CLIN. CHEM.

22/7, 972-976(1976)

Alkaline Phosphatase. I. Kinetics and Inhibition by Levamisole of Purified lsoenzymes from Humans
Herman Van Belle

I studied the kinetics and sensitivity toward inhibition by levamisoie and R 8231 of the most important human alkaline phosphatase isoenzymes. N-Ethylaminoethanol proved superior to the now widely used diethanolamine buffer, especially for the enzymes from the intestine and placenta, behaving as an uncompetitive activator. The optimum pH Iargelydepends on the substrate concentration. The addition of Mg2+ has no effect on the activities. The meaning of Km-values for alkaline phosphatases is questioned. lsoenzymes from human liver, bone, kidney, and spleen are strongly inhibited by levamisole or R 8231 at concentrations that barely affect the enzymes from intestine or placenta. The inhibition is stereospecific, uncompetitive, and not changed by Mg2. Inhibition is counteracted by increasing concentrations of N-ethylaminoethanol. The mechanism of inhibition is suggested to be formation of a complex with the phosphoenzyme.

In the accompanying serum of the findings described.

paper, the applicability to on isolated isoenzymes will be

Materials

and Methods

The human tissues were obtained from autopsy material, except the placenta, which was used immediately after delivery. The enzyme from bone was purified from
the tibia and femur of a five-month-old fetus after

spontaneous abortion. The purification involved extraction

Additional Keyphrases: nitrophenollP, release ratio

ethylaminoethanol buffer vs. diethanolamine buffer

#{149}N-

#{149} soeni

zymes of human origin #{149}Km values, and interpretation mechanism and organ specificity of inhibition

Although there are many reports on human alkaline phosphatases (for a review, see 1), few of them are concerned with the kinetics of the isoenzymes isolated from liver, kidney, bone, placenta, and intestine simultaneously-i.e., under identical conditions (2). The knowledge of their kinetics, espcially in the phosphorylatable (3) buffers NEAE (N-ethylaminoethanol) or DEA (diethanolamine), which are now widely used for the measurement of serum alkaline phosphatase activity, may contribute in defining the optimal conditions of a referee method for this enzyme in serum. Levamisole and R 8231 are known to be potent, organ-specific inhibitors of alkaline phosphatases in the dog (4) and the rat (5). A study on the sensitivity of the isoenzymes of human origin could provide useful information on the potential application of this class of inhibitors to the discrimination of the isoenzymes in serum. Such a study is the subject of this paper.
Laboratory Laboratories,
-

with n -butanol of a microsomal fraction of the tissues followed by gel chromatography through Sephadex G-200. For concentration we used Amicon-100 filters instead of the usual acetone or ammonium sulfate precipitation steps. Phosphate buffer (20 mmol/liter, pH 7.8) containing 1 mmol of Mg2+ per liter was used throughout the whole procedure, because it markedly increased the yields, either because of product protection (6) or less adsorption to the column material. Before assay, the enzymes were diluted in tris(hydroxymethyl)aminomethane (10 mmol/liter, pH 8.0) containing 1 mmol of Mg2 per liter. The specific activities-i.e., imol of nitrophenol released/mm per milligram of protein in NEAE (100 mmol/liter, pH 10.2) with ES] = 5 mmol/ liter)-were: 31.2 (intestine), 7.0 (liver), 3.6 (kidney), 2.4 (spleen), 100.6 (placenta), and 58.6 (bone). The release of p -nitrophenol or P from p -nitrophenylphosphate was measured with either the Beckman 25-Kinetic System or the Technicon AutoAnalyzer as described previously (4). Unless otherwise specified, the experiments were performed at room temperature (22 1 #{176}C). Levamisole [(- )-2,3,5,6-tetrahydro-6-phenylimidazo(2,1-b)thiazole hydrochloride, Mr 240.75] and R 8231 [()-6(m-bromophenyl)-5,6-dihydroimidazo(2,1-b)thiazole oxalate, Mr 371.22] are products of
Janssen Pharmaceutica Research Laboratories.

