Journal of Biochemical and Biophysical Methods, 25 (1992) 55-60
55
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-022X/92/$05.00
JBBM 00951
Bovine inositol monophosphatase: development of a continuous fluorescence assay of enzyme activity M.G. Gore ~, P.J. Greasley
a
and C.I. Ragan b
a Dept. of Biochemistry, School of Biological Sciences, Unicersity of Southampton, Southampton (UK) and b Merck Sharpand Dohme, Neurosciences Research Centre, Hariow, Essex (UK) (Received 2 March 1992) (Revised version received 3 April 1992) (Accepted 30 April 1992)
Summary This paper describes a continuous assay for the enzyme inositol monophosphatase which has been developed using a new substrate, the fluorescent compound 4-methylumhelliferyl phosphate. The hydrolysis of the phosphate group from this compound can be readily detected by a resultant large red shift in the emission spectrum from 390-450 nm. The kinetic constants for the enzyme using this new substrate are described. Key words: Continuous assay; Fluorescence; 4-Methylumbelliferyl phosphate
Introduction Inositol monophosphatase is a homodimer of subunit M r 30 000 which catalyses the dephosphorylation of inositol 1-phosphate, inositol 3-phosphate and inositol 4-phosphate [1] to inositol and phosphate (throughout we refer to myo-inositol, and use the numbering of 1D-myo-inositol). The supply of inositol for the production of phosphatidylinositol 4,5-bisphosphate is therefore highly dependent upon inositol monophosphatase activity, especially in brain tissue which lacks a suitable uptake system for inositol [2]. The enzyme has an absolute requirement for the divalent cation Mg 2+ and is inhibited in an uncompetitive manner by Li + ions [3], a feature which may underlie the antimanic and antidepressant actions of this ion (for review see Ref. 4). Inositol monophosphatase isolated from a number of Correspondence address: M.G. Gore, Dept. of Biochemistry, Bassett Crescent East, University of Southampton, Southampton, SO9 3TU, UK.
56 TABLE 1 Kinetic and inhibition constants at pH 8.0 and 37°C
K m (raM) Vmax+(U/mg) c K Li (raM) K ~ g2+ (raM) KiMg:+ (mM) kcat / K m ( M - I s - I) Kiei (raM) K~nhibit°r (p,M)
Ins(1)P
Mec-O-P
0.12 a 12.9 " 0.26 a 3.00 ~ 4.3 a 5.38" 10 4 0.5 a 1.1 h
0.92 0.39 16 7.19 no inhibition 2.12" 10 2 0.56 4.0
This table shows the FmCax(U -- 1 ~ m o l / m i n ) and K m for the enzyme obtained using Mec-O-P as a substrate. The K m for Mg 2+ and the K i for various ligands are also shown. The data shown using Ins(l)P as a substrate are taken from the following references: a [1]; b [16].
sources has been shown to demonstrate a broad substrate specificity. It dephosphorylates all monophosphate esters of inositol except inositol 2-phosphate, where the ester linkage is in the axial position relative to the ring. Studies have shown that the 2' and 6' hydroxyl groups of inositol 1-phosphate are independently associated with substrate binding and the mechanism of hydrolysis. The enzyme has also been shown to dephosphophorylate substrate analogues such as 2'AMP, 2'GMP, glycerol 3-phosphate as well as a number of other monophosphate esters [1,3,5-81. In all studies to date, the activity of inositol monophosphatase has been determined either by the discontinuous methods of measuring the release of Pi [9], [*4C]inositol from [14C]Ins(1)P [10] or by the coupled assay of Degroot et al. [11]. A continuous assay is preferable because it will invariably be more rapid, more accurate and the time-course of the reaction is observed directly. We have utilised the broad substrate specificity of inositol monophosphatase to identify a new substrate, 4-methylumbelliferyl phosphate, which will permit the direct observation of substrate disappearance or product appearance using fluorescence spectrophotometry. Umbelliferone derivatives have found broad use in the study of a number of enzyme systems [12-14]. 4-methylumbelliferyl phosphate (Mec-O-P) has previously been used in the study of N a ÷ / K + ATPase activity [15]. Because of the different fluorescent properties of 4-methylumbelliferone (Met) and Met-O-P, a very sensitive assay system is obtained. We have therefore investigated the possibility of using Mec-O-P as a potential substrate of inositol monophosphatase and examined its potential in the development of a continuous assay of enzyme activity. Materials and Methods
The growth of bacterial cultures and subsequent extraction and purification of the enzyme have been described elsewhere [16]. [14C]Ins(1)P was purchased from
57 Amersham, UK. All other reagents were purchased from either Sigma or BDH, Poole. The substrate O,L-Ins(1)P was synthesized as described by Billington et al. [17], and the inhibitor (1S)-phosphoryloxy-(2R,4S)-dihydroxycyelohexane as described by Baker et al. [18].
