Isolation of Tyrosylprotein Sulfotransferase from Rat Liver

ISSN 0306-3623/98 $19.00 1 .00 PII S0306-3623(97)00304-2 All rights reserved

Gen. Pharmac. Vol. 30, No. 4, pp. 555–559, 1998 Copyright  1998 Elsevier Science Inc. Printed in the USA.

Isolation of Tyrosylprotein Sulfotransferase from Rat Liver Patalapati Ramaprasad and Chinnaswamy Kasinathan* Dental Research Center, UMDNJ-NJ Dental School, University of Medicine and Dentistry of New Jersey, University Heights, 110 Bergen Street, Newark, New Jersey 07103-2400, USA [Tel: 973-972-3600; Fax: 973-972-0045; E-Mail: [email protected]] ABSTRACT. 1. Tyrosylprotein sulfotransferase (TPST) is involved in the posttranslational modification of proteins and plays a critical role in the biological activity and secretion of proteins. A simple method has been developed to isolate the TPST (28% yield) from rat liver, using polyclonal anti-TPST antibodies. 2. The protein fractions eluted from antibody affinity column showed TPST activity and revealed a 50–54 kDa protein band in the silver stained SDS-polyacrylamide gels. 3. The enzyme exhibited optimum activity at pH 5.5 with 20 mM MnCl2. Unlike the TPST activity of the Golgi membrane, the activity of the purified enzyme was not stimulated by NaF, 59-AMP, and Triton X-100. 4. The antibody was also used to study the TPST protein turnover in rat liver of animals that were given [35S]methionine. The TPST protein synthesis assessed by measuring initial rates of incorporation of [35S]methionine into TPST protein showed enzyme synthesis for up to 60 min. 35S-labeled TPST protein of rat liver was degraded with a half-life of 30 hr. 5. The immunoaffinity purification method using rat liver as an enzyme source appeared to be very simple, rapid, and easy to perform with significant enzyme recovery. Further, the antibody was also found to be useful in the study involving TPST protein metabolism. gen pharmac 30;4:555–559, 1998.  1998 Elsevier Science Inc. KEY WORDS. Tyrosylprotein sulfotransferase, antibody, isolation, purification, characterization

INTRODUCTION Tyrosine sulfation is a posttranslational modification of secretory and membrane proteins (Huttner and Baeuerle, 1988). It has been known to be required for the biological activity of caerulein (Anastasi et al., 1968) cholecystokinin (Mutt, 1980), fibrinopeptide B (Bettelheim, 1954), gastrin (Gregory et al., 1964), phyllokinin (Anastasi et al., 1966), leucosulfakinin (Nachman et al., 1986), C4 complement (Hortin et al., 1988), and hirudin (Stone and Hofsteenge, 1986). Tyrosine sulfation has been shown to regulate the secretion of yolk protein 2 in Drosophila melanogaster (Freiderich et al., 1988). In M31 hybridoma cell line, tyrosine sulfation has also been shown to serve as a signal for the secretion of IgG (Baeuerle and Huttner, 1984). Tyrosylprotein sulfotransferase (TPST), an integral membrane protein of Golgi network that catalyzes the event tyrosine sulfation was first described in 1983 in PC 12 cells by Lee and Huttner (1983). Subsequent studies have demonstrated the presence of TPST in various tissues including bovine adrenal medulla (Lee and Huttner, 1985), rat liver (Domiano and Roth, 1989), brain (Vargas and Schwartz, 1987), gastric mucosa (Kasinathan et al., 1992), and submandibular salivary glands (Sundaram et al., 1992). Recently, the TPST activity has also been detected in platelets (Sane and Baker, 1993). As the enzyme located significantly smaller amounts, *To whom correspondence should be addressed. Received 18 March 1997; accepted 17 June 1997.

there is a need for a good purification method to yield significant amounts of the enzyme. In the present work, the antibodies have been used to purify the enzyme to homogeneity by immunoaffinity chromatography. The antibody was also used to study TPST metabolism. EXPERIMENTAL PROCEDURES

Materials Male Sprague-Dawley rats (8 weeks old, with a mean weight of 175 g) and rabbits (weighing 3 kg) were obtained from Charles River Laboratories. [35S]PAPS (2 Ci/mmol) was from New England Nuclear, [35S] methionine was from Amersham, EAY (Mr 47 kDa), 59-AMP, CNBr-Sepharose 4B and Protein G sepharose beads were from Sigma. All other reagents were of analytical grade.

