Jeffrey Scholten - Academia.edu (original) (raw)
Papers by Jeffrey Scholten
Biochemistry Usa, Jul 1, 1992
The three genes encoding the 4-chlorobenzene dehalogenase polypeptides were excised from a Pseudo... more The three genes encoding the 4-chlorobenzene dehalogenase polypeptides were excised from a Pseudomonas sp. CBS-3 DNA fragment and separately cloned and expressed in Escherichia coli. The three enzymes were purified from the respective subclones by using an ammonium sulfate precipitation step followed by one or two column chromatographic steps. The 4-ch1orobenzoate:coenzyme A ligase was found to be a homcdimer (57-kDa subunit size), to require Mg2+ (Co2+ and Mn2+ are also activators) for activity, and to turn over MgATP (K, = 100 pM), coenzyme A (K, = 80 pM), and 4-chlorobenzoate ( K , = 9 pM) at a rate of 30 s-l at pH 7.5 and 25 O C . Benzoate, 4-bromobenzoate, 4-iodobenzoate, and 4-methylbenzoate were shown to be alternate substrates while 4-hydroxybenzoate, 4-aminobenzoate, 2-aminobenzoate, 2,3dihydroxybenzoate, 4-coumarate, palmate, laurate, caproate, butyrate, and phenylacetate were not substrate active. The 4-chlorobenzoate-coenzyme A dehalogenase was found to be a homotetramer (30 kDa subunit size) to have a K, = 15 pM and k,, = 0.3 s-l at pH 7.5 and 25 O C and to be catalytically inactive toward hydration of crotonyl-CoA, a-methylcrotonyl-CoA, and j3-methylcrotonyl-CoA. The 4-hydroxybenzoatecoenzyme A thioesterase was shown to be a homotetramer (16 kDa subunit size), to have a K , = 5 p M and k,,, = 7 s-l at pH 7.5 and 25 O C , and to also catalyze the hydrolyses of benzoyl-coenzyme A and I Abbreviations: 4-CBA, 4-chlorobenzoate, 4-HBA; 4-hydroxybenzoate; ATP, adenosine 5'4riphosphate; CoA, coenzyme A, NADH, dihydroniwtinamide adenine dinucleotide; Hepes, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid; Tris, tris(hydroxymethy1)aminomethane; NADP, nicotinamide adenine dinucleotide phosphate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IPTG, isopropyl @-D-thiogalactopyranoide; LMP, low-melting-point; LB, Luria-Bertani; DTT, dithiothreitol; PEP, phosphoenolpyruvate.
Protein Express Purif, 1998
Farnesyl:protein transferase (FPTase) catalyzes the transfer of a 15-carbon farnesyl isoprenoid g... more Farnesyl:protein transferase (FPTase) catalyzes the transfer of a 15-carbon farnesyl isoprenoid group from farnesyl diphosphate to the CaaX cysteine of a variety of cellular proteins. Since FPTase is a large (95-kDa) heterodimeric protein and is inactive unless the ␣and -subunits are coexpressed, large-scale overexpression of active enzyme has been challenging. We report the design of a translationally coupled expression system that will produce FPTase at levels as high as 30 mg/L Escherichia coli. Heterodimeric expression of FPTase was achieved using a translationally coupled operon from the T7 promoter of the pET23a (Novagen) expression plasmid. The -subunit-coding sequence was placed upstream of the ␣-subunit coding sequence linked by overlapping -subunit stop and ␣-subunit start codons. Additionally, the initial 88 codons of the ␣-subunit gene were altered, removing rare codons and replacing them with codons used in highly expressed proteins in E. coli. Since previous attempts at recombinantly expressing FPTase in E. coli from a translationally coupled system have demonstrated that initiation of translation of the ␣subunit is poor, we propose that the optimization of the codons at the start of the ␣-subunit gene leads to the observed high level of recombinant expression.
