Activity of Epiphyseal Cartilage Membrane Alkaline Phosphatase and the Effects of Its Inhibitors at Physiological pH*

The kinetics of epiphyseal cartilage membrane- bound alkaline phosphatase activity was studied at physiological pH using p-nitrophenylphosphate and pyrophosphate (PPi) as substrates. The effect of three general types of alkaline phosphatase inhibitors was studied on both purified and membrane-bound forms of the enzyme: 1) uncompetitive inhibitors (btetramisole and theophylline), 2) competitive inhibitors (phosphate, arsenate, and vanadate), and 3) metal ions @I&+, Ca2+, and Zn”). These studies were aimed at elucidating the physiological role of epiphyseal cartilage membrane alkaline phosphatase. only Ca*PPI, and complexes

As part of our continuing investigation of the role of alkaline phosphatase in calcification, we recently reported on the isolation and characterization of a membrane-bound alkaline phosphatase from chicken epiphyseal cartilage (16). In that study the kinetic experiments were carried out at pH 10.3, the optimal pH for the substrate used (p-nitrophenylphosphate), although it was recognized that this was far from the pH (7.5) of the extracellular fluid of epiphyseal cartilage (17).
We now report on studies of the kinetics of cartilage membrane alkaline phosphatase carried out at physiological pH (7.5). We studied the effects of several inhibitors which have been used, or are potentially useful, in investigations of the role of the enzyme as a vector in the uptake of Pi by matrix vesicles. These studies were carried out with both pNPP' (a common in vitro substrate for the enzyme) and PPi (a potentially important physiological substrate) in order to distinguish between direct effects of the inhibitors om the enzyme, and indirect effects caused by complexation with the substrate. We also studied the activity of the enzyme in its native environment, comparing inhibition of activity of the membrane-bound form with that of the purified enzyme. These experiments were designed to explore the nature and strength of inhibition of both purified and membrane-bound alkaline phosphatase at physiological pH using three general types of inhibitors: 1) heterocyclic uncompetitive inhibitors (L-tetramisole and theophylline), 2) the enzyme product and its analogues (phosphate, arsenate, and vanadate), and 3) several physiologically important divalent cations (Mg2+,Ca2',and Zn2+). 45 Ca and/or 32Pi from metastable solutions (11-15), and this

EXPERIMENTAL PROCEDURES
Purified alkaline phosphatase was prepared as previously described (16). For studies on the membrane-bound enzyme, partially purified matrix vesicles were prepared by a modification of the procedure of Watkins et al. (18). In the published procedure, a step gradient was used which was composed of 10,30,40, and 50% (w/w) sucrose in a 50 m~ TES buffer (pH 7.5) containing 0.25 m~ MgC12. In the method used here, the step gradient was composed of 15 ml of 40% (w/w) sucrose and 20 ml of 10% (w/w) sucrose, both made in the TES-MgC12 buffer. The material collected at the 10/40% interface was pooled, diluted with 2-3 volumes of the 10% sucrose buffer, and centrifuged a t 85, ooO X g for 60 min. The pellet which corresponded approximately to a combination of the A and B fractions of the earlier procedure was resuspended in 10% sucrose made in the TES-MgCl2 buffer, and used without further purification. The protein concentration both of the purified enzyme and the partially purified matrix vesicles was determined by the method of Lowry et al. (19) using bovine serum albumin as a standard.
Hydrolysis Measurements-Hydrolysis of substrates was measured in 0.25 M TES buffer adjusted to pH 7.5 with NaOH. Appearance of p-nitrophenol was monitored at 402 nm and analyzed as previously described (16) using a molar extinction coefficient of 4500 cm-W1. Hydrolysis of PPi was followed in 35-min assays as previously described (16), the rate being linear for this length of time under the conditions used. The temperature of the assays was maintained at 37 "C either by circulating water through a jacketed cuvette holder (pNPP) or by a water bath (PPi). The extent of hydrolysis was always limited to less than 10% of the available substrate, and the pH was monitored to ensure that it was not affected. All assays were initiated by the addition of substrate. Kinetic parameters (K, and V,=) were estimated by a nonlinear least squares fit (16,20), and the inhibition constants were obtained by unweighted linear least squares fits to the appropriate secondary plots.
