Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate.

BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications.     

In-gel protein phosphatase assay using fluorogenic substrates

We developed a method for the detection of phosphatase activity using fluorogenic substrates after polyacrylamide gel electrophoresis. When phosphatases such as Ca²⁺/calmodulin-dependent protein kinase phosphatase (CaMKP), protein phosphatase 2C (PP2C), protein phosphatase 5 (PP5), and alkaline phosphatase were resolved by polyacrylamide gel electrophoresis in the absence of SDS and the gel was incubated with a fluorogenic substrate such as 4-methylumbelliferyl phosphate (MUP), all of these phosphatase activities could be detected in situ. Although 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as well as MUP could be used as a fluorogenic substrate for an in-gel assay, MUP exhibited lower background fluorescence. Using this procedure, several fluorescent bands that correspond to endogenous phosphatases were observed after electrophoresis of various crude samples. The in-gel phosphatase assay could also be used to detect protein phosphatases resolved by SDS-polyacrylamide gel electrophoresis. In this case, however, the denaturation/renaturation process of resolved proteins was necessary for the detection of phosphatase activity. This procedure could be used for detection of renaturable protein phosphatases such as CaMKP and some other phosphatases expressed in cell extracts. The present fluorescent in-gel phosphatase assay is very useful, since no radioactive compounds or no special apparatus are required.     

Development of novel 2-[4-(aminoalkoxy)phenyl]-4(3H)-quinazolinone derivatives as potent and selective histamine H3 receptor inverse agonists

Novel 2-[4-(aminoalkoxy)phenyl]-4(3H)-quinazolinone derivatives were identified as potent human H(3) receptor inverse agonists. After systematic modification of lead 5a, the potent and selective analog 5r was identified. Elimination of hERG K(+) channel and human alpha(1A)-adrenoceptor activities is the main focus of the present study.     

Substrate specifity in Amoeba proteus

Three different phosphatases ("slow", "middle" and "fast") were found in Amoeba proteus (strain B) after PAGE and a subsequent gel staining in 1-naphthyl phosphate containing incubation mixture (pH 9.0). Substrate specificity of these phosphatases was determined in supernatants of homogenates using inhibitors of phosphatase activity. All phosphatases showed a broad substrate specificity. Of 10 tested compounds, p-nitrophenyl phosphate was a preferable substrate for all 3 phosphatases. All phosphatases were able to hydrolyse bis-p-nitrophenyl phosphate and, hence, displayed phosphodiesterase activity. All phosphatases hydrolysed O-phospho-L-tyrosine to a greater or lesser degree. Only little differences in substrate specificity of phosphatases were noticed: 1) "fast" and "middle" phosphatases hydrolysed naphthyl phosphates and O-phospho-L-tyrosine less efficiently than did "slow" phosphatase; 2) "fast" and "middle" phosphatases hydrolysed 2- naphthyl phosphate to a lesser degree than 1-naphthyl phosphate 3) "fast" and "middle" phosphatases hydrolysed O-phospho-L-serine and O-phospho-L-threonine with lower intensity as compared with "slow" phosphatase; 4) as distinct from "middle" and "slow" phosphatases, the "fast" phosphatase hydrolysed glucose-6-phosphate very poorly. The revealed broad substrate specificity of "slow" phosphatase together with data of inhibitory analysis and results of experiments with reactivation of this phosphatase by Zn2+-ions after its inactivation by EDTA strongly suggest that only the "slow" phosphatase is a true alkaline phosphatase (EC 3.1.3.1). The alkaline phosphatase of A. proteus is secreted into culture medium where its activity is low. The enzyme displays both phosphomono- and phosphodiesterase activities, in addition to supposed protein phosphatase activity. It still remains unknown, to which particular phosphatase class the amoeban "middle" and "fast" phosphatases (pH 9.0) may be assigned.     

