I~I. J. Biochem. Vol. 23, No. 2, pp. 175-180, 1991 Printed in Gnat Britain. All rights reserved
Copyright 0
0020-7 I 1X/91 $3.00 + 0.00 1991 Pcrgamon Press plc
EFFECT OF SOME ALKALINE PHOSPHATASE INHIBITORS ON INTESTINAL CALCIUM TRANSFER Y. DUPUIS,’ S. TARDIVEL,’ Z. POREMBSKA*and P. FOURNIER’ ‘Metabolisme mineral des Mammiferes (EPHE), Physiologie, Faculte de phatmacie, 92296, Chatenay-Malabry Cedex, France [Tel. 661-33-251 *Department of Bi~hemistry, Banacha 1, 02097 Warszawa, Poland (Received 9 April 1990)
Abstract-l. There is a good correiation between the capacity of sugars to stimulate calcium transfer and their capacity to be phosphorylated by the intestinal alkaline phosphate with a part of the phosphate liberated from an ester phosphate. 2. On the sugar dependent and sugar independent calcium transfer, inhibitors of this enzyme act differently. 3. Phosphate, a competitive inhibitor suppresses both transfers, 4. Only the dependent sugar transfer was suppressed with phloridzin acting competitively at the sugar site, or with EDTA which could react close to the active site. 5. L-phenyl~~ine and phenobarbi~l, not competitive inhibitors does not act on either type of calcium transfer, the sugar dependent or the sugar independent.
INTRODUCTION
From the numerous studies of alkaline phosphatase activity, which were based only on its phosphoesterase properties, it has not been possible to pinpoint the exact physiolo~cal role of this enzyme (McComb et al., 1979). The resolution of this interesting problem could help explain the ubiquitus localization of the phosphatase to all sites of intensive nutritient exchange such as microvilli from enterocytes or placenta and kidney tubules. However, in the bone matrix vesicles, it has been shown to play a role in the calcification process. The disparity between the number of studies of this enzyme and the paucity of results concerning its real role, suggested to us that its phosphatase activity may not be its principal function. Other properties such as tr~sphospho~lation, or its phosphorylation could be equally important. In bone and intestine, various correlations have been established between the catalytic activity of alkaline phosphatase and the amount of calcium exchange. The aim of this study is to determine whether numerous substances that affected calcium transfer, can modify three enzyme functions:
1. catalytic activity; 2. transphosphorylase 3. phosphorability,
activity;
Recent works have shown that the alkaline phosphatase is a major phosphorylable protein in the microvillar enterocyte (Dupuis et al., 1981; De Jonge et al., 1981; Crouzoulon er al., 1983; Razanamaniraka et al., 1987). Its activity increased in the presence of cations that it binds, such as Zn and Mg, as well as with Na and K (Hanna et al., 1979). This mineral dependence as well as the importance of membrane protein phosphorylation are aspects which must be considered.
MATERIALS AND METHODS
These experiments were carried out on 4-month-old male rats of the Wistar stain (A.E. Commentry), inbred in our laboratory. Intestinal calcium absorption I. Stomach tube experiments. The rats were fasted for 18 hr and then received 2 ml of a solution of 10 mM CaCl, (+ 1 LtCi45Ca) either with or without the molecule to be studied through a stomach tube. The rats were killed 24 hr follo~ng the gavage. After laparotomy, the small intestine including the wall and content was then ashed. The ashes were dissolved with HNO,. 4SCa was counted in a liquid scintillation counter. The absorption coefficient was calculated according to the formula:
(insrested ‘%a - intestinal ‘%a) x 100
2. Ileal loop experiments. After an over night fast, each rat was anesthetized with ether and the ileal loop (12cm from the ileocoecal junction) was isolated in situ-by tying off the intestine. A 1 ml sample of a 10 mM CaCl, solution (+ 0.1 &i ‘%a) either with or without the molecules to be studied, was injected into the ligated loop. The rats were killed 2 hr following- the iniection. Each loon. _ including the _ wall and content, was then ashed and the ashes were dissolved with HNOS , ‘%a was counted and the absorption coefficient was calculated as above. Semi-purified preparations phosphatases
of duodenal and ileal alkaline
The mucosa of the duodenal segment (from the pylorus to the Treitz ligament) or the mucosa of the 20 distal centimeters of the ileum was scrapped off, pooled and homogenized. Alkaline phosphatase was purified according to a modification of the method of Saini and Done (1972), acetone was substituted for ethanol in the protein precipitation.
