The direct effect of different vanadium compounds upon alkaline phosphatase (ALP) activity was investigated~ Vanadate and vanadyl inhibited both the soluble and particulate ALP activity from UMR.106 cells and front bovine intestinal ALP. We have also shown the inhibition of ALP activity in the soluble fraction of osteoblasts by peroxo and hydroperoxo vanadium compounds. ALP activity in the particulate fraction was not inhibited by these species; nor was the bovine intestinal ALP. Using inhibitors of Tyr-phosphatase (PTPases), the soluble ALP was partially characterized as a PTPase. The major activity in the particulate fraction represents the bone-specific ALPactivity. This study demonstrates that different forms of vanadium are direct inhibitors of ALP activity. This effect is dependent on the enzymatic activity investigated and on the origin of the ALP.Facultad de Ciencias Médica

Cortizo, Ana María

Etcheverry, Susana B.

Salice, Viviana C.

Biological Trace Element Research

English

LAReferencia - Red Federada de Repositorios Institucionales de Publicaciones Científicas Latinoamericanas

9 1994 by Humana Press Inc. All rights of any nature, whatsoever, reserved. 0163-4984/94/4103-0331 $03.00 Vanadium Compounds Their Action on Alkaline Phosphatase Activity ANA M. CORTIZO,"2 VIVIANA C. SALICE,1 AND SUSANA B .  FTCHEVERRY *'~'3 'Catedra de Bioquimica Patol6gica, Facultad de Ciencias Exactas, UNLP, 47 y 115, 1900 La Plata, Argentina; 2CENEXA, Facultad de Ciencias M4dicas; and 3Programa QUINOR, Facultad de Ciencias Exactas; Universidad Nacional de la Plata, La Plata, Argentina Received June 30, 1993; Accepted August 5, 1993 ABSTRACT The direct effect of different vanadium compounds upon alka- line phosphatase (ALP) activity was investigated~ Vanadate and vanadyl inhibited both the soluble and particulate ALP activity from UMR.106 cells and front bovine intestinal ALP. We have also shown the inhibition of ALP activity in the soluble fraction of osteoblasts by peroxo and hydroperoxo vanadium compounds. ALP activity in the particulate fraction was not inhibited by these species; nor was the bovine intestinal ALP. Using inhibitors of Tyr-phosphatase (PTPases), the soluble ALP was partially characterized as a PTPase. The major activity in the particulate fraction represents the bone-specific ALP- activity. This study demonstrates that different forms of vanadium are direct inhibitors of ALP activity. This effect is dependent on the enzymatic activity investigated and on the origin of the ALP. Index Entries: Vanadium; alkaline phosphatase; tyrosine-phos- phatase; osteoblast cells; bone cells; inhibitory effects. INTRODUCTION Vanad ium is a transition metal  of g roup  5 that shows  var ious  oxi- da t ion  states. It is present  in living sys tems in trace amoun t s  (1). Vana- d i u m  affects different biological processes  such as the inhibit ion of NaG *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research 331 Vol. 4I, I994 332 Cortizo, Salice, and Etcheverry K+-ATPase, glucose oxidation in adypocites, insulin-mimetic actions, and the inhibition of several Tyrosine-phosphatases (PTPases) (2,3). Phosphotyrosine accumulation is induced by vanadate inside the cultured cells (4). Vanadium +5 may be reduced to vanadyl +4 in the presence of NADPH and 02 during cellular metabolism. Addition of peroxo- or hydroperoxide vanadium compounds to permeabilized HL- 60 granulocytes greatly enhances the phosphotyrosine content of the cells (4,5). At present, the mechanism by which phosphotyrosine increases is unknown. Osteoblast cells are involved in bone formation and their differenti- ation is modulated by multiple factors such as hormones, growth fac- tors, and ions (6). Various peptide factors stimulate the tyrosine-kinases associated to the cognate receptors and subsequently they induce tyro- sine-phosphorylation of specific cellular proteins. These effects are coun- terbalanced by the presence of the PTPases, which in turn deactivate the receptors (7). PTPases have been isolated and characterized in several systems. They belong to two general families: receptorqike PTPases asso- ciated with membranes, and nonreceptor-like PTPases (8,9). The aim of this study was to investigate the direct effect of different vanadium compounds upon alkaline phosphatase (ALP) activity. We also attempted to characterize the enzymes sensitivity to vanadium