Transphosphorylation Measurements-The assay procedure was based on that of Arsenis et al. (21). A solution containing 5 M glycerol, 0.25 M TES-NaOH buffer (pH 7.54, 2 mM NADH, and 2 units of glycerol-3-phosphate dehydrogenase in a total volume of 2.0 ml was equilibrated at 37 "C. Alkaline phosphatase (1.7 pg in 0.05 m l ) was then added, and the reaction initiated by the addition of PP,. Levamisole, when used, was added from a 1 M stock solution immediately prior to the alkaline phosphatase. Absorbance was recorded at 340 rim for 10 min and the slope of this line was used for activity calculations.
Materials-The following were purchased from Sigma: p-nitrophenylphosphate (disodium hexahydrate),p-nitrophenol standard solution, TES, p-NADH (Grade III), glycerol-3-phosphate dehydrogenase (Type X), theophylline, and levamisole. The V 2 0 5 used to prepare the vanadate solutions was supplied by MCB (Cincinnati, OH). r3'P] PP, was procured from New England Nuclear. All other chemicals were of reagent grade and were obtained from Fisher. Table I presents comparative data on the K , and Vmax of the purified enzyme toward both pNPP and PPi as substrates, at both optimal and physiological pH. It is evident that both parameters are markedly decreased at pH 7.5. These data indicate that while binding to the enzyme is much tighter, hydrolytic activity toward both substrates is nevertheless greatly suppressed. Other data, not shown, reveal that the K, of the purified and membrane-bound forms of the enzyme for both substrates was essentially identical.

RESULTS
With pNPP as substrate at pH 7.5, levamisole (L-tetramisole) proved to be an uncompetitive inhibitor of both the purified and vesicular (membrane-bound) enzyme ( Fig. 1).
There was a small, but consistent difference between the K, values of the two enzyme forms (1.4 m~ for purified, 0.6 m~ for membrane-bound). Unfortunately, the effect of levamisole could not be studied with PPi as substrate because it severely interfered with the assay procedure. Theophylline (Fig. 2) also proved to be an uncompetitive inhibitor of alkaline phosphatase at this pH. Ki analysis from, secondary plots indicated that, with pNPP as substrate, there was no difference between the vesicular and the pure enzyme. With PPi as substrate, however, there was a significant difference between the values for the purified enzyme (KL = 5.6 mM) and the membrane-bound phosphatase (Ki = 3.0 m~) .
The direction of this difference was the same as that seen with levamisole inhibition of p N P P hydrolysis.
Phosphate (Fig. 3), arsenate (Fig. 4), and vanadate ( Fig. 5) were all competitive inhibitors of alkaline phosphatase activity with both the purified and membrane-bound forms of the enzyme. While all three analogues were powerful inhibitors of hydrolase activity toward both substrates, with Ki values in the low micromolar range, both arsenate and vanadate were 10 t o 20 times more effective than phosphate (Table 11).
Although slight differences between the K, values of the   membrane-bound and purified enzyme activities were evident, no consistent pattern was discernible. In the case of phosphate, the purified enzyme was more strongly inhibited with vanadate, the membrane-bound activity was more strongly affected. With arsenate, the effects depended on the substrate. These points will be discussed more fully later.
None of the metal ions tested (Zn2+, Ca2+, and Mg2+) showed any effect on alkaline phosphatase activity at pH 7.5 when pNPP was used as substrate. In contrast, with PPi as substrate, all three divalent cations inhibited enzyme activity. As will be evident, the nature of the inhibitory species differed in each case.
No apparent difference between the two forms of enzyme was evident. At the concentrations of Zn2+ studied, greater than 95% of the PP; would have been present as a Zn-PPi binary complex (Kd = 0.5 p~) , (22). Had this complex been inactive as a substrate, the hydrolytic activity should have been only 3-6% of that ob-served at low (5 p~) PPi concentration. The observed hydrolysis thus must have been of the binary Zn. PPi complex.