Discontinuous assays Inositol monophosphatase activity was determined by the discontinuous assay described by Lanzetta et al. [9] or by the detection of [~4C]inositol released upon enzyme activity as described by Ragan et al. [10]. Protein concentrations were determined by the method of Bradford [19]. Fluorescence studies Fluorescence measurements were made using a Perkin-Elmer 650S fluorescence detector. Activity of inositol monophosphatase with Mec-O-P as substrate was determined fluorimetrically by the determination of the rate of product (Mec) released (Aex = 388 nm; Aem = 450 nm). In all cases the assays were carried out using 10 ~g of enzyme buffered in 1 ml of 50 mM Tris-HCl (pH 7.8). Unless otherwise indicated in the text 0.5 mM Mec-O-P and 10 mM MgCl 2 were used in each experiment. A standard curve of Mec fluorescence (0-100 /~M) was constructed in the presence of 0.5 mM Mec-O-P and 10 mM MgCI 2 in 50 mM Tris-HCi buffer (pH 7.8). The addition of inhibitors and other ligands is as described in the text.
Results
The possible utilization of Mec-O-P as a substrate was examined initially by the discontinuous determination of phosphate released [9]. Mec-O-P was indeed utilized as a substrate, and Lineweaver-Burk analysis showed that the enzyme has a K m of approx. 1 mM for this substrate and a Vm~x of 0.38 U / m g , approx. 3% of that seen with Ins(1)P as substrate. To confirm the identity of the product of the reaction, Mec-O-P was incubated with inositol monophosphatase overnight at 37°C to allow the reaction to go to completion. A comparison of the fluorescence excitation and emission spectra of the product of this reaction with those of Mec showed both to be identical. The fluorescence emission intensity at 450 nm from the assay solution was shown to be directly proportional to the amount of Mec present, allowing the quantification of the amount of product released in the assay. The absorption of the assay solutions at all substrate concentrations was less than 0.01 and therefore corrections for inner filter effect are insignificant. Although homogeneous preparations of the enzyme were used in all experiments the possibility of contaminating phosphatase activity catalysing this reaction was eliminated by comparing the reaction rate in the absence and presence of either Li ÷ ions (an uncompetitive inhibitor of inositol monophosphatase) or (1S)-phosphoryloxy-(2 R,4S)-dihydroxycyclohexane, a competitive inhibitor of Ins
58 (1)P. In the first experiment the enzyme preparation was preincubated with 10 mM LiCI and 1 mM Ins(1)P prior to being utilized in an assay using Mec-O-P also in the presence of 10 mM LiCI. In this case, less than 5% activity was seen compared with enzyme which had not been preincubated with Ins(1)P and Li +, implying that the observed activity was dependent upon functional inositol monophosphatase. In the second experiment the presence of various concentrations of (1S).phosphoryloxy-(2R,4S)-dihydroxycyclohexane was used to inhibit the reaction and determine the nature of the inhibition and the K i. The Ki for this inhibitor with Mec-O-P as substrate was found to be 4.0/~M under the conditions of the assay (see Materials and Methods), which compares well with that measured when Ins(1)P is the substrate used. As the nature of the inhibition is not substrate-dependent, any deviation would be unexpected and it was therefore concluded that in the above experiments the dephosphorylation of the Mec-O-P was due solely to inositol monophosphatase action. The kinetic parameters of inositol monophosphatase with respect to Mec-O-P were then determined. The initial rate of product formation was linear with enzyme concentration and Lineweaver-Burk analysis gave a K m