Non-radioactive method to measure CD45 protein tyrosine phosphatase activity isolated directly from cells

JOURW OF ~tM%U&UtCAL ELSEVIER

Journal of Immunological Methods 179 (1994) 177-18.5

Non-radioactive method to measure CD45 protein tyrosine phosphatase activity isolated directly from cells David H.W. Ng a, Kenneth W. Harder b, Ian Clark-Lewis b, Frank Jirik b, Pauline Johnson a,* a Department of Microbiology and Immunology, #300-6174 lJniver& Boulevard, Vancouuer,B.C. MT 123, Canada ’ Biomedical Research Center, University of British Columbia, Vancouver,B.C.

V6T 123, Canada

Received 13 June 1994; revised 12 October 1994; accepted 19 October 1994

Abstract Preparation of radioactive phosphorylated substrates is laborious, yields a limited amount of substrate with a short half-life and generates a low percentage of phosphorylated product which then has to be separated from non-phosphorylated material. These factors limit the usefulness of radioactive phosphorylated substrates in phosphatase assays and prohibit their use for kinetic analysis, which often requires large amounts of substrate. An alternative method for the kinetic analysis of purified or recombinant soluble phosphatases uses the malachite green reagent which can detect nanomoles of phosphate released from chemically synthesized phosphorylated peptides. In this report we describe a rapid and sensitive non-radioactive method that can be used to measure protein tyrosine phosphatase (PTP) activities of both transmembrane and soluble phosphatases immunoprecipitated directly from cells. This calorimetric microassay is performed in 96 well microtitre plates and can reliably detect 100 pmol of free phosphate released, using a standard microplate reader. The phosphatase activity of CD45, a transmembrane PTP, was determined from as few as 1 X lo4 lymphoid cells. The development of this calorimetric assay to measure immunoprecipitated CD45 PTP activity isolated from very small numbers of cells has general applicability for other PTPs and will help identify the cellular situations and conditions that result in changes in PTP activity. Keywords:

Protein

tyrosine

phosphatase

assay; CD45; Malachite

green phosphate

assay

1. Introduction

Abbreviations: PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; RCM-lysozyme, reduced, carboxamidomethylated, and maleylated lysozyme; pNPP, p-nitrophenyl phosphate; EDTA, ethylenediamine tetra-acetic acid; PMSF, phenylmethylsulphonyl fluoride; BCA, bicinchoninic acid; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; BSA, bovine serum albumin. * Corresponding author. Tel.: 604-822-5995; Fax: 604-8226041.

Protein tyrosine phosphorylation is an integral component of signal transduction pathways within eukaryotic cells and is regulated by the fine interplay of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Since the purification of the first PTP in 1988 (Tonks et al., 1988a,b), several PTPs have been characterized, but little is known about the specificity or regula-

0022-1759/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDIOO22-1759(94)00281-9

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of Immunological Methods I79 (1994) 177-185

tion of these enzymes (see Walton and Dixon, 1993 for review). CD45 a transmembrane PTP, has been shown to be crucial for T cell receptor mediated T cell activation (Pingel and Thomas, 1989; Koretzky et al., 1990,1991), and may act to regulate PTK activity (Mustelin et al., 1989,1992; Mcfarland et al., 1993). Several proteins have been shown to be in vitro substrates for CD45, and ~56’“~ and ~59%” have also been shown to be potential in vivo substrates (Ostergaard et al., 1989; Hurley et al., 1993; Mcfarland et al., 1993). At present it is not known how, or if, the phosphatase activity of CD45 is regulated. A correlation between loss of serine phosphorylation of CD45 and PTP activity has been found in T cells treated with ionomycin, implying that serine phosphorylation may regulate the PTP activity of CD45 (Ostergaard and Trowbridge, 1991). Both serine and tyrosine phosphorylation of cytoplasmic PTPs have been shown to affect activity (Brautigan and Pinault, 1993; Vogel et al., 19931, suggesting that phosphorylation may represent a common means of regulating PTP activity. Under certain conditions, tyrosine phosphorylation of CD45 has been observed in T cells (Stover et al., 1991), but its affect on CD45 PTP activity is unknown. CD45 has also been reported to associate with several other proteins in lymphoid cells (reviewed in Koretzky, 1993) and it is possible that some of these interactions may influence PTP activity. In order to establish how the enzymatic activity of CD45 is regulated in cells, it is neccessary to first determine CD45’s activity under a variety of cellular conditions. Ideally, it would be best to be able to monitor the PTP activity of CD45 in situ in the cell, but as this is not yet possible, CD45 was isolated directly from cells and its PTP activity was tested in vitro. The previous method for measuring PTP activity of enzymes isolated directly from cells used radioactively labeled substrates. This required intensive work in first generating the radioactive substrate and secondly, after the completion of the phosphatase reaction, in purifying the radioactive free phosphate from the radioactive substrate (Ostergaard et al., 1989; Streuli et al., 19901. Now new technologies in peptide synthesis have made it possible to synthesize specific phos-

