The Experts below are selected from a list of 213 Experts worldwide ranked by ideXlab platform
Anne Hiltner - One of the best experts on this subject based on the ideXlab platform.
-
enzymatic degradation of poly ether urethane and poly carbonate urethane by Cholesterol Esterase
Biomaterials, 2006Co-Authors: Elizabeth M. Christenson, Sachin Patel, James M Anderson, Anne HiltnerAbstract:Abstract This study examined the effect of Cholesterol Esterase (CE) on the degradation of commercial poly(ether urethane) (PEU) and poly(carbonate urethane) (PCU). Unstrained PEU and PCU films were incubated in 400 U/mL CE solution or a buffer control for 36 days. The study used a concentration of Cholesterol Esterase that was considerably higher than the estimated physiological level in order to accelerate degradation. However, characterization of treated polyurethane films with SEM, attenuated total reflectance Fourier transform infrared (ATR-FTIR) and GPC analysis revealed only a small loss in surface soft segment content. Comparison with implanted PEU and PCU films led to the conclusion that any effect of enzymatic hydrolysis was confined to the immediate surface, and the magnitude of the effect was too small to contribute significantly to in vivo degradation. The study confirmed that oxidation, rather than enzymatic hydrolysis, is the primary mechanism responsible for the observed biodegradation of PEU and PCU. The oxidative H 2 O 2 /CoCl 2 treatment continues to accurately predict the long-term biostability of polyurethanes.
-
Enzymatic degradation of poly(ether urethane) and poly(carbonate urethane) by Cholesterol Esterase
Biomaterials, 2006Co-Authors: Elizabeth M. Christenson, Sachin Patel, James M Anderson, Anne HiltnerAbstract:This study examined the effect of Cholesterol Esterase (CE) on the degradation of commercial poly(ether urethane) (PEU) and poly(carbonate urethane) (PCU). Unstrained PEU and PCU films were incubated in 400 U/mL CE solution or a buffer control for 36 days. The study used a concentration of Cholesterol Esterase that was considerably higher than the estimated physiological level in order to accelerate degradation. However, characterization of treated polyurethane films with SEM, attenuated total reflectance Fourier transform infrared (ATR-FTIR) and GPC analysis revealed only a small loss in surface soft segment content. Comparison with implanted PEU and PCU films led to the conclusion that any effect of enzymatic hydrolysis was confined to the immediate surface, and the magnitude of the effect was too small to contribute significantly to in vivo degradation. The study confirmed that oxidation, rather than enzymatic hydrolysis, is the primary mechanism responsible for the observed biodegradation of PEU and PCU. The oxidative H2O2/CoCl2 treatment continues to accurately predict the long-term biostability of polyurethanes. © 2006 Elsevier Ltd. All rights reserved.
Linda P Dipersio - One of the best experts on this subject based on the ideXlab platform.
-
exon 11 of the rat Cholesterol Esterase gene encodes domains important for intracellular processing and bile salt modulated activity of the protein
Biochemistry, 1994Co-Authors: Linda P Dipersio, Christopher P CarterAbstract:: The rat pancreatic Cholesterol Esterase is a 74,000 molecular weight protein encoded by a gene with 10 introns and 11 exons. The last exon of the Cholesterol Esterase gene is the largest and is also the least conserved exon among the Cholesterol Esterase genes of various species. The current study investigates the functional role of the exon 11 domain in rat Cholesterol Esterase. The transfection of native Cholesterol Esterase cDNA into COS cells resulted in an enzymatically active Cholesterol Esterase that was secreted by the cells. In contrast, transfection of Cholesterol Esterase cDNA with 88% of the exon 11 residues deleted from the sequence resulted in a protein that was not secreted by the cells. The Cholesterol Esterase with deletions in the exon 11 domain retained the ability to bind bile salt but was found to be enzymatically inactive. The inefficient