Subcellular fractionation Freshly dissected rat liver was washed with cold 0.15 M NaCl and weighed. The liver was cut into small pieces, suspended in 5 vol of STKM buffer (50 mM Tris-HCl, pH 7.4, 25 mM KCl, 5 mM Mg (CH3COO)2, 5 mM 2-mercaptoethanol, containing 0.25 M sucrose, homogenized, and centrifuged at 5,0003g for 20 min. The supernatant was layered on a discontinuous gradient of 0.5 and 1.2 M sucrose in TKM buffer (Balch et al., 1984; Kasinathan et al., 1990). The gradients were centrifuged for 90 min at 33,000 rpm (100,0003g) in the SW 40 rotor. The light brown (Golgi-rich) material at the 0.5 M/1.2 M su-

556 crose interface was collected, diluted with half concentrated TKM buffer containing 0.25 M sucrose and centrifuged at 100,0003g for 1 hr. The pellet was suspended in 10 mM MES pH 6.8, containing 20% glycerol and stored at 2708C for further use. The Golgi membrane thus obtained was solubilized with Triton X-100 for 30 min in ice bath and centrifuged as described previously for 60 min. The supernatant was used for further purification of tyrosylprotein sulfotransferase.

Production and purification of anti-TPST antibody A rabbit weighing 3 kg was immunized with TPST enzyme. The TPST was isolated from submandibular salivary glands and separated on SDS-PAGE. The protein band on SDS-PAGE was located by Coomassie brilliant blue (R250, 0.05% in DDW) staining and the gel portion containing protein was excised, homogenized in PBS, and injected into rabbit subcutaneously without an adjuvant on days 1, 14, 28, and thereafter at monthly intervals (Kasinathan and William, 1995). Blood was collected from the rabbit before and after immunization at regular intervals. The serum was separated and stored at 2708C until used with the addition of sodium azide. Preimmune serum was used as a control. The sera showing antibody titer of >4 by simple double diffusion was subjected to 35% ammonium sulfate fractionation. The IgG fraction was obtained by purification of immunoglobulins by Protein G sepharose 4B column.

Purification of liver tyrosylprotein sulfotransferase by immunoaffinity chromatography The IgG fraction of rabbit anti-TPST was coupled to CNBr-activated Sepharose 4B beads. About 75 mg of IgG fraction was coupled to 7.5 ml CNBr-Sepharose 4B as described earlier (Ramaprasad and Harinath, 1987) and the beads were stored at 48C with addition of preservative. The amount of protein coupled to beads was measured by estimating protein in the supernatant before and after coupling. Two-ml beads were packed in a mini column and equilibrated with 20 mM MES pH 6.8. Five-ml vol of Triton X-100 supernatant was applied to an affinity column and equilibrated for 30 min. The column was washed with 10 vol of equilibration buffer. The bound enzyme was eluted with Gly-HCl buffer pH 2.7. About 0.5 ml fractions were collected in 0.5 ml of glycerol (final concentration 50%). The fractions were mixed thoroughly and pH was raised to 5.5 immediately with 1.0 M Tris. Aliquots of these fractions were further tested for the presence of enzyme by dot blotting. TPST activity was assayed and the purity of the enzyme was examined by SDS-PAGE followed by silver staining.