photyrosine containing peptides (Hudson, 1988; Cho et al., 1991; Clark-Lewis et al., 1991) . These peptides are purified after synthesis and are 100% phosphorylated at a specific single site. This together with the improved ability to detect small amounts of inorganic phosphate (Itaya and Ui, 1966; Lanzetta et al., 1979) has led to the use of the malachite green assay to measure PTP activity of purified soluble recombinant PTPs (Cho et al., 1991). Alternatively, the differential absorption abilities of phosphotyrosine and tyrosine residues has allowed the measurement of PTP activity by monitoring changes in absorption at 282nm during a PTP reaction (Zhao et al., 1992; Zhang et al., 1993). However, these methods have measured the activity of purified soluble phosphatases using relatively large volumes (0.4-l ml) and large amounts of substrate. We have recently scaled down the malachite green phosphate assay such that it is now more sensitive (50 pmol of inorganic phosphate can be detected using a microplate reader) and can be performed in small volumes (15-100 ~1) (Harder et al., 1994). We now report the adaptation of this calorimetric microassay for measuring the enzymatic activity of PTPs isolated directly from cells and demonstrate the feasibility of this method using the transmembrane PTP, CD45. This assay obviates the need to prepare radioactively labeled substrates and offers a considerable saving of both time and effort. It allows the determination of PTP activity to be made from different cell types and from cells undergoing differentiation or activation and allows kinetic analysis to be performed. It is a widely applicable method that can be used for any PTP that can be immunoprecipitated from cells.

2. Materials and methods 2.1. Cell lines and antibodies The T celI lines, BW5147, CD45-, CD3+, TCRf (kindly provided by B. Malissen), BW5147, CD45+, CD3+, TCR+ (Wegener et al., 1992) and EL4 (American Type Culture Collection), and two B lymphoid cell lines, RAW 253.1, and A20

D. Ng et al. /Journal of Immunological Methods I79 (1994) 177-185

(American Type Culture Collection) were used in these experiments. The I3/2 antibody reactive against all isoforms of mouse CD45 (Trowbridge, 1978) was used to immunoprecipitate CD45 and an antiserum raised against a common CD45 epitope (kindly provided by J. Marth) was used for immunoblotting. 2.2. Phosphotyrosine peptide synthesis and purification

One llmer and three 13mer phosphopeptides encompassing either the negative regulatory site or the auto-phosphorylation site of three src kinase family members were synthesized and purified essentially as described in Harder et al. (1994). Briefly, phosphotyrosine was incorporated as tBoc.Tyr PO, (phenyl),. The other amino acids were incorporated using tBoc chemistry and standard protection groups (Harder et al., 1994). They were deprotected using trimethanesulphonic acid and purified by reverse phase HPLC. Electrospray mass spectrometry results were consistent with the target sequence. The peptides used in these studies were: fyn pY531 (TATEPQpYQPGENL), src pY416 (TSTEPQpYQPGENL), and lck pY505 (TATEGQpYQPQP) which encompass the negative regulatory sites of murine p59@“, ~60”’ and and src pY416 (LIED~56’~~ respectively, NEpYTARQGA) which encompasses the autophosphorylation site of murine p60”‘“. 2.3. Phosphotyrosine peptide quantitation Quantitation of phosphopeptide amounts was achieved by determining phosphate concentrations rather than by dry weight measurements. Lyophilized peptide was dissolved in HPLC grade water and phosphopeptide concentration was quantitated (Hasegawa et al., 1982). Briefly, aliquoted amounts of peptide solution were dried by heating at 100°C. The remaining solid peptide was resuspended in 30 ~1 of a mixture of 1 part 50% sulfuric acid and 1 part 60% perchloric acid and placed in reacti-vials (Pierce) to be superheated at 160°C for 4-5 h. The acid-peptide solutions were cooled, neutralized and then

