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Evidence for the identity of alkaline phosphatase and inorganic pyrophosphatase in rat kidney
BIOCHIMICAET BIOPHYSICAACTA
93
BBA 65866 E V I D E N C E FOR T H E I D E N T I T Y OF A L K A L I N E P H O S P H A T A S E AND I N O R G A N I C P Y R O P H O S P H A T A S E IN RAT K I D N E Y *
F. MELANI AND MARTA FARNARARO Department of Biochemistry, University of Florence, Florence (Italy)
(Received October 7th, 1968) (Revised manuscript received November 29th, 1968)
SUMMARY Alkaline phosphatase, extensively purified from rat kidney, was found to hydrolyze inorganic pyrophosphate. The following experimental results suggest that alkaline phosphatase is both a phosphomonoesterase and a pyrophosphatase. I. The purification of rat kidney alkaline phosphatase was accompanied by a concomitant increase in inorganic pyrophosphatase activity and an almost constant ratio between the two enzyme activities at different purification steps was found. Moreover, both enzymatic activities were eluted together as a single peak after DEAE-Sephadex A-25 chromatography of microsomal proteins solubilized b y deoxycholate and lipase. 2. A decrease in the level of inorganic phosphate in the renal cell produces concomitant and significant increases of both enzymatic activities. 3. The amount of phosphate released in the mixed substrate experiments was less than would be expected for independent hydrolysis of the substrates. 4. Both enzyme activities were affected similarly by cysteine, a noncompetitive inhibitor, and by heating in the absence of substrate.
INTRODUCTION That mammalian alkaline phosphatase (orthophosphoric monoester phosphohydrolase (EC 3.1.3.1)) is inactive towards inorganic pyrophosphate has been known for m a n y years2, s. On the other hand there are several reports that suggest that alkaline phosphatase has inorganic pyrophosphatase activity as well 4-n. Interest in the problem was intensified by the finding that inorganic pyrophosphate is produced as a by-product in m a n y essential biosynthetic reactions including the reactions of DNA and RNA polymerization, coenzyme synthesis and amino acid and f a t t y acid activation. The hydrolysis of inorganic pyrophosphate m a y provide, as suggested by STETTENTMand by K O R N B E R G TM, a control mechanism whereby these biosynthetic reactions are made irreversible. * A preliminary report was presented at the 5th Meeting of the European Biochemical Society, Prague, 1968 (ref. i). Biochim. Biophys. Acta, 178 (1969) 93-99
94
F. MELANI, M. FARNARARO
In the present work, which was conducted with a highly purified rat kidney alkaline phosphatase, evidence that the same enzyme is responsible for alkaline phosphatase and inorganic pyrophosphatase activities is presented and discussed. EXPERIMENTAL
Enzyme preparation The procedures for purification of rat kidney alkaline phosphatase trove been described t4. In brief, alkaline phosphatase was extracted from rat kidney nficrosomes by simultaneous treatment with deoxycholate (1% solution of sodium deoxvcholate in o.o5 M Tris buffer at pH 7.8) and lipase and was purified successively by chromatography on DEAE-Sephadex A-25, eluting with a linear gradient between o.o3 and o.15 M NaCI in o.o 5 M Tris buffer (pH 8.1), and rechromatography on DEAESephadex as before. The resultant enzyme preparation gave a single coincident band when stained for enzyme activity and protein after starch-gel electrophoresis. The specific activity was 15/zlnoles of p-nitrophenol hydrolyzed from p-nitrophenylphosphate per min per mg of protein at 25 ° and pH lO.4. Other phosphomonoesters are hydrolyzed bv purified phosphatase 1'5.
Assay methods Enzyme activities were assayed at 25 ° . Alkaline phosphatase was measured spectrophotometrically at 405 nm by a continuous optical method 1" with 3 mM p-nitrophenylphosphate in 0.05 M carbonate bicarbonate buffer o.15 mM MgC12 (pH lO.4), as substrate. Alkaline inorganic pyrophosphatase was determined using 3 mM sodium pyrophosphate as substrate in 0.05 M Tris-HCl-o.I 5 mM MgCIe (pH 8). The procedure consisted of incubating o.I 1111of enzyme solution with 0. 9 ml of reaction mixture for IO rain and stopping the reaction by inlmersing the tubes in ice; phosphate release was assayed immediately by the method of LOWRY AND LOPEZ17. Other specific details are given in the legends of tables and figures. Total protein was deternfined by the biuret method, according to BEISENHERZ et al. 18, except that for individual column fractions protein was estimated from the absorbance at 280 nm as suggested by WARBURG ANI) CHRISTIAN 1'~. Intracellular inorganic phosphate was estimated by the method of LOWRY AND LOPEZ17 on a kidney perchloric extract obtained by the quick-freezing technique of HOHORST, KREUTZ AND Bi.JCHER 2°.
RESULTS
Hydrolysis of p-nitrophenylphosphate and inorganic pyrophosphate by alkaline phosphatase In Fig. I the activity pH curve for hydrolysis of p-nitrophenylphosphate is compared with that for hydrolysis of inorganic pyrophosphate : it is evident that p H has a different influence on the rates of hydrolysis of the two substrates. The curve for p-nitrophenylphosphate shows a m a x i m u m value at p H 9.8, Biochirn. Biophys. Acta, 178 (1969) 93-99
95
ALKALINE PHOSPHATASE AND INORGANIC PYROPHOSPHATASE
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Fraction No.