175
Y. DUPUISet al.
176 Enzyme arsuy
f. CuruIyric uctioir~~.Alkaline phosphatase activity was estimated at 37°C in a buffer 30 mM Na,CO,-NaHCO, pH 9.8, 5 mM p-nitrophenylphosphate (PNPP) and 2mM MgCI,. p-nitrophenol (PNP) release-d was measured at 405 nm. 2. TT~p~~phoryl~e ~r~i:y. Enzymatic hydrolysis of PNPP released as many PNP molecules as phosphate (P,) molecules. In the presence of various hydroxylated molecules, alkaline phosphatase transferred a part of P, of PNPP onto the hydroxyl. The difference between the number of estimated micromoles of PNP and P, corresponding to the P, transferred, thus represented the ~sph~pho~lase activity of the enzyme. P, was determinated according to the method of Briggs (1924). However, sorbitol interfered with the estimation of Pi. and a more concentrated molybdate reagent must be used in the presence of this molecule (Ho and Pande, 1974). Phospho~yl~ti~n of alkaline phosphatase Alkaline phosphatase phosphorylation was carried out at 30°C for 30 set in 5 FM Y-‘~P(ATP) (1 PCi) containing or no the molecule to be studied. The reaction was stopped by addition of 1ml of 10% trichloroacetic acid (TCA) followed by 0.1 ml of a bovine serum albumine solution (10 mg/ml). T’he precipitate was washed 3 times according to a modification of the method of Weller according to Schoffenniels and Dandrifosse (1980). After centrifugation at 8000g far 5 min, the precipitate was dissolved in 0.5 ml of 0.2 M NaOH, to which was added 1 ml of 10% TCA containing Na,HPCL 110 mMt and An? (0. I mM). The latter ore&itat; was &soived’in OS ml 0.i M NabH and the ‘*P As counted by the Cerenkoff effect after addition of 15 ml of distilled water. Each assay was performed in quadruplicate. Protein assay Protein content was detain by the method of Bradford (1976). using bovine serum albumin as standard. Srarisrical analysis Student test was used to compare the difference between the groups.
Alkaline phosphatase from calf intestine grade 1 was purchased from Boehringer, Hepes. EDTA, Na, ATP and PNPP were from Sigma (St Louis), y-“P(ATP) was from Amersham (France). All other chemicals were of the higher analytical grade available. RESULTS
Comparison between calcium transfer and alkaline phosphatase activity The results of this study are presented in Fig. 1. The ileal calcium transfer was markedly increased by all three sugars studied. The transfer was more than doubled by the pentose (I_-xylose) and the triholoside (raffinose), and was easily tripled by sorbitol. These three compounds had quite different effects on catalytic activity of ileal phosphor, which was enhanced by sorbitol by ca 50%. decreased by xylose whilst raffinose was inactive [Fig. l(A), (B)]. Thus, in these experiments, phosphatase activity
did not seem to be correlated to variations calcium transfer. Comparison between calcium transfer and phosphotransferase activity
The ability of the sugars to act as substrates for the transphospho~lation of P, from PNPP are shown in Fig. 1(C). The catalytic function was measured as the difference between the amount of PNP and Pi formed during the assay. Of all three sugars tested, it was in the presence of sorbitol that the degree of transphosphorylation was the greatest. This was the same relative potency that had been observed for the calcium transfer [Fig. l(A), (C)l. To con&m the existence of a correlation between the transphosphorylation power of the enzyme towards sugars, and the effect of these sugars on calcium transfer, co~nt~tio~~~n~ curves for
50
300
C
*
m
: COnttOl, 0
: sorbitol,
m
: L-xylose,
EZZ3
l
raffinose
Fig. I. Effect of the sugar on intestinal calcium absorption (A), alkaline phosphatase (AP) activity (8) and transphosphorylation on the sugar compound (C). Glucid concentrations, Xi0 mM. *Mean f: SEM of 7 rats (A) or 5 assays (B, C) arbitrarily chosen as control (100%). **Significant P < 0.001 (Student test). ***Non-significant.
177
Alkaline phosphatase inhibitor 60 absorption
coefficient
% sugar
transphosphorylation
1
A
60
60
sorbitol 0
10
30
70
orbitol
mM 0 100
200
mM
500
250
Fig. 2. Effect of increasing doses of sorbitol on ileal calcium transfer (A) and on ileal transphosphorylation (B). (A) A CaCl, solution 10mM was injected into the ileal loop with 0, 10,30,70 and 200 mM of sorbitol. *Mean + SEM (4 assays). Table I. Study of various activators or inhibitors of alkaline phosphatase, effects on calcium intestinal absorption in relation with alkaline ohosohatase Dhosohorvlation Added compound None (control) Sorbitol EDTA Phenobarbital Glycerophosphate
Concentration IIIM
Absorption coefficient in % of control
‘IP fixed omol/min~ma
‘IP % of control
1OOfll(n=8) 222f 18(n =8) 96+7(n=8) 91+IO(n=8) 12+l(n=8)
1O.V 3.1 30.2 8.6 1.2
100 35 280 80
100 10 10 50
1
*Mean of 4 assays.
sorbitol were obtained for both two parameters. The results of this experiment are shown in Fig. 2. A similar concentration dependency was noted for both phenomena, although it must be noted that the maximal effects observed in the two assays are different since only half the Pi can be transferred onto the sugar, a 50% increase in transphosphorylation, the most that can be observed. Thus, intestinal calcium transfer and transphosphorylation respond in the same way to sugars. Comparison between calcium transfer and the degree of phosphorylation of the enzyme
The effects of various compounds such as sorbitol, EDTA, phenobarbital and glycerophosphate were examined on calcium transfer and on phosphatase phosphorylation. Ingested or injected into ileal loops, the various compounds influenced the calcium transfer very differently. This was increased by sorbitol while glycerophosphate decreased it dramatically. The other molecules, EDTA and phenobarbital, did not modify the calcium transfer (Table 1). The phosphorylation of alkaline phosphatase by Y-‘~P(ATP) in the presence of these different substances also varied. However, the degree of phosphorylation measured corresponds to the transitory state of the enzyme since the phosphointermediate is continually being formed and destroyed. Unlike what was seen for calcium transfer, enzyme phosphorylation decreased with sorbitol and increased with EDTA. The enzyme phosphorylation is thus not correlated with calcium absorption.