179

aliquoted into a well containing 80 ~1 of filter sterilized malachite green reagent. Addition of malachite green reagent resulted in a colorimetric response that was directly related to the amount of free inorganic phosphate present, which was determined using a standard curve. 2.4. Immunoprecipitation of CD45 1.0 X lo6 cells were lysed in 0.5% Triton X-100, 20 mM Tris pH 7.5, 150 mM NaCI, 2.0 mM EDTA, 0.2 mM PMSF, 1.0 Fg/ml pepstatin, 1.0 pg/ml leupeptin, 1.0 pg/ml aprotinin and incubated on ice for 10 min. Immunoprecipitates from CD45- T cells were used as a negative control. Lysates were then centrifuged at 16000 x g for 10 min. The supernatant was then precleared with 10 ~1 of a 50% slurry of Sepharose CL-4B (Pharmacia) at 4°C for 1 h. 10 ~1 of 13/2 coupled Sepharose CL-4B (4.0 mg antibody conjugated/ ml packed beads) was added to lysate and incubated end over end for 2 h at 4°C. The immunoprecipitate was washed three times in 0.2% Triton X-100, 20 mM Tris pH 7.5, 150 mM NaCl, 2.0 mM EDTA, 0.2 mM PMSF, 1.0 pg/ml pepstatin, 1.0 pg/ml leupeptin, 1.0 pg/ml aprotinin, and washed twice in non-reducing PTP buffer (50 mM imidazole-Cl pH 7.2, 1.0 mM EDTA). Beads were then finally resuspended in 200 ~1 of PTP buffer (50 mM imidazole-Cl pH 7.2, 1.0 mM EDTA, 0.1% P-mercaptoethanol) such that 5 ~1 of bead suspension contained CD45 immunoprecipitated from 2.5 x lo4 cell equivalents.

2.5. Phosphatase assay of immobilized CD45 All solutions were pre-equilibrated to 30°C. 5.0 ~1 of CD45 bead suspension was added directly to 100 ~1 microtitre wells (Half-Area, Tissue Culture Treated, flat bottomed 96 well microtitre plates, Costar) for each timepoint tested. The reaction was initiated by the addition of 10.0 ~1 PTP buffer containing peptide at the defined concentration to be tested. During the reaction, the microtitre well was agitated at 120 rpm. Timepoints were taken at 0 min, 1 min, and 3 min. The reaction was stopped by the addition of

180

D. Ng et al. /Journal

of Immunological

85 ~1 of filtered malachite green reagent (1 part 0.135% malachite green-oxalate salt in distilled water, 1 part 4.2% ammonium molybdate in 4.0 M HCl, 2 parts distilled water, and Tween 20 to a final concentration of 0.01%). Release of inorganic phosphate was measured by determining the absorbance at 650 nm using a multiwell microtitre plate reader and compared to a standard curve for inorganic phosphate detection obtained using potassium dihydrogen phosphate (KH,PO,).

Methods I79 (1994) 177-185

a 2.5

2.6. Immunoblotting of CD45 0

Immunoprecipitates were electrophoresed on 7.5% SDS gels and proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore) according to the manufacturers instructions (Bio-Rad). The membranes were blocked in 5% bovine serum albumin (BSA) (Fraction V, Sigma) in TBS (10 mM Tris-HCl, pH 7.5, 1.50 mM NaCI) for approximately 1.5 h at room temperature and then washed 3 times in TBS containing 0.05% Tween 20 (TTBS). The blots were then incubated with l/1000 anti-CD45 antiserum in 5% BSA-TTBS for 1.5 h at room temperature, washed and then incubated with protein A conjugated to horseradish peroxidase (Bio-Rad, l/10000 dilution in 5% BSA-TTBS). The blot was washed and developed using enhanced chemiluminesence (ECL kit, Amersham). Relative protein amounts were calculated by comparing the intensity of the CD45 bands on the Western blot using densitometry scanning and analysis techniques (PDI systems).

3. Results 3.1. Optimum conditions for CD45 PTP assay

The enzyme reaction was performed in a total volume of 15 ~1 in one well of a 96 well microtitre plate (well capacity is 100 ~1). Malachite green reagent was added to terminate the reaction and produced a final volume of 100 ~1. The color was allowed to develop and was read at 650 nm in a microplate reader, the absorption at 650

