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A comparison of the transphosphorylating activities of human liver and intestinal alkaline phosphatases
347
Clinica Chimica Acta, 52 (1974) 341-352 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 6415
A COMPARISON OF THE TRANSPHOSPHORYLATING ACTIVITIES HUMAN LIVER AND INTESTINAL ALKALINE PHOSPHATASES
KATRINE
B. WHITAKER
Department (U.K.) (Received
and D.W. MOSS
of Chemical Pathology, Royal Postgraduate Medical School, London
January
OF
W 12 OHS
17, 1974)
Summary
The relative rates of transphosphorylation by human liver and intestinal alkaline phosphatases from p-nitrophenyl phosphate to Tris and diethanolamine have been measured. The maximum velocities of formation of diethanolamine phosphate by the two enzymes are nearly equal when preparations with equal hydrolytic activities in sodium carbonate--bicarbonate buffer are used. However, Tris phosphate is formed at a lower rate by liver alkaline phosphatase than by the intestinal enzyme. Consequently, the rstio of the activities of the solutions of liver and intestinal alkaline phosphatases varies with the nature and concentration of the buffer system.
Introduction
It has frequently been observed that alkaline phosphatases (EC 3.1.3.1) from several vertebrate tissues differ in the relative rates at which they hydrolyze various phosphate esters [l] . Alkaline phosphatases also catalyze transfer of phosphate to suitable acceptors [2], and when aminoalcohols are used as buffers in the estimation of alkaline phosphatase activity a considerable enhancement of the catalytic rate takes place since the buffer ions can act as phosphate acceptors [3,4]. Few investigations have been made into the relative transphosphorylating activities of tissue-specific forms of alkaline phosphatase. However, it seems probable that differences in this respect may contribute to the variations which have been noted when the relative activities of alkaline phosphatase preparations from various tissues measured in assay systems which contain phosphate-accepting buffers are compared with the relative activities of the same specimens determined by methods in which the only phosphate acceptor is water [5,6]. We have compared the activities of alkaline phosphatases from human liver and small intestine on p-nitrophenyl phosphate in two phosphate-accept-
348
ing buffers, Tris and diethanolamine, both with respect to formation of the products of the hydrolytic reaction and to synthesis of the buffer phosphates. The results show that the maximum velocity of formation of Tris phosphate is relatively much lower for liver phosphatase than for intestinal phosphatase, but that maximum rates of formation of diethanolamine phosphate by the two enzymes are nearly equal, when solutions of the two enzymes are used which have equal hydrolytic activities in a non-phosphorylating buffer. Consequently, the relative activities of the two enzymes as measured by the rate of appearance of p-nitrophenol appear quite dissimilar when the Tris and diethanolamine buffer systems are compared. Materials and Methods Alkaline phosphatase was extensively purified from human small intestine as described by Moss et al. [7]. Human liver phosphatase was a less purified preparation obtained by applying to the tissue the first two stages of the same procedure. All determinations of alkaline phosphatase activity were made at 37” with 15 mM p-nitrophenyl phosphate as substrate and with 5 mM MgCl* added. Tris( 2-amino-2-hydroxymethylpropane-1,3-diol) or diethanolamine were added to the final concentrations shown in the results, and in each case the pH was adjusted to 10.0 by addition of HCl. Ionic strength was maintained constant to within ?I 2% by addition of NaCl to the several reaction mixtures. Enzymic action was allowed to proceed for 10 min since previous experiments had shown that production of both p-nitrophenol and inorganic phosphate was linear during this period over the range of enzyme activities used. p-Nitrophenol was measured by its extinction at 405 nm in an aliquot of the reaction mixture transferred to 0.5 M NaOH containing 10 mM EDTA. Inorganic phosphate was measured in a further aliquot of the reaction mixture after transfer to trichloroacetic acid by the method of Delsal and Manhouri [S] . The amount of buffer phosphate formed