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Fig. I. A c t i v i t y - p H c u r v e s for h y d r o l y s i s of p - n i t r o p h e n y l p h o s p h a t e ( O - - O ) a n d i n o r g a n i c p y r o p h o s p h a t e ({b--O) b y purified alkaline p h o s p h a t a s e f r o m r a t kidney. T h e r e a c t i o n m i x t u r e s c o n t a i n e d 3 m M s u b s t r a t e , I m M MgCI~ in o.o 5 M T r i s - g l y c i n e buffer (pH 7.5-11). T h e e n z y m e a c t i v i t i e s a t o p t i m u m p H , a l t h o u g h of different v a l u e s for t h e two s u b s t r a t e s , were a r b i t r a r i l y a s s u m e d as c e n t per c e n t (hydrolysis m e a s u r e d b y release of o r t h o p h o s p h a t e ) . Fig. 2. Purification of alkaline p h o s p h a t a s e a n d inorganic p y r o p h o s p h a t a s e activities f r o m a m i c r o s o m a l e x t r a c t c o n t a i n i n g 3 ° m g of protein. T h e e x t r a c t w a s p r e p a r e d f r o m r a t k i d n e y m i c r o s o m e s b y t h e p r o c e d u r e described in t h e t e x t a n d a b s o r b e d to a c o l u m n of D E A E S e p h a d e x (2o c m x 2 cm). E l u t i o n was achieved with a linear g r a d i e n t f o r m e d f r o m ioo ml of o.o 5 M Tris-HC1 buffer (pH 8.1) in t h e m i x i n g c h a m b e r a n d t h e s a m e buffer + i M NaC1 in t h e reservoir flask. T h e eluate f r a c t i o n s (1.8 ml) were a s s a y e d for a b s o r b a n c e a t 28oo/~ ( k - - A ) , for alkaline p h o s p h a t a s e (C)--C)) a n d for inorganic p y r o p h o s p h a t a s e (O---O) activities.
whereas the pH-activity curve for inorganic pyrophosphate rises to a maximum at pH 8 and declines at higher pH values.
Purification of alkaline phosphatase and inorganic pyrophosphatase The results of a typical preparation of alkaline phosphatase and inorganic pyrophosphatase from rat kidney are summarized in Table I. As Table I shows, the TABLEI PREPARATION
OF
ALKALINE
PHOSPHATASE
AND
INORGANIC
PYROPHOSPHATASE
FROM
RAT
KIDNEY
A c t i v i t i e s are expressed a s / 2 m o l e s of p - n i t r o p h e n y l p h o s p h a t e or inorganic p y r o p h o s p h a t e h y d r o l y s e d per m i n a n d per m g of t o t a l protein. A b b r e v i a t i o n s : A, alkaline p h o s p h a t a s e ; B, inorganic pyrophosphatase.
Fraction
Total units A
Crude extract 15 ° Microsomal s u s p e n s i o n in d e o x y c h o l a t e 96 S u p e r n a t a n t after lipase digestion 64 F i r s t f r a c t i o n a t i o n on D E A E - S e p h a d e x 36 S e c o n d f r a c t i o n a t i o n o n D E A E - S e p h a d e x 34
Specific activity
B
21 11.2 8 4.9 4 .6
Purification Yield (%) A / B (fold) A B A B
A
B
0.05 0.6 1. 7 14.5 15.5
0.007 0.07 12 o.21 34 1.99 290 2.1 31o
io 30.4 285 300
ioo 64 42 24 23
IOO 53 38 23 22
7.1 8-5 8 7.3 7-4
Biochim. Biophys. Acta, 178 (1969) 9 3 - 9 9
90
F. MELANI, M. 1;ARNARARt)
T A B L E 1I EFFECT
OF A DIET LACKING
INORGANIC
IN PHOSPHATE
PYROPHOSPHATASE
ON TIlE RAT KIDNEY
AND INORGANIC
L E V E I . S O F A L K A L [ N I £ I H I t ) S I ' H A T k.SE
PIIOSPHATE
The results are means ± s t a n d a r d deviations; in parentheses the n u m b e r of rats. The content of inorganic p h o s p h a t e was determined on a kidney perchloric extract obtained bv a quick-freezing technique. The enzyme activities were determined on a fraction (15 min at 4ooo ~ ~,, s u p e r n a t a n t ) of lO°o kidney homogenate.
A~zimals
Kidney phosphate Alkaline phospkatas~ Im)rg~ie pyrophosphatase (/tmoles/g per wet weight) (/onoles/mi~ per g prolei~0 (ttmoIeslmi~z per g protei~O
Complete diet 5.~ ~ O ' 0 4 ( I O ) P h o s p h a t e free diet 3.3 ~ o.o() (lo)
82
} I2
(IO)
I I _i2 I " t
i0o t IS (io)
(IO)
23 " 2.2 (to)
purification of rat kidney alkaline phosphatase was accompanied by a concomitant increase in inorganic pyrophosphatase activity. A typical elution profile is given in Fig. 2 in which it is clear that alkaline phosphatase and inorganic pyrophosphatase activities run together through the colunm, both of them being recovered in the few initial fractions at sodium chloride concentrations ranging from o.o3 o.15 M.
Regulation by phosphate of alkaline phosphatase and inorganic pyrophosphatase activities in rat kidney A decrease in the level of inorganic phosphate in the renal cell produced by dietary treatment is accompanied by a concomitant and significant increase in alkaline phosphatase activity 2x,2~. Table II shows that in the renal cell there is also a parallel increase of inorganic pyrophosphatase activity.
Mixed-sabstrate experiments Experiments were carried out in which the rate of phosphate released fl-om TABLE III HYDROLYSIS LINE
OF p-NITROPHENYLPHOSPHATE
AND SODIUM
PYROPHOSPttATE
HY RAT KII)NEV
ALKA-
PItOSPHATASE
E n z y m e was incubated with p - n i t r o p h e n y l p h o s p h a t e (3'mM) or w i t h s o d i m n p y r o p t m s p h a t c (3 raM), or w i t h b o t h at p H 9 (o.o5 M carbonate bicarbonate buffer) for io rain at 25 °. The c o n c e n t r a t i o n of Mg 2~ was o.z mM t h r o u g h o u t . Activities are expressed in p,moles of inorganic p h o s p h a t e (Pi) or p - n i t r o p h e n o l liberated/min per mI of enzyme solution.