Mechanism of the prenyl-transfer reaction. Studies with (E)- and (Z)-3-trifluoromethyl-2-buten-1-yl pyrophosphate

The prenyl-transfer reaction catalyzed by porcine farnesyl pyrophosphate synthetase has been studied using (E)- and (Z)-3-trifluoromethyl-2-buten-1-yl pyrophosphates as substrates and inhibitors. The rate of condensation between isopentenyl pyrophosphate (IPP) and the allylic fluoro analogues is drastically depressed relative to the normal catalytic rate observed with dimethylallyl pyrophosphate (DMAPP) or geranyl pyrophosphate (GPP). A similar depression is found in the rates of solvolysis for methanesulfonate derivatives of the fluoro analogues in aqueous actone under typical SN1 reaction conditions. Prolonged incubation of [14C] IPP and (E)- or (Z)-CF3-DMAPP with the enzyme, followed by treatment with alkaline phosphatase, gave a product that comigrated with geranylgeraniol on a polystyrene column. Both fluoro analogues showed mixed linear inhibition patterns with DMAPP or GPP as the variable substrate. We interpret these results in terms of an ionization-condensation-elimination mechanism for the prenyl-transfer reaction.     

Visualization of phosphatase activity in living cells with a FRET-based calcineurin activity sensor

Protein kinases and phosphatases are organized into complex intracellular signaling networks designed to coordinate their activities in both space and time. In order to better understand the molecular mechanisms underlying the regulation of signal transduction networks, it is important to define the spatiotemporal dynamics of both protein kinases and phosphatases within their endogenous environment. Herein, we report the development of a genetically-encoded protein biosensor designed to specifically probe the activity of the Ca2+/calmodulin-dependent protein phosphatase, calcineurin. Our reporter design utilizes a phosphatase activity-dependent molecular switch based on the N-terminal regulatory domain of the nuclear factor of activated T-cells as a specific substrate of calcineurin, sandwiched between cyan fluorescent protein and yellow fluorescent protein. Using this reporter, calcineurin activity can be monitored as dephosphorylation-induced increases in fluorescence resonance energy transfer and can be simultaneously imaged with intracellular calcium dynamics. The successful design of a prototype phosphatase activity sensor lays a foundation for studying targeting and compartmentation of phosphatases.     

A novel endogenous PP2C-like phosphatase dephosphorylates casein kinase II-phosphorylated Physarum fragmin

Plasmodial fragmin, a Physarum polycephalum F-actin severing and capping protein, is phosphorylated by casein kinase II at Ser(266) (De Corte, V., Gettemans, J., De Ville, Y., Waelkens, E., and Vandekerckchove, J. (1996), Biochemistry 35, 5472-5480). In this study, we report the purification and characterization of the corresponding fragmin phosphatases. One of the enzymes was purified to near homogeneity from a cytosolic extract; it dephosphorylates CKII-phosphorylated fragmin, a peptide encompassing the CKII phosphorylation site of fragmin as well as histone 2A, CKII-phosphorylated casein and the CKII model-peptide substrate: R(3)E(3)S(P)E(3). Its activity was highly stimulated by Mn(2+) and Mg(2+), and based on its lack of sensitivity toward phosphatase effectors we could exclude similarities with PP1, PP2A and PP2B phosphatases. All biochemical properties of the phosphatase point to a PP2C-like enzyme. A second phosphatase dephosphorylating fragmin was identified as a Physarum alkaline phosphatase.     

Extracellular phosphatase activity of natural plankton studied with ELF97 phosphate: fluorescence quantification and labelling kinetics