from rat osteoblast cell line UMR.106. METHODS Materials  Vanadium (IV) oxide sulfate (vanadyl sulfate) was obtained from Merck, sodium o-vanadate, potassium superoxide, superoxide dismu- tase, p-nitrophenylphosphate (pNPP), and catalase were obtained from Sigma (St. Louis, MO), as well as the bovine intestinal alkaline phos- phatase. Tissue culture materials were provided by Corning or Falcon. DMEM and trypsin-EDTA were supplied by Gibco (Gaithersburg, MD) and FBS by Gen (Argentina). All other chemicals used were of analytical grade from Sigma. Solut ions  Fresh stock solutions of vanadyl sulfate and sodium o-vanadate were prepared in distilled water at 100 mM concentration. Peroxovana- dium and hydroperoxide vanadium compounds were prepared as previ- ously reported (4). Cell Cul ture  Rat osteosarcoma cell line (UMR.106) were grown in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine Biological Trace Element Research VoL 41, 1994 Vanadium Effects on ALP Activity 333 se rum (FBS) and antibiotics. Cells were passaged by Trypsin-EDTA treat- m e n t  to 150 cm2 flask once a week. After the cells reached confluence, the m e d i u m  was removed  and the monolayer  w a s h e d  wi th  phospha te  buffered saline (PBS) pH 7.4 and once in 50 m M  Hepes /150  m M  NaC1 buffer  conta in ing  1 m M  EDTA, 15 m M  ~3-mercaptoethanol and  1 m M  phenyle thanosul fonyl  fluoride (10). Preparation of  Cellular Fractions Cells were scraped in the above buffer and sonicated at 600 ps i /15  s. The homogena te  was centrifuged at 2500 r p m / 5  min. The supernatant  was  cen t r i fuged  at 45,000 g / 9 0  min  at 4~ The supe rna t an t  (soluble fraction) was  s tored at -70~ The pellet  was solubi l ized in 50 m M  Hepes /150  m M  NaC1/1% Triton-X100 and recentr ifuged as before. The final superna tan t  (particulate fraction) was stored at -70~ Aliquots of both fractions were used in protein assay by the Bradford me thod  (11). Alkaline Phosphatase Activity (ALP) The ALP activity from bovine intestinal mucosa  was de te rmined  as descr ibed by Crans (12). In brief, p -n i t ropheny lphospha te  (pNPP) was used  as a substrate in 20 mM Hepes buffer, pH 8.0, in the presence of 20 m M  KC1 and 30 mM MgC12. Incubat ion was carried out  at room tem- perature  for 10 min  and absorbance at 405 nm was measured  in order to de te rmine  the initial rate of the activity. Under  these experimental  con- ditions, the reaction was linear until  30 min. The initial rate of ALP activity in the soluble and particulate frac- tions of UMR.106 cells was de termined by incubation of 100 ~g protein in 20 m M  Hepes buffer pH 8.0/20 m M  KC1/30 mM MgC12 and 5 mM pNPP. Absorbance  at 405 n m  was mon i to r ed  at 37~ d u r i n g  5 min. Unde r  these conditions, the reaction proceeded linearly for 30 min. The effect of different vanad ium c o m p o u n d s  and other agents were tested in coincubations with the different enzymes.  The inhibitory effect was de te rmined  as a percentage of the basal initial velocity of the ALP activity. Statistical Analysis The results are expressed as mean _+ SEM (n -- number  of cases) and compared  using Student 's  t-test. RESULTS Effect on Bovine Intestinal ALP Activity The dependence  of the rate of the ALP activity from bovine intes- tine u p o n  vanadate and vanadyl  was first investigated. This enzyme had Biological Trace Element Research Vol. 41, 1994 334 Cortizo, Salice, and Etcheverry already been proved  to be sensitive to v a n a d i u m  by Crans et al. (12) and was used in these studies as a model .  Figure 1 shows  the dose-depen-  dent  effect of these compounds .  Both forms of v a n a d i u m  were effective inhibitors of ALP activity. Peroxo and hydroperoxo  v a n a d i u m  c o m p o u n d s  were also investi-  gated as to their ability to inhibit  ALP. However ,  the enzyme  was  not  affected at h igher  concentrat ions (100 gM) of these v a n a d i u m  species (Fig. 2). Effects  on UMR. 106 Cell A L P  A c t i v i t y  The effects of vanadate  and vanadyl  ions on the initial rate of solu- ble and particulate ALP activity of UMR.106 osteoblasts is shown  in Fig. 3. As can be seen, inhibi t ion of the soluble  e n z y m e  was of a dose-  response type (Fig. 3A)o The ED50 values show that vanadate  seems to be a more  effective inhibitor than vanadyl.  