Observed
Expectedfi)r independent kydrolysis
Sodium p y r o p h o s p h a t e (3 raM) (/tmoles of Pi/nlin) p - N i t r o p h e n y l p h o s p h a t e (3 raM) (/~moles of Pi/min) (/mmles of p - n i t r o p h e n o l / m i n )
o.3_,
Sodium p y r o p h o s p h a t e (3 mM) ! p - n i t r o p h e n y l p h o s p h a t e (3 raM) (/mloles of Pi/min) (/tmoles of p - n i t r o p h e n o l / m i n )
o.- 5
Bioehim. Biophys. Aeta, I78 (1960) 93 o~-.~
o.3o
0.2 3
o.37
97
ALKALINE PHOSPHATASE AND INORGANIC PYROPHOSPHATASE
15(
•
J
102 IO0
u
N>'-';- 5C
ge
\
"G 5 c
~.o 2~
*g
g
15
lg
20
c
Time (rain)
\
-iog[Mgct~(M)
Fig. 3. Effect of i n c u b a t i o n a t 560 a n d p H 9 (0.05 M Tris-HC1 buffer) on a l k a l i n e p h o s p h a t a s e ( O - - © ) and inorganic pyrophosphatase ( 0 - - 0 ) activities. Fig. 4- Effect of Mg 2+ on a l k a l i n e p h e s p h a t a s e ( O - - 0 ) a n d i n o r g a n i c p y r o p h o s p h a t a s e (O--11) a c t i v i t i e s . The r e a c t i o n m i x t u r e s c o n t a i n e d 3 mM s u b s t r a t e ( p - n i t r o p h e n y l p h o s p h a t e or i n o r g a n i c p y r o p h o s p h a t e ) buffered a t p H 9 w i t h 0.05 M Tris-HC1 buffer. A c t i v i t i e s , m e a s u r e d b y t h e release of o r t h o p h o s p h a t e , are g i v e n as a p e r c e n t a g e of each c o n t r o l w i t h no a d d e d MgC1 v
inorganic pyrophosphate and p-nitrophenylphosphate present together in the same concentration was compared with that from a single substrate; the concentration of Mg ~+ was I mM throughout. As shown in Table III, the amount of phosphate released in the mixed-substrate experiments was less than would be expected for independent hydrolysis of the substrates.
Heat lability of enzymes Fig. 3 shows the effect of incubation at 560 and pH 9 (0.05 M Tris-HC1 buffer) on alkaline phosphatase and inorganic pyrophosphatase activities of purified kidney enzyme; both activities show a constant ratio during heat inactivation at 56°. Treatment with amino acids COX, GILBERT AND GRIFFIN23 reported that L-cysteine caused inhibition of the alkaline phosphatase and inorganic pyrophosphatase activities of HeLa cells in tissue T A B L E IV INHIBITION
OF ALKALINE
PHOSPHATASB
AND INORGANIC PYROPHOSPHATASE
ACTIVITIES
BY CYSTBINE
The e n z y m e s o l u t i o n was p r e - i n c u b a t e d for i m i n w i t h c y s t e i n e or w i t h c y s t e i n e + Zn 2+ in a n ice b a t h . A l i q u o t s were r e m o v e d a n d a s s a y e d for a c t i v i t i e s a t p H 9 (0.05 M c a r b o n a t e - b i c a r b o n a t e buffer).
Additions
Alkaline phosphatase Inorganic pyrophosphatase (ffmoles/min per ml enzyme solution) (ffmoles/min per ml enzyme solution)
None 2 - lO -4 M c y s t e i n e 2 - lO -4 M c y s t e i n e + 2, 5 • lO -5 M Zn ~+ 2 • lO -4 M c y s t e i n e + 5 • IO-~ M Zn 2+
0.33 o.12
0.045 O.Ol 5
0.23
0.025
0.34
o.05
Biochim. Biopkys. Acta, I78 (I969) 93~ )9
98
F. MELANI, M. FARNARARO
cultures. At a cysteine concentration of 2- IO 4 M (pH 9), we observed a strong inhibition of both of these enzyme activities (Table IV). The non-competitive inhibition caused by cysteine was completely removed on the addition of Zn 2~. Other amino acids tested at the same p H in the concentration range 4 ' Io-4 M and 5 "IO 3 }[ were ineffective on either of these enzyme activities. ElFect of MgC12 o~ the e~z~,me activities In the presence of Mg 2+ the phosphomonoesterase activity of purified kidney enzyme was enhanced; with the pyrophosphate as substrate, however, and Mg 2+ concentrations up to about I mM, there was some activation, but above this concentration Mg 2+ was strongly inhibitory (Fig. 4). Similar results were obtained with intestinal and liver enzymes 24. DISCUSSION Our own data on the whole support the idea that both phosphomonoesterase and inorganic pyrophosphatase activities of rat kidney are properties of the same enzyme. In fact it is clear from Fig. z that purified kidney enzyme hydrolyzes both p-nitrophenylphosphate and inorganic pyrophosphate and that kidney preparations had inorganic pyrophosphatase activity at all stages of purification; during the purification procedure (Table I), the ratio of alkaline phosphatase activity (at pH Io.4) to pyrophosphatase activity (at p H 8) remained ahnost constant at different purification steps (7.I in crude extracts; 8. 