P. Ramaprasad and C. Kasinathan (donor), 0.1% Triton X-100, 50 mM NaF, 20 mM MnCl2, 2 mM 59-AMP, 20 mM MES pH 6.8, 50 mg Golgi membrane in a final volume of 50 ml. The standard assay mixture for the purified enzyme contained 2 mM unsulfated EAY, 5 mM [35S]PAPS (3 mM radioactive PAPS plus 2 mM nonradioactive PAPS), 20 mM MnCl2, 20 mM MES, pH 5.5, and 5 ng of purified enzyme. Assays were initiated by the addition of the enzyme and incubated for 30 min at 378C. The reactions were stopped by spotting 35 ml aliquots of the reaction mixture on 2.432.4 cm Whatman No. 3 MM filter paper (Domiano and Roth, 1989). The papers were washed three times for 15 min in 10% trichloroacetic acid/10 mM Na2SO4 and rinsed for 5 min in 95% ethanol. The dried papers were placed in vials containing scintillation solution and counted in a TriCarb-1500 scintillation counter.

SDS-Polyacrylamide gel electrophoresis Proteins were separated by electrophoresis by the method of Laemmli (Laemmli, 1970). The separating gels contained 10% (w/v) acrylamide, bis(acrylamide) in the ratio of 37.5:1, 0.375 M TrisHCl, pH 8.8, 0.1% SDS, 0.026% TEMED, and 0.026% ammonium persulfate. The stacking gel contained 3% acrylamide, bis(acrylamide) in the same ratio as before, 0.125 M Tris-HCl, pH 6.8, 0.1% SDS, 0.05% ammonium persulfate, and 0.4% TEMED. Electrophoresis was performed in a Mighty Small gel apparatus (Hoefer Scientific Instruments, San Francisco, CA) at a constant current of 20 mA per gel for approximately 1 hr. Protein bands on the gels were visualized by silver staining. 35

S Labeling and measurement of radioactivity incorporation into TPST protein For the TPST protein synthesis and degradation studies, rats were administered [35S]methionine (0.2 mCi/rat) in normal saline and killed at different intervals (15 min, 30 min, 1 hr, 2 hr, 4 hr, 12 hr,

Dot blotting The presence of TPST in all the fractions eluted was tested by dot blotting. Two ml of each fraction was spotted on a nitrocellulose membrane and dried at 378C. The membrane was blocked with 3% bovine serum albumin and washed with 25 mM phosphate buffer containing 0.05% Tween 20. The membrane was further incubated with rabbit anti-TPST antibody and then with goat anti-rabbit IgG alkaline phosphatase conjugate. The substrate for alkaline phosphatase consisted of the p-nitro blue tetrazolium and 5-bromo-4-chloro3-indolyl phosphate.

Assay of tyrosylprotein sulfotransferase The tyrosylprotein sulfotransferase assay mixture contained the following components: 3 mM unsulfated EAY (acceptor), 5 mM [35S]PAPS

FIGURE 1. Immunoaffinity chromatography of liver tyrosylprotein sulfotransferase. Triton X-100 solubilized rat liver Golgi membrane proteins were subjected to an antibody affinity column and bound proteins were eluted with Gly-HCl buffer, pH 2.7. Equal volumes of all the eluted fractions were assayed for TPST activity and protein. Insert, fraction 4 analyzed by SDS-PAGE followed by silver staining.

Rat Liver Tyrosylprotein Sulfotransferase

557

TABLE 1. Purification of liver tyrosylprotein sulfotransferase Purification step Homogenate Golgi fraction 100,000 3 g supernatant of Triton X-100 extract Immunoaffinity column

Protein (mg) 844 95 53 0.003

Specific activity (pmol/mg/min)

Total (pmol/min)

Purification (-fold)

Yield (%)

0.10 6 0.03 0.78 6 0.05

81 74

1 8.1

100 70

0.84 6 0.06 7392 6 2990

45 22

8.8 77241

55 28

Tyrosylprotein sulfotransferase was purified from one rat liver and assayed as described in Experimental Procedures, using EAY and PAPS as the sulfate acceptor and donor, respectively. Values are expressed as mean 6 SD of three experiments.