for Mec-O-P of 0.92 mM (--+_0.07 (n ffi 3)) and a Vm~, of 0.39 p m o l / m i n per mg. Thus the data obtained by the direct measurement of product released correlates well with the discontinuous determination of phosphate released. The high K m and low Vmax compared to Ins(1)P is consistent with previous observations [20] which showed that the maximal velocities with a number of different substrates depend upon the nature of the phosphate ester, and that the presence of adjacent hydroxyl groups allows hydrolysis to occur more readily. Previous workers have demonstrated that Mg 2+ is essential for catalytic activity with a K m for the enzyme from bovine brain of 3 mM with Ins(1)P as substrate. At higher concentrations it becomes an uncompetitive inhibitor. The K m and Ki for Mg 2+ has previously been shown to be dependent upon the nature of the substrate [20]. The K m for M g 2+ with Mec-O-P was shown to be 12 mM and no inhibition was observed up to 100 mM, which simplifies the kinetic analysis of reactions using saturating concentrations of this metal ion. The Ki for Li + ions, an uncompetitive inhibitor of inositoi monophosphatase has been shown to be dependent upon the nature of the substrate. Whereas the Ki for Li + ions is approx. 0.26 mM when inositol 1-phosphate is the substrate, the Ki of Li + ions was shown to be 16 mM when the substrate used is Mec-O-P. In addition, the second product of the reaction, inorganic phosphate, a competitive inhibitor of the enzyme with respect to substrate, was found to have a similar K i (0.56 or 0.50 raM) when either Mec-O-P or Ins(1)P, respectively, is used as substrate.
Discussion
The low substrate specificity of inositol monophosphatase has allowed a fluorescent compound, 4-methylumbeiliferyl phosphate, to be investigated as a potential substrate. The use of a fluorescent assay allows for the sensitive determination of
59
enzyme activity. The large difference in wavelength of maximal fluorescence emission between the spectra of the product and substrate allows the selective observation of one without the need for correction due to the contribution of the other. The kinetic parameters determined using this assay correlate well with data determined in experiments using discontinuous methods. The K m for M g 2+ and the K i for Li + are approx. 4- and 75-fold respectively higher using Mec-O-P as substrate. Both are increased as would be expected since the specificity constant (kcat/Km) for this substrate is reduced and the interactions of both metal ions have been shown to be dependent on the nature of the substrate [20]. In contrast, the substrate-independent inhibitors, inorganic phosphate and (lS)-phosphoryloxy(2R,4S)-dihydroxycyclohexane both demonstrate similar inhibition constants with either Mec-O-P or Ins(1)P. Recently an alternative continuous fluorescent assay has been published [21] based upon the use of DE-1-(o-aminobenzoyl)glycerol2-phosphate. This compound only binds to bovine serum albumin (BSA) when phosphorylated, where it fluoresces 2.75-fold more intensely than in water. The assay is therefore complex since the overall fluorescence intensity arises from both free and BSA-bound substrate. Furthermore, the assay depends upon the rapid partitioning of the substrate between the solution and BSA which may be a variable parameter that need always be considered and may limit the use of this substrate in pre-steady-state kinetic experiments. This is especially true where studies on this enzyme involve the use of chemical modification reagents which may also have an effect on the BSA and its ability to bind the substrate. The assay described in this paper is free from these drawbacks and uses a commercially available substrate.