Indeed, since the Vmax seen at aU Zn2+ concentrations tested was identical with that observed in the absence of Zn2+, and since a plot of the K,,,aPP/V,,,a~PP versus Zn2' concentration passed through the control value, the Zn.PPi complex must have been as active a substrate as free PPi.
Inhibition by CaC12 (Fig. 7) was seen at concentrations (0.5-2.0 m~) , and with Ki values (0.73-1.12 m~) , similar but not identical to those expected for inhibition by substrate depletion. A Kd of 3.4 X M for the Ca-PPi complex (22)   Inhibition of pyrophosphate activity by L-tetramisole (levamisole) could not be assayed because the drug interfered with extraction of phosphomolybic acid required for the assay method (16). D-tetramisole (dexamisole) exerted no inhibitory effect at concentrations up to 5 mM.
No measurable inhibition of pNPP hydrolysis was detected at pH 7.5 with concentrations of ZnClp up to 1 m~, CaClz up to 3 mM, and The first K, value for MgClt was as a competitive inhibitor, the MgCL UP to 1.5 mM.
second as an uncompetitive inhibitor. Inhibition of hydrolysis by calcium. See "Experimental Procedures" for assay procedures. Only PPi hydrolysis is shown since there was no effect observed on pNPP hydrolysis with Ca2+ concentrations as high as 3.0 111~. Symbols and graphing methods are as shown in Figs. 1 and 3. centration of Ca and PPi. When the expected rate of hydrolysis was calculated from these concentrations of free PPi, using the K,,, and Vmax values observed in the absence of added cations, it was noted that the calculated activity was consistently lower than that observed. When the reciprocal of the difference between the calculated (ie. from free PPi) and observed activity was plotted against the reciprocal of the calculated Ca PPi complex concentration, a linear relationship was observed. The VmeraPP so calculated was approximately one-tenth that observed in the absence of Ca2+. Thus, it was evident that while the Ca -PPi complex was capable of acting as a substrate, it was only about 10% as active as free PPi.
With MgClz as inhibitor, mixed-type inhibition was seen.
As seen in Fig. 8A, M e stimulated vesicular phosphatase activity a t low concentrations (t0.25 m), as indicated by lower s/v values. At higher concentrations, however, it was inhibitory. The intersection of the lines of this plot (Fig. 8 A ) indicated an apparent Ki' of 0.27 lll~ for the uncompetitive portion of the inhibition. Similar results were obtained with the purified enzyme (Ki' = 0.29 m~) (data not shown). Dixon plots of the inhibition data for both the vesicular (Fig. 8B) and purified phosphatase (not shown) gave a K i of 0.35 m for the competitive portion.
The results of these various inhibition studies are summarized in Table 11, where the apparent K i values for each inhibitor ahd enzyme form are shown. Although comparison of the vesicular and purified enzyme showed several differences, no clear pattern emerged. Levamisole and theophylline, which presumably share a common binding site (23), had no more than a 2-fold difference in K i value for the two substrates, or the two enzyme forms. This was also true for inhibitions by phosphate, arsenate, and vanadate. These results thus suggest that if binding of alkaline phosphatase to the membrane had an effect on enzyme conformation, it was not sufficiently large to markedly affect the kinetic parameters determined in this investigation. Finally, to test the effectiveness of levamisole as an inhibitor of one of the other functions of alkaline phosphatase, transphosphorylation using PPi as the phosphate donor and glycerol as the acceptor was measured in the presence and absence of three concentrations of the drug. The results, shown as a plot of s/v versus concentration of levamisole (Fig. 9), indicated that levamisole was an uncompetitive inhibitor of this   reaction, as well as of hydrolysis. This effect is consistent with the presumed action of levamisole in stabilizing the enzymephosphate complex (3). Dixon plots of the data showed too much scatter to allow a reliable estimation of the contribution, if any, of competitive-type binding to this inhibition.