All the inhibitors or activators of alkaline phosphatase studied previously do not interact with the enzyme by the same mechanism of action. It is possible that a certain mechanism of inhibition or activation may be correlated with calcium transfer. Effect of phenylalanine and phenobarbital on alkaline phosphatase activity from duodenum and ileum
Alkaline phosphatase activity from duodenum and ileum were reduced in the presence of L-phenylalanine in a concentration dependent manner, reaching 75% with 10 mM phenylalanine (Table 2). LineweaverBurk analysis of the alkaline phosphatase activity in the presence of phenobarbital suggested that this molecule inhibited enzyme activity in a uncompetitive manner (Fig. 3). A similar study carried out with kidney alkaline phosphatase from the rat yielded identical results: a noncompetitive inhibition for low phenobarbital concentrations and an
Table 2. Inhibitory effect of L-phenylalanine on alkaline phosphatase activity Alkaline phosphatase activity (umol of PNPP hvdrolwed/min~mn) Duodenum
None Phenylalanine 5 mM Phenylalanine 10 mM *Mean of 4 assays.
Ileum
% inhibition
Compound added 27.6’ 13.3 6.9
52 75
% inhibition 1.95 0.55 0.29
72 85
Y. DWUS et al.
178
Table 3. “CA radioactivity in the plasma of rats having received a Cdl, solution IO mM (+“Ca) done (control) or containing arabiaosc. L-phenyWonine or both “Ca in plasma (% ingested per IOOml) Time after ingestion (hr) 1
Comwunds added
None (control) L-arabinose 100 mM L-phenylalanine 100 mM L.-arabinosc 100 mM + { L-pbenylalanine 20 mM f L-arabinose 100 mhi + 1 i-phkylalaninc 100 mM
2
3
14.3 + 1.3 25.2 + 2.9
13.4 f. 0.9 28.5 + 3.5’
10.2 f 0.7 28.1 + 2.6. I I .3 + O.ZNS
25.1 + 1.3.
32.7 f 3.1.’
31.3 f 3.2.
24.6 ? 2.7
30.1 + 2.1..
27.3 f 2.9.
*Mean + SEM. P < 0.01; l*f < 0.001. Student test.
uncompetitive inhibition for upper (personal communication).
concentrations
Effect of alkaline phosphatase inhibitors on intestinal calcium transfer
The effect of these inhibitors was studied in the presence or absence of various sugar. The
results are shown in Table 3 and Fig. 4. A 1OmM CaCI, solution was ingested or injected in an intestinal loop. 20% of CaCI, was absorbed under basal conditions. In the presence of a sugar, calcium absorption was about doubled. In the presence of different inhibitors, two patterns was observed.
by min. ml
q
phenobarbital
l
phenobarbital
E phenobarbital
II 035
Fig. 3. Lineweaver-Burk
Q1 absorption
mM
200
mM
40 mM
control
l
091
400
[PNPP]
mM
1
representation of PNPP hydrolysis by alkaline phosphatase in the presence of different concentrations of phenobarbital.
coefficient
m
0 Sugar
=
+ Sugar
.; 0
inhibitor
Phosphate 20 mM
Phloridrlne 100 mM
l
Phenyl alanine 20 to 100 mM
l
&** phenobarbital” 100 mM 10mM Fig. 4. Intestinal calcium absorption in the presence or absence of the sugar. Effect of various alkaline phosphatase inhibitors. l“Ca injected by ingestion. **%a injected by ileal loop.
179
Alkaline phosphatase inhibitor 1. Phosphate. It diminished considerably basal absorption. In the presence of sorbitol, calcium transfer was also very markedly reduced. 2. Other compounds. For all the other inhibitors, basal calcium transfer was unaffected. However, in the presence of sorbitol two cases were observed: -phforidzin
and EL)TA : these two compounds abolished completely the activation of calcium transfer by sorbitol; -phenylalanine and phenobarbital: they did not modify either basal transfer nor that activated by sorbitol.