Particulate activity was inhib- ited to a lesser extent than that of the soluble form, and it was not  of a d o s e - d e p e n d e n t  type. Maximal  inhibi t ion was obta ined  at about  5-10 lJM for both v a n a d i u m  species (Fig~ 3B). Peroxo and hydroperoxo v a n a d i u m  c o m p o u n d s  effectively inhibi ted soluble ALP activity to the same extent as vanadate.  Under  similar con- ditions,  part iculate ALP activity was not  affected by these c o m p o u n d s  (Fig. 4). Partial Characterization of ALP Activity from Osteoblasts In order  to characterize ALP activity from osteoblasts UMRA06, we used  a series of c o m p o u n d s  a l ready d e m o n s t r a t e d  to be specific inhibitors of PTPases (8,10). As shown in Table 1, a m m o n i u m  mol ibdate  inhibits soluble and particulate ALP activity. The inhibit ion was about  30-40 and 10-20%, respectively. The extent of such effect was similar to that of vanadate.  Zn sulfate inhibited about  20% of the basal activity of the soluble ALP. The particulate-associated ALP activity was not  inhib- ited but  greatly enhanced by Zn2+ (Table 1). DISCUSSION There has been an increasing interest in the biological role of vana- d i u m  compounds .  Vanadium and its derivatives are capable of interact- ing wi th  var ious enzyme activities (3). Crans et al. have been work ing  with vanadate  and vanadyl  on alkaline phosphatase  activity. They have found that v a n a d i u m  interacts with the enzyme,  inducing strong inhibi- tions. Other  v a n a d i u m  derivat ives,  pe rvanada tes ,  are also potent ia l  inhibitors, as it was previously suggested (4). In this s tudy we did con- firm the results of Crans on bovine intestinal mucosa  ALP and extended the invest igat ions  on peroxo v a n a d i u m  species. The exper iments  were  Biological Trace Element Research Vol. 41, 1994 Vanadium Effects on ALP Activi ty  335 1 5 0 -  ~- I O0 <~ % <~ 50 0 - -  0 Vanadyl EDSO = 8.7MM 9 1t 1 84 .• 0 2 0  9 . . . .  QVanadate  EDS0= 15,u.M T I t - -  I t 4O 60 80  tO0 VANADIUM [ /~M ] Fig. 1. Effect of vanadate  and vanadyl  on ALP activ- ity from bovine  intestinal mucosa.  Initial rate was  deter- mined  by incubation of the e n z y m e  at 22~ for 10 min in the absence or presence of variable concentrat ions  of the inhibitors. Basal activity was  0.769 _+ 0A25 n m o l / m i n / p g  protein (n = 3). (D <:  _J <I: 03 E~ o~ 1 2 0 -  ! 0 0 -  8 0  60  40  20  0 I Basal Vanadate P < 0,ol  i f P e r a x o  v a n c ~ d [ u r n  H y d r o p e r o x o  v a n a d l u m  Fig. 2. Effect of  different v a n a d i u m  spec ies  on  intestinal ALP activity. The initial rate of reaction w a s  measured at 22~ for 10 min  w i thout  (basal) or wi th  100 ~tM vanadium c o m p o u n d s .  The basal activity was  0.871 _+ 0.094 n m o l / m i n / ~ t g  protein (n = 3). Biological Trace Element Research Vol. 41, /994 336 Cortizo, Satice, and Etcheverry 0 ~2 .< _J ~2 CD 0 (I3 l 100 ii 120-  1 0 0  80 60 A B 0 _ 0  Vanadyl EDSO= 43,u,M [ 0 0 _ _  9 Vanadate EDSO=16,u.M I I I P l I 0 20 40 60 80 100 0 - - 0  Va nadyl Q - - O  Vanadate 0 0 I ! J ~ i [ @ 20 40 60 80 1 O0 VANADIUM [ ,u.M ] Fig. 3. Effect of vanadate and vanadyl on soluble (A) and particulate (B) ALP activity from UMR.106 cells. The basal activities were 0.0262 _+ 0.002 and 0.0618 + 0.002 nmol/min/~tg protein, respectively (n -- 6). carr ied out  to compare  intest inal  ALP with  enzy mes  f rom UMR.106 osteoblasts and to unders tand  the specificity of the different v a n a d i u m  species as well as their roles in the physiology of the bone. Vanadate  and vanadyl  inhibi ted both soluble and part iculate ALP activi ty f rom UMR.106 cells and  bovine  intest inal  ALP. Vanadate  has been extensively used as specific Tyr-phosphatase inhibitor in different sys tems  (3), bu t  Se r /Thr -phospha ta ses  are not  sensit ive to these com- p o u n d s  (13). Vanadium compounds  only partially inhibited ALP activity in fraction preparat ions  from UMR osteoblasts, suggest ing that  Se r /Thr  phosphatases ,  acting at pH 8.0, are also present. It has been sugges ted  that vanada te  m a y  be r educed  to v anad y l  inside the cells and the latter species seems to be the actual inhibitor (4). However ,  in our  cell-free prepara t ions ,  the two forms were  s h o w n  to Biological Trace Element Research VoL 41, 1994 Vanadium Effects on ALP Activity 337 % t~ 120- 100 80 