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• W2000757102 abstract "An arachidonic acid-stimulated Ser/Thr phosphatase activity was detected in soluble extracts prepared from rat pituitary clonal GH4C1 cells, rat or bovine brain, and bovine heart. The enzyme activity was purified to homogeneity from bovine brain as a monomer with aM r of 63,000 and a specific activity of 32 nmol of Pi released per min/mg of protein when assayed in the presence of 10 μm phosphocasein in the absence of lipid. Arachidonic acid stimulated activity 4–14-fold, with half-maximal stimulation at 50–100 μm, when assayed in the presence of a variety of phosphosubstrates including casein, reduced carboxamidomethylated and maleylated lysozyme, myelin basic protein, and histone. Oleic acid, linoleic acid, and palmitoleic acid also stimulated activity; however, saturated fatty acids and alcohol or methyl ester derivatives of fatty acids did not significantly affect activity. The lipid-stimulated phosphatase was identified as the bovine equivalent of protein phosphatase 5 or a closely related homolog by sequence analysis of proteolytic fragments generated from the purified enzyme. When recombinant rat protein phosphatase 5 was expressed as a cleavable glutathione S-transferase fusion protein, the affinity-purified thrombin-cleaved enzyme exhibited a specific activity and sensitivity to arachidonic acid similar to those of the purified bovine brain enzyme. These results suggest that protein phosphatase 5 may be regulated in vivo by a lipid second messenger or another endogenous activator. An arachidonic acid-stimulated Ser/Thr phosphatase activity was detected in soluble extracts prepared from rat pituitary clonal GH4C1 cells, rat or bovine brain, and bovine heart. The enzyme activity was purified to homogeneity from bovine brain as a monomer with aM r of 63,000 and a specific activity of 32 nmol of Pi released per min/mg of protein when assayed in the presence of 10 μm phosphocasein in the absence of lipid. Arachidonic acid stimulated activity 4–14-fold, with half-maximal stimulation at 50–100 μm, when assayed in the presence of a variety of phosphosubstrates including casein, reduced carboxamidomethylated and maleylated lysozyme, myelin basic protein, and histone. Oleic acid, linoleic acid, and palmitoleic acid also stimulated activity; however, saturated fatty acids and alcohol or methyl ester derivatives of fatty acids did not significantly affect activity. The lipid-stimulated phosphatase was identified as the bovine equivalent of protein phosphatase 5 or a closely related homolog by sequence analysis of proteolytic fragments generated from the purified enzyme. When recombinant rat protein phosphatase 5 was expressed as a cleavable glutathione S-transferase fusion protein, the affinity-purified thrombin-cleaved enzyme exhibited a specific activity and sensitivity to arachidonic acid similar to those of the purified bovine brain enzyme. These results suggest that protein phosphatase 5 may be regulated in vivo by a lipid second messenger or another endogenous activator. Four major types of protein-Ser/Thr phosphatases have been described: PP1, 1The abbreviations used are: PP, protein phosphatase; AA, arachidonic acid; TPR, tetratricopeptide repeat; RCML, reduced carboxamidomethylated and maleylated lysozyme; MBP, myelin basic protein; CMC, critical micellar concentration; HPLC, high performance liquid chromatography; GST, glutathioneS-transferase; pNPP, para-nitrophenyl phosphate; SM-PP1M, smooth muscle myosin light chain phosphatase 1.1The abbreviations used are: PP, protein phosphatase; AA, arachidonic acid; TPR, tetratricopeptide repeat; RCML, reduced carboxamidomethylated and maleylated lysozyme; MBP, myelin basic protein; CMC, critical micellar concentration; HPLC, high performance liquid chromatography; GST, glutathioneS-transferase; pNPP, para-nitrophenyl phosphate; SM-PP1M, smooth muscle myosin light chain phosphatase 1. PP2A, calcineurin, and PP2C. The catalytic subunits of PP1, PP2A, and calcineurin constitute a large family of structurally related enzymes (1Barton G.J. Cohen P.T.W. Barford D. Eur. J. Biochem. 