ELF(R)97 phosphate (ELFP) is a phosphatase substrate which produces ELF(R)97 alcohol (ELFA), a fluorescent water-insoluble product, upon hydrolysis. We studied the kinetics of ELFA precipitation in freshwater samples at levels of total plankton and single phytoplankton cells, and tested the suitability of ELFP for measurement of surface-bound algal extracellular phosphatases. Samples from acidic Plesné Lake (pH approximately 5; high phosphatase activity) and eutrophic Rímov reservoir (pH approximately 7-10; moderate phosphatase activity) were incubated with ELFP for 5-300 min, fixed with HgCl2 and filtered through polycarbonate filters. Relative fluorescence of filter-retained ELFA precipitates was quantified with image analysis. Time-courses of ELFA formation exhibited lag periods followed by finite periods of linear increase. In Plesné Lake, lag-times were shorter (1-18 min) and rates of increase in ELFA fluorescence higher (by approximately 2 orders of magnitude) than in Rímov reservoir (lag-times 30-200 min). Similar patterns of ELFA formation kinetics were also observed in Plesné Lake samples in cuvette spectrofluorometer measurements (which failed in Rímov reservoir). Linear regression of seasonal data on rates of increase in ELFA fluorescence from image cytometry and spectrofluorometry (r2 = 0.65, n = 10) allowed for calibration of image cytometry in terms of amount of cell-associated ELFA. Preliminary measurements of extracellular phosphatase activities of several algae resulted in rates (10-2260 fmol cell-1 h-1) which are comparable to data reported in the literature for algal cultures.     

Specific activity of cell-surface acid phosphatase in different bacterioplankton morphotypes in an acidified mountain lake

Activity of extracellular acid phosphatases was measured at single-cell level in bacterioplankton groups defined by their morphology and size, in acidified mountain Lake Certovo, during the 2003 season, with a method based on use of the substrate ELF97 phosphate which provides fluorescent precipitates upon hydrolysis by phosphatases. The bacterial cell-associated precipitates were quantified by image analysis. A specific, conspicuous, apparently homogeneous morphotype of curved cells of approximately 5 microm average length, despite its low total biomass (average of 4%), contributed significantly (in average by 31%) to the total bacterioplankton phosphatase activity in Lake Certovo (ranging from 1.0 to 12.7 micromol l(-1) h(-1), using ELF97 phosphate as a substrate). Bacterial filaments (> 10 microm), although comprising in average 85% of bacterioplankton biomass, contributed to the total bacterioplankton activity only by 45%. Biomass-specific activity of extracellular (cell-surface) phosphatases of the main bacterioplankton morphotypes increased in the order filaments < cocci and rods < curved cells. The biomass-specific activity of bacterioplankton extracellular phosphatases (0-300 nmol microgC(-1) h(-1)) was generally highest in the spring and decreased gradually during summer. These changes could result from seasonal changes in the phosphorus status of the lake and from subsequent regulation of enzyme expression by bacteria.     

Assay of phosphotyrosyl protein phosphatase using synthetic peptide 1142-1153 of the insulin receptor

Synthetic peptide 1142-1153 of the insulin receptor was phosphorylated on tyrosine by the insulin receptor and found to be a potent substrate for dephosphorylation by rat liver particulate and soluble phosphotyrosyl protein phosphatases. Apparent Km values were approximately 5 microM. Vm values (nmol phosphate removed/min per mg protein) were 0.62 (particulate) and 0.2 (soluble). This corresponds to 80% of total activity being membrane-associated, indicating that membrane-bound phosphatases are important receptor phosphatases. The phosphatase activities were distinct from acid and alkaline phosphatase. In conclusion peptide 1142-1153 provides a useful tool for the further study and characterization of phosphotyrosyl protein phosphatases.     

Ultrastructural localization of alkaline phosphatases in rat incisor odontoblasts.

The localization of alkaline phosphatases in dentinogenically active rat incisor odontoblasts was studied by means of subcellular fractionation and electron microscopical histochemistry. Subcellular fractionation revealed the predominant phosphatase activity to be present in the microsome fraction and to a lesser extent in the mitochondrial fraction. Adenosine triphosphate degrading enzyme activity was determined in the presence or absence of (+/-)-6(m-bromophenyl)-5, 6-dihydroimidazo(le) (2,1-b) thiazole oxalate (R 8231). Before the histochemical study, the effects on phosphatase activities by aldehyde fixation were studied by biochemical assay. A method of fixation for optimal preservation of phosphatase activity is presented. Phosphatase electron microscopic histochemistry was performed by using ATP as a substrate and with or without addition of the inhibitor R 82319 Precipitates were seen in the membranes of vesicles present in the odontoblast process and the Golgi region. When there were signs of insufficient fixation, precipitates were also seen in the outer membranes of mitochondria. No phosphatase activity was seen in the cell membrane. ATP degrading enzyme activities mediated by nonspecific alkaline phosphatase (APase) and Ca2+ -adenosine triphosphatase thus have the same morphological localization. This close association is consistent with earlier biochemical studies.     