60- 40 P( 0.001 [ - -  P{ 0.002 Soluble Part iculate Basal ~5~ Peraxa vanadium Vanadate ~ Hydroperoxo vanadium Fig. 4. Effect of different vanadium species on sol- uble and particulate ALP activity from UMR.106 ceils. The concentration of the different compounds was 100 btM and the basal activities were 0.0273 + 0.001 and 0.0202 _+ 0.002 nmol/min/btg protein (n = 6) for soluble and particulate fractions, respectively. directly interact with ALE Furthermore,  in the soluble fraction, vanadate  shows greater inhibition than vanadyl  (Fig. 3A). Peroxo and hydroperoxo v a n a d i u m  c o m p o u n d s  were sh own  to be more  effective inductors  of pro te in  p h o s p h o r y l a t i o n  in cu l tu red  cells than vanadate  (4). Trudel et al suggest  but  do not  prove that the mecha- n ism m a y  take place through direct inhibit ion of Tyr-phosphatases .  In our  experiments ,  we have shown inhibition of ALP activity in the solu- ble fraction of osteoblasts by pervanadate  forms. ALP activity in the par- t iculate fract ion was not  inhibi ted by these species,  nor  was  bovine  intestinal ALP. In other  systems it has been shown  that pe rvanada tes  seem to be selective for the Tyr-phosphatase investigated (14). Fantus et al. working with bovine intestine ALP and rat liver microsomes found  greater inhi- bition of tyrosine dephosphoryla t ion  with pervanadate  than with vana- date. They have also shown a little inhibition of ALP activity wi th  10-100 wV/pervanadate .  In our studies, ALP activity in the particulate fraction behaves in a different manner,  because it is not inhibited at all. The assay we used measures total ALP activity, that is Se r /Thr  plus Tyr-phosphatases.  Moreover, the major componen t  in the particulate frac- tion is bone ALP, which is a Zn-metal loenzyme previously characterized in membranes  of osteoblasts (15). ALP in this fraction fullfilled the crite- ria of Zn-meta l loenzyme because it showed great activation by this ion (Table 1). Additionally, in preparat ions wi thou t  ~3-mercaptoethanol, the Biological Trace Element Research Vol. 41, 1994 338 Cortizo, Salice, and Etcheverry Table 1 Effect of Cations on ALP Activity from UMR.106 Cells a Percent basal activity Compound Conc., ~tM Soluble Particulate - -  - -  1 0 0  • 8 1 0 0  • 7 V a n a d a t e  10 78 + 1 86 _+ 3 50 74 __+ I b 79 • 1 b 100 66 • lc  91 • 1 N H 4  m o l y b d a t e  10 78 _+ lb  87  + 2c 50 73 _+ 20 - -  100 61 _+ 70 73 • ld  Z n  s u l f a t e  10 96 • 4 161 • 24 50 85 • 7 - -  100 78 +_ 4O 242 _+ 8e aResul ts  are expressed  as m e a n  +_ SEM, are: bp < 0.05 cp < 0.02 dp < 0.01 ep < 0.001. n = 3. Differences  vs basa l  effect of Zn was partially lost (data not shown), suggesting that the -SH groups are impor tant  for the role of Zn upon  the ALP activity of osteoblasts as it has been shown for other Zn-metalloenzymes (17). In the soluble fraction, approx 40% of the ALP activity was charac- terized as a Tyr-phosphatase. This part of the activity was also inhibited by vanadate,  vanadyl, and pervanadate to the same extent. The remain- ing ALP activity should be associated with Ser /Thr  phosphatases. In summary,  this study demonstrates that different forms of vana- d ium are direct inhibitors of ALP activity. This effect is dependent  on the enzymatic activity investigated and on the origin of the ALP and it can account for the stimulation of tyrosine phosphorylat ion in several sys- tems. The data presented will serve for future investigation of peroxo v a n a d i u m  compounds  in the p h o s p h o r y l a t i o n / d e p h o s p h o r y l a t i o n  process associated with cell physiology. ACKNOWLEDGMENTS The authors would  like to thank E. J. Baran for the support given to this research and Nyria Fenoglio for revision of the manuscript  language. AMC is Member  of the Carrera del Investigador CICPBA; SBE is Mem- ber of the Carrera del Investigador CONICET. This work was partially supported by The Third World Academy of Sciences. Biological Trace Element Research Vol. 41, 1994 Vanadium Effects on ALP Activity 339 REFERENCES 1. B. R. Nechay, Lo B. Nanninga, P. S. E. Nechay, Ro L. Post, J. J. Grantham, I. G. Macara, L. F. Kubena, T. Do Phillips, E H~ Nielsen, Fed. proc, 45, 123 (1986). 2. I. G. Macara, K. Kustin, L. C., Jr. Cantley, Biochim. Biophys. Acta, 629, 95 (1980). 3. D. Rehder, Angew. Chem. Int. Ed. Engl., 30, 148 (1991). 