1994; 220: 225-237Crossref PubMed Scopus (154) Google Scholar, 2Wera S. Hemmings B.A. Biochem. J. 1995; 311: 17-29Crossref PubMed Scopus (600) Google Scholar, 3Barford D. Trends Biochem. Sci. 1996; 21: 407-412Abstract Full Text PDF PubMed Scopus (313) Google Scholar). Cloning studies have also revealed several novel protein-Ser/Thr phosphatases related to PP1 and PP2A, the properties and functions of which are not yet understood (2Wera S. Hemmings B.A. Biochem. J. 1995; 311: 17-29Crossref PubMed Scopus (600) Google Scholar). In addition to a catalytic subunit, PP1, PP2A, and calcineurin each contain one or more regulatory subunits. Mechanisms regulating the activity of these enzymes include the control of localization and substrate specificity by regulatory subunits; direct phosphorylation; the binding of a second messenger; and inhibition by regulatory domains, subunits, and interacting proteins (2Wera S. Hemmings B.A. Biochem. J. 1995; 311: 17-29Crossref PubMed Scopus (600) Google Scholar). Several members of the PP1/PP2A family have been shown to be sensitive to putative lipid second messengers (4Gong M.C. Fuglsang A. Alessi D. Kobayashi S. Cohen P. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1992; 267: 21492-21498Abstract Full Text PDF PubMed Google Scholar, 5Dobrowsky R.T. Hannun Y.A. J. Biol. Chem. 1992; 267: 5048-5051Abstract Full Text PDF PubMed Google Scholar, 6Dobrowsky R.T. Kamibayashi C. Mumby M.C. Hannun Y.A. J. Biol. Chem. 1993; 268: 15523-15530Abstract Full Text PDF PubMed Google Scholar, 7Law B. Rossie S. J. Biol. Chem. 1995; 270: 12808-12813Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 8Nickels J.T. Broach J.R. Genes Dev. 1996; 10: 382-394Crossref PubMed Scopus (176) Google Scholar). Arachidonic acid and several of its lipoxygenase or cytochrome P-450 metabolites have been reported to modulate the activity of a variety of ion channels (9Meves H. Prog. Neurobiol. ( Oxf. ). 1994; 43: 175-186Crossref PubMed Scopus (195) Google Scholar, 10Petrou S. Ordway R.W. Kirber M.T. Dopico A.M. Hamilton J.A. Walsh Jr., J.V. Singer J.J. Prostaglandins Leukotrienes Essent. Fatty Acids. 1995; 52: 173-178Abstract Full Text PDF PubMed Scopus (33) Google Scholar, 11Yu S.P. J. Physiol. ( Lond. ). 1995; 487: 797-811Crossref PubMed Scopus (31) Google Scholar, 12Petitjacques J. Hartzell H.C. J. Physiol. ( Lond. ). 1996; 493: 67-81Crossref PubMed Scopus (49) Google Scholar, 13Duerson K. White R.E. Jiang F. Schonbrunn A. Armstrong D.L. Neuropharmacology. 1996; 35: 949-961Crossref PubMed Scopus (61) Google Scholar). Although ion channel responses may be directly regulated by lipids (9Meves H. Prog. Neurobiol. ( Oxf. ). 1994; 43: 175-186Crossref PubMed Scopus (195) Google Scholar, 10Petrou S. Ordway R.W. Kirber M.T. Dopico A.M. Hamilton J.A. Walsh Jr., J.V. Singer J.J. Prostaglandins Leukotrienes Essent. Fatty Acids. 1995; 52: 173-178Abstract Full Text PDF PubMed Scopus (33) Google Scholar), in several cases, lipid-dependent responses can be blocked by Ser/Thr phosphatase inhibitors such as microcystin, okadaic acid, and calyculin (11Yu S.P. J. Physiol. ( Lond. ). 1995; 487: 797-811Crossref PubMed Scopus (31) Google Scholar, 12Petitjacques J. Hartzell H.C. J. Physiol. ( Lond. ). 1996; 493: 67-81Crossref PubMed Scopus (49) Google Scholar, 13Duerson K. White R.E. Jiang F. Schonbrunn A. Armstrong D.L. Neuropharmacology. 