secretion and the loss of enzyme activity for the truncated protein were not due to deletion of the proline-rich repeating units located in the exon 11 domain at the carboxyl terminus of the Cholesterol Esterase. The expression of rat Cholesterol Esterase with zero or one proline-rich units resulted in a truncated protein that was secreted by the transfected COS cells. The Cholesterol Esterases with reducing numbers of the proline-rich repeating units were also active in hydrolyzing p-nitrophenyl butyrate and cholesteryl oleate. The Cholesterol Esterase with fewer proline-rich repeating units were more active than the native enzyme in substrate hydrolysis at low bile salt concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)
-
aspartic acid 320 is required for optimal activity of rat pancreatic Cholesterol Esterase
Journal of Biological Chemistry, 1993Co-Authors: Linda P DipersioAbstract:Abstract The acidic amino acid residue required for the catalytic activity of rat pancreatic Cholesterol Esterase has been identified in this study by sequence comparison with other serine Esterases and by site-directed mutagenesis experiments. The sequence comparison studies identified 3 acidic residues in homologous domains between Cholesterol Esterase, acetylcholinEsterase, cholinEsterase, and Geotrichum candida lipase that may potentially be the catalytic acidic residue in these proteins. The role of Glu78, Asp79, and Asp320 in the catalytic activity of rat Cholesterol Esterase was then addressed by mutagenesis and expression of the cDNA. Results showed that replacement of Glu78 or Asp79 with alanine has no effect on the ability of the Cholesterol Esterase to hydrolyze the artificial water-soluble substrate p-nitrophenyl butyrate. In contrast, the Asp320-->Ala320 substitution abolished the enzyme activity of the Cholesterol Esterase. The specific requirement of Asp320 for optimal enzyme activity was demonstrated by substitution of the aspartic acid with glutamic acid, thus retaining the charge unit at this position. The Asp320-->Glu320 substitution resulted in an enzyme that displayed normal interaction with bile salt. However, catalytic activity of this mutagenized protein was reduced by approximately 50%. These results strongly suggested that aspartic acid 320 is an important component of the catalytic triad of pancreatic Cholesterol Esterase. The specific requirement of aspartic acid, instead of glutamic acid, for optimal activity is different from that of other members of the serine Esterase gene family.
-
Purification of pancreatic Cholesterol Esterase expressed in recombinant baculovirus-infected Sf9 cells.
Protein Expression and Purification, 1992Co-Authors: Linda P Dipersio, James A KisselAbstract:A cDNA clone encoding the entire coding sequence of rat pancreatic Cholesterol Esterase (bile salt-stimulated lipase) was subcloned into the Baculovirus transfer vector pVL1392 and used to co-transfect Spodoptera frugiperda (Sf9) insect cells with wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. Two recombinant proteins ( M r 74 kDa and 64 kDa) reactive with anti-Cholesterol Esterase IgG were produced and secreted by the infected Sf9 cells in large quantities in a time-dependent manner. The 74-kDa protein was detectable in the cultured medium at the second day post-infection and increased progressively, reaching a level of 50 μ g/ml of culture medium after 8 days. Amino-terminal sequencing of this recombinant protein showed that the signal peptide of Cholesterol Esterase was correctly cleaved, resulting in the production of mature protein. The 64-kDa recombinant protein was not detected in the medium until Day 5 post-infection and accumulated to a level of 25 μ g/ml at Day 8. Both the 74- and the 64-kDa Cholesterol Esterases were biologically active and hydrolyzed the artificial substrate p -nitrophenyl butyrate. Results of this study demonstrated that Baculovirus-infected Sf9 cells can be used for high-level expression of pancreatic Cholesterol Esterase. The recombinant enzyme will be useful for further characterization of this protein.