4. S. Trudel, M. R. Paquet, S. Grinstein, Biochem. J., 276, 611 (1991). 5. S. Trudel, G. P. Downey, S. Grinstein, M. R. Paquet, Biochem. J., 269, 127 (1990). 6. R. Bouillon, Horm. Res., 36, (suppl 1) 49 (1991). 7. Y. Yarden, A. Ullrich, Ann. Rev. Biochemo, 57, 443 (1988). 8. N. K. Tonks, C. D. Diltz, and E. H. Fischer, J. Biol. Chem., 263, 6731 (1988). 9. K. L. Guan, and J. E. Dixon, J. Biol. Chem., 266, 17026 (1991). 10. P. Peraldi, S. Hauguel-De Mouzon, and E Alengrin, Biochem. J., 285, 71 (1992). 11. M. Bradford, Anal. Biochem. 72, 248 (1976). 12. D. C. Crans, R. L. Bunch, and L. A. Theisen, J. Am. Chem. Soc., 111, 7597 (1989). 13. P. Cohen, Annu. Rev. Biochem., 58, 453 (1989). 14. I. G. Fantus, S. Kadota, G. Deragon, B. Foster, and B. I. Posner, Biochemistnj, 28, 8864 (1989). 15. T. Stingbrand, and W. H. Fishman, Human Alkaline Phosphatase, New York, AR Liss (1984). 16. K.-H. W. Lau, J. R. Farley, and D. J. Baylink, Biochem. J., 257, 23 (1989). 17. D. R. Bevan, P. Bodlaender, and D. Shemin, J. Biol. Chem., 255; 2030 (1980). Biological Trace Element Research Vol. 41, 1994 

Vanadium Compounds : Their Action on Alkaline Phosphatase Activity

El Servicio de Difusión de la Creación Intelectual

9 1994 by Humana Press Inc. 
All rights of any nature, whatsoever, eserved. 
0163-4984/94/4103-0331 $03.00 
Vanadium Compounds 
Their Action on Alkaline Phosphatase Activity 
ANA M. CORTIZO,"2 VIVIANA C. SALICE,1 
AND SUSANA B .  FTCHEVERRY *'~'3 
'Catedra de Bioquimica Patol6gica, Facultad de Ciencias 
Exactas, UNLP, 47 y 115, 1900 La Plata, Argentina; 
2CENEXA, Facultad de Ciencias M4dicas; and 3Programa 
QUINOR, Facultad de Ciencias Exactas; Universidad Nacional 
de la Plata, La Plata, Argentina 
Received June 30, 1993; Accepted August 5, 1993 
ABSTRACT 
The direct effect of different vanadium compounds upon alka- 
line phosphatase (ALP) activity was investigated~ Vanadate and 
vanadyl inhibited both the soluble and particulate ALP activity from 
UMR.106 cells and front bovine intestinal ALP. We have also shown 
the inhibition of ALP activity in the soluble fraction of osteoblasts by 
peroxo and hydroperoxo vanadium compounds. ALP activity in the 
particulate fraction was not inhibited by these species; nor was the 
bovine intestinal ALP. Using inhibitors of Tyr-phosphatase (PTPases), 
the soluble ALP was partially characterized as a PTPase. The major 
activity in the particulate fraction represents the bone-specific ALP- 
activity. This study demonstrates that different forms of vanadium 
are direct inhibitors of ALP activity. This effect is dependent on the 
enzymatic activity investigated and on the origin of the ALP. 
Index Entries: Vanadium; alkaline phosphatase; tyrosine-phos- 
phatase; osteoblast cells; bone cells; inhibitory effects. 
INTRODUCTION 
Vanadium is a transition metal of group 5 that shows various oxi- 
dation states. It is present in living systems in trace amounts (1). Vana- 
d ium affects different biological processes uch as the inhibition of NaG 
*Author to whom all correspondence and reprint requests hould be addressed. 
Biological Trace Element Research 331 Vol. 4I, I994 
332 Cortizo, Salice, and Etcheverry 
K+-ATPase, glucose oxidation in adypocites, insulin-mimetic a tions, and 
the inhibition of several Tyrosine-phosphatases (PTPases) (2,3). 
Phosphotyrosine accumulation is induced by vanadate inside the 
cultured cells (4). Vanadium +5 may be reduced to vanadyl +4 in the 
presence of NADPH and 02 during cellular metabolism. Addition of 
peroxo- or hydroperoxide vanadium compounds to permeabilized HL- 
60 granulocytes greatly enhances the phosphotyrosine content of the 
cells (4,5). At present, the mechanism by which phosphotyrosine 
increases i unknown. 
Osteoblast cells are involved in bone formation and their differenti- 
ation is modulated by multiple factors such as hormones, growth fac- 
tors, and ions (6). Various peptide factors stimulate the tyrosine-kinases 
associated to the cognate receptors and subsequently they induce tyro- 
sine-phosphorylation of specific cellular proteins. These effects are coun- 
terbalanced by the presence of the PTPases, which in turn deactivate the 
receptors (7). PTPases have been isolated and characterized in several 
systems. They belong to two general families: receptorqike PTPases asso- 
ciated with membranes, and nonreceptor-like PTPases (8,9). 