1996; 35: 949-961Crossref PubMed Scopus (61) Google Scholar). This suggests that a Ser/Thr phosphatase related to PP1 or PP2A may mediate the effect of some lipids on ion channel activity (14Armstrong D.L. White R.E. Trends Neurosci. 1992; 15: 403-408Abstract Full Text PDF PubMed Scopus (45) Google Scholar). The possibility that a lipid-activated Ser/Thr phosphatase may in turn regulate ion channel function prompted us to look for such an enzyme. In this report, we describe the purification of a fatty acid-stimulated Ser/Thr phosphatase from bovine brain. The purified enzyme contains a single M r 63,000 polypeptide and is identical or closely related to the recently cloned Ser/Thr phosphatase PP5 (15Becker W. Kentrup H. Klumpp S. Schultz J.E. Joost H.G. J. Biol. Chem. 1994; 269: 22586-22592Abstract Full Text PDF PubMed Google Scholar, 16Chen M.X. McPartlin A.E. Brown L. Chen Y.H. Barker H.M. Cohen P.T.W. EMBO J. 1994; 13: 4278-4290Crossref PubMed Scopus (252) Google Scholar, 17Chinkers M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11075-11079Crossref PubMed Scopus (135) Google Scholar). 2Protein phosphatase 5 cloned from a human teratocarcinoma cell cDNA library (16Chen M.X. McPartlin A.E. Brown L. Chen Y.H. Barker H.M. Cohen P.T.W. EMBO J. 1994; 13: 4278-4290Crossref PubMed Scopus (252) Google Scholar), PPT cloned from a rat fat cell cDNA library (15Becker W. Kentrup H. Klumpp S. Schultz J.E. Joost H.G. J. Biol. Chem. 1994; 269: 22586-22592Abstract Full Text PDF PubMed Google Scholar), and PPK cloned from a mouse lung cDNA library (17Chinkers M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11075-11079Crossref PubMed Scopus (135) Google Scholar) are >90% identical in amino acid sequence, but PP5 is only 40% identical to PPT1 cloned from S. cerevisiae (16Chen M.X. McPartlin A.E. Brown L. Chen Y.H. Barker H.M. Cohen P.T.W. EMBO J. 1994; 13: 4278-4290Crossref PubMed Scopus (252) Google Scholar). We will therefore refer to mammalian forms of this enzyme as PP5 and reserve the term PPT for the yeast form of the enzyme. This nomenclature is consistent with the majority of other reports on this enzyme (18Yong W.H. Ueki K. Chou D. Reeves S.A. von Deimling A. Gusella J.F. Mohrenweiser H.W. Buckler A.J. Louis D.N. Genomics. 1995; 29: 533-536Crossref PubMed Scopus (28) Google Scholar, 19Xu X.L. Lagercrantz J. Zickert P. Bajalicalagercrantz S. Zetterberg A. Biochem. Biophys. Res. Commun. 1996; 218: 514-517Crossref PubMed Scopus (9) Google Scholar, 20Becker W. Buttini M. Limonta S. Boddeke H. Joost H.G. Mol. Brain Res. 1996; 36: 23-28Crossref PubMed Scopus (12) Google Scholar, 21Fukuda H. Shima H. Vesonder R.F. Tokuda H. Nishino H. Katoh S. Tamura S. Sugimura T. Nagao M. Biochem. Biophys. Res. Commun. 1996; 220: 160-165Crossref PubMed Scopus (51) Google Scholar, 22Ansai T. Dupuy L.C. Barik S. J. Biol. Chem. 1996; 271: 24401-24407Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 23Chen M.-S. Silverstein A.M. Pratt W.B. Chinkers M. J. Biol. Chem. 1996; 271: 32315-32320Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar) following the initial cloning studies.2Protein phosphatase 5 cloned from a human teratocarcinoma cell cDNA library (16Chen M.X. McPartlin A.E. Brown L. Chen Y.H. Barker H.M. Cohen P.T.W. EMBO J. 1994; 13: 4278-4290Crossref PubMed Scopus (252) Google Scholar), PPT cloned from a rat fat cell cDNA library (15Becker W. Kentrup H. Klumpp S. Schultz J.E. Joost H.G. J. Biol. Chem. 1994; 269: 22586-22592Abstract Full Text PDF PubMed Google Scholar), and PPK cloned from a mouse lung cDNA library (17Chinkers M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11075-11079Crossref PubMed Scopus (135) Google Scholar) are >90% identical in amino acid sequence, but PP5 is only 40% identical to PPT1 cloned from S. cerevisiae (16Chen M.X. McPartlin A.E. Brown L. Chen Y.H. Barker H.M. Cohen P.T.W. EMBO J. 1994; 13: 4278-4290Crossref PubMed Scopus (252) Google Scholar). We will therefore refer to mammalian forms of this enzyme as PP5 and reserve the term PPT for the yeast form of the enzyme. This nomenclature is consistent with the majority of other reports on this enzyme (18Yong W.H. Ueki K. Chou D. Reeves S.A. von Deimling A. Gusella J.F. Mohrenweiser H.W. Buckler A.J. Louis D.N. Genomics. 