DISCUSSION
The primary focus of these experiments was to determine the effects at physiological pH of various inhibitors of alkaline phosphatase activity from epiphyseal cartilage membranes, using two distinctive substrates. Inhibition was examined with both the purified and membrane-bound forms of the enzyme. We wished to examine these parameters both for reasons of kinetic importance ( i e . mechanism of inhibitor and preferred substrate form) and to determine the feasibility of using these inhibitors to elucidate the possible role of alkaline phosphatase in endochondral calcification. The kinetic considerations will be discussed first.
Four kinetically interesting conclusions can be drawn from the data obtained. 1) In agreement with data on the bone enzyme studied by Farley et al. (23), the monovalent anion is the kinetically relevant inhibitory form of phosphate (2.e. H2P04-) and arsenate (H2AsO4-). 2) Vanadate binds to the enzyme in a manner similar to that observed with other phosphohydrolases which incorporate a covalent phosphoryl enzyme intermediate in the mechanism. 3) There are slight, if any, changes in the kinetic properties of the membranebound enzyme when it is solubilized and purified by our reported method. And 4) the suitability of metal ion. PPi complexes as substrates for alkaline phosphatase depends strongly on the nature of the metal ion involved.
The monovalent form of substrate anion has been implicated as the active form in both alkaline (23) and acid (24) phosphatases. Table I11 shows the Ki values for phosphate and arsenate at pH 7.5 and 10.3, expressed as concentrations of the monovalent form of anion. The resulting values, while not constant, are much more alike than those derived from the total concentration of inhibitor . Farley et al. (23) reported that the K, value of phosphate also depends on the nature of the buffer used. With an amino-alcohol buffer (Tris) the K, was 6-10-fold less than with a non-amino-alcohol buffer (carbonate). This difference is approximately the same change we * Calculations based on a pK2 value of 6.9 (22).
observed in going from 2-amino-2-methyl-1-propanol at pH 10.3 to TES at pH 7.5. Our data are therefore basically consistent with the monovalent anion being the active inhibitor species. Inhibition by vanadate has been hypothesized to be more potent in enzymes involving a stable covalent phosphoryl enzyme intermediate than in those in which the product is formed without covalent involvement of the enzyme (25). Thus, it is particularly active against Na+/K+-ATPase (26), human alkaline phosphatase (27), Escherichia coli alkaline phosphatase (28), and less so against sarcoplasmic reticulum Caz'-ATPase (29). The vanadate (from VZOs) used in this study is known to exist in the form of H2V04-and HV04'-in dilute neutral solutions (30). The Ki of vanadate (1.4-2.6 p ) thus is within the range expected for phosphoryl-enzyme mechanisms. Further, we have evidence that, in agreement with other alkaline phosphatases, a stable 32Pi-alkaline phosphatase form of the cartilage enzyme exists at acid pH.
The presence of the membrane is known to affect the properties of several membrane-derived enzymes. The data presented here, however, do not show any consistent major change in the kinetic properties of the membrane-bound cartilage enzyme as a result of solubilization and purification. In several cases (levamisole versus pNPP theophylline versus PPi; phosphate versus pNPP; and vanadate versus PPi) there was about a 2-fold difference between the Ki of the membranebound and the purified enzyme form. These rather small differences, and the lack of a consistent pattern, indicate that the membrane environment has little effect on the activity of the enzyme. A similar lack of effect of the membrane was also noted for the calf intestinal alkaline phosphatase (31).
Each of the three metal ions investigated showed different effects on PPi hydrolysis. As noted before, Ca" seemed to act primarily by way of substrate depletion, with the Ca-PPi complex being only one-tenth as active as free PPi. By contrast, Zn.PPi complex appeared to bind to the enzyme and undergo hydrolysis much like free PPi. This interpretation is based on the fact that Vmx was unchanged, and plots of KmapP/ VmaxsPP versus Zn2+ concentration extrapolated to the value seen in the absence of added cations, although essentially no free PPI should have been present under the conditions of these assays. Several workers have reported inhibition of mammalian alkaline phosphatases by excess Zn2+ (32)(33)(34)(35). They proposed that the inhibition is caused by Zn2+ replacing M e at one site on the enzyme. This does not appear to be the mechanism involved here, since we observed no effect of ZnCb on pNPP hydrolysis at pH 7.5. In this regard, it is interesting to note that ZnCh was a powerful inhibitor of pNPP hydrolysis (K, = 19 p t ) at pH 10.3 (16). This suggests that the active form of zinc at alkaline pH is not Zn2+, but ZnOH+. It is known that Zn2+ forms a soluble Znz -PPi complex (22) and it is likely that this ternary complex is the form directly involved in the competitive inhibitory process.