was calculated from the difference between the amounts of p-nitrophenol and inorganic phosphate thus determined. A reaction mixture consisting of 15 mM p-nitrophenyl phosphate and 5 mM MgClz in 0.05 M Na, CO3 -NaHC03 buffer, pH 10.0, was included with each experiment. This buffer does not act as a phosphate acceptor so that the enzymic reaction is entirely hydrolytic: the molar amounts of p-nitrophenol and inorganic phosphate formed in this buffer are therefore equal. All the activities measured in Tris or diethanolamine buffers have been expressed in terms of an enzyme activity equivalent to the production of 1 pmole of p-nitrophenol (or inorganic phosphate) per minute per ml of enzyme solution in the sodium carbonate-bicarbonate buffer. Results The rates of formation of p-nitrophenol, inorganic phosphate and buffer phosphate by liver and intestinal alkaline phosphatases are plotted as functions of Tris or diethanolamine concentration in Figs 1 and 2. Increasing concentrations of Tris or diethanolamine accelerate the release of p-nitrophenol by both
349
0.5
Tris
concentration(M)
1.0
0.5 Oiethonolamine
1.0 concentration(M)
Fig. 1. Rates of formation of p-nitrophenol (a$). inorganic phosphate (0.A) and Tris phosphate (*,A) by human liver alkaline phosphatase (circular symbols) and intestinal alkaline phosphatase (triangular symbols) at different concentrations of Tris. The substrate was 15 mM p-nitrophenyl phosphate at pH 10.0 in each case, and the velocities shown are those for activities of each phosphatase corresponding to the production of 1 pmole of p-nitrophenoljmin per ml enzyme solution in Na~C03-NaHC03 buffer at pH 10.0. Fig. 2. Rates Of formation of p-nitrophenol (?.A), inorganic phosphate (,.A) and diethanolamine phosphate (*,A) by human liver alkaline phosphatase (circular symbols) and intestinal alkaline phosphatase (triangular symbols), at different concentrations of diethanolamine. The substrate was 15 mM p-nitrophenyl phosphate at pH 10.0 in each case, and the velocities shown are those for activities of each phosphatase corresponding to the production of 1 Mmole of p-nitrophenollmin per ml enzyme solution in Na~C03---NaHC03 buffer at pH 10.0.
enzymes, as described in earlier reports. However, the magnitude of this effect differs for the two enzymes and differences are also present in relative rates of buffer phosphate formation. Thus, formation of Tris phosphate by liver phosphatase is appreciably less rapid than by intestinal phosphatase at each concentration of Tris, when the two enzymes are present in amounts which have equal activities in sodium carbonate-bicarbonate buffer. On the other hand, rates of formation of diethanolamine phosphate by liver and intestinal alkaline phosphatases are very nearly equal. Formation of buffer phosphate is a two-substrate reaction and, with the donor substrate (p-nitrophenyl phosphate) present in excess as in these experiments, values for the Michaelis constant and V,., can be obtained in terms of the acceptor substrate, the buffer. These values have been derived from plots of reciprocals of the velocity of buffer phosphate formation against reciprocals of buffer concentration (Fig. 3). For the formation bf Tris phosphate, Km values for liver and intestinal phosphatases were similar at 1.17 M, but Vmax was 1.48 pmoles/min per ml enzyme solution for liver phosphatase compared with 2.78 pmoles/min per ml for intestinal phosphatase. The two enzymes showed rather similar Km values for diethanolamin’e phosphate forma-
350
5-
1
0
0 or diethanolamine]
(M”)
‘/Tris [
2
4 or
6
8
diethanolamine]
Fig. 3. Plots of l/u against reciprocal of buffer concentration (Tris or diethanolamine) of Tris phosphate (0.A) and diethanolamine phosphate (*.a) by human liver alkaline and intestinal alkaline phosphatase ( A,A) by transphosphorylation from p-nitrophenyl 10.0.
10 CM-‘)
for the formation phosphatase (13.0) phosphate at pH
Fig. 4. Plots of l/(ua--rrg) against reciprocal of Tris (“,‘+) or diethanolamine (0-A) concentration for the activation of release of p-nitrophenol from p-nitrophenyl phosphate (15 mM. pH 10.0) by human liver alkaline phosphatase (Q.*) and intestinal alkaline phosphatase ( a,.). ua represents the observed velocity in the presence of Tris or diethanolamine and “0 the apparent velocity at zero concentrations of these buffers.