Results
Effect of Buffer Composition
Borate, glycine, and NEAE buffers, all at 50 mmol/ liter, were compared at 1.25, 2.5, 5, and 10 mmol/liter substrate concentrations and at pH 9.8 and 10.4. Under all these conditions the NEAE buffer proved to be su-

of Biochemistry, Janssen Pharmaceutica, Research 2340-Beerse, Belgium. Nonstandard abbreviations used: NEAE, N-ethylaminoethanol; DEA, diethanolamine; and P, inorganic phosphate. In accord with convention, brackets signify concentration; e.g., [SI means concen-

tration of substrate. Received Jan. 27, 1976; accepted

April 6, 1976

972

CLINICALCHEMISTRY,Vol. 22, No. 7, 1976

ReLActivity 100 a
a a-

NP ratio r5

[53 V

F.
,

NEAE

100 mo(/titer m
1-3 1 xlO6mot/titer
R8231

so
1-2

I-I

100

200

#{163}00

600

600

1000

[NEAEJmmot/titer

S
[N AJ

Ret. ctivity A
100

NIP ratio i

mmol/titer

-100

-1.

200 400 800

a a--a-

1-3
50
-

1-2
-a.
S.

-v I-I

I -

-5.0

1.25 2.5

5.0

10.0
[S] mmol/liter

100 200

#{163}00

600

600

1000

[NEAEJmm0I/Iiter

Fig. 1. Plots of the relative activities and the ratio nitrophenol

released/P1 released (NIP) vs. the concentration of NEAE for the isoenzymes from (a) intestine (S) and placenta (0) and (b) bone
and liver(mean for both isoenzymes, which behae almost

Fig.2. Plot of [SI/V vs. ES],showing uncompetiti inhibition by R 8231 and uncompetitive activation by NEAE of human liver
alkaline

phosphatase

identically)
pH of the buffer, 10; substrate concentration, 5 mmol/Ilter

perior, greater

the enzyme than

activity

being buffers.

from two- to fivefold

creasing the substrate concentration lessens the effect insofar that, at the lowest substrate concentrations tested, increasing concentrations of NEAE even seem to inhibit enzyme activity; that is, the ratio activity NEAE

in the other

activity

NEAE

1000 mmol/liter 100 mmol/liter

<

Effect of Buffer Concentration

At pH 10 and 5 mmol/liter substrate concentration, all isoenzyme activities strongly depend on the NEAE concentration (Figure 1). This effect is largely due to the well-known transphosphorylation, as apparent from the ratio nitrophenol released/Ps released. Moreover, the effect of increasing concentrations of the buffer is influenced by both pH and substrate concentration. Decreasing the pH enhances the activation while de-

This holds true for all isoenzymes studied, but it is most pronounced for the intestinal isoenzyme as illustrated in Table 1. A plot of [S]/V vs. ES] at various concentrations of NEAE reveals that all curves have a common intercept at the [S]/V axis (Figure 2). NEAE behaves as an uncompetitive activator. The effect of NEAE also depends on the source of the enzyme, being most pronounced with the intestinal

isoenzyme.

Table 1. Ratios of Intestinal Enzymatic Activity NEAE 1000 mmol/iiter/NEAE at Different pH and [SI
ES) mmol/ii1.r pH .16 .32 .63 1.25 2.5

100 mmol/Iiter

5.

10.

10.2
9.8

0.63
1.29

0.88
2.01

1.43
3.15

2.21
4.45

3.41
6.47

4.57
7.92

5.90
9.30

9.4
9.0

2.62
4.71

4.62
7.24

6.23
8.75

8.38
10.02

9.59
12.00

10.86
12.69

12.38
15.00

CLINICAL CHEMISTRY,

Vol. 22, No. 7, 1976

973

placental enzyme. At 6.4 mmol/liter the optimum lies at 10.4 for all isoenzymes. At 3.2 mmol/liter it is 10.1 and 10.4 for the nonplacental and the placental isoenzymes. At pH 8.6 the enzyme activities are completely independent of substrate concentrations above 0.1 mmolfliter whereas at pH 10.4 the reaction proceeds by Michaelis-Menten kinetics to concentrations of up to 12.8 mmol/liter.