24 hr, 48 hr, 96 hr). The Golgi membranes were isolated from liver as described earlier (Kasinathan et al., 1993). The radioactivity incorporated into TPST protein was determined by incubating Triton X-100 supernatant of Golgi membrane with TPST antibody coupled to CNBr-Sepharose 4B beads. The beads were washed three times with 25 mM PBS and radioactivity in TPST was determined by liquid scintillation counting or by SDS-PAGE and autoradiography. RESULTS AND DISCUSSION In the present study, the polyclonal antibodies to TPST were used to isolate TPST from the Triton X-100 solubilized Golgi membranes of rat liver. The TPST protein bound to antibody Sepharose 4B column was eluted with Gly-HCl buffer. The initial fractions eluted from the immunocolumn showed significant activity when aliquots of these fractions were assayed for TPST activity. Figure 1 represents the elution profile of TPST and enzyme activity. Table 1 presents the overall purification scheme of rat liver TPST. This procedure resulted in a 77241 fold purification of the TPST activity. The fold purification of liver enzyme (Table 1, Fig. 1) differs from the fold purification reported for adrenal enzyme, suggesting that the liver rep-

resents a better enriched source of this enzyme. Under the optimized assay condition the purified enzyme, exhibited 7392 pmol/mg protein/min, which is in the range of activity expressed by the adrenal enzyme (Niehrs and Huttner, 1990). However, our method demonstrated 28% yield of pure enzyme, which is 80 times more than the 0.35% yield obtained form the adrenal medulla (Niehrs and Huttner, 1990). SDS-PAGE analysis of the protein followed by a silver staining revealed a protein of 50-54 Kda (appearing as a doublet) in the fractions showing TPST (Fig. 1 insert). A similar 50–54 kDa doublet protein was observed for the TPST purified from bovine adrenal medulla (Niehrs and Huttner, 1990). The effect of pH on EAY sulfation by the purified enzyme is shown in Figure 2. The optimum pH for tyrosine sulfation was found to be 5.5–6.0 for the purified enzyme, whereas Golgi preparation of rat liver tyrosylprotein sulfotransferase displayed optimum pH at 6.7 (Domiano and Roth, 1989). Tyrosylprotein sulfotransferase purified from bovine adrenal medulla exhibited optimum pH at 6.0 (Lee and Huttner, 1985). MnCl2 is required for the purified tyrosylprotein sulfotransferase, whereas MgCl2 is not effective in activating the enzyme when displaced by MnCl2. The metal ion Mn21 requirement for the tyrosylprotein sulfotransferase activity of Golgi membrane from rat liver was also noticed by Domiano and Roth (1989). Further, as shown in Table 2, some of the requirements for the purified enzyme were different from those described earlier for the crude enzyme. NaF and 59-AMP were routinely included in the assay mixture to prevent degradation of the sulfate donor, PAPS, and enhance the measurement of rat liver tyrosylprotein sulfotransferase in Golgi membrane preparations. Domiano and Roth (1989) reported that the addition of both NaF and 59-AMP to the incubation mixture increased TPST activity by 10-fold in rat liver Golgi membrane. However, use of NaF as well as 59-AMP was observed to inhibit the activity of purified enzyme. As there is no contamination

TABLE 2. Requirements for rat liver tyrosylprotein sulfotransferase activity Incubation mixture Standard Triton X-100 (0.25%) 59-AMP (2 mM) Minus MnCl2 Minus MnCl2, plus MgCl2 (20 mM) NaF (50 mM) FIGURE 2. Effect of pH on the activity of tyrosylprotein sulfotransferase purified by immunoaffinity column. The tyrosylprotein sulfotransferase activity was measured using 20 mM MES (d) and 75 mM phosphate buffer (s).