tion, 0.83 M (liver phosphatase) and 0.66 M (intestinal phosphatase), but in this case the values of V,,, were also nearly equal, being 3.70 pmoles/min per ml and 3.64 pmoles/min per ml for the liver and intestinal enzymes respectively. Buffers such as Tris or diethanolamine can also be considered as activators of p-nitrophenol release by alkaline phosphatase, whether or not this is due solely to formation of buffer phosphates. Thus, plots of reciprocals of the enhancement of p-nitrophenol release against reciprocals of buffer concentration can be used to derive activator constants (K,) for Tris and diethanolamine values for maximum activation. Plots of this type are shown in and Vmax Fig. 4, in which enhancement of the velocity due to the phosphate-accepting buffer has been obtained by subtracting the apparent velocity at zero Tris or diethanolamine concentration (u. ) from each observed velocity (u, ). Values of K, obtained in this way were 0.74 M (liver phosphatase) and 0.41 M (intestinal phosphatase) for Tris, and 0.29 M (liver phosphatase) and 0.23 M (intestinal phosphatase) for diethanolamine. These values are of the same order of magni-
351
tude as the K, values for buffer phosphate formation but are not identical with them, as would be expected if the transphosphorylation Gaction is the only process involving combination between the enzyme and buffer ions. The rather lower values for K, compared with K, suggest that some activation of p-nitrophenol release may occur which is distinct from formation of buffer phosphate. However, the similarity between the I’,.. values for formation of the phosphates and those for activation of p-nitrophenol release, mentioned below, implies that, if any such activation occurs, it must do so only at low buffer concentrations. Moreover, the present data do not exclude the possibility that the apparent differences between K, and K, values lie within the experimental error. Maximum values of u, (pmoles p-nitrophenol released/min per ml enzyme solution) calculated from Fig. 4 were quite dissimilar for the two enzymes in Tris buffer as for the formation of buffer phosphate, being 2.57 for liver phosphatase and 3.40 for intestinal phosphatase. In diethanolamine buffer more similar values, 4.08 and 4.45, respectively, were obtained. When the basal rates of hydrolysis of p-nitrophenyl phosphate (uO ) in the absence of either Tris or diethanolamine are subtracted, the remaining velocities represent the maximum activating effects of Tris or diethanolamine for the two enzymes. Comparison of the velocities obtained in this way with the maximum velocities of buffer phosphate formation calculated previously shows that the increased rates of release of p-nitrophenol in the presence of Tris or diethanolamine are accounted for within the limits of experimental error by formation of buffer phosphates. The calculated maximum additional velocities of p-nitrophenol release for the various combinations of enzymes and buffers (with V,.. values for buffer phosphate formation in parentheses) are: liver phosphatase in Tris 1.67 (1.48), and in diethanolamine 3.08 (3.70); intestinal phosphatase in Tris 3.00 (2.78), and in diethanolamine 3.45 (3.64). Discussion These results show an additional point of difference between the alkaline phosphatases of liver and small intestine in the rates at which they are able to form Tris phosphate, which further emphasizes their distinctiveness [9]. This difference in reaction with Tris does not appear to reside in markedly dissimilar affinities for Tris ions on the part of the enzymes Jrom the two tissues, since they have similar K, values for Tris phosphate formation, but in differences in rates of transfer of phosphate from the presumed phosphoryl-enzyme intermediate to the buffer ions. With diethanolamine as the phosphate acceptor, however, transfer of phosphate from the phosphoryl-phosphatases to the acceptor appears to take place at similar rates for both enzymes. The relatively lower maximum rate of transphosphorylation to Tris on the part of liver alkaline phosphatase compared with the intestinal enzyme, and consequently its relatively lower activity in this buffer in terms of rate of p-nitrophenol release, may be due in part to inhibition of the liver enzyme by Tris or one of its ionic forms, as has been suggested to occur with E. coli alkaline phosphatase in this buffer [ 31. One practical consequence of the different behaviour of liver and in-
352
testinal alkaline phosphatases in these buffer systems is that an alteration in the apparent relative activities of solutions of these two enzymes can result from a change in the nature of the buffer or its concentration, without any change in the nature of the substrate. In our experiments, for example, a change from 1 M Tris to 1 M diethanolamine altered the ratio of the rates of release of p-nitrophenol by liver and intestinal phosphatases from 0.72:1 to 0.85:1 (the ratio was l.OO:l in sodium carbonate-bicarbonate buffer). Similarly, when the concentration of Tris was increased from 0.1 M to 1.0 M this ratio decreased from 1.02:1 to 0.76:1. However, a corresponding increase in the concentration of diethanolamine did not have a significant effect on the ratio of activities of the two enzymes. An alteration in the relative activities of intestinal and nonintestinal alkaline phosphatase when glycine buffer is replaced by 2-amino-2methyl-1-propanol buffer has recently been reported [ 61. The absolute molecular activities of alkaline phosphatases from different human tissues are not yet known, so that relative activities of the several tissue-spedific forms of the enzyme can only be referred to at present in terms of some arbitrarily chosen set of experimental conditions; e.g. with respect to their activities in sodium carbonate--bicarbonate buffer as has been done in the present experiments. Thus the observation of a ratio differing from unity for the relative activities of liver and intestinal phosphatases in a given buffer system does not necessarily disqualify that system from use in measurements of enzymic activity. Nevertheless, with phosphate-accepting buffers under active consideration for inclusion in proposed reference methods for alkaline phosphatase assay, our results and those already reported indicate difficulties which may be encountered in comparing activities obtained with reference methods incorporating phosphate-accepting buffers with those derived from older procedures, in the case of ill-defined mixtures of tissue alkaline phosphatases such as occur in serum. References 1
W.H.
