l t a n e o u s l y i n t o t h e r e a c t i o n cells t o i n i t i a t e h y d r o l y s i s . T h e t e m p e r a t u r e w a s 25 ° . T h e h e a t e v o l u t i o n w a s r e c o r d e d g r a p h i c a l l y a n d t h e t o t a l h e a t p r o d u c t i o n d e t e r m i n e d b y i n t e g r a t i o n of t h e h e a t - t i m e c u r v e . T h e r e a c t i o n w a s s t o p p e d b y t h e a d d i t i o n of I m l o f I M N a O H a f t e r a p p r o x . 7 o % of e s t e r p h o s p h a t e g r o u p s h a d b e e n h y d r o l y s e d ( 4 0 - 9 ° r a i n ) . T h e P t f o r m e d w a s d e t e r m i n e d b y t h e m e t h o d of W E t L - M A L H E R B E AND GrREEN 8.
Compound
Pi formed (tzmoles)
Heat (mcal)
Heat of hydrolysis (cal/mole of Pi)
3/Iean AHap~
-- 2720 --285o --2830 -- 313 ° --3 °20 --297 ° -- 261o --329 ° --2560 --320o --257 ° --2760 --4300 --381o -- 3900 o o o --221o --224 ° -- 6000 --5900
--2800
AHapp Inositol hexaphosphate
Inositol pentaphosphate
Inositol tetraphosphate
Inositol triphosphate
Inositol diphosphate
Inositol monophosphate
Glucose 6-phosphate ATP
6.06 5.96 5.96 6.75 5.93 5.83 8.09 6.71 7.51 4.25 4.55 5.4 ° 5.21 5+9o 5.97 3.12 2.81 3.41 5.3 7.6 13.6 12.4
16. 5 17.o 16.9 2 I. i 17-9 17.3 21. I 22.1 19.2 13.6 11.7 14.9 22. 4 22.5 23.3 o o o i1. 7 17.o 8 i. 7 73.2
-- 304 °
-- 282o
--2840
--4000
--222o -- 595 °
The phosphate esters of myo-inositol were prepared from sodium phytate by controlled hydrolysis with phytase at p H 5.2. The various phosphate esters were separated b y ion-exchange chromatography using Dowex AGI-X8 resin (C1- form) and isolated as the barium salts 4. Each of the isolated esters appeared as a single major spot after paper electrophoresisS; the penta- and tetraphosphate esters showed additional minor spots which were presumably different isomers. The barium salts were converted to the free acid form b y using Dowex-5o resin (H + form), neutralized with NaOH and adjusted to 0.2 M with respect to sodium acetate buffer (pH 5.2). Phytase was prepared from wheat bran and purified as described b y lXTAGAIAND FUNAHASHI~ up to the stage of methanol fractionation. The heat evolved and the amount of Pt liberated b y the action of phytase is shown in Table I. The calculated heat of hydrolysis is expressed as callmole of Pt formed. No allowance has been made for the heat of ionization of the buffer, consequently the heat of hydrolysis can only be considered as an apparent value (AHa~p). Biochim. Biophys. Aeta,
17 ° (1968) 4 4 8 - 4 5 1
45 °
SHORT COMMUNICATIONS
Since the heat of ionization of acetate buffer is small compared to other buffer systems 9 the correction which would be applied if a H ÷ were produced would be approx. 2 mcal which would reduce the AHapp by approx. 2oo cal/mole. Because of the relatively small quantities of heat produced, the estimated error involved is of the order of zh 3oo cal/mole. Thus there is no significant difference in AHapp of hydrolysis of the inositol hexa-, penta-, tetra-, or triphosphate esters of inositol. There is, however, a significant increase in the value of AHapp of hydrolysis of the inositol diphosphate compared to the other esters. No heat evolution could be detected with the samples of inositol monophosphate, although Pl was liberated as shown in Table I. The AHaop for the hydrolysis of ATP ( 595 ° cal/mole) shown in Table I represents mainly the mean for each of the two terminal phosphate bonds since Pl analysis and thin-layer chromatography of the reaction mixture showed that 91% of the phosphate was hydrolyzed and the only nucleoside phosphate present after hydrolysis was AMP. This figure is similar to the value obtained by PODOLSKV AND STURTEYANT10 ( 5300 ± 600 cal./nlole) for the hydrolysis of the terminal phosphate of ATP by myosin (ATP phosphohydrolase, EC 3.6.1.3) at pH 8. The AHapp obtained for the samples of inositol hexa-, penta-, tetra-, and triphosphate esters show that the phosphate-ester bonds of these compounds have heats of hydrolysis similar to sugar monophosphate esters such as