NEAE 1 mol/liter

vs. DEA 1 mol/liter

5.1

02

04

OS

IS

3.2

5.1. 12.5

(S] ..m.IlIit.c

FIg.3. Relativeactivityf human liver o alkaline phosphatase as affected by pH and substrateconcentrations

I00

When one compares NEAE (1 mol/liter, pH 10.2) to DEA (1 mol/liter, pH 9.8, +0.5 mmol of Mg2 per liter) at different substrate concentrations, remarkable differences are seen between both buffers on the several isoenzymes (Figure 4). At 10 mmol/liter substrate concentration the placental and intestinal isoenzymes are much more activated by NEAE than by DEA, whereas the other isoenzymes are almost equally active in both buffers. Another difference is that the increase in activity in NEAE with increasing substrate concentration is much more pronounced than it is in DEA. When compared at different pH values and 10 mmol/liter substrate, the pH optimum in DEA is 9.5 for the intestinal isoenzyme, between 9.8 and 10.1 for the isoenzymes from the liver, kidney, spleen, and bone, and 10.4 for the placental isoenzyme. In NEAE the pH optimum is ?10.4 for all isoenzymes except for the intestinal one (optimum, 9.8-10.1). Neither the optimum pH nor the differences in activation are changed by increasing the temperature from 25 to 37 #{176}C.

.0

so

PL AC CS TA

5,4 ii,

AT INC CST

_.o.
0
- -

-o -o
,

___________________
125 25

Ill

2S

50

(sJ,s.iiir

TOO

TOO

.7
0

Effect of Addition of Mg2

The addition of Mg2 (1 mmol/liter) to either borate or NEAE has a negligible effect on the activities of all isoenzymes at pH values between 9.5 and 10.4 at 25 or 37 #{176}C.
Km
-o

so

LIVIS

so

5054! I(I0550 IPLO SN

0#{149} ISO 21

SO

io

25

2 5

10 (n,.hhu1

Fig. 4. Relative activities of human alkaline phosphatase isoenzymes in (0 - - - 0) borate buffer (50 mmol/liter;pH 10.2), (x--x) NEAE (1 mol/liter; pH 10.2), and (0 - . - 0) DEA (1 mol/liter, pH 9.8,+0.5 mmol/literMg2)

Optimum

pH and Substrate

Concentration

We measured the release of nitrophenol for the different enzymes in 100 mmol/liter NEAE at pH values ranging from 8.6 to 10.7 and at substrate concentrations increasing from 0.1 to 12.8 mmol/liter. The results for the liver enzyme are illustrated in Figure 3. Similar results were obtained for the isoenzymes from intestine and bone. The placental isoenzyme differed in that the optimum pH at 12.8 mmol/liter substrate was 10.7 instead of 10.4. Evidently the optimum pH for the various isoenzymes largely depends on the substrate concentration. At 12.8 mmollliter substrate the optimal pH is 10.4 for the nonplacental isoenzymes and 10.7 for the
974 CLINICAL CHEMISTRY, Vol.22,No. 7, 1978

In the light of the foregoing it is evident that values of Km highly depend on the reaction conditions. Thus the lowering of the pH by 0.4 units from 10.2 to 9.8 results in a decrease of the Km value for the bone enzyme from 0.65 to 0.12 mmol/liter. Increasing the NEAE concentration from 100 to 1 mol/liter increases the Km for the intestinal enzyme from 0.23 to 2.97 mmol/liter at pH 10.2. The increase was less pronounced for the other enzymes. An increase in temperature from 25 to 37 #{176}C decreases the Km for the liver enzyme from 2.06 to 1.41 mmol/liter in 1 mol/liter NEAE, pH 10.2. Under similar conditions the Km for the placental enzyme remained constant at 1.3 mmol/liter. In DEA buffer (1 mol/liter, pH 9.8) the Km values are independent of changes in the temperature except for the placental enzyme, in which case the Km increased from 0.24 mmol/liter at 25 #{176}C to 0.57 mmol/liter at 37 #{176}C.