5 in microsomal suspension in deoxycholate; 8 in supernatant after lipase digestion; 7.3 after the first fractionation; 7.4 after the second fractionation). Furthermore, the alkaline phosphatase and inorganic pyrophosphatase were eluted together as a single peak after DEAE-Sephadex A-25 chromatography of the microsomal proteins (Fig. 2). Additional evidence is provided b y studies of the mechanism by which inorganic phosphate regulates the level of alkaline phosphatase: a decrease in inorganic phosphate level in the renal cell by dietary treatment is accompanied by concomitant and significant increases in both alkaline phosphatase and inorganic pyrophosphatase activities. ROCHE2'~has reported that alkaline phosphatase activity is less stable than pyrophosphatase activity in extracts of pig liver, but our thermal inactivation experiments with rat kidney enzyme did not reveal any such difference in stability (Fig. 3)Furthermore, there was no summation of rates of Pi formation when saturating concentrations of p-nitrophenylphosphate and inorganic pyrophosphate were mixed (Table III). In addition the inhibition by cysteine of alkaline phosphatase and inorganic pyrophosphatase activities proceeded in a parallel fashion (Table IV). It seems less probable that the two types of phosphatase activity are the result of the presence of two enzymes, each of restricted specificity, since experimental results were obtained with an enzyme preparation which appears substantially homogeneous. The last experimental result, which requires further mention, is that hydrolysis of inorganic pyrophosphate m a y be suppressed in a selective fashion by relatively high concentrations of Mg 2+ (Fig. 4). This inhibition m a y have been responsible for MORTON'S observation a that highly purified calf intestinal alkaline phosphatase was free from inorganic pyrophosphatase activity, since his experimental conditions Bfochim. Bfophys. Acta, I78 (I969) 9399
ALKALINE PHOSPHATASE AND INORGANIC PYROPHOSPHATASE
99
included a large excess of Mgz+ (2u-fold) which in our experience would be highly inhibitory. Our interpretation that in rat kidney both orthophosphatase and pyrophosphatase activities are due to the same enzyme, is in agreement with observations concerning alkaline phosphatase from calf intestine n and from several human tissuesS,934. Since purified inorganic pyrophosphatase from yeast 27 and from E s c h e r i chia coli 28 does not exhibit alkaline phosphatase activity, it is probable that the above conclusions may apply essentially to mammalian enzymes. ACKNOWLEDGMENT
We are grateful to Prof. VINCENZO BACCARI for his interest and helpful advice in this work. This work was supported by a grant from the Italian Consiglio Nazionale delle Ricerche. REFERENCES I F. MELANI AND M. FARNARARO,Abstr. 5th Meeting Federation European Biochem. Soc., Prague, i968 , A 947, P- 237. 2 S. K. VOLLEY AND H. D. KAY, Ergeb. Enzymforsch., 5 (1936) 159. 3 R. K. MORTON, Biochem. J., 61 (1955) 232. 4 V. BACCARI AND L. DE ROSA, Boll. Soc. Ital. Biol. Sper., 23 (1947) lO65. 5 H. R. PERKINS AND P. G. WALKER, J. Bone Joint Surg., 4DE (1958) 3336 H. FLEISCH AND S. BISAZ, Nature, 195 (1962) 911. 7 R. G. G. RUSSEL, Lancet, ii (1965) 461. 8 R. P. Cox AND M. J. GRIFFIN, Lancet, ii (1965) lO18. 9 D. W. Moss, R. K. EATON, J. K. SMITH AND L. G. WHITBY, Bioehem. J., lO2 (1967) 53. IO R. K. EATON AND D. W. Moss, Biochem. J., lO 5 (1967) 307 . I I H. N. FERNLEY AND P. G. WALKER, Biochem. J., lO 4 (1967) IOli. 12 D. STETTEN, Am. J. Med., 28 (196o) 867. 13 A. KORNBERG, in M. KASHA AND B. PULLMAN, Horizons in Biochemistry, Academic Press New York, 1962, p. 251. 14 F. MELANI, M. FARNARARO AND G. SGARAGLI,Experientia, 24 (i968) 114. 15 F. MELANI, M. FARNARARO AND G. SGARAGLI, Arch. Biochem. Biophys., 122 (1967) 417 . 16 F. MELANI AND A. GUERRITORE, Experientia, 2o (1964) 464 . 17 O. H. LOWRY AND J. A. LOPEZ, J. Biol. Chem., 162 (1964) 421. 18 G. BEISENHERTZ, H. J. BOLTZE, TH. BOCHER, R. CZOK, K. H. GARBADE, E. MEYER-ARENDT AND G. PFLEIDERER, Z. Naturforsch., 8b (1953) 555. 19 O. WARBURG AND W. CHRISTIAN,Biochem. Z., 31o (1941) 384. 20 H. J. HOHORST, F. H. KREUTZ AND TH. BtDCHER, Biochem. Z., 332 (1959) 18. 21 F. MELANI, G. RAMPONI, A. GUERRITORE AND V. BACCARI, Nature, 2Ol (1964) 71o. 22 F. MELANI, G. RAMPONI, M. FARNARARO, E. C o c u c c I AND A. GUERRITORE, Biochim. Biophys. Acta, 138 (1967) 411. 23 R. P. Cox, P. GILBERT AND M. J. GRIFFIN, Biochem. J., lO 5 (1967) 155. 24 R. H. EATON AND D. W. MOSS, Bioehem. J., lO2 (1967) 917. 25 J. ROCHE, in P. D. BUYER, H. LARDY AND K. MYRBACK, The Enzymes, Vol. I, Academic Press, New York, 195 o, p. 493. 26 H. H. SUSSMAN AND E. LAGA, Biochim. Biophys. Acta, 151 (1968) 281. 27 L. A. HEPPEL AND R. J. HILMOE, J. Biol. Chem., 192 (1951) 87. 28 J JossE, J . Biol. Chem., 241 (1966) 1938.