Control % 100 ND 30 ND ND ND

The standard assay contained 20 mM MES pH 5.5, 20 mM MnCl2, 5 mM [35S]PAPS, 3 mM EAY, and 5 ng immunopurified TPST. Enzyme activities were measured as described in Experimental Procedures. The standard assay gave the specific activity of 3475 pmol/mg protein/min. Values are the average of two independent preparations. ND 5 not detected.

558

FIGURE 3. Isolation of [35S] methionine labeled tyrosylprotein sulfotransferase from rat liver. Triton X-100 solubilized rat liver Golgi membrane proteins were applied to an antibody affinity column and bound proteins were eluted with Gly-HCl buffer, pH 2.7. Radioactivity in equal aliquot volume of eluted fractions was measured by liquid scintillation counting. Insert, fraction 4 analyzed by SDS-PAGE followed by autoradiography.

FIGURE 4. In vivo synthesis of liver tyrosylprotein sulfotransferase. Rats were injected with [35S]methionine to radiolabel TPST protein. The animals were killed and liver Golgi membranes were prepared 15, 30, 60, and 120 min after radioactive methionine injection. 35S radioactivity in TPST was measured by liquid scintillation counting after immunoprecipitation of the protein from the rat liver Golgi membrane with antibody coupled anti-TPST antibody beads.

P. Ramaprasad and C. Kasinathan

FIGURE 5. In vivo turnover of rat liver TPST protein. Rats were injected with [35S]methionine to radiolabel TPST protein. The animals were killed and liver Golgi membranes were prepared 2, 4, 12, 24, and 48 hr after radioactive methionine injection. [35S] radioactivity in TPST was measured by liquid scintillation counting after immunoprecipitation of the protein from the rat liver Golgi membrane with antibody coupled anti-TPST antibody beads. Degradation half-life was estimated from the slopes of straight lines fitted by regression analysis of specific radioactivity versus time.

of Golgi membrane components in the purified fractions, both NaF and 59-AMP are not required to prevent PAPS degradation. This observation further affirms the purity of enzyme preparation. Further, unlike the membrane-associated enzyme, the affinity purified rat liver TPST did not require a detergent (Triton X-100) for its activity. In addition to its use in enzyme isolation, the TPST antibody was also used in the present study to evaluate the turnover of TPST protein. Figure 3 shows the immunopurification of [35S]-labeled TPST from rat liver Golgi membranes labeled with [35S]methionine. A single labeled protein of 50-54 kDa was observed on the autoradiogram of TPST fraction (Fig. 3 insert). These results confirm the specificity of antibody to TPST. To study the in vivo turnover of TPST, rats were killed at the indicated time periods after the simultaneous intraperitoneal injection of [35S]methionine. Figure 4 depicts the rate of incorporation of [35S]methionine and Fig. 5 depicts the degradation of TPST. The enzyme synthesis was proceeded for 60 min followed by a gradual degradation, which continued for 4 days. Highest incorporation of [35S]methionine into Golgi TPST protein was observed at 1 hr. As shown in Fig. 5, the TPST protein was degraded in a monophasic fashion in vivo. The half-life of liver TPST was 30 hr. Tyrosylprotein sulfotransferase is an enzyme involved in protein secretion, and it also regulates the biological activity of certain proteins. We have earlier shown the stimulation of TPST activity in alcoholics (Sundaram et al., 1992) and also in rats subjected to chronic alcohol feeding (Kasinathan et al., 1994). Our current results show an increase in the level of TPST protein in alcohol treated rats compared with the controls (unpublished observation). The increase in TPST content may result from an increase in pro-

Rat Liver Tyrosylprotein Sulfotransferase tein synthesis or by a decrease in TPST degradation. The present TPST protein turnover study will be useful in evaluating the effect of ethanol on TPST turnover. The method could also be used in evaluating TPST metabolism in different pathological conditions. In conclusion, the availability of specific antibody to TPST enabled us to develop a simple purification method for the isolation of liver enzyme with high recovery and the anti-TPST antibodies were further explored for their use in TPST metabolism. This work was supported by grant #AA09191 from the National Institute on Alcohol Abuse and Alcoholism.

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