Inhibition

by Levamisole

and R 8231

The isoenzymes from human liver, bone, kidney, and spleen are very sensitive to levamisole and R 8231,

Ish,bi

lion

Table 2. Effect of [NEAEI on the Inhibition by Levamisole and L-Phenylaianine

% kth4t1on Lsvamlsols, X 10 mol/Ilts,
Uy.r

L-ph.nyl.IanIns, 2 X i0 mol/Ilts, Placsnta Intostin.

[NEAEI,
mmo4/lItsr 50 Boos

100 150

46 54 51

49 51 48

64 66 63

68 68 69

I0 [int] mol/Ilt.r

200 400 600

800 1000

47 38 31
25 22

45 36 29
24 21

62 61 53
50 47

68 67 65
58 55

Fig.5. Organ specificity of the inhibition levamisole(bottom) by

and A 8231 (top)

Buffer, 100 mmol/Ilter NEAE;

[Sj, 5

mmol/Iiter pH. 9.8; [S], 5 mmol/llter

whereas the phosphatases from the intestine or placenta are inhibited only at the highest concentrations (Figure
is stereospecific, devoid of any effect below 2 X 10 5). The inhibition the D-isomer being mol/liter. At pH 9.8

and a buffer concentration of 50 mmol/liter, the inhibition is similar, whether borate, glycine, or NEAE is used. However, higher concentrations of NEAE progressively relieve the inhibition, as shown in Table 2. The same applies to the inhibition of human placental
or intestinal enzyme by L-phenylalanine,

Table 3. Effect of ES],pH, and [NEAE] on Percentage Inhibition by R 8231 (1 X 10-6 mol/ liter) of Liver Alkaline Phosphatase
INEAEJ, minol!
IItsr

[SI. mmol/llts,
0.16 0.32
0.64

1.28

2.5

5.

10.

about three times more inhibitor NEAE than in 100 mmol/liter

same extent of inhibition.

for which is needed in 1 mol/liter

100
1000

40
0

pH 10.2 43 47 52
0 0
pH 9.8 56 13 pH 9.4 61 34 2

56
3

58
13

63
14

NEAE

to achieve

the
100 1000 53 0 56 14

At a given

pH and buffer

concentration the inhibition increases with increasing substrate concentrations, a situation typical for uncompetitive inhibition. A plot of [S]/V vs. [S1 clearly reveals that type of inhibition, as illustrated in Figure 2 for the liver enzyme. Similar plots were found for the enzymes from bone, kidney, and spleen. At 100 mmol of NEAE per liter and high substrate concentrations, the inhibition is unaffected by changes in pH from 9.0 to 10.2. At the lower substrate concentrations, the inhibition increases with decreasing pH. In 1 mol/liter NEAE the latter applies to all substrate concentrations (Table 3). The inhibition is not affected by the addition of magnesium ion (up to 2 mmol/liter). Discussion In agreement with previous findings on human total serum alkaline phosphatase (3, 7), we found NEAE to be a strongly activating buffer of the isolated isoenzymes. The activation is most pronounced for the isoenzymes from placenta and intestine, and depends on pH and substrate concentrations. From a plot of [S]/V
vs. [5], NEAE can be regarded as an uncompetitive

60 20
61

61 24
64

63 29
66

65 34
68

100 1000

60 32

61 26

36
61 49

48
60 53

51
62 57

53
63 61
or-

pH 9.Oa
100 1000 57 .66 60 59 61 62

#{149} actlvftles the ratIos are strongly affected At low rds.