References I Gee, N.S., Ragan C.I., Watling, K.J., Aspley, S., Jackson, R.G,, Reid, G.R., Gani, D. and Shute, J.K. (1988) Biochem. J. 249, 883-889. 2 Spector, R. (1988) Neurochem. Res. 13, 785-787. 3 Hallcher, L.M. and Sherman, W.R. (1980) J. Biol. Chem. 255, 10986-10901. 4 Nahorski, S.R., Ragan, C.I. and Challis, R.A.J. (1991)TIPS 12, 297-3{)3. 5 Roth, S.C., Harkness, D.R. and Isaacks, R.E. (1981) Arch. Biochem. Biophys. 210, 465-473. 6 Takimoto, K., Okada, M., Matsuda, Y. and Nakagawa, H. (1985)J. Biochem. 98, 363-37{}. 7 Attwood, P.V., Ducep, J.-V. and Chanal, M.C. (1988) Biochem. J. 253, 387-394. 8 Baker, R., Kulagowski, J.J., Biilington, D.C., Leeson, P.D., Lennon, I.C. and Liverton, N. (1989) J. Chem. Soc. Chem. Commun. 1383-1385. 9 Lanzetta, P.A., Alvarez, L.J., Reinach, P.S. and Candia, O.A. (1979) Anal. Biochem. I{l{},95-97. 10 Ragan, C.I., Watling, K.J., Gee, N.S., Aspley, S. Jackson, R.G., Reid, G.G., Baker, R., Billington, B.C., Barnaby, R.J. and Leeson, P.D. (1988) Biochem. J. 249, 143-148. 11 De Groot, H., De Groot, H. and Noll, T. (1985) Biochem. J. 23{I, 255-26{). 12 Hultberg, B. and Ocherman, P.A. (1972) Clin. Chim. Acta. 39, 49-58. 13 Rosenthal, A.L. and Sailer, A. (1973) Anal. Biochem. 55, 85-92. 14 Sandman, R., Margules, R.M. and Kowitz, S. (1973) Clin. Chim. Acta 45, 349-359. 15 Pitts, J.R. and Askari, A. (1971) Biochim. Biophys. Acta. 227, 453-459. 16 McAllister, G. Whiting, P.J., Hammond, E.A., Knowles, M.R., Atack, J.R., Bailey, F.J., Maigetler, R. and Ragan, C.I. (1992) Biochem. J. (In Press).
60 17 Billington, D.C., Baker, R., Kulagowski, J.J. and Mawer, I.M. (1987) J. Chem. Soc. Chem. Commun. 314-316. 18 Baker, R., Leeson, P.D., Liverton, N.J. and Kulagowski, J.J. (1990) J. Chem. Soc. Chem. Commun. 462 -464. 19 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 20 Ganzhorn, A.J. and Chanal, M.C. (1990) Biochemistry 29, 6065-6071. 21 Churchich, J.E. and Kwok, F. (1991) Anal. Biochem, 198, 375-378.
55
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-022X/92/$05.00
JBBM 00951
Bovine inositol monophosphatase: development of a continuous fluorescence assay of enzyme activity M.G. Gore ~, P.J. Greasley
a
and C.I. Ragan b
a Dept. of Biochemistry, School of Biological Sciences, Unicersity of Southampton, Southampton (UK) and b Merck Sharpand Dohme, Neurosciences Research Centre, Hariow, Essex (UK) (Received 2 March 1992) (Revised version received 3 April 1992) (Accepted 30 April 1992)
Summary This paper describes a continuous assay for the enzyme inositol monophosphatase which has been developed using a new substrate, the fluorescent compound 4-methylumhelliferyl phosphate. The hydrolysis of the phosphate group from this compound can be readily detected by a resultant large red shift in the emission spectrum from 390-450 nm. The kinetic constants for the enzyme using this new substrate are described. Key words: Continuous assay; Fluorescence; 4-Methylumbelliferyl phosphate
Introduction Inositol monophosphatase is a homodimer of subunit M r 30 000 which catalyses the dephosphorylation of inositol 1-phosphate, inositol 3-phosphate and inositol 4-phosphate [1] to inositol and phosphate (throughout we refer to myo-inositol, and use the numbering of 1D-myo-inositol). The supply of inositol for the production of phosphatidylinositol 4,5-bisphosphate is therefore highly dependent upon inositol monophosphatase activity, especially in brain tissue which lacks a suitable uptake system for inositol [2]. The enzyme has an absolute requirement for the divalent cation Mg 2+ and is inhibited in an uncompetitive manner by Li + ions [3], a feature which may underlie the antimanic and antidepressant actions of this ion (for review see Ref. 4). Inositol monophosphatase isolated from a number of Correspondence address: M.G. Gore, Dept. of Biochemistry, Bassett Crescent East, University of Southampton, Southampton, SO9 3TU, UK.