Biochemistry Usa, Feb 1, 1996
Cobalamin-dependent methionine synthase from Escherichia coli is a monomeric 136 kDa protein comp... more Cobalamin-dependent methionine synthase from Escherichia coli is a monomeric 136 kDa protein composed of multiple functional regions. The X-ray structure of the cobalamin-binding region of methionine synthase reveals that the cofactor is sandwiched between an R-helical domain that contacts the upper face of the cobalamin and an R/ (Rossmann) domain that interacts with the lower face. An unexpected conformational change accompanies binding of the methylcobalamin cofactor. The dimethylbenzimidazole ligand to the lower axial position of the cobalt in the free cofactor is displaced by histidine In order to facilitate studies of the roles of amino acid residues in the cobalamin-binding region of methionine synthase, we have constructed a synthetic module corresponding to nucleotides (nt) 1741-2668 in the metH gene and incorporated it into the wild-type metH gene. This module contains unique restriction sites at ∼80 base pair intervals and was synthesized by overlap extension of 22 synthetic oligonucleotides ranging in length from 70 to 105 nt and subsequent amplification using two sets of primers. Expression of methionine synthase from a plasmid containing the modified gene was shown to be unaffected by the introduction of the synthetic module. E. coli does not synthesize cobalamin, and overexpression of MetH holoenzyme requires accelerated cobalamin transport. Growth conditions are described that enable the production of holoenzyme rather than apoenzyme. We describe the construction and initial characterization of seven mutants. Four mutations (His759Gly, Asp757Glu, Asp757Asn, and Ser810Ala) alter residues in the hydrogen-bonded network His-Asp-Ser that connects the histidine ligand of the cobalt to solvent. Three mutations (Phe708Ala, Phe714Ala, and Leu715Ala) alter residues in the cap region that covers the upper face of the cobalamin. The His759Gly mutation has profound effects, essentially abolishing steady-state activity, while the Asp757, Ser810, Phe708, and Leu715 mutations lead to decreases in activity. These mutations assess the importance of individual residues in modulating cobalamin reactivity. †
Biochemistry Usa, Aug 1, 1990
In this paper the purification and characterization of the Tetrahymena pyriformis enzyme phosphoe... more In this paper the purification and characterization of the Tetrahymena pyriformis enzyme phosphoenolpyruvate phosphomutase are described. PEP phosphomutase was first fractionated from T.
J Biol Chem, 1997
Protein geranylgeranyltransferase-I (PGGT-I) and protein farnesyltransferase (PFT) attach geranyl... more Protein geranylgeranyltransferase-I (PGGT-I) and protein farnesyltransferase (PFT) attach geranylgeranyl and farnesyl groups, respectively, to the C termini of eukaryotic cell proteins. In vitro, PGGT-I and PFT can transfer both geranylgeranyl and farnesyl groups from geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP) to their protein or peptide prenyl acceptor substrates. In the present study it is shown that PGGT-I binds GGPP 330-fold tighter than FPP and that PFT binds FPP 15-fold tighter than GGPP. Therefore, in vivo, where both GGPP and FPP compete for the binding to prenyltransferases, PGGT-I and PFT will likely be bound predominantly to GGPP and FPP, respectively. Previous studies have shown that K-Ras4B and the Ras-related GTPase TC21 are substrates for both PGGT-I and PFT in vitro. It is shown that TC21 can compete with the C-terminal peptide of the gamma subunit of heterotrimeric G proteins and with the C-terminal peptide of lamin B for geranylgeranylation by PGGT-I and for farnesylation by PFT, respectively. K-Ras4B competes in both cases but is almost exclusively farnesylated by PFT in the presence of the lamin B peptide competitor. Rapid and single turnover kinetic studies indicate that the rate constant for the PGGT-I-catalyzed geranylgeranyl transfer step of the reaction cycle is 14-fold larger than the steady-state turnover number, which indicates that the rate of the overall reaction is limited by a step subsequent to prenyl transfer such as release of products from the enzyme. PGGT-I-catalyzed farnesylation is 37-fold slower than geranylgeranylation and is limited by the farnesyl transfer step. These results together with earlier studies provide a paradigm for the substrate specificity of PGGT-I and PFT and provide information that is critical for the design of prenyltransferase inhibitors as anti-cancer agents.