1995; 29: 533-536Crossref PubMed Scopus (28) Google Scholar, 19Xu X.L. Lagercrantz J. Zickert P. Bajalicalagercrantz S. Zetterberg A. Biochem. Biophys. Res. Commun. 1996; 218: 514-517Crossref PubMed Scopus (9) Google Scholar, 20Becker W. Buttini M. Limonta S. Boddeke H. Joost H.G. Mol. Brain Res. 1996; 36: 23-28Crossref PubMed Scopus (12) Google Scholar, 21Fukuda H. Shima H. Vesonder R.F. Tokuda H. Nishino H. Katoh S. Tamura S. Sugimura T. Nagao M. Biochem. Biophys. Res. Commun. 1996; 220: 160-165Crossref PubMed Scopus (51) Google Scholar, 22Ansai T. Dupuy L.C. Barik S. J. Biol. Chem. 1996; 271: 24401-24407Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 23Chen M.-S. Silverstein A.M. Pratt W.B. Chinkers M. J. Biol. Chem. 1996; 271: 32315-32320Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar) following the initial cloning studies. We also demonstrate that recombinant rat PP5 is similarly activated by the unsaturated fatty acid arachidonic acid (AA). Protein phosphatase 5 and its yeast homolog (PPT1) contain a C-terminal catalytic domain that is structurally related to PP2A and PP1 and an N-terminal domain consisting of several tetratricopeptide repeats (TPRs) that is not shared with other members of the PP1/PP2A family (15Becker W. Kentrup H. Klumpp S. Schultz J.E. Joost H.G. J. Biol. Chem. 1994; 269: 22586-22592Abstract Full Text PDF PubMed Google Scholar, 16Chen M.X. McPartlin A.E. Brown L. Chen Y.H. Barker H.M. Cohen P.T.W. EMBO J. 1994; 13: 4278-4290Crossref PubMed Scopus (252) Google Scholar, 17Chinkers M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11075-11079Crossref PubMed Scopus (135) Google Scholar). The stimulation of PP5 in vitro by unsaturated fatty acids suggests that this enzyme may be regulated in vivo and raises the possibility that it is a target for activation by a lipid second messenger or some other endogenous effector. All materials were purchased from Sigma unless otherwise noted. The catalytic subunit of cAMP-dependent protein kinase was purified from bovine heart (24Kaczmarek L.K. Jennings K.R. Strumwasser F. Nairn A.C. Walter U. Wilson F.D. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7487-7491Crossref PubMed Scopus (172) Google Scholar). Protein phosphatase 2A containing aM r 55,000 B′ subunit (25Ruediger R. Van Wart Hood J.E. Mumby M. Walter G. Mol. Cell. Biol. 1991; 11: 4282-4285Crossref PubMed Scopus (71) Google Scholar, 26Csortos C. Zolnierowicz S. Bako E. Durbin S.D. DePaoli-Roach A.A. J. Biol. Chem. 1996; 271: 2578-2588Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) was purified from rat brain. 3B. Law, J. Skinner, and S. Rossie, manuscript in preparation. All steps were carried out at 4 °C unless otherwise noted. Frozen bovine brain cortex (615 g) was thawed and homogenized in a Waring blender with 1.2 liters of 20 mmTris-HCl, pH 7.6 (4 °C), 1 mm EDTA, 1 mmEGTA, 0.1% β-mercaptoethanol, 0.5 μg/ml leupeptin, 1 mm benzamidine, 0.5 mm phenylmethylsulfonyl fluoride, and 1 μm pepstatin A (buffer A). The homogenization buffer also included 10 μg/ml trypsin inhibitor. The homogenate was centrifuged at 15,000 × g for 40 min, and the supernatant was filtered through glass wool, diluted to 3 liters, and then mixed with 500 ml of DE52 (Whatman) for 90 min. The DE52 was