Fishman,
2
R.K.
Morton.
3
I.B.
4
G.N.
Bowers.
5
A.W.
Skillen
6
W.G.
Haije.
7
D.W.
8
J.L.
9
D.W.
Wilson,
Moss, Delsal Moss,
S. Green Nature.
and
172
J. Dayan
and
Jr and
R.B.
Chim.
Acta,
R.H.
Eaton.
J.K.
H. Manhouri. N.Y.
Acad.
Clin. 48
Sci.,
Biophys.
J. Biol.
Chem.,
239
Clin.
Chem.,
18
Chim. (1973)
Smith Bull.
Biochim.
Acta,
62
(1962)
363
65
K. Cyr,
and J. Harrison.
Ann.
Inglis.
McComb,
Clin. and
N.I.
(1953)
and Sot. 166
Acta,
45
(1964)
4182
(1972)
(1973)
97 287
23 L.G.
Whitby.
Chim.
Biol..
(1969)
641
Biochem. 40
(1958)
J.. 102 1623
(1967)
53
Clinica Chimica Acta, 52 (1974) 341-352 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 6415
A COMPARISON OF THE TRANSPHOSPHORYLATING ACTIVITIES HUMAN LIVER AND INTESTINAL ALKALINE PHOSPHATASES
KATRINE
B. WHITAKER
Department (U.K.) (Received
and D.W. MOSS
of Chemical Pathology, Royal Postgraduate Medical School, London
January
OF
W 12 OHS
17, 1974)
Summary
The relative rates of transphosphorylation by human liver and intestinal alkaline phosphatases from p-nitrophenyl phosphate to Tris and diethanolamine have been measured. The maximum velocities of formation of diethanolamine phosphate by the two enzymes are nearly equal when preparations with equal hydrolytic activities in sodium carbonate--bicarbonate buffer are used. However, Tris phosphate is formed at a lower rate by liver alkaline phosphatase than by the intestinal enzyme. Consequently, the rstio of the activities of the solutions of liver and intestinal alkaline phosphatases varies with the nature and concentration of the buffer system.