-
site specific mutagenesis of an essential histidine residue in pancreatic Cholesterol Esterase
Journal of Biological Chemistry, 1991Co-Authors: Linda P Dipersio, Robert N FontaineAbstract:Abstract The histidine residue essential for the catalytic activity of pancreatic Cholesterol Esterase (carboxylester lipase) has been identified in this study using sequence comparison and site-specific mutagenesis techniques. In the first approach, comparison of the primary structure of rat pancreatic Cholesterol Esterase with that of acetylcholinEsterase and cholinEsterase revealed two conserved histidine residues located at positions 420 and 435. The sequence in the region around histidine 420 is quite different between the three enzymes. However, histidine 435 is located in a 22-amino acid domain that is 47% homologous with other serine Esterases. Based on this sequence homology, it was hypothesized that histidine 435 is the histidine residue essential for catalytic activity of Cholesterol Esterase. The role of His435 in the catalytic activity of pancreatic Cholesterol Esterase was then studied by the site-specific mutagenesis technique. Substitution of the histidine in position 435 with glutamine, arginine, alanine, serine, or aspartic acid abolished the ability of Cholesterol Esterase to hydrolyze p-nitrophenyl butyrate and Cholesterol [14C]oleate. In contrast, mutagenesis of the histidine residue at position 420 to glutamine had no effect on Cholesterol Esterase enzyme activity. The results of this study strongly suggested that histidine 435 may be a component of the catalytic triad of pancreatic Cholesterol Esterase.
-
identification of the active site serine in pancreatic Cholesterol Esterase by chemical modification and site specific mutagenesis
Journal of Biological Chemistry, 1990Co-Authors: Linda P Dipersio, Robert N FontaineAbstract:Abstract Chemical modification and site-specific mutagenesis approaches were used in this study to identify the active site serine residue of pancreatic Cholesterol Esterase. In the first approach, purified porcine pancreatic Cholesterol Esterase was covalently modified by incubation with [3H]diisopropylfluorophosphate (DFP). The radiolabeled Cholesterol Esterase was digested with CNBr, and the peptides were separated by high performance liquid chromatography. A single 3H-containing peptide was obtained for sequence determination. The results revealed the binding of DFP to a serine residue within the serine Esterase homologous domain of the protein. Furthermore, the DFP-labeled serine was shown to correspond to serine residue 194 of rat Cholesterol Esterase (Kissel, J. A., Fontaine, R. N., Turck, C. W., Brockman, H. L., and Hui, D. Y. (1989) Biochim. Biophys. Acta 1006, 227-236). The codon for serine 194 in rat Cholesterol Esterase cDNA was then mutagenized to ACT or GCT to yield mutagenized Cholesterol Esterase with either threonine or alanine, instead of serine, at position 194. Expression of the mutagenized cDNA in COS-1 cells demonstrated that substitution of serine 194 with threonine or alanine abolished enzyme activity in hydrolyzing the water-soluble substrate, p-nitrophenyl butyrate, and the lipid substrates cholesteryl [14C]oleate and [14C] lysophosphatidylcholine. These studies definitively identified serine 194 in the catalytic site of pancreatic Cholesterol Esterase.
Robert N Fontaine - One of the best experts on this subject based on the ideXlab platform.
-
structure of the rat pancreatic Cholesterol Esterase gene
Biochemistry, 1991Co-Authors: Robert N Fontaine, Christopher P CarterAbstract:: The gene encoding the rat pancreatic Cholesterol Esterase has been isolated and characterized. Analysis of overlapping genomic clones showed that the Cholesterol Esterase gene spans approximately 8 kb, containing 11 exons interrupted by 10 introns. The exons ranged in size from 83 to 201 bp except for the last exon, which was 548 bp in length. A TAAATA sequence was present at -31 nucleotides from the transcriptional initiation site. A putative pancreas-specific enhancer sequence was found at -90 bp upstream from the CAP site. Although Cholesterol Esterase shares three domains of similarity with cholinEsterase and acetylcholinEsterase, these domains were found to be localized in distinct exons of the Cholesterol Esterase gene. The organization of the Cholesterol Esterase gene suggests its divergent evolution with other members of the serine Esterase gene family.