by small analytical

enzymes are almost twice as active in 1 mol/liter NEAE as they are in 1 mol/liter DEA. The importance of this finding is further stressed in the paper that follows. In 0.1 mol/liter NEAE the pH optimum for all human
isoenzymes decreases with decreasing concentrations of the substrate, which is in agreement with previous findings by Moss et al. (2) in Na2CO3-NaHCO3 buffers and 13-naphthylphosphate as substrate (0.05-1 mmol/ liter). At micromolar (physiological?) substrate concentrations, alkaline phosphatase should no longer be regarded as alkaline (4). When the enzymes are kept

activator. DEA has been recommended (8) for determination of serum alkaline phosphatase activity based on comparative studies of buffers on total serum enzyme activity (7, 9). However, as we have shown, the activation
by NEAE or DEA strongly depends on the isoenzyme

in a Mg2+containing

medium,

there

is no effect of

under study. Indeed,

the placental

and intestinal

iso-

Mg2, added to the incubate, on the enzymic activities, either in borate or in NEAE, at pH values between 9.5 and 10.4. The lack of effect of Mg2 on human isoenzymes from bone, liver, and intestine has already been reported (3), although it is generally assumed that Mg2 should be added for maximal activity (10).
CLINICAL CHEMISTRY, Vol.22,No. 7, 1976 975

The Km values strongly depend on slight variations in pH, substrate concentration, buffer and buffer concentration, and temperature, so that a comparison with
data from others does not make sense at all. Furthermore, for alkaline phosphatase the interpretation of Km is difficult, because not all the assumptions involved in the formulation of the Michaelis-Menten equation are necessarily validated (11). Thus the simple sequence E + S ES E + P does not hold, because it is well known (2, 10) that the enzyme is phosphorylated during the reaction. Then the rate equation in its simplest form becomes:E+R-PER-PE-PRE+P+ R. The situation is even more complex when NEAE or another phosphorylatable buffer is used that, by acting as an acceptor, affects the rate constant for the reaction E PR E + P + R. The Km defined in this kind of buffers should therefore be rather described as an apparent Km (Kma). Levamisole and R 8231 are potent inhibitors of the alkaline phosphatase from bone, kidney, liver, and spleen. The human intestinal enzyme is not sensitive. In contrast to the rat or the dog, the placental isoenzyme is not affected in the human. The reverse has been found for the inhibition by L-phenylalanine (12). It looks as if those isoenzymes that are inhibited by L-phenylalanine are insensitive to levamisole and, at least for the mammalian enzymes, vice
-

pendent on the E + RP ERP step. Both levamisole and NEAE behave as uncompetitive effectors, acting in the opposite direction. The inhibition by levamisole is most pronounced at high pH and high substrate concentrations or at the lower pH values, i.e.,
-

under the conditions for maximal NEAE activation. At high pH and low substrate concentrations both NEAE and levamisole have negligible effects. The inhibition by levamisole is also relieved by increasing concentrations of NEAE. Taking this information together, it may be suggested that levamisole acts by inhibiting the dephosphorylation of the phosphoenzyme (= the opposite of the activation by NEAE) through a complex formation (= uncompetitive inhibition, 11). A similar mechanism has been proposed for the inhibition by L-phenylalanine of human placental alkaline phosphatase based on experiments in which a rapid mixing and quenching technique was used (15).

I am indebted to Dr. Paul A. J. Janssen, director of the laboratory, for his continuous encouragement. I thank Mrs. M. Raeves-Van Reusel for her technical assistance. This work was supported by a grant from the I.W.0.N.L. (Instituut voor bevordering van Wetenschappelijk Onderzoek in Nijverheid en Landbouw).

versa. Levamisole and R 8231 have a similar organ specificity as L-homoarginine (13, 14) but they are inhibitory at concentrations that are 100-1000 times lower.
The inhibition by levamisole is stereospecific, the D-isomer being inactive. The inhibition is uncompetitive and is not affected by the addition of Mg2 but is counteracted by increasing concentrations of NEAE. In 100 mmol/liter NEAE and at the lower substrate concentrations there is no effect of changes in pH from 9.0 to 10.2 but at the higher substrate concentrations or in 1 mel/liter NEAE the inhibition increases with de-

References
1. Fishman, Am. J. Med.