Introduction
It has frequently been observed that alkaline phosphatases (EC 3.1.3.1) from several vertebrate tissues differ in the relative rates at which they hydrolyze various phosphate esters [l] . Alkaline phosphatases also catalyze transfer of phosphate to suitable acceptors [2], and when aminoalcohols are used as buffers in the estimation of alkaline phosphatase activity a considerable enhancement of the catalytic rate takes place since the buffer ions can act as phosphate acceptors [3,4]. Few investigations have been made into the relative transphosphorylating activities of tissue-specific forms of alkaline phosphatase. However, it seems probable that differences in this respect may contribute to the variations which have been noted when the relative activities of alkaline phosphatase preparations from various tissues measured in assay systems which contain phosphate-accepting buffers are compared with the relative activities of the same specimens determined by methods in which the only phosphate acceptor is water [5,6]. We have compared the activities of alkaline phosphatases from human liver and small intestine on p-nitrophenyl phosphate in two phosphate-accept-
348
ing buffers, Tris and diethanolamine, both with respect to formation of the products of the hydrolytic reaction and to synthesis of the buffer phosphates. The results show that the maximum velocity of formation of Tris phosphate is relatively much lower for liver phosphatase than for intestinal phosphatase, but that maximum rates of formation of diethanolamine phosphate by the two enzymes are nearly equal, when solutions of the two enzymes are used which have equal hydrolytic activities in a non-phosphorylating buffer. Consequently, the relative activities of the two enzymes as measured by the rate of appearance of p-nitrophenol appear quite dissimilar when the Tris and diethanolamine buffer systems are compared. Materials and Methods Alkaline phosphatase was extensively purified from human small intestine as described by Moss et al. [7]. Human liver phosphatase was a less purified preparation obtained by applying to the tissue the first two stages of the same procedure. All determinations of alkaline phosphatase activity were made at 37” with 15 mM p-nitrophenyl phosphate as substrate and with 5 mM MgCl* added. Tris( 2-amino-2-hydroxymethylpropane-1,3-diol) or diethanolamine were added to the final concentrations shown in the results, and in each case the pH was adjusted to 10.0 by addition of HCl. Ionic strength was maintained constant to within ?I 2% by addition of NaCl to the several reaction mixtures. Enzymic action was allowed to proceed for 10 min since previous experiments had shown that production of both p-nitrophenol and inorganic phosphate was linear during this period over the range of enzyme activities used. p-Nitrophenol was measured by its extinction at 405 nm in an aliquot of the reaction mixture transferred to 0.5 M NaOH containing 10 mM EDTA. Inorganic phosphate was measured in a further aliquot of the reaction mixture after transfer to trichloroacetic acid by the method of Delsal and Manhouri [S] . The amount of buffer phosphate formed was calculated from the difference between the amounts of p-nitrophenol and inorganic phosphate thus determined. A reaction mixture consisting of 15 mM p-nitrophenyl phosphate and 5 mM MgClz in 0.05 M Na, CO3 -NaHC03 buffer, pH 10.0, was included with each experiment. This buffer does not act as a phosphate acceptor so that the enzymic reaction is entirely hydrolytic: the molar amounts of p-nitrophenol and inorganic phosphate formed in this buffer are therefore equal. All the activities measured in Tris or diethanolamine buffers have been expressed in terms of an enzyme activity equivalent to the production of 1 pmole of p-nitrophenol (or inorganic phosphate) per minute per ml of enzyme solution in the sodium carbonate-bicarbonate buffer. Results The rates of formation of p-nitrophenol, inorganic phosphate and buffer phosphate by liver and intestinal alkaline phosphatases are plotted as functions of Tris or diethanolamine concentration in Figs 1 and 2. Increasing concentrations of Tris or diethanolamine accelerate the release of p-nitrophenol by both
349
0.5
Tris
concentration(M)
1.0
0.5 Oiethonolamine
1.0 concentration(M)
Fig. 1. Rates of formation of p-nitrophenol (a$). inorganic phosphate (0.A) and Tris phosphate (*,A) by human liver alkaline phosphatase (circular symbols) and intestinal alkaline phosphatase (triangular symbols) at different concentrations of Tris. The substrate was 15 mM p-nitrophenyl phosphate at pH 10.0 in each case, and the velocities shown are those for activities of each phosphatase corresponding to the production of 1 pmole of p-nitrophenoljmin per ml enzyme solution in Na~C03-NaHC03 buffer at pH 10.0. Fig. 2. Rates Of formation of p-nitrophenol (?.A), inorganic phosphate (,.A) and diethanolamine phosphate (*,A) by human liver alkaline phosphatase (circular symbols) and intestinal alkaline phosphatase (triangular symbols), at different concentrations of diethanolamine. The substrate was 15 mM p-nitrophenyl phosphate at pH 10.0 in each case, and the velocities shown are those for activities of each phosphatase corresponding to the production of 1 Mmole of p-nitrophenollmin per ml enzyme solution in Na~C03---NaHC03 buffer at pH 10.0.
