This suggests that these mutations resulted in a misfolded protein that was degraded intracellularly. the catalytic domain name of murine GPI-PLD resulted in three general phenotypes: not secreted or retained (His56 or His88), secreted with catalytic activity (His34, His81, His98 or His219) and secreted without catalytic activity (His29, His125, His133 or His158). Changing His133 but not His29, His125 or His158 to Cys resulted in a mutant that retained catalytic activity, suggesting that at least His133 is usually involved in Zn2+ binding. His133 and His158 also retained the biochemical properties of wild-type GPI-PLD including trypsin cleavage pattern and phosphorylation by protein kinase A. Hence, His29, His125, His133 and His158 are required for GPI-PLD catalytic activity. mutagenesis system (Promega, Madison, WI, U.S.A.). Histidine was mutated to asparagine because asparagine provides a polar amide group that does not participate in Zn2+ binding and was used to study the catalytic site of phosphatidylcholine phospholipase D [17,20]. All the mutations were verified by sequencing (Biochemistry and Biotechnology Facilities, Indiana University or college). Additional nucleotides that were not reported in our initial murine pancreatic GPI-PLD cDNA sequence due to a sequencing error were recognized [5]. These nucleotides corresponded to four additional amino acids (Ile, Glu, Gln and Gly) after Gly136 and matched those for the murine liver GPI-PLD reported by others [21,22]. Wild-type and mutated GPI-PLD cDNAs were subcloned into the expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA, U.S.A.) at the EcoRI and XbaI sites. COS-I cells (A.T.C.C., Rockville, MD, U.S.A.) (60?mm dishes) were transiently transfected with wild-type or each mutant (5?g of plasmid cDNA) using 20?g of Lipofectamine? (Invitrogen) in Opti-MEM? I (Reduced Serum Medium) according to the manufacturer’s methods. After 24?h, the medium was replaced with Dulbecco’s modified Eagle’s medium containing 100?mg/dl of glucose with 0.5?mg/ml of fatty acid-free BSA (SigmaCAldrich, St. Louis, MO, U.S.A.). After an additional 24?h, the medium was removed and centrifuged for 5?min (200? em g /em ) at 4?C to remove any suspended cells/debris. The cells were harvested and sonicated in ice-cold PBS made up of 0.1% (v/v) NP40, 1?mM benzamidine, 5?g/ml leupeptin, 0.2?mM PMSF and 5?g/ml aprotinin. Lysates were centrifuged (16000? em g /em ) at 4?C for 10?min. GPI-PLD activity was decided in both the medium and the cell lysates as described above except that this incubation time was increased to 1?h and the final NP40 concentration was 0.01% (v/v). Proteins in the medium were precipitated with ice-cold acetone and then separated by SDS/PAGE (7% polyacrylamide). GPI-PLD mass was analysed by Western blotting using anti-GPI-PLD771 antibody as previously described [12]. Characterization of GPI-PLD mutants To examine protein kinase A phosphorylation of wild-type and mutated GPI-PLD, conditioned media (10?ml) from transfected COS-I cells were concentrated approx.?25-fold using an Amicon Ultra (Millipore, Billerica, MA, U.S.A.) with a 100?kDa molecular mass cut-off and washed five times with 5?ml of 20?mM Tris (pH?7.5) and 50?mM NaCl. The 100?kDa cut-off was chosen to minimize the amount of BSA in the concentrate. The amount of GPI-PLD in the concentrated medium was estimated by Western blotting using purified murine serum GPI-PLD as the standard. Secreted GPI-PLD was phosphorylated by protein kinase A (Calbiochem, San Diego, CA, U.S.A.) using an equivalent amount of GPI-PLD from your conditioned medium of COS-I cells transfected with wild-type or mutated GPI-PLD as previously described [23]. Phosphorylated proteins were separated by SDS/PAGE (7% polyacrylamide) and visualized by autoradiography. Trypsin cleavage of secreted GPI-PLD was examined by using conditioned medium prepared as described above and was incubated with or without trypsin (4?g/ml for 15?min) as previously described [23]. Fragments were separated by SDS/PAGE (7C15% polyacrylamide) and fragments containing the C-terminal of GPI-PLD were identified with anti-GPI-PLD771 antibody by Western blotting as previously described [23]. RESULTS AND DISCUSSION Identification of histidine residues required for GPI-PLD catalytic activity As an initial approach to determine if histidine residue(s) play a role in GPI-PLD catalytic activity, we examined the effect of DEPC on GPI-PLD activity since DEPC modifies histidine and lysine residues. DEPC inhibited GPI-PLD Bleomycin hydrochloride catalytic activity