DISCUSSION Sugars, as activators, and phosphate, as an inhibitor, both modulates intestinal calcium transfer. This modulation seems to be controlled e~mati~ally, and in particular, by alkaline phosphatase. This enzyme can transphosphorylate Pi, an inhibitor of calcium transfer, from an ester phosphate onto the sugar, which activates calcium transfer. The effects of Pi and glucids on calcium transfer are mutually antago~sti~ (Dupuis et af., 19’77, 1978). Among the different properties of the enzyme, two cannot be correlated with calcium transfer: catalytic activity and the degree of autophosphorylation. However, transphorylation of Pi, like calcium transfer, is increased by sugars. Pi, a com~titive inhibitor of alkaline phosphatase, also inhibits calcium transfer. Pi interacts with the catalytic site of the enzyme. Thus this site could be implicated in calcium transfer. When a sugar is present simultaneously with the Pi, transphosphorylation is possible and the amount of calcium absorption absorbed depends on the relative amounts of sugar and phosphate present. We can suppose that the sugar binds to a site topographically close to the catalytic site. In contrast, phenylalanine and phenobarbital which are not competitive inhibitors do not modify basal calcium transfer, the catalytic site not being implicated in this type of inhibition. These two molecules do not interfere with the effect of the sugar. The glucoside, phloridzin, which inhibits alkaline phosphatase activity (Kalckar, 1936) and EDTA, uncom~titive inhibitors of alkafine phosphatase did not affect calcium transfer but abolished the effect of the sugar. It can be supposed that basal calcium transfer, which requires the integrity of the active site is conserved, but that the transphospho~lation process is disturbed. Phloridzin could bind to the sugar site. Transphosphorylation onto the sugar is blocked and sugar stimulated calcium transfer is inhibited. Another glycoside, gentamicin, has also been reported to decrease kidney alkaline phosphatase activity as well as glucose transfer. The authors propose that the inositol phosphate cycle is involved (Takahashi et al., 1987). EDTA acts in different ways. It chelates the metal from the enzyme. However, the apoenzyme can also bind EDTA, although not to at the active site (Csopak et al., 1972; Csopak and Szahn, 1973). From our results, we can suppose that in the presence of EDTA, the transphosphorylation onto the sugar is perturbed.
Two main conclusions can be drawn from this study. Firstly, that basal calcium transfer requires a functional active site. Secondly, the transphospho~lating capacity of the enzyme must be intact for the sugar to increase calcium transfer. An inhibitor blocking transphosphorylation also blocks sugarinduced calcium transfer. These ideas may imply that the active site is involved since the presence of the sugar increases the absorption duration of the cation (Lengemann et al., 1959). The role of the cation is, however, not clear, nor is the significance of the multication-dependency of the enzyme. This suggests that alkaline phosphatase like any protein, but to a particularly high degree, possesses cation exchange properties.
SUMMARY
The property of phosphate acceptors, principally, sugars, to increase intestinal calcium transfer, allows a sugar-dependent transfer to be differentiated from a sugar-independent transfer. Sugars and alkaline phosphatase are functionally related. By transphosphorylation, the alkaline phosphatase transfers to the sugar a part of the phosphate liberated by hydrolysis of the phosphate ester. There is a very good correlation between the capacity of various sugars to stimulate the calcium transfer and their capacity to be phospho~Iat~ by the enzyme. Both calcium transfer and the rate of transphosphorylation increase with the concentration of any given sugars. We have examined to what extent inhibitors of the enzyme modify calcium transfer, especially the sugar-dependent component of the transfer. These inhibitors can be divided into three groups. -Phosphate, a competitive inhibitor, suppresses both the sugar-de~ndent and the sugar independent transfer. This inhibition may correspond, in this case, to the occupation of the active site by phosphate. -The dependent sugar transfer was suppressed with other inhibitors which did not modify the sugar-independent transfer. This was the case for phloridzin which competes with the sugar. Indeed, phloridzin binds competitively to alkaline phosphatase at the sugar site, a site most likely near the active site, since the enzyme can transphospho~late the sugar. In fact, similar observations have been made in another functional system, “phosphatase-aminoglucoside-glucose” for transfer of the glucose. This is also valid for EDTA. Its affinity for the zinc atom requires it to be near the active site, but not to occupy it. -A third group of inhibitors of alkaline phosphatase does not act on either type of transfer, the sugar-dependent or the sugarinde~ndent. Thus, L-phenylalanine and phenobarbital, which are not competitive inhibitors, do not act directly at the active site of the enzyme.
180
Y. DUP‘uts et al. lysine explain the activity of the latter on calcium transfer?
REFERENCES
ht. J. Biochem. 13, 1170-1181. Bradford M. A. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254.
Briggs A. P. (1924) Some applications of the coloriJ. biol. Chem. 59, metric phosphate method.
Biochem. 60, 413-416.
255-264.
Crouzoulon G., Dupuis Y., Rey D. and Foumier P. (1983) Phosphorylated proteins from rat intestinal microvilli membranes. Electrophoretic similarities with alkaline phosphatase. Molec. Physiol. 4, 423-429. Csopak H. and Szajn H. (1973) Factors affecting the zinc content of E. coli alkaline phosphatase. Archs Biochem. Biophys. 157, 374-379.
Csopak H., Falk K. E. and Szajn H. (1972) Effect of EDTA on Escherichia coli alkaline phosphatase. Biochem. biophys. Acta. 258, 466-472.
Hanna S. D., Mircheff A. K. and Wright E. M. (1979) Alkaline phosphatase of basal lateral and brush border plasma membranes from intestinal epithelium. J. Su@amolec. Str. 11, 451-466. Ho C. H. and Pande S. V. (1974) Grthoohosohate determination interference by mannitol and soibitol. Analyt.
_
De Jonee H.. Ghiisen W. and Van OS C. H. (1981) Phosphorylated intermediates of Ca++ ATPase and alkaline phosphatase in plasma membranes from rat duodenal epithelium. Biochem. biophys. Acta. 647, 140-149.
Dupuis Y., Digaud A. and Fontaine N. (1977) Further observations in favour of the role of alkaline phosphatase in calcium absorption. Calc. Tiss. Res. 22, 556-560.