-
site specific mutagenesis of an essential histidine residue in pancreatic Cholesterol Esterase
Journal of Biological Chemistry, 1991Co-Authors: Linda P Dipersio, Robert N FontaineAbstract:Abstract The histidine residue essential for the catalytic activity of pancreatic Cholesterol Esterase (carboxylester lipase) has been identified in this study using sequence comparison and site-specific mutagenesis techniques. In the first approach, comparison of the primary structure of rat pancreatic Cholesterol Esterase with that of acetylcholinEsterase and cholinEsterase revealed two conserved histidine residues located at positions 420 and 435. The sequence in the region around histidine 420 is quite different between the three enzymes. However, histidine 435 is located in a 22-amino acid domain that is 47% homologous with other serine Esterases. Based on this sequence homology, it was hypothesized that histidine 435 is the histidine residue essential for catalytic activity of Cholesterol Esterase. The role of His435 in the catalytic activity of pancreatic Cholesterol Esterase was then studied by the site-specific mutagenesis technique. Substitution of the histidine in position 435 with glutamine, arginine, alanine, serine, or aspartic acid abolished the ability of Cholesterol Esterase to hydrolyze p-nitrophenyl butyrate and Cholesterol [14C]oleate. In contrast, mutagenesis of the histidine residue at position 420 to glutamine had no effect on Cholesterol Esterase enzyme activity. The results of this study strongly suggested that histidine 435 may be a component of the catalytic triad of pancreatic Cholesterol Esterase.
-
identification of the active site serine in pancreatic Cholesterol Esterase by chemical modification and site specific mutagenesis
Journal of Biological Chemistry, 1990Co-Authors: Linda P Dipersio, Robert N FontaineAbstract:Abstract Chemical modification and site-specific mutagenesis approaches were used in this study to identify the active site serine residue of pancreatic Cholesterol Esterase. In the first approach, purified porcine pancreatic Cholesterol Esterase was covalently modified by incubation with [3H]diisopropylfluorophosphate (DFP). The radiolabeled Cholesterol Esterase was digested with CNBr, and the peptides were separated by high performance liquid chromatography. A single 3H-containing peptide was obtained for sequence determination. The results revealed the binding of DFP to a serine residue within the serine Esterase homologous domain of the protein. Furthermore, the DFP-labeled serine was shown to correspond to serine residue 194 of rat Cholesterol Esterase (Kissel, J. A., Fontaine, R. N., Turck, C. W., Brockman, H. L., and Hui, D. Y. (1989) Biochim. Biophys. Acta 1006, 227-236). The codon for serine 194 in rat Cholesterol Esterase cDNA was then mutagenized to ACT or GCT to yield mutagenized Cholesterol Esterase with either threonine or alanine, instead of serine, at position 194. Expression of the mutagenized cDNA in COS-1 cells demonstrated that substitution of serine 194 with threonine or alanine abolished enzyme activity in hydrolyzing the water-soluble substrate, p-nitrophenyl butyrate, and the lipid substrates cholesteryl [14C]oleate and [14C] lysophosphatidylcholine. These studies definitively identified serine 194 in the catalytic site of pancreatic Cholesterol Esterase.
Elizabeth M. Christenson - One of the best experts on this subject based on the ideXlab platform.
-
enzymatic degradation of poly ether urethane and poly carbonate urethane by Cholesterol Esterase
Biomaterials, 2006Co-Authors: Elizabeth M. Christenson, Sachin Patel, James M Anderson, Anne HiltnerAbstract:Abstract This study examined the effect of Cholesterol Esterase (CE) on the degradation of commercial poly(ether urethane) (PEU) and poly(carbonate urethane) (PCU). Unstrained PEU and PCU films were incubated in 400 U/mL CE solution or a buffer control for 36 days. The study used a concentration of Cholesterol Esterase that was considerably higher than the estimated physiological level in order to accelerate degradation. However, characterization of treated polyurethane films with SEM, attenuated total reflectance Fourier transform infrared (ATR-FTIR) and GPC analysis revealed only a small loss in surface soft segment content. Comparison with implanted PEU and PCU films led to the conclusion that any effect of enzymatic hydrolysis was confined to the immediate surface, and the magnitude of the effect was too small to contribute significantly to in vivo degradation. The study confirmed that oxidation, rather than enzymatic hydrolysis, is the primary mechanism responsible for the observed biodegradation of PEU and PCU. The oxidative H 2 O 2 /CoCl 2 treatment continues to accurately predict the long-term biostability of polyurethanes.