6

b

2

2.5T

z E

2f

0

/

0.5

1

1.5

2

2.5

3

3.5

Time (minutes) Fig. 1. a: standard curve of OD at 650 nm vs. inorganic phosphate concentration. Absorbance readings (0) at a wavelength of 650 nm were taken at different concentrations of potassium dihydrogen phosphate (KH,POJ. b: effect of enzyme concentration on CD45 PTP activity using saturating amounts of substrate (10 mM fyn pY531). CD45 was immunoprecipitated from BW5147 CD45+ cells at the following cell equivalents: (0) 1 X 105, ( n ) 5 X 104, (0) 2 X 104, (X) 1 X 104, (+ ) 5 X 103, and (A) 2 X 103. CD45 was also immunoprecipitated from 1 X lo5 BW5147 CD45- cells and tested for PTP activity ( A 1. Assay readings were taken at 0 min, 1 min and 3 min 20 s.

nm being proportional to the amount of free phosphate generated over a range of O-5 nmol (Fig. la). The sensitivity of the assay was such that 100 pmol phosphate generated an OD read-

D. Ng et al. /Journal

of Immunological Methods 179 (1994) 177-185

1001 801

3

I

E

J_,+_,+ 5.5

6

.I~

6.5

7

7.5

8

8.5

-_j

9

9.5

PH Fig. 2. pH optimum for immunoprecipitated CD45. PTPase activity was measured using standard assay conditions with the following buffers: 50 mM imidazole-Cl at pH 6.0, 6.6, 6.9, 7.2, 7.5, 7.8 and 9.0.

ing that was significantly above background levels (inset to Fig. la). In order to determine the optimum enzyme concentration for this range, enzyme concentration was varied and the initial rate of reaction determined. CD45 immunoprecipitated from 1 X 105, 5 x 104, 2 x 104, 1 x 104, 5 X 103, and 2 X lo3 BW5147 CD45+ cells was tested (Fig. lb). Initial rates of activity could be measured from CD45 isolated from 1 x lo4 cells. CD45 isolated from 2.5 x lo4 cells was used for all subsequent experiments. Identical immunoprecipitation experiments were done with 1 X lo5 BW5147

181

CD45- cells and showed no observable PTP activity (Fig. lb), illustrating that the generation of inorganic phosphate was solely due to the PTP activity of CD45. pH dependence of immunoprecipitated CD45 was determined. It had previously been shown that recombinant cytoplasmic CD45 PTP activity reaches an optimum at pH 7.2 (Itoh et al., 1992). It should be noted that at alkaline pH values, the malachite green reagent is more strongly absorptive at 650 nm. This was observed by comparing background absorption values at 650 nm through the use of different pH buffers. We therefore wished to use an optimal pH that would not compromise the effectiveness of the calorimetric capabilities of malachite green. Results indicated that, like recombinant CD45, immunoprecipitated CD45 had a narrow pH range, being virtually inactive at pH below 6 and above 8 in an imidazole buffer (Fig. 2). 3.2. Effect of Sepharose beads in malachite green PTP assay Since the amount of phosphate released is measured by optical density at 650 nm, it was possible that the presence of the Sepharose beads used to immunoprecipitate the PTP may interfere with the spectrophotometric readings. In a PTP assay of CD45 immunoprecipitated from 2.5 X lo4 cells, 0.125 ~1 of packed Sepharose beads were present in the assay. This amount was deter-

Table 1 Comparison of CD45 PTP activity isolated from various lymphoma cell lines Cell line

Cell type

PTP activity/ 5 X lo4 cells (nmol/min)

Relative amounts of CD45 (OD x mm’)

PTP activity/ equiv. amounts of CD45 (nmol, min _ ‘, OD-‘, mm-‘)

BW5147 EL4 RAW 253.1 A20

T lymphoma T lymphoma B lymphoma B lymphoma

0.77 0.57 0.00 0.19

4.86 3.61 n/d 1.20

0.16 0.16 n/d 0.16

Amount of CD45 immunoprecipitated from each cell line was compared by Western blot analysis. Undetectable levels of CD45 were immunoprecipitated from RAW 253 cells, hence a comparative rate could not be determined (n/d). Reaction rate was measured in nmol phosphate released per min per 5 X lo4 cell equivalents using saturating amounts of the src phosphopeptide (1.6 mM src pY416) as substrate. The adjusted reaction rate of CD45 from equivalent amounts of CD45 was obtained by dividing the PTP activity by the relative amount of CD45 present.