washed until the effluent absorbance at 280 nm was equal to that of the wash buffer, and then bound proteins were eluted with 1 liter of buffer A containing 200 mm NaCl and 10 μg/ml trypsin inhibitor. Eluted protein was dialyzed against buffer A and then applied to a 5 × 30-cm DEAE-Sepharose CL-6B column. The column was eluted with a 1-liter gradient of 0–0.3 m NaCl in buffer A (including 10 μg/ml trypsin inhibitor) at a flow rate of 7.5 ml/min. Fractions of 7.5 ml were collected and assayed as described below. A peak of AA-stimulated phosphatase activity eluting at 60 mm NaCl was pooled, dialyzed in buffer A, and then applied to a 2.5 × 18-cm CM-Sepharose CL-6B column and eluted with a 300-ml gradient of 0–0.3 m NaCl in buffer A at a flow rate of 2 ml/min. Fractions of 2.5 ml were collected and assayed. A single peak of phosphatase activity eluting at 100 mm NaCl was pooled and diluted with buffer A to contain ∼25 mm NaCl and then loaded onto a 1 × 14-cm heparin-agarose column. The column was washed with 15 ml of buffer A containing 100 mmNaCl and then eluted with a 10-ml gradient of 0.1–1 m NaCl in buffer A. Fractions of 0.5 ml were collected at 0.5 ml/min and assayed. Active fractions were pooled and subjected to gel filtration on a 1.5 × 95-cm Sephadex G-100 superfine column (Pharmacia Biotech Inc.) in buffer A containing 100 mm NaCl. Standards used to calibrate the column included aldolase (158 kDa), bovine serum albumin (66.2 kDa), carbonic anhydrase (29 kDa), cytochromec (12.4 kDa), and aprotinin (6.5 kDa). The column flow rate was 0.1 ml/min, and 1-ml fractions were collected and assayed. Active fractions were pooled and loaded onto a 1.5 × 16-cm α-casein-agarose column. This column was washed and then eluted with a 100-ml gradient of 0–0.3 m NaCl in buffer A at 1 ml/min; fractions of 1 ml were collected and assayed. A single peak of AA-stimulated phosphatase activity eluting at 140 mm NaCl was pooled and concentrated on a 1-ml heparin-agarose column and then subjected to cation-exchange chromatography on a Mono S HR 5/5 column (Pharmacia Biotech Inc.) at 25 °C. The column was washed and eluted with a 110-ml gradient of 0–186 mm NaCl in buffer A (pH 7.6, 25 °C) at a flow rate of 1 ml/min. Fractions of 1 ml were collected at 4 °C and assayed for phosphatase activity. Pooled fractions were dialyzed against buffer A containing 50% glycerol and stored at −20 °C. Purification was monitored by SDS-polyacrylamide gel electrophoresis (27Maizel J.V. Methods Virol. 1971; 5: 179-246Crossref Google Scholar) using a 4% stacking gel and a 10% resolving gel, and proteins were detected by staining with Coomassie Blue or silver (28Blum H. Beier H. Gross H.J. Electrophoresis. 1987; 8: 93-99Crossref Scopus (3738) Google Scholar). Casein, RCML (Life Technologies, Inc.), and lysine-rich histone (type III-S from calf thymus, Sigma) were phosphorylated overnight at 30 °C in a reaction containing 20 mm Tris-HCl, pH 7.5, 5 mm magnesium acetate, 0.1% β-mercaptoethanol, 750 μm [γ-32P]ATP (3.5 × 1014 cpm/mol ATP), 5 mg/ml substrate, and 4 μg/ml purified catalytic subunit of cAMP-dependent protein kinase (29Sheng Z. Charbonneau H. J. Biol. Chem. 1993; 268: 4728-4733Abstract Full Text PDF PubMed Google Scholar). Each reaction was terminated by adding trichloroacetic acid to a final concentration of 12% (w/v) and then left on ice for 2 h and centrifuged at 12,000 × g for 10 min at 4 °C. The resulting pellet was resuspended in 1 m Tris-HCl, pH 7.6, and dialyzed against 50 mm Tris-HCl, pH 7.6, and 0.1 mm EGTA at 4 °C. Myelin basic protein (Life Technologies, Inc.) was phosphorylated as described above in a 500-μl reaction, and then the reaction was terminated with 800 μl of ice-cold 20% (w/v) trichloroacetic acid in 20 mmNaH2PO4. The