glucose 6-phosphate (AHapp --22oo ± 3oo cal/mole, see Table I), rather than the so-called "high-energy" compounds such as ATP 1° or creatine phosphate; AH-.9ooo ± 5oo cal/mole (ref. II). The only inositol phosphate ester which showed a significantly higher AHapp was inositol diphosphate. The inositol diphosphate formed by hydrolysis of inositol hexaphosphate with phytase is predominantly inositol 1,2-diphosphate~; further hydrolysis yields the inositol 2-monophosphate and finally inositol. The phosphate group at position 2 is the only axially oriented phosphate group and is considered to be relatively inaccessible to enzymic hydrolysis, it has been proposed that the phosphate group at position 2 of inositol would have a high phosphoryl transfer potential, considering the high charge repulsion exerted on this phosphate group by the adjacent cis-phosphates ~a. The inability to detect heat evolution during the hydrolysis of the inositol monophosphate m a y be due to the relatively low velocity of the hydrolysis of this ester compared to the higher phosphate esters of inositol. With the calorimeter used in these studies small quantities of heat produced over a long period of time (in the case of the monophosphate ester, 9 ° min) would be difficult to detect. In terms of heat available on hydrolysis the results here would suggest that inositol hexaphosphate does not possess a phosphate group of sufficient "high energy" to act as a donor of a phosphoryl group in the formation of a nucleoside triphosphate. The results do not, however, exclude the possibility that the more thermodynamically significant free energy change of hydrolysis is not sufficient to allow for the transfer of a phosphoryl group from phytate to a nucleoside diphosphate, as indicated in reactions carried out at higher p H values and in the presence of Mg2+ (refs. 2, 3). GELLERT ANI) STURTEVANT11 have shown that the AH of hydrolysis of creatine phosphate is --9000 cal/mole (compared to the AH of ATP of --53oo cal/mole), but the standard free energy of hydrolysis of these two compounds is nearly equal. Thus the standard entropy of hydrolysis of creatine phosphate must be more negative than
Biochim. Biophys. Aria, I7o (1968) 448-451
SHORT COMMUNICATIONS
451
that of ATP by about 13 entropy units/mole (ref. II). The same considerations could be applied to inositol hexaphosphate. Using [a2P]inositol hexaphosphate it has been shown that the transfer of a phosphoryl group from inositol hexaphosphate to GDP forming GTP is reversible under physiological conditions 3, indicating that the standard free energy of hydrolysis of inositol hexaphosphate is of the same order as the standard free energy of hydrolysis of GTP. Since the enthalpy of hydrolysis of inositol hexaphosphate, as determined here, is less negative than the enthalpy of hydrolysis of ATP, the entropy of hydrolysis of inositol hexaphosphate is probably more positive than that of ATP by approx. 8 entropy units/mole. On the basis of NMR studies, TATE14 recently proposed that inositol hexaphosphate consists of a hexaorthophosphate structure rather than a tripyrophosphate structure suggested by other workers 1~. If a pyrophosphate structure was present in inositol hexaphosphate the AH of hydrolysis as determined here would have included the AH of hydrolysis of both the pyrophosphate and the Pl bonds, since phytase is capable of hydrolysing both types of phosphate bonds 1~.The AH of hydrolysis of the pyrophosphate bond is of the order of --3000 cal/mole 17. The AH of hydrolysis of inositol hexaphosphate containing a pyrophosphate linkage should thus exceed the AH of hydrolysis of inositol pentaphosphate by --3000 cal/mole. Since there was no significant difference in the AH of hydrolysis of the inositol hexa-, penta-, tetra-, or triphosphate the results reported here support the hexaorthophosphate structure for inositol hexaphosphate. One of the authors (J. K. R.) is a Post-doctoral Research Associate.
Seed Protein Laboratory, U.S. Department of Agriculture, New Orleans, La. (U.S.A.)