Biochim. Biophys. Acta, 178 (1969) 93-99
93
BBA 65866 E V I D E N C E FOR T H E I D E N T I T Y OF A L K A L I N E P H O S P H A T A S E AND I N O R G A N I C P Y R O P H O S P H A T A S E IN RAT K I D N E Y *
F. MELANI AND MARTA FARNARARO Department of Biochemistry, University of Florence, Florence (Italy)
(Received October 7th, 1968) (Revised manuscript received November 29th, 1968)
SUMMARY Alkaline phosphatase, extensively purified from rat kidney, was found to hydrolyze inorganic pyrophosphate. The following experimental results suggest that alkaline phosphatase is both a phosphomonoesterase and a pyrophosphatase. I. The purification of rat kidney alkaline phosphatase was accompanied by a concomitant increase in inorganic pyrophosphatase activity and an almost constant ratio between the two enzyme activities at different purification steps was found. Moreover, both enzymatic activities were eluted together as a single peak after DEAE-Sephadex A-25 chromatography of microsomal proteins solubilized b y deoxycholate and lipase. 2. A decrease in the level of inorganic phosphate in the renal cell produces concomitant and significant increases of both enzymatic activities. 3. The amount of phosphate released in the mixed substrate experiments was less than would be expected for independent hydrolysis of the substrates. 4. Both enzyme activities were affected similarly by cysteine, a noncompetitive inhibitor, and by heating in the absence of substrate.
INTRODUCTION That mammalian alkaline phosphatase (orthophosphoric monoester phosphohydrolase (EC 3.1.3.1)) is inactive towards inorganic pyrophosphate has been known for m a n y years2, s. On the other hand there are several reports that suggest that alkaline phosphatase has inorganic pyrophosphatase activity as well 4-n. Interest in the problem was intensified by the finding that inorganic pyrophosphate is produced as a by-product in m a n y essential biosynthetic reactions including the reactions of DNA and RNA polymerization, coenzyme synthesis and amino acid and f a t t y acid activation. The hydrolysis of inorganic pyrophosphate m a y provide, as suggested by STETTENTMand by K O R N B E R G TM, a control mechanism whereby these biosynthetic reactions are made irreversible. * A preliminary report was presented at the 5th Meeting of the European Biochemical Society, Prague, 1968 (ref. i). Biochim. Biophys. Acta, 178 (1969) 93-99
94
F. MELANI, M. FARNARARO
In the present work, which was conducted with a highly purified rat kidney alkaline phosphatase, evidence that the same enzyme is responsible for alkaline phosphatase and inorganic pyrophosphatase activities is presented and discussed. EXPERIMENTAL
Enzyme preparation The procedures for purification of rat kidney alkaline phosphatase trove been described t4. In brief, alkaline phosphatase was extracted from rat kidney nficrosomes by simultaneous treatment with deoxycholate (1% solution of sodium deoxvcholate in o.o5 M Tris buffer at pH 7.8) and lipase and was purified successively by chromatography on DEAE-Sephadex A-25, eluting with a linear gradient between o.o3 and o.15 M NaCI in o.o 5 M Tris buffer (pH 8.1), and rechromatography on DEAESephadex as before. The resultant enzyme preparation gave a single coincident band when stained for enzyme activity and protein after starch-gel electrophoresis. The specific activity was 15/zlnoles of p-nitrophenol hydrolyzed from p-nitrophenylphosphate per min per mg of protein at 25 ° and pH lO.4. Other phosphomonoesters are hydrolyzed bv purified phosphatase 1'5.
Assay methods Enzyme activities were assayed at 25 ° . Alkaline phosphatase was measured spectrophotometrically at 405 nm by a continuous optical method 1" with 3 mM p-nitrophenylphosphate in 0.05 M carbonate bicarbonate buffer o.15 mM MgC12 (pH lO.4), as substrate. Alkaline inorganic pyrophosphatase was determined using 3 mM sodium pyrophosphate as substrate in 0.05 M Tris-HCl-o.I 5 mM MgCIe (pH 8). The procedure consisted of incubating o.I 1111of enzyme solution with 0. 9 ml of reaction mixture for IO rain and stopping the reaction by inlmersing the tubes in ice; phosphate release was assayed immediately by the method of LOWRY AND LOPEZ17. Other specific details are given in the legends of tables and figures. Total protein was deternfined by the biuret method, according to BEISENHERZ et al. 18, except that for individual column fractions protein was estimated from the absorbance at 280 nm as suggested by WARBURG ANI) CHRISTIAN 1'~. Intracellular inorganic phosphate was estimated by the method of LOWRY AND LOPEZ17 on a kidney perchloric extract obtained by the quick-freezing technique of HOHORST, KREUTZ AND Bi.JCHER 2°.
RESULTS
Hydrolysis of p-nitrophenylphosphate and inorganic pyrophosphate by alkaline phosphatase In Fig. I the activity pH curve for hydrolysis of p-nitrophenylphosphate is compared with that for hydrolysis of inorganic pyrophosphate : it is evident that p H has a different influence on the rates of hydrolysis of the two substrates. The curve for p-nitrophenylphosphate shows a m a x i m u m value at p H 9.8, Biochirn. Biophys. Acta, 178 (1969) 93-99
95
ALKALINE PHOSPHATASE AND INORGANIC PYROPHOSPHATASE
! l.i / : °
lO£ 9£ ,-_o~ ~~ .____~ 8£
~ "6 601
U
kd
g
~ pN at 25 °
Ib
$
Fraction No.