182

D. Ng et ai. /Journal of lmmunofogical Methods I79 (1994) I77-185

4205 4116.5 4106

Fig. 3. Immunoblot of CD45 immunoprecipitated from two T and two B cell lines. CD45 was immunoprecipitated from 1 x 106 cells of BW5147 and EL4 T lymphoma cell lines and from RAW 253 and A20 B cell lines. The immunoprecipitate was run on SDS-PAGE and the protein transferred to PVDF membrane. CD45 protein was detected by Western blot analysis using an anti-CD45 antisera specific for an extracellular epitope common to all CD45 isoforms.

mined to be sufficient to precipitate all CD45 from the cells. Bead volumes five times this amount were found to have no significant absorption at this wavelength (data not shown). Because CD45 was immobilized to Sepharose beads, the effect of agitation on immobilized enzyme was examined. It was seen that at lower peptide concentrations a linear reaction rate was only observed with agitation of the microtitre plate at 120 rpm or higher. However, at saturating peptide concentrations, agitation did not have a significant effect (data not shown). 3.3. Use of assay to compare CD45 PTP actiuity isolated from different lymphoid cell lines CD45 immunoprecipitated from four different T or B lymphoma cell lines (BW5147, EL4, RAW 253.1, and A20) was tested for its PTP activity (Table 1). Immunoprecipitated CD45 was also run on SDS-PAGE and immunoblotted with an antiserum raised against a common CD45 epitope (Fig. 3). CD45 exists in different molecular weight isoforms ranging from 180-220 kDa and this is reflected in the CD45 immunoprecipitated from these cell lines. As undetectable levels of CD45 was immunoprecipitated from the RAW 253 cell line, the specific PTP activity was not

determined. Relative protein amounts were calculated by comparing the intensity of the CD45 bands on the Western blot using densitometry scanning and analysis techniques. Amounts of CD45 were expressed as a function of the optical density of the band and the area which the band occupied. Table 1 shows the PTP activity of CD45 isolated from each cell line, comparing PTP activity obtained from equal cell numbers and from equivalent amounts of CD45 protein. .3.4. Use of assay to make kinetic measurements Initial rates of phosphate hydrolysis were determined for CD45 immunoprecipitated from 2.5 X lo4 BW5147 cells using four peptide substrates (fyn pY531, src pY527, lck pY505 and src pY416) (Fig. 4al. By varying peptide concentration, conditions of substrate saturation and maximum velocity values were obtained (Table 2). K, values were calculated from a Lineweaver Burk plot as shown for lck p 505 (Fig. 4b).

4. Discussion This work demonstrates the feasibility of using immunoprecipitated CD45 PTP isolated from as few as 10000 lymphoid cells in a non-radioactive, calorimetric, micro assay to quantitate enzymatic activity by measuring the release of inorganic phosphate. It demonstrates that the Sepharose beads used to immunoprecipitate the PTP do not interfere with the assay and that comparative enzymatic studies can be performed on CD45 isolated from different cell types. In addition, kinetic analysis determining substrate affinity (K,) and maximum rates of reaction can be determined and compared. The advantages of this method over the existing radioactive method are that it is quick, easy to perform and sensitive. No preparation of substrate is required after initial synthesis and enzymatic activity can be determined by measuring the amount of free phosphate present without prior separation from phosphorylated substrate. The assay is very sensitive allowing CD45 PTP activity to be determined from as few as 1 x lo4

D. Ng et al. /Journal

of Immunological Methods I79 (1994) 177-185

cells and can easily detect 100 pmol of free phosphate. Use of a phosphorylated peptide as a substrate instead of simple phosphotyrosine analogs such as p-nitrophenyl phosphate and Ophosphotyrosine is preferred as phosphorylated peptides have been shown to have a higher affinity (lower K, value) for CD45 (Cho et al., 1993). Although phosphotyrosine containing peptides may not exactly mimic the intact protein, they do contain key amino acids that determine substrate specificity (Zhang et al., 1994). Analysis of PTP peptide specificity together with the recent 3D structure of PTPlB (Barford et al., 1994) suggests that 4-5 residues adjacent to the phosphotyrosine influence the efficiency of binding and hydrolysis of substrate. Other calorimetric methods for measuring PTP activity have all used purified, soluble recombinant PTPs. In general, these methods tend to use larger assay volumes (0.4-l ml) and large amounts of substrate, but have provided useful kinetic analysis and insights into PTP substrate specificity (Daum et al., 1991; Cho et al., 1993; Zhang et al., 1994). Recently, we and others (Harder et al., 1994; Fisher and Higgins, 1994) scaled down the malachite green assay such that lower volumes (40-100 ~1) could be used in microtitre wells and measured using an ELISA plate reader. Robotization of this method allows one to screen thousands of chemicals that may act as either phosphatase inhibitors or activators (Fisher and Higgins, 1994). These assays using recombinant soluble protein do not however, provide information on how these PTP enzymes may be regulated in the cell. This is especially true for many transmembrane PTPs which are normally anchored in the cell membrane and may be regulated by celluTable 2 Kinetic measurements substrates

of CD45 immunoprecipitated

Substrate

fyn pY53l(TATEPQpYQPGENL) STC pY527 (TSTEPQpYQPGENL) Ick pY505 (TATEGQpYQPQP) src pY416 (LIEDNEpYTARQGA)

from K, 0.9 0.9 0.8 0.6

BW5147

(mM)