J O H N K. RAISON* WILLIAM J. EVANS
I P. K. STUMPF, in W. D. MCELROY AND B. GLASS, Phosphorus Metabolism, Vol. 2, J o h n s Hopkins, Baltimore, 1952, p 29. 2 R. K. MORTON AND J. K. RAISON, Nature, 200 (1963) 429. 3 S. BlswAs AND B. B. BlSWAS, Biochim. Biophys. Acta, lO8 (1965) 71o. 4 D. J. COSGROVE, Australian J. Soil Res., i (1963) 203. 5 U. B. SEIFFERT AND B. W. AGRANOFF, Biochim. Biophys. Acta, 98 (1965) 574. 6 Y. NAGAI AND S. FUNAHASHI, Agr. Biol. Chem. Tokyo, 26 (1962) 7947 W. J. EVANS AND W. B. CARNEY,Anal. Biochem., I I (1965) 449. 8 H. WEIL-MALHERBE AND R. H. GREEN, Biochem. J., 49 (1951) 286. 9 I(. S. PITZER, J. Am. Chem. Soc., 59 (1937) 2365 . io R. J. PODOLSKY AND J. M. STURTEVANT, J. Biol. Chem., 217 (1955) 603. 11 M. GELLERT AND J. M. STURTEVANT, J. Am. Chem. Soc., 82 (196o) 1497. 12 R. V. TOMLINSON AND C. E. BALLOU, Biochemistry, i (1962) 166. 13 M. R. ATKINSON AND R. K. MORTON, in M. FLORKIN AND H. MASON, Comparative Biochemistry, Vol. 2, Academic Press, N e w York, 196o, p. i. i4 M. E. TATE, Can. J. Chem., in t h e press. 15 C. NEUBERG, Biochem. Z., 9 (19o8) 557. 16 T. IKAWA, K. NISlZAWA AND T. MIWA, Nature, 203 (1964) 939. 17 N. S. GING AND J. M. STURTEVANT, ,f. An~. Chem. Soc., 76 (1954) 2087.
Received September I6th, 1968 * P r e s e n t address: C.S.I.R.O. P l a n t Physiology Unit, School of Biological Sciences, S y d n e y University, N.S.W., 2006, Australia.
Biochim. Biophys. Acta, 17o (1968) 448-45 x
SHORT COMMUNICATIONS
two lines of evidence. First, when the sialic acids isolated from HeLa S 3 cells grown in presence of radioactive glucose were subjected to paper chromatography, a zone of radioactivity corresponding to the position of authentic NGNA was detected on the chromatogram (Fig. I). Second, hydrolysis of the material eluted from the NGNA zone of the paper chromatogram yielded radioactive glycolic acid as shown by recrystallizing a derivative to constant specific activity (Table I). HeLa Sa cells, a tissueculture line derived from a cervical cancer, represents the first material of human origin to be reported to contain NGNA. This investigation was supported in part by National Science Foundation Grant GB 6o43 and National Institutes of Health Grants AI o4954 and CA lO614. R.C. is recipient of a Research Career Development Award (I-K3-AM-38649) from the U.S. Public Health Service.
Oklahoma Medical Research Foundation and Department of Biochemistry, School of Medicine, University of Oklahoma, Oklahoma City, Okla. (U.S.A.)
R. CARUBELLI
M. J. GRIFFIN
I R. CARUBELLI A N D iV[. J. GRIFFIN, Science, 157 (1967) 693. 2 A. GOTTSCHALK,The Chemistry and Biology of Sialic Acids and Related S~tbstances, C a m b r i d g e U n i v e r s i t y Press, L o n d o n , 196o, p. 31. 3 L. SVENNERHOLM, Acta Chem. Scan&, 12 (1958) 547. 4 F~. SVENNERHOLM AND L. SVENNERHOLM, Nature, 181 (1958) 1154. 5 L. %VARREN, Nature, 186 (196o) 237. 6 E. I~LENK AND G. UHLENBRUCK,Z. Physiol. Chem., 307 (1957) 266. 7 G. TETTAMANTI, L. BERTONA, g . BERRA AND V. ZAMBOTTI, Italian J. Biochem., 13 (1964) 315 . 8 R. L. SHRINER AND R. C. FUSON, Identification of Organic Compounds, J o h n W i l e y a n d Sons, N e w York, 3rd ed., 1948, p. 157.