Phosphatase of Chlamydomonas reinhardi: biochemical and cytochemical approach with specific mutants.

The unicellular alga Chlamydomonas reinhardi produces two constitutive acid phosphatases and three depressible phosphatases (a neutral and two alkaline ones) that can utilize napthyl phosphate as a substrate. Specific mutants depressible phosphatase were used to investigate biochemical properties and the cytochemical localization of these enzymes. The two constitutive phosphatases show similar pH optima (about 5.0) and Km values (2 x 10(-3) to 3.3 x 10(-3) M) but differ in their heat sensitivity and affinity for glycerophosphate.     

Determination of taurine in plasma by high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a fluorescent labeling reagent

A sensitive high-performance liquid chromatography method for the determination of taurine in human plasma was developed. Taurine and N-methyltaurine (internal standard) were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride to produce fluorescent sulfonamides. The labeling reaction was carried out at 70 degrees C for 20 min at pH 7.5. The fluorescent derivatives were separated on a reversed-phase column by a stepwise elution using (A) acidic phosphate buffer/acetonitrile (83/17) and (B) acetonitrile and detected by fluorescence measurement at excitation and emission wavelengths of 318 and 392 nm, respectively. The detection limit (signal-to-noise ratio=3) of taurine was 3 fmol per injection. The within-day and day-to-day relative standard deviations were 3.0-4.8 and 2.5-4.7%, respectively. The concentration (means) of taurine in normal human plasma was 48.9+/-7.5 microM.     

Dephosphorylation of cardiac proteins in vitro - a matter of phosphatase specificity

Protein phosphorylation is reversibly regulated by the interplay between kinases and phosphatases. Recent developments within the field of proteomics have revealed the extent of this modification in nature. To date there is still a lack of information about phosphatase specificity for different proteomes and their conditions to achieve maximum enzyme activity. This information is important per se, and in addition often requested in functional and biochemical in vitro studies, where a dephosphorylated sample is needed as a negative control to define baseline conditions. In this study, we have addressed the effectiveness of two phosphatases endogenously present in the heart (protein phosphatases 1 and 2A) and two generic phosphatases (alkaline phosphatase and lambda protein phosphatase) on three cardiac subproteomes known to be regulated by phosphorylation. We optimized the dephoshorylating conditions on a cardiac tissue fraction comprising cytosolic and myofilament proteins using 2DE and MS. The two most efficient conditions were further investigated on a mitochondrial-enriched fraction. Dephosphorylation of specific proteins depends on the phosphatase, its concentration, as well as sample preparation including buffer composition. Finally, we analyzed the efficiency of alkaline phosphatase, the phosphatase with the broadest substrate specificity, using TiO(2) peptide enrichment and 2DLC-MS/MS. Under these conditions, 95% of the detected cardiac cytoplasmic-enriched phospho-proteome was dephosphorylated. In summary, targeting dephosphorylation of the cardiac muscle subproteomes or a specific protein will drive the selection of the specific phosphatase, and each requires different conditions for optimal performance.     

Phosphatase activity in Trypanosoma cruzi. Phosphate removal from ATP, phosphorylated proteins and other phosphate compounds

T. cruzi epimastigotes have a lysosomal acid phosphatase (pH 4.0) and acid and alkaline phosphatases (pH 5.5 and 8.0) localized in the cytosolic fraction. The levels of the lysosomal acid phosphatase increase with the age of the cultures, but the cytosolic phosphatases decline after the logarithmic phase of growth. The lysosomal phosphatase preferentially hydrolyses low mol. wt phosphate esters; whereas, the cytosolic alkaline phosphatases primarily act on phosphorylated proteins, and both the cytosolic acid and alkaline phosphatases on uridine nucleotide derivatives. The parasite also contains a microsomal glucose 6-phosphatase, and ATPases (Mg2+ and Ca2+-activated) derived from plasma membranes and mitochondria.     