J Am Chem Soc, 1988
'All reactions performed in 50 mM KCI/DMSO (l: l), pH 8.8. Initial velocities de... more 'All reactions performed in 50 mM KCI/DMSO (l: l), pH 8.8. Initial velocities determined by Radiometer pH-Stat at four or more substrate concentrations. Enzyme concentration 29 pM. Parameters determined by Lineweaver-Burke plots, with correlation coefficients of 0.98 or ...
Methods in Enzymology, 1999
American Peptide Symposia, 2002
... Kevon R. Shuler, Daniele M. Leonard, Dennis J. McNamara, Jeffrey D. Scholten, Judith S. Sebol... more ... Kevon R. Shuler, Daniele M. Leonard, Dennis J. McNamara, Jeffrey D. Scholten, Judith S. Sebolt-Leopold, Annette M. Doherty Parke-Davis Pharmaceutical Research ... 4. Bolton, GL, Creswell, MW, Hodges, JC, Gowan, RC, Scholten, J., and Sebolt-Leopold, JS, (209th ACS Natl. ...
Science, 1991
Microbial enzyme systems may be used in the biodegradation of persistent environmental pollutants... more Microbial enzyme systems may be used in the biodegradation of persistent environmental pollutants. The three polypeptide components of one such system, the 4-chlorobenzoate dehalogenase system, have been isolated, and the chemical steps of the 4-hydroxybenzoate-forming reaction that they catalyze have been identified. The genes contained within a 4.5-kilobase Pseudomonas sp. strain CBS3 chromosomal DNA fragment that encode dehalogenase activity were selectively expressed in transformed Escherichia coli. Oligonucleotide sequencing revealed a stretch of homology between the 57-kilodalton (kD) polypeptide and several magnesium adenosine triphosphate (MgATP)-cleaving enzymes that allowed MgATP and coenzyme A (CoA) to be identified as the dehalogenase cosubstrate and cofactor, respectively. The dehalogenase activity arises from two components, a 4-chlorobenzoate:CoA ligase-dehalogenase (an alpha beta dimer of the 57- and 30-kD polypeptides) and a thioesterase (the 16-kD polypeptide).
Protein Expression and Purification, 1998
Farnesyl:protein transferase (FPTase) catalyzes the transfer of a 15-carbon farnesyl isoprenoid g... more Farnesyl:protein transferase (FPTase) catalyzes the transfer of a 15-carbon farnesyl isoprenoid group from farnesyl diphosphate to the CaaX cysteine of a variety of cellular proteins. Since FPTase is a large (95-kDa) heterodimeric protein and is inactive unless the ␣and -subunits are coexpressed, large-scale overexpression of active enzyme has been challenging. We report the design of a translationally coupled expression system that will produce FPTase at levels as high as 30 mg/L Escherichia coli. Heterodimeric expression of FPTase was achieved using a translationally coupled operon from the T7 promoter of the pET23a (Novagen) expression plasmid. The -subunit-coding sequence was placed upstream of the ␣-subunit coding sequence linked by overlapping -subunit stop and ␣-subunit start codons. Additionally, the initial 88 codons of the ␣-subunit gene were altered, removing rare codons and replacing them with codons used in highly expressed proteins in E. coli. Since previous attempts at recombinantly expressing FPTase in E. coli from a translationally coupled system have demonstrated that initiation of translation of the ␣subunit is poor, we propose that the optimization of the codons at the start of the ␣-subunit gene leads to the observed high level of recombinant expression.