183

lar events or extracellular interactions. In this paper, CD45 is immunoprecipitated from both T and B cell lines and their phosphatase activities compared. It was shown that CD45 had similar phosphatase activity when equivalent protein amounts were isolated from the B cell line, RAW253 or the T cell lines, EL4 and BW5147. This method thus provides a means to assay phosphatase activity of enzymes isolated directly from cells and allows the comparison of enzymatic activities to be made from phosphatases isolated from different cell populations. This method can also be used to perform kinetic analysis on PTPs isolated from cells. Unlike the method that uses radioactively labeled substrate, the availability of large amounts of chemically synthesised phosphorylated substrate allows kinetic measurements to be made and rates of reaction compared. To illustrate this point, K, and Kl,, values were determined for CD45 isolated from 2.5 X lo4 BW5147 T lymphoma cells using four src family phosphotyrosine peptides as substrates. The estimated K, values (Table 21, were somewhat higher. than the reported values for soluble purified CD45 (12 PM for the substrate RCM-lysozyme, 0.15 mM for fyn 531 peptide) and for recombinant cytoplasmic CD45 against phosphopeptide substrates (0.13 mM for lck 505 peptide, 0.17 mM for fyn 531 peptide, 0.21 mM for CD35 peptide). However, they were lower than K, values (4.8 mM) reported using pNPP as a substrate (Tanks et al., 1990; Pacitti et al., 1994; Cho et al., 1993). It is possible that the presence of the anchoring effect of the protein to the beads or the presence of the antibody attatched to the extracellular domain may affect K, values. T cells using

four different

KU, (nmol/min/2.5

src family

phospho-peptides

as

X 10’ cells)

0.54 0.53 0.57 0.53

Enzyme reactions were performed in phosphatase buffer, pH 7.2 at 30°C. K, values are given in mM and comparative are given in nmol phosphate per min per 2.5 X lo4 cell equivalents of immunoprecipitated CD45.

V,,,

values

184

D. Ng et al. /Journal

of immunological Methods 179 (1994) 177-185

a

at different stages of development or activation. The feasibility of this method was demonstrated using the transmembrane PTP, CD45. The enzymatic activity of CD45 isolated from 2.5 x 104 BW5147 T lymphoma cells was measured, initial rates of reaction compared and kinetic values determined. It is a sensitive assay that is quick and easy to perform and will provide a means of investigating the cellular regulation of a variety of protein tyrosine phosphatases.

i 0

0.5

1

2.5 1.5 2 Time (minutes)

3

3.5

Acknowledgements

This work was supported by the National Cancer Institute with funds from the Canadian Cancer Society to P.J. We thank Pasquale Melito for technical assistance during the preliminary stages of this work and Dr. Jamey Marth for providing the anti-CD45 rabbit antiserum. We also thank Peter Borowski, Philip Owen, and Luan Vo for assistance with the phosphopeptide synthesis.

b

References

-4

-2

0

6

8

I0

Fig. 4. a: comparison of initial rates of reaction for CD45 using four src family phosphopeptides as substrates. CD45 immunoprecipitated from 2.5 x lo4 BW5147 T cells was tested for activity using saturating amounts (10 mM) of the following peptides: (0) fyn pY531, (0) src pY527, (+I Ick pY505 and ( X) src pY416. Assay readings were taken at 0 min, 1 min and 3 min. b: Lineweaver Burk plot for CD45, comparing the initial rates of reaction 0’) in nmol of inorganic phosphate produced per min per 2.5~ lo4 cell equivalents versus Ick pY505 substrate concentration (5) in mM.

In conclusion, this method has widespread application for measuring and comparing specific PTP activities and for performing kinetic analysis on PTP enzymes, such as CD45, isolated directly from cells. In particular, this assay can be used to determine the enzymatic activity of PTPs immunoprecipitated from cells which have undergone specific manipulations or from cells that are

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of Immunological Methods I79 (1994) 177-185

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