DNA hybridization assay using ATTOPHOS, a fluorescent substrate for alkaline phosphatase.

A fluorometric procedure for the detection of DNA-DNA hybrids is described. The procedure involved the detection of probe-bound alkaline phosphatase with the fluorescent substrate ATTOPHOS. This substrate is converted to ATTOFLUOR by alkaline phosphatase and fluoresces strongly at 550 nm when excited with a wavelength of 440 nm. DNA hybridization assays were performed both with dilutions of purified target plasmid DNA (pSE9 or PBR322) and whole bacterial cells. Streptavidin-alkaline phosphatase conjugates were added to react with bound probe. Fluorometric assays, as well as colorimetric assays, using 5-bromo-4-chloro-3-indolylphosphate + nitroblue tetrazolium for alkaline phosphatase activity were performed. The fluorescence of the substrate was measured at time intervals, and the slope of the regression line calculated. A slope four times greater than that of background was considered positive. One hundred femtograms or 2.2 x 10(4) molecules of homologous DNA were detected with the fluorescent assay as compared with 10,000 femtograms or 2.2 x 10(6) molecules of homologous DNA with the colorimetric assay. Similar results were obtained with whole cells. Approximately 1 x 10(3) homologous cells were detected fluorometrically and 1 x 10(5) cells were detected colorimetrically. Based on these results, we conclude that, in our hands, the DNA hybridization assay described here using ATTOPHOS as the substrate for alkaline phosphatase is a very sensitive assay for the detection of DNA-DNA hybrids.     

Naphthol AS-BI (7-bromo-3-hydroxy-2-naphtho-o-anisidine) phosphatase and naphthol AS-BI beta-D-glucuronidase in Chinese hamster ovary cells: biochemical and flow cytometric studies.

Conditions for the biochemical and flow cytometric assay of 7-bromo-3-hydroxy-2-naphtho-o-anisidine phosphatase and beta-D-glucuronidase activities in Chinese hamster ovary cells were studied. In the biochemical assay, the pH optimum for the phosphatase activity was pH 4.6 with a Km of 10(-5) M; the pH optimum for beta-D-glucuronidase activity was pH 5.0 with a Km of 2 x 10(-5) M. For intact cells the derived constants were 3 to 10 times higher. The rate of hydrolysis of both substrates was also examined by flow cytometry. Cellular fluorescence increased linearly for only about 15 min. Diffusion of the fluorescent product probably caused nonlinearity of the fluorescence increase and was demonstrated by mixing cells incubated with substrate with those that had not been incubated. After 15 min, cells that had not been exposed previously to product or substrate contained the fluorescent product. Cells fractionated into size classes by centrifugal elutriation also were analyzed by flow cytometry for beta-D-glucuronidase activity. The activity increased linearly with the increase in cell size corresponding to the progression from G1 through S and into G2-M phases of the cell cycle.     

Appearance of different phosphatase forms and phosphorus partitioning in nodules of chickpea (Cicer arietinum L.) during development