in a concentration- and time-dependent manner (Figure 1). Modification of histidine but not lysine residues can be reversed by hydroxylamine [24]. To differentiate a histidine versus lysine residue modification, GPI-PLD was incubated with DEPC for 30?min and then incubated with or without hydroxylamine for 5C10?min. Hydroxylamine reversed DEPC inhibition of GPI-PLD catalytic activity (Figure 1B). These results suggest that a histidine modification accounts for the DEPC.This suggests that GPI-PLD utilizes a different catalytic mechanism although further experimentation is required. that retained catalytic activity, suggesting that at least His133 is involved in Zn2+ binding. His133 and His158 also retained the biochemical properties of wild-type GPI-PLD including trypsin cleavage pattern and phosphorylation by protein kinase A. Hence, His29, His125, His133 and His158 are required for GPI-PLD catalytic activity. mutagenesis system (Promega, Madison, WI, U.S.A.). Histidine was mutated to asparagine because asparagine provides a polar amide group that does not participate in Zn2+ binding and was used to study the catalytic site of phosphatidylcholine phospholipase D [17,20]. All the mutations were verified by sequencing (Biochemistry and Biotechnology Facilities, Indiana University). Additional nucleotides that were not reported in our original murine pancreatic GPI-PLD cDNA sequence due to a sequencing error were identified [5]. These nucleotides corresponded to four additional amino acids (Ile, Glu, Gln and Gly) after Gly136 and matched those for the murine liver GPI-PLD reported by others [21,22]. Wild-type and mutated GPI-PLD cDNAs were subcloned into the expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA, U.S.A.) at the EcoRI and XbaI sites. COS-I cells (A.T.C.C., Rockville, MD, U.S.A.) (60?mm dishes) were transiently transfected with wild-type or each mutant (5?g of plasmid cDNA) using 20?g of Lipofectamine? (Invitrogen) in Opti-MEM? I (Reduced Serum Medium) according to the manufacturer’s methods. After 24?h, the medium was replaced with Dulbecco’s modified Eagle’s medium containing 100?mg/dl of glucose with 0.5?mg/ml of fatty acid-free BSA (SigmaCAldrich, St. Louis, MO, U.S.A.). After an additional 24?h, the medium was removed and centrifuged for 5?min (200? em g /em ) at 4?C to remove any suspended cells/debris. The cells were harvested and sonicated in ice-cold PBS containing 0.1% (v/v) NP40, 1?mM benzamidine, 5?g/ml leupeptin, 0.2?mM PMSF and 5?g/ml aprotinin. Lysates were centrifuged (16000? em g /em ) at 4?C for 10?min. GPI-PLD activity was determined in both the medium and the cell lysates as described above except that this incubation time was increased to 1?h and the final NP40 concentration was 0.01% (v/v). Proteins in the medium were precipitated with ice-cold acetone and then separated by SDS/PAGE (7% polyacrylamide). GPI-PLD mass was analysed by Western blotting using anti-GPI-PLD771 antibody as previously described [12]. Characterization of GPI-PLD mutants To examine protein kinase A phosphorylation of wild-type and mutated GPI-PLD, conditioned media (10?ml) from transfected COS-I cells were concentrated approx.?25-fold using an Amicon Ultra (Millipore, Billerica, MA, U.S.A.) with a 100?kDa molecular mass cut-off and washed five times with 5?ml of 20?mM Tris (pH?7.5) and 50?mM NaCl. The 100?kDa cut-off was chosen to minimize the amount of BSA in the concentrate. The amount of GPI-PLD in the concentrated medium was estimated by Western blotting using purified murine serum GPI-PLD as the standard. Secreted GPI-PLD was phosphorylated by protein kinase A (Calbiochem, San Bleomycin hydrochloride Diego, CA, U.S.A.) using an equivalent amount of GPI-PLD from your conditioned medium of COS-I cells transfected with wild-type or mutated GPI-PLD as previously described [23]. Phosphorylated proteins were separated by SDS/PAGE (7% polyacrylamide) and visualized by autoradiography. Trypsin cleavage of secreted GPI-PLD was examined by using conditioned medium prepared as described above and was incubated with or without trypsin (4?g/ml for 15?min) as previously described [23]. Fragments were separated by SDS/PAGE (7C15% polyacrylamide) and fragments containing the C-terminal of GPI-PLD.Mutating His88 to Asn may interfere with this role for Glu85. This model also predicts the involvement of five histidine residues (His29, His88, His125, His133 and His158) in co-ordinating three bound Zn2+ atoms. histidine residues, inhibits GPI-PLD catalytic activity. Individual mutation of the ten histidine