Finally, Mg-PPI may be a better substrate for the enzyme than is free PPi. This is evidenced by the observation of stimulation of PPi hydrolysis by low concentrations of MgC12.
we observed no effect of added Mg2f on pNpp hydrolysis, suggesting that this stimulation is the result of Mg2+-PPi interactions rather than Mg2+-enzyme interactions. Since Mg2' is also capable of forming soluble Mgz -PPi complexes (22), this may be the mechanism of competitive inhibition seen at higher MgClz concentrations, much as was observed with Zn". The uncompetitive portion of the inhibition could be due to stabilization of the phosphoryl-enzyme complex by free M2+. Exact elucidation of the complexes and mechanisms involved must, however, await more detailed experiments using constant metal ion:PPi ratios, and competitive inhibitions of pNPP hydrolysis by metal-ion:PPi complexes.
In this study we chose conditions which would ensure extrapolation of the observed effects on the activity of alkaline phosphatase to the physiological situation. For instance, TES buffer was used in order to avoid the known stimulation by amino-and alcohol-containing buffers such as Tris and 2- amino-2-methyl-1-propanol (21,36). Second, the activity was studied using phosphate monoester and anhydride substrates since both types are present in epiphyseal cartilage, and the metal ions might be expected to differ in their effects on the two types of substrates. Further, as discussed above, we determined the effect of the membrane environment on alkaline phosphatase activity. Finally, the effect of all of these parameters was studied at physiological pH.
In terms of physiological function, these findings, in conjunction with previously published information on the weak activity of alkaline phosphatase at this pH (16), are not supportive of this enzyme acting as a hydrolase in vivo.
Several observations support this conclusion. First, the specific activity of alkaline phosphatase at pH 7.5 was only onetenth to one-hundredth (depending on substrate) of that seen at optimal pH. Thus, whereas the specific activity of pNPP hydrolysis at pH 10.3 in 2-amino-2-methyl-~-propanol buffer was approximately 200-220 pmol/min/mg of enzyme (16), the Vmax observed at pH 7.5 was only about 0.5 pmol/min/mg. With PPi the observed V,,, was on the order of 0.2 pmol/min/ mg. These activities are such that only very limited hydrolysis would occur, even in the absence of inhibitors. However, the concentration of phosphate in the extracellular fluid of epiphyseal cartilage (2.2-2.3 m) (17), is approximately two orders of magnitude above the K, determined for both phosphate monoesters and pyrophosphate in this study. Since both nucleotide phosphates (37) and pyrophosphate (38) are present in the extracellular fluid in only micromolar amounts, it is unlikely that they could compete effectively for the enzyme in vivo.
Finally, it should be noted that extracellular cartilage fluid also contains 1.5-1.7 ~l l~ Ca2+ (17). This concentration, coupled with the fact that PPI is present in only micromolar concentrations as indicated above, leads to the conclusion that PPi would be largely complexed with Ca", a form which was shown above to be almost completely inactive as a substrate. These considerations thus lead to the conclusion that alkaline phosphatase in vivo would be almost entirely without hydrolytic activity toward any likely substrate. Of the proposed roles of the protein, that of phosphate transport across the membrane seems most likely. For example, the K , for phosphate indicates that alkaline phosphatase has a high affinity for this anion at physiological pH. Secondly, the role of alkaline phosphatase as a Pi vector has been recently investigated using vesicles isolated by non-protease-dependent methods (15). Those studies showed that levamisole strongly and selectively inhibited 32Pi uptake, lending credence to this idea. The strong competitive inhibition by arsenate and vanadate observed in this study make it likely that these ions will prove useful in the study of phosphate transport, since they should be effective competitors of phosphate in the uptake system.