Dupuis Y., Digaud A. and Fournier P. (1978) Relationship between intestinal alkaline phosphatase and carbohydrates to their connexion with calcium absorption. krchs Int. Physiol. Biochem. 86, 543-556.
_
Dupuis Y., Crouzoulon G. and Fourier P. (1981) Does the inhibition of microvillar protein phosphorylation by
Kalckar H. (1936) Inhibitory effect of phloridzin and phloretin on kidney phosphatase. Nature (London) 138, 289-290.
Lengemann F. W., Wasserman R. H. and Comar C. L. (1959) Studies on the enhencement of radiocalcium and radiostrontium absorption by lactose in rat. J. Nutr. 68, 443-446. MC Comb R., Bowers G. and Posen S. (1979) Alkaline Phosphatase, pp. l-986. Plenum Press, New York. Razanamaniraka L., Tardivel S., Porembska Z., Dupuis Y. and Crouzoulon G. (1987) Phosphorylable proteins and alkaline phosphatase of brush border membranes from different part of the rat small intestine. Znr. J. Biochem. 19, 1075-1084.
Saini P. K. and Done J. (1972) The diversity of alkaline phosphatase from rat intestine. Isolation and purification of the enzymes. Biochim. biophys. Acta 258, 147-153. Schoffeniels E. and Dandrifosse G. (1980) Protein phosphorylation and sodium conductance in nerve membranes. Proc. natn. Acad. Sci. U.S.A. 77, 812-816. Takahashi M., Aramaki Y., Inaba A. and Tsuchiya (1987) Inhibition of alkaline phosphatase activity and o-glucose uptake in rat renal brush border membrane vesicles by aminoglycoside. Biochim. biophys. Acta 903, 3 l-36.
Copyright 0
0020-7 I 1X/91 $3.00 + 0.00 1991 Pcrgamon Press plc
EFFECT OF SOME ALKALINE PHOSPHATASE INHIBITORS ON INTESTINAL CALCIUM TRANSFER Y. DUPUIS,’ S. TARDIVEL,’ Z. POREMBSKA*and P. FOURNIER’ ‘Metabolisme mineral des Mammiferes (EPHE), Physiologie, Faculte de phatmacie, 92296, Chatenay-Malabry Cedex, France [Tel. 661-33-251 *Department of Bi~hemistry, Banacha 1, 02097 Warszawa, Poland (Received 9 April 1990)
Abstract-l. There is a good correiation between the capacity of sugars to stimulate calcium transfer and their capacity to be phosphorylated by the intestinal alkaline phosphate with a part of the phosphate liberated from an ester phosphate. 2. On the sugar dependent and sugar independent calcium transfer, inhibitors of this enzyme act differently. 3. Phosphate, a competitive inhibitor suppresses both transfers, 4. Only the dependent sugar transfer was suppressed with phloridzin acting competitively at the sugar site, or with EDTA which could react close to the active site. 5. L-phenyl~~ine and phenobarbi~l, not competitive inhibitors does not act on either type of calcium transfer, the sugar dependent or the sugar independent.
INTRODUCTION
From the numerous studies of alkaline phosphatase activity, which were based only on its phosphoesterase properties, it has not been possible to pinpoint the exact physiolo~cal role of this enzyme (McComb et al., 1979). The resolution of this interesting problem could help explain the ubiquitus localization of the phosphatase to all sites of intensive nutritient exchange such as microvilli from enterocytes or placenta and kidney tubules. However, in the bone matrix vesicles, it has been shown to play a role in the calcification process. The disparity between the number of studies of this enzyme and the paucity of results concerning its real role, suggested to us that its phosphatase activity may not be its principal function. Other properties such as tr~sphospho~lation, or its phosphorylation could be equally important. In bone and intestine, various correlations have been established between the catalytic activity of alkaline phosphatase and the amount of calcium exchange. The aim of this study is to determine whether numerous substances that affected calcium transfer, can modify three enzyme functions:
1. catalytic activity; 2. transphosphorylase 3. phosphorability,
activity;
Recent works have shown that the alkaline phosphatase is a major phosphorylable protein in the microvillar enterocyte (Dupuis et al., 1981; De Jonge et al., 1981; Crouzoulon er al., 1983; Razanamaniraka et al., 1987). Its activity increased in the presence of cations that it binds, such as Zn and Mg, as well as with Na and K (Hanna et al., 1979). This mineral dependence as well as the importance of membrane protein phosphorylation are aspects which must be considered.