residues to asparagine in the catalytic domain name of murine GPI-PLD resulted in three general phenotypes: not secreted or retained (His56 or His88), secreted with catalytic activity (His34, His81, His98 or His219) and secreted without catalytic activity (His29, His125, His133 or His158). Changing His133 but not His29, His125 or His158 to Cys resulted in a mutant that retained catalytic activity, suggesting that at least His133 is usually involved in Zn2+ binding. His133 and His158 also retained the biochemical properties of wild-type GPI-PLD including trypsin cleavage pattern and phosphorylation by protein kinase A. Hence, His29, His125, His133 and His158 are required for GPI-PLD catalytic activity. mutagenesis system (Promega, Madison, WI, U.S.A.). Histidine was mutated to asparagine because asparagine provides a polar amide group that does not participate in Zn2+ binding and was used to study the catalytic site of phosphatidylcholine phospholipase D [17,20]. All the mutations were verified by sequencing (Biochemistry and Biotechnology Facilities, Indiana University or college). Additional nucleotides that were not reported in our initial murine pancreatic GPI-PLD cDNA sequence due to a sequencing error were recognized [5]. These nucleotides corresponded to four additional amino acids (Ile, Glu, Gln and Gly) after Gly136 and matched those for the murine liver organ GPI-PLD reported by others [21,22]. Wild-type and mutated GPI-PLD cDNAs had been subcloned in to the manifestation vector pcDNA3.1 (Invitrogen, Carlsbad, CA, U.S.A.) in the EcoRI and XbaI sites. COS-I cells (A.T.C.C., Rockville, MD, U.S.A.) (60?mm dishes) were transiently transfected with wild-type or each mutant (5?g of plasmid cDNA) using 20?g of Lipofectamine? (Invitrogen) in Opti-MEM? I (Reduced Serum Medium) based on the manufacturer’s methods. After 24?h, the medium was replaced with Dulbecco’s modified Eagle’s medium containing 100?mg/dl of glucose with 0.5?mg/ml of fatty acid-free BSA (SigmaCAldrich, St. Louis, MO, U.S.A.). After yet another 24?h, the medium was removed and centrifuged for Bleomycin hydrochloride 5?min (200? em g /em ) at 4?C to eliminate any suspended cells/debris. The cells were harvested and sonicated Vegfa in ice-cold PBS containing 0.1% (v/v) NP40, 1?mM benzamidine, 5?g/ml leupeptin, 0.2?mM PMSF and 5?g/ml aprotinin. Lysates were centrifuged (16000? em g /em ) at 4?C for 10?min. GPI-PLD activity was determined in both medium as well as the cell lysates as described above except how the incubation time was risen to 1?h and the ultimate NP40 concentration was 0.01% (v/v). Proteins in the medium were precipitated with ice-cold acetone and separated by SDS/PAGE (7% polyacrylamide). GPI-PLD mass was analysed by Western blotting using anti-GPI-PLD771 antibody as previously described [12]. Characterization of GPI-PLD mutants To examine protein kinase A phosphorylation of wild-type and mutated GPI-PLD, conditioned media (10?ml) from transfected COS-I cells were concentrated approx.?25-fold using an Amicon Ultra (Millipore, Billerica, MA, U.S.A.) having a 100?kDa molecular mass cut-off and washed five times with 5?ml of 20?mM Tris (pH?7.5) and 50?mM NaCl. The 100?kDa cut-off was chosen to reduce the quantity of BSA in the concentrate. The quantity of GPI-PLD in the concentrated medium was estimated by Western blotting using purified murine serum GPI-PLD as the typical. Secreted GPI-PLD was phosphorylated by protein kinase A (Calbiochem, NORTH PARK, CA, U.S.A.) using an equivalent amount of GPI-PLD through the conditioned medium of COS-I cells transfected with wild-type or mutated Bleomycin hydrochloride GPI-PLD as previously described [23]. Phosphorylated proteins were separated by SDS/PAGE (7% polyacrylamide) and visualized by autoradiography. Trypsin cleavage of secreted GPI-PLD was examined through the use of conditioned medium prepared as described above and was incubated with or without trypsin (4?g/ml for 15?min) as previously described [23]. Fragments were separated by SDS/PAGE (7C15% polyacrylamide) and fragments containing the C-terminal of GPI-PLD were.Since cysteine is involved with some Zn2+-binding sites, we hypothesize that if the histidine residue was involved with Zn2+ binding, mutating the residue to cysteine wouldn’t normally result in the increased loss of catalytic activity. not secreted or retained (His56 or His88), secreted with catalytic activity (His34, His81, His98 or His219) and secreted without catalytic activity (His29, His125, His133 or His158). Changing His133 however, not His29, His125 