mixture was left for 15 min on ice and centrifuged for 15 min at 4 °C in a microcentrifuge. The pellet was washed four times; resuspended in 1 m Tris-HCl, pH 7.6 (4 °C); and dialyzed as described above. Kemptide was phosphorylated in the same manner as MBP and purified by binding to phosphocellulose P-81 paper (29Sheng Z. Charbonneau H. J. Biol. Chem. 1993; 268: 4728-4733Abstract Full Text PDF PubMed Google Scholar). Phosphorylase a was phosphorylated as described by Cohen et al. (30Cohen P. Alemany S. Hemmings B.A. Resink T.J. Strålfors P. Tung H.Y.L. Methods Enzymol. 1988; 159: 390-408Crossref PubMed Scopus (387) Google Scholar). The average stoichiometry of phosphorylation for these substrates (expressed as mol of phosphate/mol of protein) was 0.27 for casein, 0.21 for RCML, 0.2 for histone, 0.96 for MBP, 0.33 for Kemptide, and 0.4 for phosphorylasea. During purification, casein was phosphorylated with 100 μm [γ-32P]ATP (3.5 × 1015 cpm/mol ATP) to a stoichiometry of 0.05 mol of phosphate/mol of casein and used to assay column fractions and pools. Phosphatase reactions conducted during purification were carried out for 15 min at 30 °C with 0.6 μm32P-casein in the presence and absence of 50 μm AA (Calbiochem) in 60 mm Tris, pH 7.6 (25 °C), 1 mm EDTA, 1 mm EGTA, and 0.1% β-mercaptoethanol (buffer B). Reactions were initiated by adding 20 μl of substrate containing lipid or ethanol vehicle to 10 μl of an enzyme sample. Once the phosphatase was purified, the assay conditions were modified slightly. All solutions were pre-equilibrated to room temperature just before assay. Ten microliters of lipid or ethanol vehicle solution were mixed with 10 μl of phosphatase, and then the reaction was immediately initiated by adding 10 μl of substrate and incubated for 10 min at 30 °C in buffer B. Reactions were terminated with 100 μl of ice-cold 10% (w/v) trichloroacetic acid and 20 μl of 7.5 mg/ml bovine serum albumin and then microcentrifuged at 12,000 × g for 5 min. Reactions with32P-RCML were terminated instead with 100 μl of ice-cold 20% (w/v) trichloroacetic acid. Assays using 32P-Kemptide,32P-histone, and 32P-MBP were quenched with 450 μl of 4% (w/v) activated charcoal in 0.6 m HCl, 90 mm Na4P2O7, and 2 mm NaH2PO4 and then centrifuged at 12,000 × g for 5 min. Acid-soluble32Pi was quantified by liquid scintillation counting. The phosphate released from each substrate was <20% of the total present. The final ethanol concentration in lipid-containing reactions was 0.8–1.7% and had no significant effect on phosphatase activity. The CMCs of lipid solutions in 60 mmTris, pH 7.6 (25 °C), 1 mm EDTA, 1 mm EGTA, 0.1% β-mercaptoethanol, 1.3% ethanol, and a 2 μmconcentration of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene were determined by fluorescence spectroscopy (31Chattopadhyay A. London E. Anal. Biochem. 1984; 139: 408-412Crossref PubMed Scopus (370) Google Scholar). Lipids were dispersed by sonication or vigorous vortexing, equilibrated to room temperature, and then incubated with fluorescent probe in the dark for at least 30 min. Fluorescence intensity was measured at excitation and emission wavelengths of 358 and 430 nm, respectively, on a Hitachi F-2000 fluorescence spectrophotometer. Fifty micrograms of purified phosphatase were concentrated by trichloroacetic acid precipitation, resuspended in 8 m urea and 0.4m NH4HCO3, reduced and carboxymethylated (32Stone K.L. Williams K.R. A Practical Guide to Proteins & Peptide Purification for Microsequencing. 