enzymes, as described in earlier reports. However, the magnitude of this effect differs for the two enzymes and differences are also present in relative rates of buffer phosphate formation. Thus, formation of Tris phosphate by liver phosphatase is appreciably less rapid than by intestinal phosphatase at each concentration of Tris, when the two enzymes are present in amounts which have equal activities in sodium carbonate-bicarbonate buffer. On the other hand, rates of formation of diethanolamine phosphate by liver and intestinal alkaline phosphatases are very nearly equal. Formation of buffer phosphate is a two-substrate reaction and, with the donor substrate (p-nitrophenyl phosphate) present in excess as in these experiments, values for the Michaelis constant and V,., can be obtained in terms of the acceptor substrate, the buffer. These values have been derived from plots of reciprocals of the velocity of buffer phosphate formation against reciprocals of buffer concentration (Fig. 3). For the formation bf Tris phosphate, Km values for liver and intestinal phosphatases were similar at 1.17 M, but Vmax was 1.48 pmoles/min per ml enzyme solution for liver phosphatase compared with 2.78 pmoles/min per ml for intestinal phosphatase. The two enzymes showed rather similar Km values for diethanolamin’e phosphate forma-
350
5-
1
0
0 or diethanolamine]
(M”)
‘/Tris [
2
4 or
6
8
diethanolamine]
Fig. 3. Plots of l/u against reciprocal of buffer concentration (Tris or diethanolamine) of Tris phosphate (0.A) and diethanolamine phosphate (*.a) by human liver alkaline and intestinal alkaline phosphatase ( A,A) by transphosphorylation from p-nitrophenyl 10.0.
10 CM-‘)
for the formation phosphatase (13.0) phosphate at pH
Fig. 4. Plots of l/(ua--rrg) against reciprocal of Tris (“,‘+) or diethanolamine (0-A) concentration for the activation of release of p-nitrophenol from p-nitrophenyl phosphate (15 mM. pH 10.0) by human liver alkaline phosphatase (Q.*) and intestinal alkaline phosphatase ( a,.). ua represents the observed velocity in the presence of Tris or diethanolamine and “0 the apparent velocity at zero concentrations of these buffers.
tion, 0.83 M (liver phosphatase) and 0.66 M (intestinal phosphatase), but in this case the values of V,,, were also nearly equal, being 3.70 pmoles/min per ml and 3.64 pmoles/min per ml for the liver and intestinal enzymes respectively. Buffers such as Tris or diethanolamine can also be considered as activators of p-nitrophenol release by alkaline phosphatase, whether or not this is due solely to formation of buffer phosphates. Thus, plots of reciprocals of the enhancement of p-nitrophenol release against reciprocals of buffer concentration can be used to derive activator constants (K,) for Tris and diethanolamine values for maximum activation. Plots of this type are shown in and Vmax Fig. 4, in which enhancement of the velocity due to the phosphate-accepting buffer has been obtained by subtracting the apparent velocity at zero Tris or diethanolamine concentration (u. ) from each observed velocity (u, ). Values of K, obtained in this way were 0.74 M (liver phosphatase) and 0.41 M (intestinal phosphatase) for Tris, and 0.29 M (liver phosphatase) and 0.23 M (intestinal phosphatase) for diethanolamine. These values are of the same order of magni-
351
tude as the K, values for buffer phosphate formation but are not identical with them, as would be expected if the transphosphorylation Gaction is the only process involving combination between the enzyme and buffer ions. The rather lower values for K, compared with K, suggest that some activation of p-nitrophenol release may occur which is distinct from formation of buffer phosphate. However, the similarity between the I’,.. values for formation of the phosphates and those for activation of p-nitrophenol release, mentioned below, implies that, if any such activation occurs, it must do so only at low buffer concentrations. Moreover, the present data do not exclude the possibility that the apparent differences between K, and K, values lie within the experimental error. Maximum values of u, (pmoles p-nitrophenol released/min per ml enzyme solution) calculated from Fig. 4 were quite dissimilar for the two enzymes in Tris buffer as for the formation of buffer phosphate, being 2.57 for liver phosphatase and 3.40 for intestinal phosphatase. In diethanolamine buffer more similar values, 4.08 and 4.45, respectively, were obtained. When the basal rates of hydrolysis of p-nitrophenyl phosphate (uO ) in the absence of either Tris or diethanolamine are subtracted, the remaining velocities represent the maximum activating effects