MATERIALS AND METHODS
These experiments were carried out on 4-month-old male rats of the Wistar stain (A.E. Commentry), inbred in our laboratory. Intestinal calcium absorption I. Stomach tube experiments. The rats were fasted for 18 hr and then received 2 ml of a solution of 10 mM CaCl, (+ 1 LtCi45Ca) either with or without the molecule to be studied through a stomach tube. The rats were killed 24 hr follo~ng the gavage. After laparotomy, the small intestine including the wall and content was then ashed. The ashes were dissolved with HNO,. 4SCa was counted in a liquid scintillation counter. The absorption coefficient was calculated according to the formula:
(insrested ‘%a - intestinal ‘%a) x 100
2. Ileal loop experiments. After an over night fast, each rat was anesthetized with ether and the ileal loop (12cm from the ileocoecal junction) was isolated in situ-by tying off the intestine. A 1 ml sample of a 10 mM CaCl, solution (+ 0.1 &i ‘%a) either with or without the molecules to be studied, was injected into the ligated loop. The rats were killed 2 hr following- the iniection. Each loon. _ including the _ wall and content, was then ashed and the ashes were dissolved with HNOS , ‘%a was counted and the absorption coefficient was calculated as above. Semi-purified preparations phosphatases
of duodenal and ileal alkaline
The mucosa of the duodenal segment (from the pylorus to the Treitz ligament) or the mucosa of the 20 distal centimeters of the ileum was scrapped off, pooled and homogenized. Alkaline phosphatase was purified according to a modification of the method of Saini and Done (1972), acetone was substituted for ethanol in the protein precipitation.
175
Y. DUPUISet al.
176 Enzyme arsuy
f. CuruIyric uctioir~~.Alkaline phosphatase activity was estimated at 37°C in a buffer 30 mM Na,CO,-NaHCO, pH 9.8, 5 mM p-nitrophenylphosphate (PNPP) and 2mM MgCI,. p-nitrophenol (PNP) release-d was measured at 405 nm. 2. TT~p~~phoryl~e ~r~i:y. Enzymatic hydrolysis of PNPP released as many PNP molecules as phosphate (P,) molecules. In the presence of various hydroxylated molecules, alkaline phosphatase transferred a part of P, of PNPP onto the hydroxyl. The difference between the number of estimated micromoles of PNP and P, corresponding to the P, transferred, thus represented the ~sph~pho~lase activity of the enzyme. P, was determinated according to the method of Briggs (1924). However, sorbitol interfered with the estimation of Pi. and a more concentrated molybdate reagent must be used in the presence of this molecule (Ho and Pande, 1974). Phospho~yl~ti~n of alkaline phosphatase Alkaline phosphatase phosphorylation was carried out at 30°C for 30 set in 5 FM Y-‘~P(ATP) (1 PCi) containing or no the molecule to be studied. The reaction was stopped by addition of 1ml of 10% trichloroacetic acid (TCA) followed by 0.1 ml of a bovine serum albumine solution (10 mg/ml). T’he precipitate was washed 3 times according to a modification of the method of Weller according to Schoffenniels and Dandrifosse (1980). After centrifugation at 8000g far 5 min, the precipitate was dissolved in 0.5 ml of 0.2 M NaOH, to which was added 1 ml of 10% TCA containing Na,HPCL 110 mMt and An? (0. I mM). The latter ore&itat; was &soived’in OS ml 0.i M NabH and the ‘*P As counted by the Cerenkoff effect after addition of 15 ml of distilled water. Each assay was performed in quadruplicate. Protein assay Protein content was detain by the method of Bradford (1976). using bovine serum albumin as standard. Srarisrical analysis Student test was used to compare the difference between the groups.
Alkaline phosphatase from calf intestine grade 1 was purchased from Boehringer, Hepes. EDTA, Na, ATP and PNPP were from Sigma (St Louis), y-“P(ATP) was from Amersham (France). All other chemicals were of the higher analytical grade available. RESULTS
Comparison between calcium transfer and alkaline phosphatase activity The results of this study are presented in Fig. 1. The ileal calcium transfer was markedly increased by all three sugars studied. The transfer was more than doubled by the pentose (I_-xylose) and the triholoside (raffinose), and was easily tripled by sorbitol. These three compounds had quite different effects on catalytic activity of ileal phosphor, which was enhanced by sorbitol by ca 50%. decreased by xylose whilst raffinose was inactive [Fig. l(A), (B)]. Thus, in these experiments, phosphatase activity
did not seem to be correlated to variations calcium transfer. Comparison between calcium transfer and phosphotransferase activity
The ability of the sugars to act as substrates for the transphospho~lation of P, from PNPP are shown in Fig. 1(C). The catalytic function was measured as the difference between the amount of PNP and Pi formed during the assay. Of all three sugars tested, it was in the presence of sorbitol that the degree of transphosphorylation was the greatest. This was the same relative potency that had been observed for the calcium transfer [Fig. l(A), (C)l. To con&m the existence of a correlation between the transphosphorylation power of the enzyme towards sugars, and the effect of these sugars on calcium transfer, co~nt~tio~~~n~ curves for
50
300
C
*
m
: COnttOl, 0
: sorbitol,
m
: L-xylose,
EZZ3
l
raffinose
Fig. I. Effect of the sugar on intestinal calcium absorption (A), alkaline phosphatase (AP) activity (8) and transphosphorylation on the sugar compound (C). Glucid concentrations, Xi0 mM. *Mean f: SEM of 7 rats (A) or 5 assays (B, C) arbitrarily chosen as control (100%). **Significant P < 0.001 (Student test). ***Non-significant.