Acid and alkaline phosphatase and phytase activities were determined in the bacteroid free fractions of chickpea (Cicer arietinum L.) nodules at 15 days intervals, from 40 days after sowing (DAS) to 85 DAS. In general, the activities and specific activity of both the acid and alkaline phosphatases declined at 55 DAS. Out of the various substrates studied, ATP was the best substrate for both phosphatases. Activities of phosphatases with glucose-6-phosphate and fructose-6-phosphate were low in comparison to these with fructose 1,6 bisphosphate. The efficiency of acid phosphatase for utilizing fructose 1,6 bis phosphate as a substrate increased with nodule development. A fructose 1,6 bis phosphate specific acid phosphatase with elution volume to void volume (Ve/Vo) ratio of around 2.0 was observed in mature nodules (80 DAS). Acid phosphatase at 40 DAS was resolved into two peaks which were eluted at Ve/Vo of about 1.5 and 1.8. However, at 60 DAS the peak with Ve/Vo of 1.5 could not be detected. With ATP as substrate, a high (Ve/Vo of 1.2) and low MM form (Ve/Vo of 2.1) alkaline phosphatases were observed at 40 DAS however at 60 DAS stage only one peak with Ve/Vo of 1.7 was detected. Although, a low activity of acid phytase was observed in nodules at all stages of development but neither alkaline phytase nor phytic acid could be detected. It appears that the nodules acquire inorganic phosphate from the roots. The higher content of water soluble organic phosphorus in mature nodules could be due to the low activities of phosphatases at maturity.     

Partial purification and characterization of phosphotyrosyl-protein phosphatase from Ehrlich ascites tumor cells

We have previously described a phosphotyrosylprotein phosphatase in membrane vesicles from human epidermoid carcinoma A431 cells which is inhibited by micromolar concentration of Zn2+ and is insensitive to ethylenediaminetetraacetic acid (EDTA) and NaF [Brautigan, D. L., Bornstein, P., & Gallis, B. (1981) J. Biol. Chem. 256, 6519-6522]. Here we present the identification and partial purification of a similar enzyme from lysates of Ehrlich ascites tumor cells. the enzyme was purified by using diethylaminoethyl-Sephadex, Zn2+ affinity, and Sephadex G-75 chromatography. During purification, the phosphatase was separated into at least three fractions, all of which exhibited very similar properties and an apparent molecular weight of 40 000 upon gel filtration. The enzyme dephosphorylated phosphotyrosine (P-Tyr)-containing carboxymethylated and succinylated (CM-SC) phosphorylase with an apparent Km of 0.8 microM, as well as P-Tyr containing casein and epidermal growth factor (EGF) receptor kinase, but did not dephosphorylate P-Ser-phosphorylase. The phosphatase was inhibited by Zn2+ at micromolar concentrations (K0.5 with EGF receptor kinase = 5 X 10(-6) M; with CM-SC phosphorylase = 3.3 X 10(-5) M) but not by millimolar concentrations of EDTA and NaF. No inhibition was seen with 1 mM tetramisole, a specific inhibitor of alkaline phosphatases. P-Tyr inhibited the enzyme by 50% at 0.4 X 10(-3) M, while Tyr, Pi, PPi, and p-nitrophenyl phosphate, an excellent substrate for alkaline phosphatases and structurally very similar to P-Tyr, exerted partial inhibition at concentrations above 10(-3) M. The pH optimum was found to be 6.5-7, depending on the substrate used. Very little activity was seen below pH 5 and above pH 8.5. These properties clearly distinguish this enzyme from alkaline phosphatases, as well as the neutral and acidic protein phosphatases so far described, and therefore define it as a new enzyme of the phosphatase family--a phosphotyrosyl-protein phosphatase.     

Differential theophylline inhibition of alkaline phosphatase and 5'-nucleotidase of bovine milk fat globule membranes

1. The effects of theophylline (1,3-dimethylxanthine) on alkaline phosphatase and 5'-nucleotidase activities of bovine milk fat globule membranes (MFGM) were examined. 2. Theophylline inhibited MFGM alkaline phosphatase in a concentration-dependent manner with 50% inhibition produced by 99 +/- 28 microM theophylline. 3. The 5'-nucleotidase activity was resistant to theophylline inhibition with 50% inhibition produced by 33.9 +/- 3.1 mM theophylline. 4. Theophylline was an uncompetitive inhibitor of MFGM alkaline phosphatase with a Ki of 126 +/- 15 microM. 5. The extent of theophylline inhibition of alkaline phosphatase activity was independent of the substrate utilized in the assay. 6. The effect of theophylline on bovine MFGM alkaline phosphatase was similar to theophylline effects on other mammalian alkaline phosphatases of liver/bone isoenzyme origin.