Molecular Neurobiology, 1994
Inhibition of brain acetylcholinesterase (AChE) can provide relief from the cognitive loss associ... more Inhibition of brain acetylcholinesterase (AChE) can provide relief from the cognitive loss associated with Alzheimer's disease (AD). However, unwanted peripheral side effects often limit the usefulness of the available anticholinesterases. Recently, we identified a dihydroquinazoline compound, PD 142676 (CI 1002) that is a potent anticholinesterase and a functional muscarinic antagonist at higher concentrations. Peripherally, PD 142676, unlike other anticholinesterases, inhibits gastrointestinal motility in rats, an effect consistent with its muscarinic antagonist properties. Centrally, the compound acts as a cholinomimetic. In rats, PD 142676 decreases core body temperature. It also increases neocortical arousal, as measured by quantitative electroencephalography, and cortical acetylcholine levels, measured by in vivo microdialysis. The compound improves the performance of C57/B10j mice in a water maze task and of aged rhesus monkeys in a delayed match-to-sample task involving short-term memory. The combined effect of AChE inhibition and muscarinic antagonism distinguishes PD 142676 from other anticholinesterases, and may be useful in treating the cognitive dysfunction of AD and produce fewer peripheral side effects.
Journal of the American Chemical Society, 1988
'All reactions performed in 50 mM KCI/DMSO (l: l), pH 8.8. Initial velocities de... more 'All reactions performed in 50 mM KCI/DMSO (l: l), pH 8.8. Initial velocities determined by Radiometer pH-Stat at four or more substrate concentrations. Enzyme concentration 29 pM. Parameters determined by Lineweaver-Burke plots, with correlation coefficients of 0.98 or ...
Journal of the American Chemical Society, 1988
Journal of the American Chemical Society, 1995
Biochemistry Usa, Jul 1, 1992
The three genes encoding the 4-chlorobenzene dehalogenase polypeptides were excised from a Pseudo... more The three genes encoding the 4-chlorobenzene dehalogenase polypeptides were excised from a Pseudomonas sp. CBS-3 DNA fragment and separately cloned and expressed in Escherichia coli. The three enzymes were purified from the respective subclones by using an ammonium sulfate precipitation step followed by one or two column chromatographic steps. The 4-ch1orobenzoate:coenzyme A ligase was found to be a homcdimer (57-kDa subunit size), to require Mg2+ (Co2+ and Mn2+ are also activators) for activity, and to turn over MgATP (K, = 100 pM), coenzyme A (K, = 80 pM), and 4-chlorobenzoate ( K , = 9 pM) at a rate of 30 s-l at pH 7.5 and 25 O C . Benzoate, 4-bromobenzoate, 4-iodobenzoate, and 4-methylbenzoate were shown to be alternate substrates while 4-hydroxybenzoate, 4-aminobenzoate, 2-aminobenzoate, 2,3dihydroxybenzoate, 4-coumarate, palmate, laurate, caproate, butyrate, and phenylacetate were not substrate active. The 4-chlorobenzoate-coenzyme A dehalogenase was found to be a homotetramer (30 kDa subunit size) to have a K, = 15 pM and k,, = 0.3 s-l at pH 7.5 and 25 O C and to be catalytically inactive toward hydration of crotonyl-CoA, a-methylcrotonyl-CoA, and j3-methylcrotonyl-CoA. The 4-hydroxybenzoatecoenzyme A thioesterase was shown to be a homotetramer (16 kDa subunit size), to have a K , = 5 p M and k,,, = 7 s-l at pH 7.5 and 25 O C , and to also catalyze the hydrolyses of benzoyl-coenzyme A and I Abbreviations: 4-CBA, 4-chlorobenzoate, 4-HBA; 4-hydroxybenzoate; ATP, adenosine 5'4riphosphate; CoA, coenzyme A, NADH, dihydroniwtinamide adenine dinucleotide; Hepes, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid; Tris, tris(hydroxymethy1)aminomethane; NADP, nicotinamide adenine dinucleotide phosphate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IPTG, isopropyl @-D-thiogalactopyranoide; LMP, low-melting-point; LB, Luria-Bertani; DTT, dithiothreitol; PEP, phosphoenolpyruvate.