177
Alkaline phosphatase inhibitor 60 absorption
coefficient
% sugar
transphosphorylation
1
A
60
60
sorbitol 0
10
30
70
orbitol
mM 0 100
200
mM
500
250
Fig. 2. Effect of increasing doses of sorbitol on ileal calcium transfer (A) and on ileal transphosphorylation (B). (A) A CaCl, solution 10mM was injected into the ileal loop with 0, 10,30,70 and 200 mM of sorbitol. *Mean + SEM (4 assays). Table I. Study of various activators or inhibitors of alkaline phosphatase, effects on calcium intestinal absorption in relation with alkaline ohosohatase Dhosohorvlation Added compound None (control) Sorbitol EDTA Phenobarbital Glycerophosphate
Concentration IIIM
Absorption coefficient in % of control
‘IP fixed omol/min~ma
‘IP % of control
1OOfll(n=8) 222f 18(n =8) 96+7(n=8) 91+IO(n=8) 12+l(n=8)
1O.V 3.1 30.2 8.6 1.2
100 35 280 80
100 10 10 50
1
*Mean of 4 assays.
sorbitol were obtained for both two parameters. The results of this experiment are shown in Fig. 2. A similar concentration dependency was noted for both phenomena, although it must be noted that the maximal effects observed in the two assays are different since only half the Pi can be transferred onto the sugar, a 50% increase in transphosphorylation, the most that can be observed. Thus, intestinal calcium transfer and transphosphorylation respond in the same way to sugars. Comparison between calcium transfer and the degree of phosphorylation of the enzyme
The effects of various compounds such as sorbitol, EDTA, phenobarbital and glycerophosphate were examined on calcium transfer and on phosphatase phosphorylation. Ingested or injected into ileal loops, the various compounds influenced the calcium transfer very differently. This was increased by sorbitol while glycerophosphate decreased it dramatically. The other molecules, EDTA and phenobarbital, did not modify the calcium transfer (Table 1). The phosphorylation of alkaline phosphatase by Y-‘~P(ATP) in the presence of these different substances also varied. However, the degree of phosphorylation measured corresponds to the transitory state of the enzyme since the phosphointermediate is continually being formed and destroyed. Unlike what was seen for calcium transfer, enzyme phosphorylation decreased with sorbitol and increased with EDTA. The enzyme phosphorylation is thus not correlated with calcium absorption.
All the inhibitors or activators of alkaline phosphatase studied previously do not interact with the enzyme by the same mechanism of action. It is possible that a certain mechanism of inhibition or activation may be correlated with calcium transfer. Effect of phenylalanine and phenobarbital on alkaline phosphatase activity from duodenum and ileum
Alkaline phosphatase activity from duodenum and ileum were reduced in the presence of L-phenylalanine in a concentration dependent manner, reaching 75% with 10 mM phenylalanine (Table 2). LineweaverBurk analysis of the alkaline phosphatase activity in the presence of phenobarbital suggested that this molecule inhibited enzyme activity in a uncompetitive manner (Fig. 3). A similar study carried out with kidney alkaline phosphatase from the rat yielded identical results: a noncompetitive inhibition for low phenobarbital concentrations and an
Table 2. Inhibitory effect of L-phenylalanine on alkaline phosphatase activity Alkaline phosphatase activity (umol of PNPP hvdrolwed/min~mn) Duodenum
None Phenylalanine 5 mM Phenylalanine 10 mM *Mean of 4 assays.
Ileum
% inhibition
Compound added 27.6’ 13.3 6.9
52 75
% inhibition 1.95 0.55 0.29
72 85
Y. DWUS et al.
178
Table 3. “CA radioactivity in the plasma of rats having received a Cdl, solution IO mM (+“Ca) done (control) or containing arabiaosc. L-phenyWonine or both “Ca in plasma (% ingested per IOOml) Time after ingestion (hr) 1
Comwunds added
None (control) L-arabinose 100 mM L-phenylalanine 100 mM L.-arabinosc 100 mM + { L-pbenylalanine 20 mM f L-arabinose 100 mhi + 1 i-phkylalaninc 100 mM
2
3
14.3 + 1.3 25.2 + 2.9
13.4 f. 0.9 28.5 + 3.5’
10.2 f 0.7 28.1 + 2.6. I I .3 + O.ZNS
25.1 + 1.3.
32.7 f 3.1.’
31.3 f 3.2.
24.6 ? 2.7
30.1 + 2.1..
27.3 f 2.9.
*Mean + SEM. P < 0.01; l*f < 0.001. Student test.
uncompetitive inhibition for upper (personal communication).
concentrations
Effect of alkaline phosphatase inhibitors on intestinal calcium transfer
The effect of these inhibitors was studied in the presence or absence of various sugar. The
results are shown in Table 3 and Fig. 4. A 1OmM CaCI, solution was ingested or injected in an intestinal loop. 20% of CaCI, was absorbed under basal conditions. In the presence of a sugar, calcium absorption was about doubled. In the presence of different inhibitors, two patterns was observed.
by min. ml
q
phenobarbital
l
phenobarbital
E phenobarbital
II 035
Fig. 3. Lineweaver-Burk
Q1 absorption
mM
200
mM
40 mM
control
l
091
400
[PNPP]
mM
1
representation of PNPP hydrolysis by alkaline phosphatase in the presence of different concentrations of phenobarbital.
coefficient
m
0 Sugar
=
+ Sugar
.; 0
inhibitor
Phosphate 20 mM
Phloridrlne 100 mM
l
Phenyl alanine 20 to 100 mM
l
&** phenobarbital” 100 mM 10mM Fig. 4. Intestinal calcium absorption in the presence or absence of the sugar. Effect of various alkaline phosphatase inhibitors. l“Ca injected by ingestion. **%a injected by ileal loop.