2nd Ed. Academic Press, Inc., San Diego, CA1993: 45-73Google Scholar), and then digested with 1.8 μg of endoproteinase Lys-C (Waco Chemicals) at 37 °C overnight. Peptides were purified by reverse-phase HPLC on a Vydac C18 column (2.1 × 250 mm) using a gradient of 0–30% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 150 μl/min. The amino acid sequences of selected peptides were determined by automated Edman degradation performed with either an Applied Biosystems 470A or 491 gas-phase sequencer. A BLAST search (33Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70319) Google Scholar) was performed to compare the peptide sequences obtained with those in the GenBank™, SwissProt, and PIR data bases. Messenger RNA was isolated from rat GH4C1 cells and used to generate cDNA. The cDNA was then used as a template in polymerase chain reaction together with PP5-specific oligonucleotide primers, and the major polymerase chain reaction product was cloned into theMluI-NotI sites of the mammalian expression vector pCI (Promega). The 1.5-kilobase pair cDNA containing the entire PP5 coding region was then amplified by polymerase chain reaction using the pCI-PP5 clone as a template. The 5′-sense primer (5′-GACTGGATCCATGGCGATGGCGGAGGGCGAG) and the 3′-antisense primer (5′-GACTGAATTCTTACATCATTCCTAGCTG) utilized in this polymerase chain reaction contained initiation and stop codons, respectively, as well asBamHI and EcoRI restriction linkers. The major product containing the full-length PP5 coding region was then cloned into the BamHI-EcoRI sites of pBluescript II (pBII) KS− (Stratagene) and sequenced using an ALFexpress automatic DNA sequencer (Pharmacia Biotech Inc.) to verify its authenticity. To perform bacterial expression, the entire PP5 cDNA was excised from the pBII-PP5 construct using BamHI andEcoRI restriction enzymes and cloned immediately downstream of the GST coding region into the BamHI-EcoRI sites of the pET-21a GST plasmid. 4Constructed as described by Taylor et al. (G. Taylor, Y. Liu, C. Baskerville, and H. Charbonneau, submitted for publication). The newly constructed plasmid, termed pET GST-PP5, was then transformed intoEsherichia coli strain BL21(DE3). Bacterial cultures were grown in Luria broth supplemented with 50 μg/ml ampicillin and 4 mm MnCl2, and protein expression was induced with 50 μm isopropylthiogalactoside. Cells were lysed in 50 mm Tris, pH 7.6, 0.1% β-mercaptoethanol, 2 mm EDTA, 4 mm MnCl2, 1 mm phenylmethylsulfonyl fluoride, and 1 mg/ml each aprotinin, leupeptin, and pepstatin. The GST-PP5 fusion protein was affinity-purified with glutathione-agarose and then treated with thrombin (34Smith D.B. Johnson K.S. Gene ( Amst. ). 1988; 67: 31-40Crossref PubMed Scopus (5046) Google Scholar, 353rd Ed.16211622John Wiley & Sons, Inc.New YorkAusubel, F., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1995)Short Protocols in Molecular Biology (Matsudaira, P., ed) 3rd Ed., pp. 1621–1622, John Wiley & Sons, Inc., New York.Google Scholar). Proteolysis was terminated with 1 mmphenylmethylsulfonyl fluoride, and recombinant PP5 was dialyzed in 50% glycerol, 1 mm EGTA, 0.1% β-mercaptoethanol, 20 mm Tris, pH 7.6 (4 °C), and 4 mmMnCl2 overnight and then stored at −20 °C. The amino acid sequence of recombinant PP5 is identical to that reported by Becker et al. (15Becker W. Kentrup H. Klumpp S. Schultz J.E. Joost H.G. J. Biol. Chem. 1994; 269: 22586-22592Abstract Full Text PDF PubMed Google Scholar) except for four residues (GSGS) remaining at the N terminus after thrombin cleavage. Recombinant PP5 was assayed for para-nitrophenylphosphatase activity in a 50-μl reaction containing 10–150 mm pNPP, 500 ng of enzyme, and 0–40 μm AA in 20 mm Tris, pH 7.4, 1 mm EDTA, 1 mm EGTA, 0.1% β-mercaptoethanol, and 0.1% ethanol. Aliquots of enzyme were preincubated for 1 min at 30 °C, and then reactions were initiated by adding a mixture of substrate and lipid prewarmed to 30 °C. Control reactions were assayed without enzyme to determine the spontaneous hydrolysis of pNPP at each