of Tris or diethanolamine for the two enzymes. Comparison of the velocities obtained in this way with the maximum velocities of buffer phosphate formation calculated previously shows that the increased rates of release of p-nitrophenol in the presence of Tris or diethanolamine are accounted for within the limits of experimental error by formation of buffer phosphates. The calculated maximum additional velocities of p-nitrophenol release for the various combinations of enzymes and buffers (with V,.. values for buffer phosphate formation in parentheses) are: liver phosphatase in Tris 1.67 (1.48), and in diethanolamine 3.08 (3.70); intestinal phosphatase in Tris 3.00 (2.78), and in diethanolamine 3.45 (3.64). Discussion These results show an additional point of difference between the alkaline phosphatases of liver and small intestine in the rates at which they are able to form Tris phosphate, which further emphasizes their distinctiveness [9]. This difference in reaction with Tris does not appear to reside in markedly dissimilar affinities for Tris ions on the part of the enzymes Jrom the two tissues, since they have similar K, values for Tris phosphate formation, but in differences in rates of transfer of phosphate from the presumed phosphoryl-enzyme intermediate to the buffer ions. With diethanolamine as the phosphate acceptor, however, transfer of phosphate from the phosphoryl-phosphatases to the acceptor appears to take place at similar rates for both enzymes. The relatively lower maximum rate of transphosphorylation to Tris on the part of liver alkaline phosphatase compared with the intestinal enzyme, and consequently its relatively lower activity in this buffer in terms of rate of p-nitrophenol release, may be due in part to inhibition of the liver enzyme by Tris or one of its ionic forms, as has been suggested to occur with E. coli alkaline phosphatase in this buffer [ 31. One practical consequence of the different behaviour of liver and in-
352
testinal alkaline phosphatases in these buffer systems is that an alteration in the apparent relative activities of solutions of these two enzymes can result from a change in the nature of the buffer or its concentration, without any change in the nature of the substrate. In our experiments, for example, a change from 1 M Tris to 1 M diethanolamine altered the ratio of the rates of release of p-nitrophenol by liver and intestinal phosphatases from 0.72:1 to 0.85:1 (the ratio was l.OO:l in sodium carbonate-bicarbonate buffer). Similarly, when the concentration of Tris was increased from 0.1 M to 1.0 M this ratio decreased from 1.02:1 to 0.76:1. However, a corresponding increase in the concentration of diethanolamine did not have a significant effect on the ratio of activities of the two enzymes. An alteration in the relative activities of intestinal and nonintestinal alkaline phosphatase when glycine buffer is replaced by 2-amino-2methyl-1-propanol buffer has recently been reported [ 61. The absolute molecular activities of alkaline phosphatases from different human tissues are not yet known, so that relative activities of the several tissue-spedific forms of the enzyme can only be referred to at present in terms of some arbitrarily chosen set of experimental conditions; e.g. with respect to their activities in sodium carbonate--bicarbonate buffer as has been done in the present experiments. Thus the observation of a ratio differing from unity for the relative activities of liver and intestinal phosphatases in a given buffer system does not necessarily disqualify that system from use in measurements of enzymic activity. Nevertheless, with phosphate-accepting buffers under active consideration for inclusion in proposed reference methods for alkaline phosphatase assay, our results and those already reported indicate difficulties which may be encountered in comparing activities obtained with reference methods incorporating phosphate-accepting buffers with those derived from older procedures, in the case of ill-defined mixtures of tissue alkaline phosphatases such as occur in serum. References 1
W.H.
Fishman,
2
R.K.
Morton.
3
I.B.
4
G.N.
Bowers.
5
A.W.
Skillen
6
W.G.
Haije.
7
D.W.
8
J.L.
9
D.W.
Wilson,
Moss, Delsal Moss,
S. Green Nature.
and
172
J. Dayan
and
Jr and
R.B.
Chim.
Acta,
R.H.
Eaton.
J.K.
H. Manhouri. N.Y.
Acad.
Clin. 48
Sci.,
Biophys.
J. Biol.
Chem.,
239
Clin.
Chem.,
18
Chim. (1973)
Smith Bull.
Biochim.
Acta,
62
(1962)
363
65
K. Cyr,
and J. Harrison.
Ann.
Inglis.
McComb,
Clin. and
N.I.
(1953)
and Sot. 166
Acta,
45
(1964)
4182
(1972)
(1973)
97 287
23 L.G.
Whitby.
Chim.
Biol..
(1969)
641
Biochem. 40
(1958)
J.. 102 1623
(1967)
53