56 TABLE 1 Kinetic and inhibition constants at pH 8.0 and 37°C
K m (raM) Vmax+(U/mg) c K Li (raM) K ~ g2+ (raM) KiMg:+ (mM) kcat / K m ( M - I s - I) Kiei (raM) K~nhibit°r (p,M)
Ins(1)P
Mec-O-P
0.12 a 12.9 " 0.26 a 3.00 ~ 4.3 a 5.38" 10 4 0.5 a 1.1 h
0.92 0.39 16 7.19 no inhibition 2.12" 10 2 0.56 4.0
This table shows the FmCax(U -- 1 ~ m o l / m i n ) and K m for the enzyme obtained using Mec-O-P as a substrate. The K m for Mg 2+ and the K i for various ligands are also shown. The data shown using Ins(l)P as a substrate are taken from the following references: a [1]; b [16].
sources has been shown to demonstrate a broad substrate specificity. It dephosphorylates all monophosphate esters of inositol except inositol 2-phosphate, where the ester linkage is in the axial position relative to the ring. Studies have shown that the 2' and 6' hydroxyl groups of inositol 1-phosphate are independently associated with substrate binding and the mechanism of hydrolysis. The enzyme has also been shown to dephosphophorylate substrate analogues such as 2'AMP, 2'GMP, glycerol 3-phosphate as well as a number of other monophosphate esters [1,3,5-81. In all studies to date, the activity of inositol monophosphatase has been determined either by the discontinuous methods of measuring the release of Pi [9], [*4C]inositol from [14C]Ins(1)P [10] or by the coupled assay of Degroot et al. [11]. A continuous assay is preferable because it will invariably be more rapid, more accurate and the time-course of the reaction is observed directly. We have utilised the broad substrate specificity of inositol monophosphatase to identify a new substrate, 4-methylumbelliferyl phosphate, which will permit the direct observation of substrate disappearance or product appearance using fluorescence spectrophotometry. Umbelliferone derivatives have found broad use in the study of a number of enzyme systems [12-14]. 4-methylumbelliferyl phosphate (Mec-O-P) has previously been used in the study of N a ÷ / K + ATPase activity [15]. Because of the different fluorescent properties of 4-methylumbelliferone (Met) and Met-O-P, a very sensitive assay system is obtained. We have therefore investigated the possibility of using Mec-O-P as a potential substrate of inositol monophosphatase and examined its potential in the development of a continuous assay of enzyme activity. Materials and Methods
The growth of bacterial cultures and subsequent extraction and purification of the enzyme have been described elsewhere [16]. [14C]Ins(1)P was purchased from
57 Amersham, UK. All other reagents were purchased from either Sigma or BDH, Poole. The substrate O,L-Ins(1)P was synthesized as described by Billington et al. [17], and the inhibitor (1S)-phosphoryloxy-(2R,4S)-dihydroxycyelohexane as described by Baker et al. [18].
Discontinuous assays Inositol monophosphatase activity was determined by the discontinuous assay described by Lanzetta et al. [9] or by the detection of [~4C]inositol released upon enzyme activity as described by Ragan et al. [10]. Protein concentrations were determined by the method of Bradford [19]. Fluorescence studies Fluorescence measurements were made using a Perkin-Elmer 650S fluorescence detector. Activity of inositol monophosphatase with Mec-O-P as substrate was determined fluorimetrically by the determination of the rate of product (Mec) released (Aex = 388 nm; Aem = 450 nm). In all cases the assays were carried out using 10 ~g of enzyme buffered in 1 ml of 50 mM Tris-HCl (pH 7.8). Unless otherwise indicated in the text 0.5 mM Mec-O-P and 10 mM MgCl 2 were used in each experiment. A standard curve of Mec fluorescence (0-100 /~M) was constructed in the presence of 0.5 mM Mec-O-P and 10 mM MgCI 2 in 50 mM Tris-HCi buffer (pH 7.8). The addition of inhibitors and other ligands is as described in the text.