The aim of this study was to investigate the direct effect of different 
vanadium compounds upon alkaline phosphatase (ALP) activity. We also 
attempted to characterize the enzymes ensitivity to vanadium from rat 
osteoblast cell line UMR.106. 
METHODS 
Materials 
Vanadium (IV) oxide sulfate (vanadyl sulfate) was obtained from 
Merck, sodium o-vanadate, potassium superoxide, superoxide dismu- 
tase, p-nitrophenylphosphate (pNPP), and catalase were obtained from 
Sigma (St. Louis, MO), as well as the bovine intestinal alkaline phos- 
phatase. Tissue culture materials were provided by Corning or Falcon. 
DMEM and trypsin-EDTA were supplied by Gibco (Gaithersburg, MD) 
and FBS by Gen (Argentina). All other chemicals used were of analytical 
grade from Sigma. 
Solut ions 
Fresh stock solutions of vanadyl sulfate and sodium o-vanadate 
were prepared in distilled water at 100 mM concentration. Peroxovana- 
dium and hydroperoxide vanadium compounds were prepared as previ- 
ously reported (4). 
Cell Culture 
Rat osteosarcoma cell line (UMR.106) were grown in Dulbecco's 
Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine 
Biological Trace Element Research VoL 41, 1994 
Vanadium Effects on ALP Activity 333 
serum (FBS) and antibiotics. Cells were passaged by Trypsin-EDTA treat- 
ment  to 150 cm2 flask once a week. After the cells reached confluence, 
the med ium was removed and the monolayer washed with phosphate 
buffered saline (PBS) pH 7.4 and once in 50 mM Hepes/150 mM NaC1 
buffer containing 1 mM EDTA, 15 mM ~3-mercaptoethanol and 1 mM 
phenylethanosulfonyl fluoride (10). 
Preparation of Cellular Fractions 
Cells were scraped in the above buffer and sonicated at 600 psi/15 
s. The homogenate was centrifuged at 2500 rpm/5  min. The supernatant 
was centr i fuged at 45,000 g/90 min at 4~ The supernatant  (soluble 
fraction) was stored at -70~ The pellet was solubil ized in 50 mM 
Hepes/150 mM NaC1/1% Triton-X100 and recentrifuged as before. The 
final supernatant (particulate fraction) was stored at -70~ Aliquots of 
both fractions were used in protein assay by the Bradford method (11). 
Alkaline Phosphatase Activity (ALP) 
The ALP activity from bovine intestinal mucosa was determined as 
described by Crans (12). In brief, p-n i trophenylphosphate (pNPP) was 
used as a substrate in 20 mM Hepes buffer, pH 8.0, in the presence of 20 
mM KC1 and 30 mM MgC12. Incubation was carried out at room tem- 
perature for 10 min and absorbance at 405 nm was measured in order to 
determine the initial rate of the activity. Under these experimental con- 
ditions, the reaction was linear until 30 min. 
The initial rate of ALP activity in the soluble and particulate frac- 
tions of UMR.106 cells was determined by incubation of 100 ~g protein 
in 20 mM Hepes buffer pH 8.0/20 mM KC1/30 mM MgC12 and 5 mM 
pNPP. Absorbance at 405 nm was monitored at 37~ dur ing 5 min. 
Under  these conditions, the reaction proceeded linearly for 30 min. 
The effect of different vanadium compounds and other agents were 
tested in coincubations with the different enzymes. The inhibitory effect 
was determined as a percentage of the basal initial velocity of the ALP 
activity. 
Statistical Analysis 
The results are expressed as mean _+ SEM (n -- number of cases) and 
compared using Student's t-test. 
RESULTS 
Effect on Bovine Intestinal ALP Activity 
The dependence of the rate of the ALP activity from bovine intes- 
tine upon  vanadate and vanadyl was first investigated. This enzyme had 
Biological Trace Element Research Vol. 41, 1994 
334 Cortizo, Salice, and Etcheverry 
already been proved to be sensitive to vanadium by Crans et al. (12) and 
was used in these studies as a model. Figure 1 shows the dose-depen- 
dent effect of these compounds. Both forms of vanadium were effective 
inhibitors of ALP activity. 
Peroxo and hydroperoxo vanad ium compounds were also investi- 
gated as to their ability to inhibit ALP. However, the enzyme was not 
affected at higher concentrations (100 gM) of these vanad ium species 
(Fig. 2). 
Effects on UMR. 106 Cell ALP Act iv i ty  
The effects of vanadate and vanadyl ions on the initial rate of solu- 
ble and particulate ALP activity of UMR.106 osteoblasts i shown in Fig. 