W. H.,
56, 617

Perspectives
(1974).

on alkaline

phosphatase

isoenzymes.

2. Moss, D. W., Campbell, D. M., Anagnoeton-Kakaras, E., and King, E. J., Characterization of tissue alkaline phosphatases and their partial purification by starch-gel electrophoresis. Biochem. J. 81,441 (1961). 3. Amador, E., and Urban, J., Transphosphorylation by human alkaline phosphatases. Am. J. Clin. Pat ho!. 57, 167 (1972).

4. Van Belle, H., Kinetics and inhibition of alkaline phosphatases from canine tissues. Biochim. Biophys. Acta 289, 158 (1972). 5. Van Belle, H., Kinetics and inhibition of rat and avian alkaline
phosphatases. Gen. Pharmacol. 7, 53 (1976).

creasing

pH.

Several data from this study provide indirect evidence on the mechanism of inhibition. The dephosphorylation of the phosphoenzyme, known to be slowed down by lowering the pH, is generally assumed not to be rate-limiting above pH 7.0 (10). However, the results from Figure 3 clearly show that at the lower pH values the rate of reaction becomes more and more independent of substrate concentration. Thus, at pH 8.6, increasing the substrate concentration above 0.1 mmol/ liter does not result in increased velocities as should be expected if E + RP ERP were rate-limiting. Even at
-

6. Brown, E. M., Ulmer, D. D., and Valler, B. L., Hydrogen-tritium exchange of partially and fully reconstituted zinc and cobalt alkaline phosphatase of Escherichia coli. Biochemistry 13, 5328 (1974).
7. McComb, R. B., and Bowers, G. N., Jr., Study conditions for measuring alkaline phosphatase serum. Clin. Chem. 18, 97 (1972). of optimum buffer activity in human

8. Recommendations of the German Society for Clinical Chemistry. Z. Kim. Chem. Kliri. Biochem. 10, 281 (1972). 9. Hausamen, T., Helger, R., Rick, W., and Gross, W., Optimal conditions kinetic for the determination of serum alkaline phosphatase method. Clin. Chim. Acta 15, 241 (1967). by a new

10. Fernley, H. N., Mammalian alkaline phosphatases. In The Enzymes. chap. 18, P. D. Boyer, Ed. Academic Press, New York, N.Y.,
1971, pp 417-447. 11. Webb, J. L., Enzyme and Metabolic Inhibitors. Press, New York and London, 1963, pp 17-27. Vol. 1, Academic

this relatively

high pH the dephosphorylation

still

seems to be the rate-limiting step. This is further strengthened by the results with NEAE, which, by acting as an acceptor, accelerates the dephosphorylation of the enzyme. Indeed, the activation by NEAE is apparent only under these conditions where EPR E + P + R becomes rate limiting, i.e., at high pH and the higher substrate concentrations or at the lower pH values. There is no activation at high pH and low substrate concentrations where the overall reaction is de-#{247}

12. Fishman, W. H., Inglis, N. R., and Ghosh, N. K., Distinctions between intestinal and placental isoenzymes of alkaline phosphatase. Clin. Chim. Acta 19, 71(1968). 13. Fishman, W. H., and Sie, H. G., L-Homoarginine an inhibitor of serum bone and liver alkaline phosphatase. Clin. Chim. Acta 29,339

(1970).
14. Lin, C. W., and Fishman, W. H., L-Homoarginine: An organspecific uncompetitive inhibitor of human liver and bone alkaline phosphohydrolases. J. Biol. Chem. 247, 3082 (1972). 15. Byers, D. A., Fernley, H. N., and Walker, P. G., Studies on alkaline phosphatase. Inhibition of human placental phosphoryl phosphatase by L-phenylalanine. Eur. J. Biochem. 29, 197 (1972).

976

CLINICAL CHEMISTRY, Vol. 22, No. 7, 1976