lb
1.5
Fig. I. A c t i v i t y - p H c u r v e s for h y d r o l y s i s of p - n i t r o p h e n y l p h o s p h a t e ( O - - O ) a n d i n o r g a n i c p y r o p h o s p h a t e ({b--O) b y purified alkaline p h o s p h a t a s e f r o m r a t kidney. T h e r e a c t i o n m i x t u r e s c o n t a i n e d 3 m M s u b s t r a t e , I m M MgCI~ in o.o 5 M T r i s - g l y c i n e buffer (pH 7.5-11). T h e e n z y m e a c t i v i t i e s a t o p t i m u m p H , a l t h o u g h of different v a l u e s for t h e two s u b s t r a t e s , were a r b i t r a r i l y a s s u m e d as c e n t per c e n t (hydrolysis m e a s u r e d b y release of o r t h o p h o s p h a t e ) . Fig. 2. Purification of alkaline p h o s p h a t a s e a n d inorganic p y r o p h o s p h a t a s e activities f r o m a m i c r o s o m a l e x t r a c t c o n t a i n i n g 3 ° m g of protein. T h e e x t r a c t w a s p r e p a r e d f r o m r a t k i d n e y m i c r o s o m e s b y t h e p r o c e d u r e described in t h e t e x t a n d a b s o r b e d to a c o l u m n of D E A E S e p h a d e x (2o c m x 2 cm). E l u t i o n was achieved with a linear g r a d i e n t f o r m e d f r o m ioo ml of o.o 5 M Tris-HC1 buffer (pH 8.1) in t h e m i x i n g c h a m b e r a n d t h e s a m e buffer + i M NaC1 in t h e reservoir flask. T h e eluate f r a c t i o n s (1.8 ml) were a s s a y e d for a b s o r b a n c e a t 28oo/~ ( k - - A ) , for alkaline p h o s p h a t a s e (C)--C)) a n d for inorganic p y r o p h o s p h a t a s e (O---O) activities.
whereas the pH-activity curve for inorganic pyrophosphate rises to a maximum at pH 8 and declines at higher pH values.
Purification of alkaline phosphatase and inorganic pyrophosphatase The results of a typical preparation of alkaline phosphatase and inorganic pyrophosphatase from rat kidney are summarized in Table I. As Table I shows, the TABLEI PREPARATION
OF
ALKALINE
PHOSPHATASE
AND
INORGANIC
PYROPHOSPHATASE
FROM
RAT
KIDNEY
A c t i v i t i e s are expressed a s / 2 m o l e s of p - n i t r o p h e n y l p h o s p h a t e or inorganic p y r o p h o s p h a t e h y d r o l y s e d per m i n a n d per m g of t o t a l protein. A b b r e v i a t i o n s : A, alkaline p h o s p h a t a s e ; B, inorganic pyrophosphatase.
Fraction
Total units A
Crude extract 15 ° Microsomal s u s p e n s i o n in d e o x y c h o l a t e 96 S u p e r n a t a n t after lipase digestion 64 F i r s t f r a c t i o n a t i o n on D E A E - S e p h a d e x 36 S e c o n d f r a c t i o n a t i o n o n D E A E - S e p h a d e x 34
Specific activity
B
21 11.2 8 4.9 4 .6
Purification Yield (%) A / B (fold) A B A B
A
B
0.05 0.6 1. 7 14.5 15.5
0.007 0.07 12 o.21 34 1.99 290 2.1 31o
io 30.4 285 300
ioo 64 42 24 23
IOO 53 38 23 22
7.1 8-5 8 7.3 7-4
Biochim. Biophys. Acta, 178 (1969) 9 3 - 9 9
90
F. MELANI, M. 1;ARNARARt)
T A B L E 1I EFFECT
OF A DIET LACKING
INORGANIC
IN PHOSPHATE
PYROPHOSPHATASE
ON TIlE RAT KIDNEY
AND INORGANIC
L E V E I . S O F A L K A L [ N I £ I H I t ) S I ' H A T k.SE
PIIOSPHATE
The results are means ± s t a n d a r d deviations; in parentheses the n u m b e r of rats. The content of inorganic p h o s p h a t e was determined on a kidney perchloric extract obtained bv a quick-freezing technique. The enzyme activities were determined on a fraction (15 min at 4ooo ~ ~,, s u p e r n a t a n t ) of lO°o kidney homogenate.
A~zimals
Kidney phosphate Alkaline phospkatas~ Im)rg~ie pyrophosphatase (/tmoles/g per wet weight) (/onoles/mi~ per g prolei~0 (ttmoIeslmi~z per g protei~O
Complete diet 5.~ ~ O ' 0 4 ( I O ) P h o s p h a t e free diet 3.3 ~ o.o() (lo)
82
} I2
(IO)
I I _i2 I " t
i0o t IS (io)
(IO)
23 " 2.2 (to)
purification of rat kidney alkaline phosphatase was accompanied by a concomitant increase in inorganic pyrophosphatase activity. A typical elution profile is given in Fig. 2 in which it is clear that alkaline phosphatase and inorganic pyrophosphatase activities run together through the colunm, both of them being recovered in the few initial fractions at sodium chloride concentrations ranging from o.o3 o.15 M.
Regulation by phosphate of alkaline phosphatase and inorganic pyrophosphatase activities in rat kidney A decrease in the level of inorganic phosphate in the renal cell produced by dietary treatment is accompanied by a concomitant and significant increase in alkaline phosphatase activity 2x,2~. Table II shows that in the renal cell there is also a parallel increase of inorganic pyrophosphatase activity.
Mixed-sabstrate experiments Experiments were carried out in which the rate of phosphate released fl-om TABLE III HYDROLYSIS LINE
OF p-NITROPHENYLPHOSPHATE
AND SODIUM
PYROPHOSPttATE
HY RAT KII)NEV
ALKA-
PItOSPHATASE
E n z y m e was incubated with p - n i t r o p h e n y l p h o s p h a t e (3'mM) or w i t h s o d i m n p y r o p t m s p h a t c (3 raM), or w i t h b o t h at p H 9 (o.o5 M carbonate bicarbonate buffer) for io rain at 25 °. The c o n c e n t r a t i o n of Mg 2~ was o.z mM t h r o u g h o u t . Activities are expressed in p,moles of inorganic p h o s p h a t e (Pi) or p - n i t r o p h e n o l liberated/min per mI of enzyme solution.