179
Alkaline phosphatase inhibitor 1. Phosphate. It diminished considerably basal absorption. In the presence of sorbitol, calcium transfer was also very markedly reduced. 2. Other compounds. For all the other inhibitors, basal calcium transfer was unaffected. However, in the presence of sorbitol two cases were observed: -phforidzin
and EL)TA : these two compounds abolished completely the activation of calcium transfer by sorbitol; -phenylalanine and phenobarbital: they did not modify either basal transfer nor that activated by sorbitol.
DISCUSSION Sugars, as activators, and phosphate, as an inhibitor, both modulates intestinal calcium transfer. This modulation seems to be controlled e~mati~ally, and in particular, by alkaline phosphatase. This enzyme can transphosphorylate Pi, an inhibitor of calcium transfer, from an ester phosphate onto the sugar, which activates calcium transfer. The effects of Pi and glucids on calcium transfer are mutually antago~sti~ (Dupuis et af., 19’77, 1978). Among the different properties of the enzyme, two cannot be correlated with calcium transfer: catalytic activity and the degree of autophosphorylation. However, transphorylation of Pi, like calcium transfer, is increased by sugars. Pi, a com~titive inhibitor of alkaline phosphatase, also inhibits calcium transfer. Pi interacts with the catalytic site of the enzyme. Thus this site could be implicated in calcium transfer. When a sugar is present simultaneously with the Pi, transphosphorylation is possible and the amount of calcium absorption absorbed depends on the relative amounts of sugar and phosphate present. We can suppose that the sugar binds to a site topographically close to the catalytic site. In contrast, phenylalanine and phenobarbital which are not competitive inhibitors do not modify basal calcium transfer, the catalytic site not being implicated in this type of inhibition. These two molecules do not interfere with the effect of the sugar. The glucoside, phloridzin, which inhibits alkaline phosphatase activity (Kalckar, 1936) and EDTA, uncom~titive inhibitors of alkafine phosphatase did not affect calcium transfer but abolished the effect of the sugar. It can be supposed that basal calcium transfer, which requires the integrity of the active site is conserved, but that the transphospho~lation process is disturbed. Phloridzin could bind to the sugar site. Transphosphorylation onto the sugar is blocked and sugar stimulated calcium transfer is inhibited. Another glycoside, gentamicin, has also been reported to decrease kidney alkaline phosphatase activity as well as glucose transfer. The authors propose that the inositol phosphate cycle is involved (Takahashi et al., 1987). EDTA acts in different ways. It chelates the metal from the enzyme. However, the apoenzyme can also bind EDTA, although not to at the active site (Csopak et al., 1972; Csopak and Szahn, 1973). From our results, we can suppose that in the presence of EDTA, the transphosphorylation onto the sugar is perturbed.
Two main conclusions can be drawn from this study. Firstly, that basal calcium transfer requires a functional active site. Secondly, the transphospho~lating capacity of the enzyme must be intact for the sugar to increase calcium transfer. An inhibitor blocking transphosphorylation also blocks sugarinduced calcium transfer. These ideas may imply that the active site is involved since the presence of the sugar increases the absorption duration of the cation (Lengemann et al., 1959). The role of the cation is, however, not clear, nor is the significance of the multication-dependency of the enzyme. This suggests that alkaline phosphatase like any protein, but to a particularly high degree, possesses cation exchange properties.
SUMMARY
The property of phosphate acceptors, principally, sugars, to increase intestinal calcium transfer, allows a sugar-dependent transfer to be differentiated from a sugar-independent transfer. Sugars and alkaline phosphatase are functionally related. By transphosphorylation, the alkaline phosphatase transfers to the sugar a part of the phosphate liberated by hydrolysis of the phosphate ester. There is a very good correlation between the capacity of various sugars to stimulate the calcium transfer and their capacity to be phospho~Iat~ by the enzyme. Both calcium transfer and the rate of transphosphorylation increase with the concentration of any given sugars. We have examined to what extent inhibitors of the enzyme modify calcium transfer, especially the sugar-dependent component of the transfer. These inhibitors can be divided into three groups. -Phosphate, a competitive inhibitor, suppresses both the sugar-de~ndent and the sugar independent transfer. This inhibition may correspond, in this case, to the occupation of the active site by phosphate. -The dependent sugar transfer was suppressed with other inhibitors which did not modify the sugar-independent transfer. This was the case for phloridzin which competes with the sugar. Indeed, phloridzin binds competitively to alkaline phosphatase at the sugar site, a site most likely near the active site, since the enzyme can transphospho~late the sugar. In fact, similar observations have been made in another functional system, “phosphatase-aminoglucoside-glucose” for transfer of the glucose. This is also valid for EDTA. Its affinity for the zinc atom requires it to be near the active site, but not to occupy it. -A third group of inhibitors of alkaline phosphatase does not act on either type of transfer, the sugar-dependent or the sugarinde~ndent. Thus, L-phenylalanine and phenobarbital, which are not competitive inhibitors, do not act directly at the active site of the enzyme.
180
Y. DUP‘uts et al. lysine explain the activity of the latter on calcium transfer?
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