Results
The possible utilization of Mec-O-P as a substrate was examined initially by the discontinuous determination of phosphate released [9]. Mec-O-P was indeed utilized as a substrate, and Lineweaver-Burk analysis showed that the enzyme has a K m of approx. 1 mM for this substrate and a Vm~x of 0.38 U / m g , approx. 3% of that seen with Ins(1)P as substrate. To confirm the identity of the product of the reaction, Mec-O-P was incubated with inositol monophosphatase overnight at 37°C to allow the reaction to go to completion. A comparison of the fluorescence excitation and emission spectra of the product of this reaction with those of Mec showed both to be identical. The fluorescence emission intensity at 450 nm from the assay solution was shown to be directly proportional to the amount of Mec present, allowing the quantification of the amount of product released in the assay. The absorption of the assay solutions at all substrate concentrations was less than 0.01 and therefore corrections for inner filter effect are insignificant. Although homogeneous preparations of the enzyme were used in all experiments the possibility of contaminating phosphatase activity catalysing this reaction was eliminated by comparing the reaction rate in the absence and presence of either Li ÷ ions (an uncompetitive inhibitor of inositol monophosphatase) or (1S)-phosphoryloxy-(2 R,4S)-dihydroxycyclohexane, a competitive inhibitor of Ins
58 (1)P. In the first experiment the enzyme preparation was preincubated with 10 mM LiCI and 1 mM Ins(1)P prior to being utilized in an assay using Mec-O-P also in the presence of 10 mM LiCI. In this case, less than 5% activity was seen compared with enzyme which had not been preincubated with Ins(1)P and Li +, implying that the observed activity was dependent upon functional inositol monophosphatase. In the second experiment the presence of various concentrations of (1S).phosphoryloxy-(2R,4S)-dihydroxycyclohexane was used to inhibit the reaction and determine the nature of the inhibition and the K i. The Ki for this inhibitor with Mec-O-P as substrate was found to be 4.0/~M under the conditions of the assay (see Materials and Methods), which compares well with that measured when Ins(1)P is the substrate used. As the nature of the inhibition is not substrate-dependent, any deviation would be unexpected and it was therefore concluded that in the above experiments the dephosphorylation of the Mec-O-P was due solely to inositol monophosphatase action. The kinetic parameters of inositol monophosphatase with respect to Mec-O-P were then determined. The initial rate of product formation was linear with enzyme concentration and Lineweaver-Burk analysis gave a K m for Mec-O-P of 0.92 mM (--+_0.07 (n ffi 3)) and a Vm~, of 0.39 p m o l / m i n per mg. Thus the data obtained by the direct measurement of product released correlates well with the discontinuous determination of phosphate released. The high K m and low Vmax compared to Ins(1)P is consistent with previous observations [20] which showed that the maximal velocities with a number of different substrates depend upon the nature of the phosphate ester, and that the presence of adjacent hydroxyl groups allows hydrolysis to occur more readily. Previous workers have demonstrated that Mg 2+ is essential for catalytic activity with a K m for the enzyme from bovine brain of 3 mM with Ins(1)P as substrate. At higher concentrations it becomes an uncompetitive inhibitor. The K m and Ki for Mg 2+ has previously been shown to be dependent upon the nature of the substrate [20]. The K m for M g 2+ with Mec-O-P was shown to be 12 mM and no inhibition was observed up to 100 mM, which simplifies the kinetic analysis of reactions using saturating concentrations of this metal ion. The Ki for Li + ions, an uncompetitive inhibitor of inositoi monophosphatase has been shown to be dependent upon the nature of the substrate. Whereas the Ki for Li + ions is approx. 0.26 mM when inositol 1-phosphate is the substrate, the Ki of Li + ions was shown to be 16 mM when the substrate used is Mec-O-P. In addition, the second product of the reaction, inorganic phosphate, a competitive inhibitor of the enzyme with respect to substrate, was found to have a similar K i (0.56 or 0.50 raM) when either Mec-O-P or Ins(1)P, respectively, is used as substrate.