Received July 29th, I968 Biochim. Biophys. Acta, 17o (1968) 4 4 6 - 4 4 8
BBA 23472
The enthalpy change in the hydrolysis of the phosphate esters of myo-inositol Most of the phosphate in plant seeds is stored in the form of the hexaphosphate ester of myo-inositol (phytate) 1. The phytate is hydrolysed during germination to provide Pl for the developing embryo 1. Recent data indicate that in addition to its role as a reservoir of phosphate, phytate might also function as a phosphoryl donor in reactions with nueleoside diphosphates forming nucleoside triphosphates~, a. These recent findings indicate that at least one of the phosphate groups of phytate has a high phosphoryl transfer potential (so-called "high-energy" phosphate) and that phytate might function as a plant phosphagen 2. In an attempt to verify this proposal, measurements have been made of the enthalpy change during hydrolysis of inositol polyphosphates by phytase (myo-inositol hexaphosphate phosphohydrolase, EC 3.1.3.8). Biochim. Biophys. Acta, I7O (1968) 448-451
449
SHORT COMMUNICATIONS TABLE
I
THE HEAT OF HYDROLYSIS ~JHapp FOR THE PHOSPHATE ESTERS OF INOSITOL H e a t e v o l u t i o n w a s m e a s u r e d w i t h a m i c r o - c a l o r i m e t e r of t h e t y p e d e s c r i b e d b y EVANS AND CARNEY 7. O n e of t h e t w i n t a n t a l u m r e a c t i o n c e l l s c o n t a i n e d I m l of p h y t a s e s o l u t i o n i n o.2 M a c e t a t e b u f f e r ( p H 5.2), t h e o t h e r c o n t a i n e d b u f f e r o n l y . T h e s u b s t r a t e f o r e a c h r e a c t i o n (o.I m l o f t h e i n o s i t o l p h o s p h a t e e s t e r , e q u i v a l e n t t o 6 - 2 0 / * m o l e s of P t i n 0.2 M a c e t a t e b u f f e r ( p H 5.2), w a s p l a c e d i n t w o s y r i n g e s a n d d i s c h a r g e d s i m u l t a n e o u s l y i n t o t h e r e a c t i o n cells t o i n i t i a t e h y d r o l y s i s . T h e t e m p e r a t u r e w a s 25 ° . T h e h e a t e v o l u t i o n w a s r e c o r d e d g r a p h i c a l l y a n d t h e t o t a l h e a t p r o d u c t i o n d e t e r m i n e d b y i n t e g r a t i o n of t h e h e a t - t i m e c u r v e . T h e r e a c t i o n w a s s t o p p e d b y t h e a d d i t i o n of I m l o f I M N a O H a f t e r a p p r o x . 7 o % of e s t e r p h o s p h a t e g r o u p s h a d b e e n h y d r o l y s e d ( 4 0 - 9 ° r a i n ) . T h e P t f o r m e d w a s d e t e r m i n e d b y t h e m e t h o d of W E t L - M A L H E R B E AND GrREEN 8.