-
Enzymatic degradation of poly(ether urethane) and poly(carbonate urethane) by Cholesterol Esterase
Biomaterials, 2006Co-Authors: Elizabeth M. Christenson, Sachin Patel, James M Anderson, Anne HiltnerAbstract:This study examined the effect of Cholesterol Esterase (CE) on the degradation of commercial poly(ether urethane) (PEU) and poly(carbonate urethane) (PCU). Unstrained PEU and PCU films were incubated in 400 U/mL CE solution or a buffer control for 36 days. The study used a concentration of Cholesterol Esterase that was considerably higher than the estimated physiological level in order to accelerate degradation. However, characterization of treated polyurethane films with SEM, attenuated total reflectance Fourier transform infrared (ATR-FTIR) and GPC analysis revealed only a small loss in surface soft segment content. Comparison with implanted PEU and PCU films led to the conclusion that any effect of enzymatic hydrolysis was confined to the immediate surface, and the magnitude of the effect was too small to contribute significantly to in vivo degradation. The study confirmed that oxidation, rather than enzymatic hydrolysis, is the primary mechanism responsible for the observed biodegradation of PEU and PCU. The oxidative H2O2/CoCl2 treatment continues to accurately predict the long-term biostability of polyurethanes. © 2006 Elsevier Ltd. All rights reserved.
James M Anderson - One of the best experts on this subject based on the ideXlab platform.
-
enzymatic degradation of poly ether urethane and poly carbonate urethane by Cholesterol Esterase
Biomaterials, 2006Co-Authors: Elizabeth M. Christenson, Sachin Patel, James M Anderson, Anne HiltnerAbstract:Abstract This study examined the effect of Cholesterol Esterase (CE) on the degradation of commercial poly(ether urethane) (PEU) and poly(carbonate urethane) (PCU). Unstrained PEU and PCU films were incubated in 400 U/mL CE solution or a buffer control for 36 days. The study used a concentration of Cholesterol Esterase that was considerably higher than the estimated physiological level in order to accelerate degradation. However, characterization of treated polyurethane films with SEM, attenuated total reflectance Fourier transform infrared (ATR-FTIR) and GPC analysis revealed only a small loss in surface soft segment content. Comparison with implanted PEU and PCU films led to the conclusion that any effect of enzymatic hydrolysis was confined to the immediate surface, and the magnitude of the effect was too small to contribute significantly to in vivo degradation. The study confirmed that oxidation, rather than enzymatic hydrolysis, is the primary mechanism responsible for the observed biodegradation of PEU and PCU. The oxidative H 2 O 2 /CoCl 2 treatment continues to accurately predict the long-term biostability of polyurethanes.
-
Enzymatic degradation of poly(ether urethane) and poly(carbonate urethane) by Cholesterol Esterase
Biomaterials, 2006Co-Authors: Elizabeth M. Christenson, Sachin Patel, James M Anderson, Anne HiltnerAbstract:This study examined the effect of Cholesterol Esterase (CE) on the degradation of commercial poly(ether urethane) (PEU) and poly(carbonate urethane) (PCU). Unstrained PEU and PCU films were incubated in 400 U/mL CE solution or a buffer control for 36 days. The study used a concentration of Cholesterol Esterase that was considerably higher than the estimated physiological level in order to accelerate degradation. However, characterization of treated polyurethane films with SEM, attenuated total reflectance Fourier transform infrared (ATR-FTIR) and GPC analysis revealed only a small loss in surface soft segment content. Comparison with implanted PEU and PCU films led to the conclusion that any effect of enzymatic hydrolysis was confined to the immediate surface, and the magnitude of the effect was too small to contribute significantly to in vivo degradation. The study confirmed that oxidation, rather than enzymatic hydrolysis, is the primary mechanism responsible for the observed biodegradation of PEU and PCU. The oxidative H2O2/CoCl2 treatment continues to accurately predict the long-term biostability of polyurethanes. © 2006 Elsevier Ltd. All rights reserved.