Discussion
The low substrate specificity of inositol monophosphatase has allowed a fluorescent compound, 4-methylumbeiliferyl phosphate, to be investigated as a potential substrate. The use of a fluorescent assay allows for the sensitive determination of
59
enzyme activity. The large difference in wavelength of maximal fluorescence emission between the spectra of the product and substrate allows the selective observation of one without the need for correction due to the contribution of the other. The kinetic parameters determined using this assay correlate well with data determined in experiments using discontinuous methods. The K m for M g 2+ and the K i for Li + are approx. 4- and 75-fold respectively higher using Mec-O-P as substrate. Both are increased as would be expected since the specificity constant (kcat/Km) for this substrate is reduced and the interactions of both metal ions have been shown to be dependent on the nature of the substrate [20]. In contrast, the substrate-independent inhibitors, inorganic phosphate and (lS)-phosphoryloxy(2R,4S)-dihydroxycyclohexane both demonstrate similar inhibition constants with either Mec-O-P or Ins(1)P. Recently an alternative continuous fluorescent assay has been published [21] based upon the use of DE-1-(o-aminobenzoyl)glycerol2-phosphate. This compound only binds to bovine serum albumin (BSA) when phosphorylated, where it fluoresces 2.75-fold more intensely than in water. The assay is therefore complex since the overall fluorescence intensity arises from both free and BSA-bound substrate. Furthermore, the assay depends upon the rapid partitioning of the substrate between the solution and BSA which may be a variable parameter that need always be considered and may limit the use of this substrate in pre-steady-state kinetic experiments. This is especially true where studies on this enzyme involve the use of chemical modification reagents which may also have an effect on the BSA and its ability to bind the substrate. The assay described in this paper is free from these drawbacks and uses a commercially available substrate.
References I Gee, N.S., Ragan C.I., Watling, K.J., Aspley, S., Jackson, R.G,, Reid, G.R., Gani, D. and Shute, J.K. (1988) Biochem. J. 249, 883-889. 2 Spector, R. (1988) Neurochem. Res. 13, 785-787. 3 Hallcher, L.M. and Sherman, W.R. (1980) J. Biol. Chem. 255, 10986-10901. 4 Nahorski, S.R., Ragan, C.I. and Challis, R.A.J. (1991)TIPS 12, 297-3{)3. 5 Roth, S.C., Harkness, D.R. and Isaacks, R.E. (1981) Arch. Biochem. Biophys. 210, 465-473. 6 Takimoto, K., Okada, M., Matsuda, Y. and Nakagawa, H. (1985)J. Biochem. 98, 363-37{}. 7 Attwood, P.V., Ducep, J.-V. and Chanal, M.C. (1988) Biochem. J. 253, 387-394. 8 Baker, R., Kulagowski, J.J., Biilington, D.C., Leeson, P.D., Lennon, I.C. and Liverton, N. (1989) J. Chem. Soc. Chem. Commun. 1383-1385. 9 Lanzetta, P.A., Alvarez, L.J., Reinach, P.S. and Candia, O.A. (1979) Anal. Biochem. I{l{},95-97. 10 Ragan, C.I., Watling, K.J., Gee, N.S., Aspley, S. Jackson, R.G., Reid, G.G., Baker, R., Billington, B.C., Barnaby, R.J. and Leeson, P.D. (1988) Biochem. J. 249, 143-148. 11 De Groot, H., De Groot, H. and Noll, T. (1985) Biochem. J. 23{I, 255-26{). 12 Hultberg, B. and Ocherman, P.A. (1972) Clin. Chim. Acta. 39, 49-58. 13 Rosenthal, A.L. and Sailer, A. (1973) Anal. Biochem. 55, 85-92. 14 Sandman, R., Margules, R.M. and Kowitz, S. (1973) Clin. Chim. Acta 45, 349-359. 15 Pitts, J.R. and Askari, A. (1971) Biochim. Biophys. Acta. 227, 453-459. 16 McAllister, G. Whiting, P.J., Hammond, E.A., Knowles, M.R., Atack, J.R., Bailey, F.J., Maigetler, R. and Ragan, C.I. (1992) Biochem. J. (In Press).
60 17 Billington, D.C., Baker, R., Kulagowski, J.J. and Mawer, I.M. (1987) J. Chem. Soc. Chem. Commun. 314-316. 18 Baker, R., Leeson, P.D., Liverton, N.J. and Kulagowski, J.J. (1990) J. Chem. Soc. Chem. Commun. 462 -464. 19 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 20 Ganzhorn, A.J. and Chanal, M.C. (1990) Biochemistry 29, 6065-6071. 21 Churchich, J.E. and Kwok, F. (1991) Anal. Biochem, 198, 375-378.