Compound
Pi formed (tzmoles)
Heat (mcal)
Heat of hydrolysis (cal/mole of Pi)
3/Iean AHap~
-- 2720 --285o --2830 -- 313 ° --3 °20 --297 ° -- 261o --329 ° --2560 --320o --257 ° --2760 --4300 --381o -- 3900 o o o --221o --224 ° -- 6000 --5900
--2800
AHapp Inositol hexaphosphate
Inositol pentaphosphate
Inositol tetraphosphate
Inositol triphosphate
Inositol diphosphate
Inositol monophosphate
Glucose 6-phosphate ATP
6.06 5.96 5.96 6.75 5.93 5.83 8.09 6.71 7.51 4.25 4.55 5.4 ° 5.21 5+9o 5.97 3.12 2.81 3.41 5.3 7.6 13.6 12.4
16. 5 17.o 16.9 2 I. i 17-9 17.3 21. I 22.1 19.2 13.6 11.7 14.9 22. 4 22.5 23.3 o o o i1. 7 17.o 8 i. 7 73.2
-- 304 °
-- 282o
--2840
--4000
--222o -- 595 °
The phosphate esters of myo-inositol were prepared from sodium phytate by controlled hydrolysis with phytase at p H 5.2. The various phosphate esters were separated b y ion-exchange chromatography using Dowex AGI-X8 resin (C1- form) and isolated as the barium salts 4. Each of the isolated esters appeared as a single major spot after paper electrophoresisS; the penta- and tetraphosphate esters showed additional minor spots which were presumably different isomers. The barium salts were converted to the free acid form b y using Dowex-5o resin (H + form), neutralized with NaOH and adjusted to 0.2 M with respect to sodium acetate buffer (pH 5.2). Phytase was prepared from wheat bran and purified as described b y lXTAGAIAND FUNAHASHI~ up to the stage of methanol fractionation. The heat evolved and the amount of Pt liberated b y the action of phytase is shown in Table I. The calculated heat of hydrolysis is expressed as callmole of Pt formed. No allowance has been made for the heat of ionization of the buffer, consequently the heat of hydrolysis can only be considered as an apparent value (AHa~p). Biochim. Biophys. Aeta,
17 ° (1968) 4 4 8 - 4 5 1
45 °
SHORT COMMUNICATIONS
Since the heat of ionization of acetate buffer is small compared to other buffer systems 9 the correction which would be applied if a H ÷ were produced would be approx. 2 mcal which would reduce the AHapp by approx. 2oo cal/mole. Because of the relatively small quantities of heat produced, the estimated error involved is of the order of zh 3oo cal/mole. Thus there is no significant difference in AHapp of hydrolysis of the inositol hexa-, penta-, tetra-, or triphosphate esters of inositol. There is, however, a significant increase in the value of AHapp of hydrolysis of the inositol diphosphate compared to the other esters. No heat evolution could be detected with the samples of inositol monophosphate, although Pl was liberated as shown in Table I. The AHaop for the hydrolysis of ATP ( 595 ° cal/mole) shown in Table I represents mainly the mean for each of the two terminal phosphate bonds since Pl analysis and thin-layer chromatography of the reaction mixture showed that 91% of the phosphate was hydrolyzed and the only nucleoside phosphate present after hydrolysis was AMP. This figure is similar to the value obtained by PODOLSKV AND STURTEYANT10 ( 5300 ± 600 cal./nlole) for the hydrolysis of the terminal phosphate of ATP by myosin (ATP phosphohydrolase, EC 3.6.1.3) at pH 8. The AHapp obtained for the samples of inositol hexa-, penta-, tetra-, and triphosphate esters show that the phosphate-ester bonds of these compounds have heats of hydrolysis similar to sugar monophosphate esters such as glucose 6-phosphate (AHapp --22oo ± 3oo cal/mole, see Table I), rather than the so-called "high-energy" compounds such as ATP 1° or creatine phosphate; AH-.9ooo ± 5oo cal/mole (ref. II). The only inositol phosphate ester which showed a significantly higher AHapp was inositol diphosphate. The inositol diphosphate formed by hydrolysis of inositol hexaphosphate with phytase is predominantly inositol 1,2-diphosphate~; further hydrolysis yields the inositol 2-monophosphate and finally inositol. The phosphate group at position 2 is the only axially oriented phosphate group and is considered to be relatively inaccessible to enzymic hydrolysis, it has been proposed that the phosphate group at position 2 of inositol would have a high phosphoryl transfer potential, considering the high charge repulsion exerted on this phosphate group by the adjacent cis-phosphates ~a. The inability to detect heat evolution during the hydrolysis of the inositol monophosphate m a y be due to the relatively low velocity of the hydrolysis of this ester compared to the higher phosphate