3. As can be seen, inhibition of the soluble enzyme was of a dose- 
response type (Fig. 3A)o The ED50 values show that vanadate seems to 
be a more effective inhibitor than vanadyl. Particulate activity was inhib- 
ited to a lesser extent than that of the soluble form, and it was not of a 
dose-dependent  type. Maximal inhibit ion was obtained at about 5-10 
lJM for both vanadium species (Fig~ 3B). 
Peroxo and hydroperoxo vanadium compounds effectively inhibited 
soluble ALP activity to the same extent as vanadate. Under similar con- 
ditions, particulate ALP activity was not affected by these compounds  
(Fig. 4). 
Partial Characterization of ALP Activity from Osteoblasts 
In order to characterize ALP activity from osteoblasts UMRA06, we 
used a series of compounds  already demonstrated to be specific 
inhibitors of PTPases (8,10). As shown in Table 1, ammonium molibdate 
inhibits soluble and particulate ALP activity. The inhibition was about 
30-40 and 10-20%, respectively. The extent of such effect was similar to 
that of vanadate. Zn sulfate inhibited about 20% of the basal activity of 
the soluble ALP. The particulate-associated ALP activity was not inhib- 
ited but greatly enhanced by Zn2+ (Table 1). 
DISCUSSION 
There has been an increasing interest in the biological role of vana- 
d ium compounds.  Vanadium and its derivatives are capable of interact- 
ing with various enzyme activities (3). Crans et al. have been working 
with vanadate and vanadyl on alkaline phosphatase activity. They have 
found that vanadium interacts with the enzyme, inducing strong inhibi- 
tions. Other vanad ium derivatives, pervanadates,  are also potential  
inhibitors, as it was previously suggested (4). In this study we did con- 
firm the results of Crans on bovine intestinal mucosa ALP and extended 
the investigations on peroxo vanadium species. The experiments were 
Biological Trace Element Research Vol. 41, 1994 
Vanadium Effects on ALP Activity 335 
150-  
~- I O0 
<~ 
% 
<~ 50 
0 - -  0 Vanadyl EDSO = 8.7MM 
9 1t 
1 84 .
• 
0 20  
9 . . . .  QVanadate EDS0= 15,u.M 
T 
I t - -  I t 
4O 60 80 tO0 
VANADIUM [ /~M ] 
Fig. 1. Effect of vanadate and vanadyl on ALP activ- 
ity from bovine intestinal mucosa. Initial rate was deter- 
mined by incubation of the enzyme at 22~ for 10 min in 
the absence or presence of variable concentrations of the 
inhibitors. Basal activity was 0.769 _+ 0A25 nmol /min /pg  
protein (n = 3). 
(D 
<: 
_J 
<I: 
03 
E~ 
o~ 
120-  
!00-  
80  
60 
40 
20 
0 
I Basal 
Vanadate 
P < 0,ol 
i 
f 
Peraxo  vanc~d[urn  
Hydroperoxo  vanad lum 
Fig. 2. Effect of different vanad ium species on 
intestinal ALP activity. The initial rate of reaction was 
measured at 22~ for 10 min without  (basal) or with 
100 ~tM vanadium compounds.  The basal activity was 
0.871 _+ 0.094 nmol /min /~tg  protein (n = 3). 
Biological Trace Element Research Vol. 41, /994 
336 Cortizo, Satice, and Etcheverry 
0 
~2 
.< 
_J 
~2 
CD 
0 
(I3 
l 100 
ii 
120- 
100 
80 
60 
A 
B 
0_0  Vanadyl EDSO= 43,u,M 
[ 0 0__  9 Vanadate EDSO=16,u.M 
I I I P l I 
0 20 40 60 80 100 
0 - -0  Va nadyl 
Q- -O  Vanadate 
0 0 
I ! J ~ i [ 
@ 20 40 60 80 1 O0 
VANADIUM [ ,u.M ] 
Fig. 3. Effect of vanadate and vanadyl on soluble (A) 
and particulate (B) ALP activity from UMR.106 cells. The 
basal activities were 0.0262 _+ 0.002 and 0.0618 + 0.002 
nmol/min/~tg protein, respectively (n -- 6). 
carried out to compare intestinal ALP with enzymes from UMR.106 
osteoblasts and to understand the specificity of the different vanadium 
species as well as their roles in the physiology of the bone. 
Vanadate and vanadyl inhibited both soluble and particulate ALP 
activity from UMR.106 cells and bovine intestinal ALP. Vanadate has 
been extensively used as specific Tyr-phosphatase inhibitor in different 
systems (3), but Ser/Thr-phosphatases are not sensitive to these com- 
pounds (13). Vanadium compounds only partially inhibited ALP activity 
in fraction preparations from UMR osteoblasts, uggesting that Ser/Thr 
phosphatases, acting at pH 8.0, are also present. 