substrate concentration. Assays were terminated after 15 min with 450 μl of 0.25 n NaOH. Sample absorbance was measured at A 410 to quantitate the release of para-nitrophenolate, after subtracting the appropriate background (29Sheng Z. Charbonneau H. J. Biol. Chem. 1993; 268: 4728-4733Abstract Full Text PDF PubMed Google Scholar). The catalytic parametersK m and V max were determined by nonlinear regression and Lineweaver-Burk analysis using the computer software program EnzymeKinetics (Trinity Software). When soluble extracts prepared from rat brain were subjected to Mono Q anion-exchange chromatography and fractions were assayed for phosphatase activity using 32P-casein as substrate in the presence of 50 μm AA, a peak of activity eluting at ∼110–125 mm NaCl was observed (data not shown). Its activity ranged from undetectable to very low in the absence of AA. A similar peak of lipid-stimulated phosphatase activity was observed during chromatography of soluble extracts from bovine brain or heart and from rat clonal GH4C1 pituitary cells. After Mono Q ion-exchange chromatography, the lipid-stimulated activity was inhibited >80% by 100 nm okadaic acid, suggesting that it was related to PP1 or PP2A (36Holmes C.F.B. Boland M.P. Curr. Opin. Struct. Biol. 1993; 3: 934-943Crossref Scopus (64) Google Scholar). To purify the fatty acid-stimulated phosphatase, a soluble extract was prepared from bovine brain and subjected to batch chromatography on DE52-cellulose, followed by column chromatography on DEAE-Sepharose CL-6B, CM-Sepharose CL-6B, heparin-agarose, Sephadex G-100 superfine, α-casein-agarose, and Mono S (Table I). Phosphatase activity was monitored in the presence and absence of 50 μm AA using 32P-casein as a substrate. A single peak of AA-stimulated phosphatase activity eluting at 60 mm NaCl was resolved from the majority of phosphatase activity during chromatography on DEAE-Sepharose CL-6B. During gel filtration, the lipid-stimulated phosphatase eluted with an apparentM r of 63,000. After Mono S chromatography, the purified phosphatase contained a single M r63,000 band when analyzed by SDS-polyacrylamide gel electrophoresis and staining with silver (Fig. 1) or Coomassie Blue (data not shown). These results suggested that the lipid-stimulated phosphatase is a monomer of M r63,000 and that it is distinct from PP1 and PP2A, which are typically multisubunit enzymes containing catalytic subunits of 36–37 kDa (1Barton G.J. Cohen P.T.W. Barford D. Eur. J. Biochem. 1994; 220: 225-237Crossref PubMed Scopus (154) Google Scholar, 2Wera S. Hemmings B.A. Biochem. J. 1995; 311: 17-29Crossref PubMed Scopus (600) Google Scholar, 3Barford D. Trends Biochem. Sci. 1996; 21: 407-412Abstract Full Text PDF PubMed Scopus (313) Google Scholar). Four-hundred micrograms of pure phosphatase were obtained from 615 g of bovine brain (Table I). The pure enzyme exhibited a specific activity of 6 nmol of Pi released per min/mg of enzyme when assayed in the presence of 50 μm AA and 0.6 μm32P-casein for 15 min at 30 °C (TableI).Table IPurification of a fatty acid-stimulated protein-Ser/Thr phosphatase from bovine brainPurification stepTotal protein1-aProtein was estimated by the Bradford method (51).Total activity1-bPhosphatase activity was assayed toward 0.6 μm32P-casein in the presence of 50 μm arachidonic acid.Specific activityYield1-cYield and purification were compared to the DEAE pool since this was the first step at which the lipid-act" @default.
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• W2000757102 title "Purification of a Fatty Acid-stimulated Protein-serine/threonine Phosphatase from Bovine Brain and Its Identification as a Homolog of Protein Phosphatase 5" @default.
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