esters of inositol. With the calorimeter used in these studies small quantities of heat produced over a long period of time (in the case of the monophosphate ester, 9 ° min) would be difficult to detect. In terms of heat available on hydrolysis the results here would suggest that inositol hexaphosphate does not possess a phosphate group of sufficient "high energy" to act as a donor of a phosphoryl group in the formation of a nucleoside triphosphate. The results do not, however, exclude the possibility that the more thermodynamically significant free energy change of hydrolysis is not sufficient to allow for the transfer of a phosphoryl group from phytate to a nucleoside diphosphate, as indicated in reactions carried out at higher p H values and in the presence of Mg2+ (refs. 2, 3). GELLERT ANI) STURTEVANT11 have shown that the AH of hydrolysis of creatine phosphate is --9000 cal/mole (compared to the AH of ATP of --53oo cal/mole), but the standard free energy of hydrolysis of these two compounds is nearly equal. Thus the standard entropy of hydrolysis of creatine phosphate must be more negative than
Biochim. Biophys. Aria, I7o (1968) 448-451
SHORT COMMUNICATIONS
451
that of ATP by about 13 entropy units/mole (ref. II). The same considerations could be applied to inositol hexaphosphate. Using [a2P]inositol hexaphosphate it has been shown that the transfer of a phosphoryl group from inositol hexaphosphate to GDP forming GTP is reversible under physiological conditions 3, indicating that the standard free energy of hydrolysis of inositol hexaphosphate is of the same order as the standard free energy of hydrolysis of GTP. Since the enthalpy of hydrolysis of inositol hexaphosphate, as determined here, is less negative than the enthalpy of hydrolysis of ATP, the entropy of hydrolysis of inositol hexaphosphate is probably more positive than that of ATP by approx. 8 entropy units/mole. On the basis of NMR studies, TATE14 recently proposed that inositol hexaphosphate consists of a hexaorthophosphate structure rather than a tripyrophosphate structure suggested by other workers 1~. If a pyrophosphate structure was present in inositol hexaphosphate the AH of hydrolysis as determined here would have included the AH of hydrolysis of both the pyrophosphate and the Pl bonds, since phytase is capable of hydrolysing both types of phosphate bonds 1~.The AH of hydrolysis of the pyrophosphate bond is of the order of --3000 cal/mole 17. The AH of hydrolysis of inositol hexaphosphate containing a pyrophosphate linkage should thus exceed the AH of hydrolysis of inositol pentaphosphate by --3000 cal/mole. Since there was no significant difference in the AH of hydrolysis of the inositol hexa-, penta-, tetra-, or triphosphate the results reported here support the hexaorthophosphate structure for inositol hexaphosphate. One of the authors (J. K. R.) is a Post-doctoral Research Associate.
Seed Protein Laboratory, U.S. Department of Agriculture, New Orleans, La. (U.S.A.)
J O H N K. RAISON* WILLIAM J. EVANS
I P. K. STUMPF, in W. D. MCELROY AND B. GLASS, Phosphorus Metabolism, Vol. 2, J o h n s Hopkins, Baltimore, 1952, p 29. 2 R. K. MORTON AND J. K. RAISON, Nature, 200 (1963) 429. 3 S. BlswAs AND B. B. BlSWAS, Biochim. Biophys. Acta, lO8 (1965) 71o. 4 D. J. COSGROVE, Australian J. Soil Res., i (1963) 203. 5 U. B. SEIFFERT AND B. W. AGRANOFF, Biochim. Biophys. Acta, 98 (1965) 574. 6 Y. NAGAI AND S. FUNAHASHI, Agr. Biol. Chem. Tokyo, 26 (1962) 7947 W. J. EVANS AND W. B. CARNEY,Anal. Biochem., I I (1965) 449. 8 H. WEIL-MALHERBE AND R. H. GREEN, Biochem. J., 49 (1951) 286. 9 I(. S. PITZER, J. Am. Chem. Soc., 59 (1937) 2365 . io R. J. PODOLSKY AND J. M. STURTEVANT, J. Biol. Chem., 217 (1955) 603. 11 M. GELLERT AND J. M. STURTEVANT, J. Am. Chem. Soc., 82 (196o) 1497. 12 R. V. TOMLINSON AND C. E. BALLOU, Biochemistry, i (1962) 166. 13 M. R. ATKINSON AND R. K. MORTON, in M. FLORKIN AND H. MASON, Comparative Biochemistry, Vol. 2, Academic Press, N e w York, 196o, p. i. i4 M. E. TATE, Can. J. Chem., in t h e press. 15 C. NEUBERG, Biochem. Z., 9 (19o8) 557. 16 T. IKAWA, K. NISlZAWA AND T. MIWA, Nature, 203 (1964) 939. 17 N. S. GING AND J. M. STURTEVANT, ,f. An~. Chem. Soc., 76 (1954) 2087.
Received September I6th, 1968 * P r e s e n t address: C.S.I.R.O. P l a n t Physiology Unit, School of Biological Sciences, S y d n e y University, N.S.W., 2006, Australia.
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