It has been suggested that vanadate may be reduced to vanadyl  
inside the cells and the latter species seems to be the actual inhibitor (4). 
However,  in our cell-free preparations, the two forms were shown to 
Biological Trace Element Research VoL 41, 1994 
Vanadium Effects on ALP Activity 337 
% 
t~ 
120- 
100 
80 
60- 
40 
P( 0.001 [ - -  P{ 0.002 
Soluble Particulate 
Basal ~5~ Peraxa vanadium 
Vanadate ~ Hydroperoxo vanadium 
Fig. 4. Effect of different vanadium species on sol- 
uble and particulate ALP activity from UMR.106 ceils. 
The concentration of the different compounds was 100 
btM and the basal activities were 0.0273 + 0.001 and 
0.0202 _+ 0.002 nmol/min/btg protein (n = 6) for soluble 
and particulate fractions, respectively. 
directly interact with ALE Furthermore, in the soluble fraction, vanadate 
shows greater inhibition than vanadyl (Fig. 3A). 
Peroxo and hydroperoxo vanadium compounds were shown to be 
more effective inductors of protein phosphory lat ion i cultured cells 
than vanadate (4). Trudel et al suggest but do not prove that the mecha- 
nism may take place through direct inhibition of Tyr-phosphatases. In 
our experiments, we have shown inhibition of ALP activity in the solu- 
ble fraction of osteoblasts by pervanadate forms. ALP activity in the par- 
ticulate fraction was not inhibited by these species, nor was bovine 
intestinal ALP. 
In other systems it has been shown that pervanadates seem to be 
selective for the Tyr-phosphatase investigated (14). Fantus et al. working 
with bovine intestine ALP and rat liver microsomes found greater inhi- 
bition of tyrosine dephosphorylation with pervanadate than with vana- 
date. They have also shown a little inhibition of ALP activity with 10-100 
wV/pervanadate. In our studies, ALP activity in the particulate fraction 
behaves in a different manner, because it is not inhibited at all. 
The assay we used measures total ALP activity, that is Ser/Thr plus 
Tyr-phosphatases. Moreover, the major component in the particulate frac- 
tion is bone ALP, which is a Zn-metalloenzyme previously characterized 
in membranes of osteoblasts (15). ALP in this fraction fullfilled the crite- 
ria of Zn-metal loenzyme b cause it showed great activation by this ion 
(Table 1). Additionally, in preparations without ~3-mercaptoethanol, the 
Biological Trace Element Research Vol. 41, 1994 
338 Cortizo, Salice, and Etcheverry 
Table 1 
Effect of Cations on ALP Activity from UMR.106 Cells a 
Percent basal activity 
Compound Conc., ~tM Soluble Particulate 
- -  - -  100  • 8 100  • 7 
Vanadate  10 78 + 1 86 _+ 3 
50 74 __+ I b 79 • 1 b 
100 66 • lc 91 • 1 
NH4 molybdate  10 78 _+ lb 87 + 2c 
50 73 _+ 20 - -  
100 61 _+ 70 73 • ld 
Zn  su l fa te  10 96 • 4 161 • 24 
50 85 • 7 - -  
100 78 +_ 4O 242 _+ 8e 
aResults are expressed as mean +_ SEM, 
are: 
bp < 0.05 
cp < 0.02 
dp < 0.01 
ep < 0.001. 
n = 3. Differences vs basal 
effect of Zn was partially lost (data not shown), suggesting that the -SH 
groups are important for the role of Zn upon the ALP activity of 
osteoblasts as it has been shown for other Zn-metalloenzymes (17). 
In the soluble fraction, approx 40% of the ALP activity was charac- 
terized as a Tyr-phosphatase. This part of the activity was also inhibited 
by vanadate, vanadyl, and pervanadate to the same extent. The remain- 
ing ALP activity should be associated with Ser/Thr phosphatases. 
In summary, this study demonstrates that different forms of vana- 
dium are direct inhibitors of ALP activity. This effect is dependent on the 
enzymatic activity investigated and on the origin of the ALP and it can 
account for the stimulation of tyrosine phosphorylation i several sys- 
tems. The data presented will serve for future investigation of peroxo 
vanadium compounds in the phosphory lat ion/dephosphory lat ion 
process associated with cell physiology. 
ACKNOWLEDGMENTS 
The authors would like to thank E. J. Baran for the support given to 
this research and Nyria Fenoglio for revision of the manuscript language. 
AMC is Member of the Carrera del Investigador CICPBA; SBE is Mem- 
ber of the Carrera del Investigador CONICET. This work was partially 
supported by The Third World Academy of Sciences. 
Biological Trace Element Research Vol. 41, 1994 
Vanadium Effects on ALP Activity 339 
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Biological Trace Element Research Vol. 41, 1994 


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