Observed
Expectedfi)r independent kydrolysis
Sodium p y r o p h o s p h a t e (3 raM) (/tmoles of Pi/nlin) p - N i t r o p h e n y l p h o s p h a t e (3 raM) (/~moles of Pi/min) (/mmles of p - n i t r o p h e n o l / m i n )
o.3_,
Sodium p y r o p h o s p h a t e (3 mM) ! p - n i t r o p h e n y l p h o s p h a t e (3 raM) (/mloles of Pi/min) (/tmoles of p - n i t r o p h e n o l / m i n )
o.- 5
Bioehim. Biophys. Aeta, I78 (1960) 93 o~-.~
o.3o
0.2 3
o.37
97
ALKALINE PHOSPHATASE AND INORGANIC PYROPHOSPHATASE
15(
•
J
102 IO0
u
N>'-';- 5C
ge
\
"G 5 c
~.o 2~
*g
g
15
lg
20
c
Time (rain)
\
-iog[Mgct~(M)
Fig. 3. Effect of i n c u b a t i o n a t 560 a n d p H 9 (0.05 M Tris-HC1 buffer) on a l k a l i n e p h o s p h a t a s e ( O - - © ) and inorganic pyrophosphatase ( 0 - - 0 ) activities. Fig. 4- Effect of Mg 2+ on a l k a l i n e p h e s p h a t a s e ( O - - 0 ) a n d i n o r g a n i c p y r o p h o s p h a t a s e (O--11) a c t i v i t i e s . The r e a c t i o n m i x t u r e s c o n t a i n e d 3 mM s u b s t r a t e ( p - n i t r o p h e n y l p h o s p h a t e or i n o r g a n i c p y r o p h o s p h a t e ) buffered a t p H 9 w i t h 0.05 M Tris-HC1 buffer. A c t i v i t i e s , m e a s u r e d b y t h e release of o r t h o p h o s p h a t e , are g i v e n as a p e r c e n t a g e of each c o n t r o l w i t h no a d d e d MgC1 v
inorganic pyrophosphate and p-nitrophenylphosphate present together in the same concentration was compared with that from a single substrate; the concentration of Mg ~+ was I mM throughout. As shown in Table III, the amount of phosphate released in the mixed-substrate experiments was less than would be expected for independent hydrolysis of the substrates.
Heat lability of enzymes Fig. 3 shows the effect of incubation at 560 and pH 9 (0.05 M Tris-HC1 buffer) on alkaline phosphatase and inorganic pyrophosphatase activities of purified kidney enzyme; both activities show a constant ratio during heat inactivation at 56°. Treatment with amino acids COX, GILBERT AND GRIFFIN23 reported that L-cysteine caused inhibition of the alkaline phosphatase and inorganic pyrophosphatase activities of HeLa cells in tissue T A B L E IV INHIBITION
OF ALKALINE
PHOSPHATASB
AND INORGANIC PYROPHOSPHATASE
ACTIVITIES
BY CYSTBINE
The e n z y m e s o l u t i o n was p r e - i n c u b a t e d for i m i n w i t h c y s t e i n e or w i t h c y s t e i n e + Zn 2+ in a n ice b a t h . A l i q u o t s were r e m o v e d a n d a s s a y e d for a c t i v i t i e s a t p H 9 (0.05 M c a r b o n a t e - b i c a r b o n a t e buffer).
Additions
Alkaline phosphatase Inorganic pyrophosphatase (ffmoles/min per ml enzyme solution) (ffmoles/min per ml enzyme solution)
None 2 - lO -4 M c y s t e i n e 2 - lO -4 M c y s t e i n e + 2, 5 • lO -5 M Zn ~+ 2 • lO -4 M c y s t e i n e + 5 • IO-~ M Zn 2+
0.33 o.12
0.045 O.Ol 5
0.23
0.025
0.34
o.05
Biochim. Biopkys. Acta, I78 (I969) 93~ )9
98
F. MELANI, M. FARNARARO
cultures. At a cysteine concentration of 2- IO 4 M (pH 9), we observed a strong inhibition of both of these enzyme activities (Table IV). The non-competitive inhibition caused by cysteine was completely removed on the addition of Zn 2~. Other amino acids tested at the same p H in the concentration range 4 ' Io-4 M and 5 "IO 3 }[ were ineffective on either of these enzyme activities. ElFect of MgC12 o~ the e~z~,me activities In the presence of Mg 2+ the phosphomonoesterase activity of purified kidney enzyme was enhanced; with the pyrophosphate as substrate, however, and Mg 2+ concentrations up to about I mM, there was some activation, but above this concentration Mg 2+ was strongly inhibitory (Fig. 4). Similar results were obtained with intestinal and liver enzymes 24. DISCUSSION Our own data on the whole support the idea that both phosphomonoesterase and inorganic pyrophosphatase activities of rat kidney are properties of the same enzyme. In fact it is clear from Fig. z that purified kidney enzyme hydrolyzes both p-nitrophenylphosphate and inorganic pyrophosphate and that kidney preparations had inorganic pyrophosphatase activity at all stages of purification; during the purification procedure (Table I), the ratio of alkaline phosphatase activity (at pH Io.4) to pyrophosphatase activity (at p H 8) remained ahnost constant at different purification steps (7.I in crude extracts; 8. 