or His158 to Cys led to a mutant that retained catalytic activity, suggesting that at least His133 is involved with Zn2+ binding. His133 and His158 also retained the biochemical properties of wild-type GPI-PLD including trypsin cleavage pattern and phosphorylation by protein kinase A. Hence, His29, His125, His133 and His158 are necessary for GPI-PLD catalytic activity. mutagenesis system (Promega, Madison, WI, U.S.A.). Histidine was mutated to asparagine because asparagine offers a polar amide group that will not take part in Zn2+ binding and was used to review the catalytic site of phosphatidylcholine phospholipase D [17,20]. All of the mutations were verified by sequencing (Biochemistry and Biotechnology Facilities, Indiana University). Additional nucleotides which were not reported inside our original murine pancreatic GPI-PLD cDNA sequence because of a sequencing error were identified [5]. These nucleotides corresponded to four additional proteins (Ile, Glu, Gln and Gly) after Gly136 and matched those for the murine liver GPI-PLD reported by others [21,22]. Wild-type and mutated GPI-PLD cDNAs were subcloned in to the expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA, U.S.A.) in the EcoRI and XbaI sites. COS-I cells (A.T.C.C., Rockville, MD, U.S.A.) (60?mm dishes) were transiently transfected with wild-type or each mutant (5?g of plasmid cDNA) using 20?g of Lipofectamine? (Invitrogen) in Opti-MEM? I (Reduced Serum Medium) based on the manufacturer’s methods. After 24?h, the medium was replaced with Dulbecco’s modified Eagle’s medium containing 100?mg/dl of glucose with 0.5?mg/ml of fatty acid-free BSA (SigmaCAldrich, St. Louis, MO, U.S.A.). After yet another 24?h, the medium was removed and centrifuged for 5?min (200? em g /em ) at 4?C to eliminate any suspended cells/debris. The cells were harvested and sonicated in ice-cold PBS containing 0.1% (v/v) NP40, 1?mM benzamidine, 5?g/ml leupeptin, 0.2?mM PMSF and 5?g/ml aprotinin. Lysates were centrifuged (16000? em g /em ) at 4?C for 10?min. GPI-PLD activity was determined in both medium as well as the cell lysates as described above except how the incubation time was risen to 1?h and the ultimate NP40 concentration was 0.01% (v/v). Proteins in the medium were precipitated with ice-cold acetone and separated by SDS/PAGE (7% polyacrylamide). GPI-PLD mass was analysed by Western blotting using anti-GPI-PLD771 antibody as previously described [12]. Characterization of GPI-PLD mutants To examine protein kinase A phosphorylation of wild-type and mutated GPI-PLD, conditioned media (10?ml) from transfected COS-I cells were concentrated approx.?25-fold using an Amicon Ultra (Millipore, Billerica, MA, U.S.A.) having a 100?kDa molecular mass cut-off and washed five times with 5?ml of 20?mM Tris (pH?7.5) and 50?mM NaCl. The 100?kDa cut-off was chosen to reduce the quantity of BSA in the concentrate. The quantity of GPI-PLD in the concentrated medium was estimated by Western blotting using purified murine serum GPI-PLD as the typical. Secreted GPI-PLD was phosphorylated by protein kinase A (Calbiochem, NORTH PARK, CA, U.S.A.) using an equivalent amount of GPI-PLD through the conditioned medium of COS-I cells transfected with wild-type or mutated GPI-PLD as previously described [23]. Phosphorylated proteins were separated by SDS/PAGE (7% polyacrylamide) and visualized by autoradiography. Trypsin cleavage of secreted GPI-PLD was examined through the use of conditioned medium prepared as described above and was incubated with or without trypsin (4?g/ml for 15?min) as previously described [23]. Fragments were separated by SDS/PAGE (7C15% polyacrylamide) and fragments containing the C-terminal of GPI-PLD were identified with anti-GPI-PLD771 antibody by Western blotting as previously described [23]. RESULTS AND DISCUSSION Identification of histidine residues necessary for GPI-PLD catalytic activity As a short approach to see whether histidine residue(s) are likely involved in GPI-PLD catalytic activity, we examined the result of DEPC on GPI-PLD activity since DEPC modifies histidine and lysine residues. DEPC inhibited GPI-PLD catalytic activity inside a concentration- and time-dependent manner (Figure 1). Modification of histidine however, not lysine residues could be reversed by hydroxylamine [24]. To differentiate a histidine versus lysine residue modification, GPI-PLD was incubated with DEPC for 30?min and incubated with or without hydroxylamine for 5C10?min. Hydroxylamine reversed DEPC inhibition of GPI-PLD catalytic activity (Figure 1B). These total results.