5 in microsomal suspension in deoxycholate; 8 in supernatant after lipase digestion; 7.3 after the first fractionation; 7.4 after the second fractionation). Furthermore, the alkaline phosphatase and inorganic pyrophosphatase were eluted together as a single peak after DEAE-Sephadex A-25 chromatography of the microsomal proteins (Fig. 2). Additional evidence is provided b y studies of the mechanism by which inorganic phosphate regulates the level of alkaline phosphatase: a decrease in inorganic phosphate level in the renal cell by dietary treatment is accompanied by concomitant and significant increases in both alkaline phosphatase and inorganic pyrophosphatase activities. ROCHE2'~has reported that alkaline phosphatase activity is less stable than pyrophosphatase activity in extracts of pig liver, but our thermal inactivation experiments with rat kidney enzyme did not reveal any such difference in stability (Fig. 3)Furthermore, there was no summation of rates of Pi formation when saturating concentrations of p-nitrophenylphosphate and inorganic pyrophosphate were mixed (Table III). In addition the inhibition by cysteine of alkaline phosphatase and inorganic pyrophosphatase activities proceeded in a parallel fashion (Table IV). It seems less probable that the two types of phosphatase activity are the result of the presence of two enzymes, each of restricted specificity, since experimental results were obtained with an enzyme preparation which appears substantially homogeneous. The last experimental result, which requires further mention, is that hydrolysis of inorganic pyrophosphate m a y be suppressed in a selective fashion by relatively high concentrations of Mg 2+ (Fig. 4). This inhibition m a y have been responsible for MORTON'S observation a that highly purified calf intestinal alkaline phosphatase was free from inorganic pyrophosphatase activity, since his experimental conditions Bfochim. Bfophys. Acta, I78 (I969) 9399
ALKALINE PHOSPHATASE AND INORGANIC PYROPHOSPHATASE
99
included a large excess of Mgz+ (2u-fold) which in our experience would be highly inhibitory. Our interpretation that in rat kidney both orthophosphatase and pyrophosphatase activities are due to the same enzyme, is in agreement with observations concerning alkaline phosphatase from calf intestine n and from several human tissuesS,934. Since purified inorganic pyrophosphatase from yeast 27 and from E s c h e r i chia coli 28 does not exhibit alkaline phosphatase activity, it is probable that the above conclusions may apply essentially to mammalian enzymes. ACKNOWLEDGMENT
We are grateful to Prof. VINCENZO BACCARI for his interest and helpful advice in this work. This work was supported by a grant from the Italian Consiglio Nazionale delle Ricerche. REFERENCES I F. MELANI AND M. FARNARARO,Abstr. 5th Meeting Federation European Biochem. Soc., Prague, i968 , A 947, P- 237. 2 S. K. VOLLEY AND H. D. KAY, Ergeb. Enzymforsch., 5 (1936) 159. 3 R. K. MORTON, Biochem. J., 61 (1955) 232. 4 V. BACCARI AND L. DE ROSA, Boll. Soc. Ital. Biol. Sper., 23 (1947) lO65. 5 H. R. PERKINS AND P. G. WALKER, J. Bone Joint Surg., 4DE (1958) 3336 H. FLEISCH AND S. BISAZ, Nature, 195 (1962) 911. 7 R. G. G. RUSSEL, Lancet, ii (1965) 461. 8 R. P. Cox AND M. J. GRIFFIN, Lancet, ii (1965) lO18. 9 D. W. Moss, R. K. EATON, J. K. SMITH AND L. G. WHITBY, Bioehem. J., lO2 (1967) 53. IO R. K. EATON AND D. W. Moss, Biochem. J., lO 5 (1967) 307 . I I H. N. FERNLEY AND P. G. WALKER, Biochem. J., lO 4 (1967) IOli. 12 D. STETTEN, Am. J. Med., 28 (196o) 867. 13 A. KORNBERG, in M. KASHA AND B. PULLMAN, Horizons in Biochemistry, Academic Press New York, 1962, p. 251. 14 F. MELANI, M. FARNARARO AND G. SGARAGLI,Experientia, 24 (i968) 114. 15 F. MELANI, M. FARNARARO AND G. SGARAGLI, Arch. Biochem. Biophys., 122 (1967) 417 . 16 F. MELANI AND A. GUERRITORE, Experientia, 2o (1964) 464 . 17 O. H. LOWRY AND J. A. LOPEZ, J. Biol. Chem., 162 (1964) 421. 18 G. BEISENHERTZ, H. J. BOLTZE, TH. BOCHER, R. CZOK, K. H. GARBADE, E. MEYER-ARENDT AND G. PFLEIDERER, Z. Naturforsch., 8b (1953) 555. 19 O. WARBURG AND W. CHRISTIAN,Biochem. Z., 31o (1941) 384. 20 H. J. HOHORST, F. H. KREUTZ AND TH. BtDCHER, Biochem. Z., 332 (1959) 18. 21 F. MELANI, G. RAMPONI, A. GUERRITORE AND V. BACCARI, Nature, 2Ol (1964) 71o. 22 F. MELANI, G. RAMPONI, M. FARNARARO, E. C o c u c c I AND A. GUERRITORE, Biochim. Biophys. Acta, 138 (1967) 411. 23 R. P. Cox, P. GILBERT AND M. J. GRIFFIN, Biochem. J., lO 5 (1967) 155. 24 R. H. EATON AND D. W. MOSS, Bioehem. J., lO2 (1967) 917. 25 J. ROCHE, in P. D. BUYER, H. LARDY AND K. MYRBACK, The Enzymes, Vol. I, Academic Press, New York, 195 o, p. 493. 26 H. H. SUSSMAN AND E. LAGA, Biochim. Biophys. Acta, 151 (1968) 281. 27 L. A. HEPPEL AND R. J. HILMOE, J. Biol. Chem., 192 (1951) 87. 28 J JossE, J . Biol. Chem., 241 (1966) 1938.
Biochim. Biophys. Acta, 178 (1969) 93-99