Protein Express Purif, 1998
Farnesyl:protein transferase (FPTase) catalyzes the transfer of a 15-carbon farnesyl isoprenoid g... more Farnesyl:protein transferase (FPTase) catalyzes the transfer of a 15-carbon farnesyl isoprenoid group from farnesyl diphosphate to the CaaX cysteine of a variety of cellular proteins. Since FPTase is a large (95-kDa) heterodimeric protein and is inactive unless the ␣and -subunits are coexpressed, large-scale overexpression of active enzyme has been challenging. We report the design of a translationally coupled expression system that will produce FPTase at levels as high as 30 mg/L Escherichia coli. Heterodimeric expression of FPTase was achieved using a translationally coupled operon from the T7 promoter of the pET23a (Novagen) expression plasmid. The -subunit-coding sequence was placed upstream of the ␣-subunit coding sequence linked by overlapping -subunit stop and ␣-subunit start codons. Additionally, the initial 88 codons of the ␣-subunit gene were altered, removing rare codons and replacing them with codons used in highly expressed proteins in E. coli. Since previous attempts at recombinantly expressing FPTase in E. coli from a translationally coupled system have demonstrated that initiation of translation of the ␣subunit is poor, we propose that the optimization of the codons at the start of the ␣-subunit gene leads to the observed high level of recombinant expression.
Biochemistry Usa, Feb 1, 1996
Cobalamin-dependent methionine synthase from Escherichia coli is a monomeric 136 kDa protein comp... more Cobalamin-dependent methionine synthase from Escherichia coli is a monomeric 136 kDa protein composed of multiple functional regions. The X-ray structure of the cobalamin-binding region of methionine synthase reveals that the cofactor is sandwiched between an R-helical domain that contacts the upper face of the cobalamin and an R/ (Rossmann) domain that interacts with the lower face. An unexpected conformational change accompanies binding of the methylcobalamin cofactor. The dimethylbenzimidazole ligand to the lower axial position of the cobalt in the free cofactor is displaced by histidine In order to facilitate studies of the roles of amino acid residues in the cobalamin-binding region of methionine synthase, we have constructed a synthetic module corresponding to nucleotides (nt) 1741-2668 in the metH gene and incorporated it into the wild-type metH gene. This module contains unique restriction sites at ∼80 base pair intervals and was synthesized by overlap extension of 22 synthetic oligonucleotides ranging in length from 70 to 105 nt and subsequent amplification using two sets of primers. Expression of methionine synthase from a plasmid containing the modified gene was shown to be unaffected by the introduction of the synthetic module. E. coli does not synthesize cobalamin, and overexpression of MetH holoenzyme requires accelerated cobalamin transport. Growth conditions are described that enable the production of holoenzyme rather than apoenzyme. We describe the construction and initial characterization of seven mutants. Four mutations (His759Gly, Asp757Glu, Asp757Asn, and Ser810Ala) alter residues in the hydrogen-bonded network His-Asp-Ser that connects the histidine ligand of the cobalt to solvent. Three mutations (Phe708Ala, Phe714Ala, and Leu715Ala) alter residues in the cap region that covers the upper face of the cobalamin. The His759Gly mutation has profound effects, essentially abolishing steady-state activity, while the Asp757, Ser810, Phe708, and Leu715 mutations lead to decreases in activity. These mutations assess the importance of individual residues in modulating cobalamin reactivity. †
Biochemistry Usa, Aug 1, 1990
In this paper the purification and characterization of the Tetrahymena pyriformis enzyme phosphoe... more In this paper the purification and characterization of the Tetrahymena pyriformis enzyme phosphoenolpyruvate phosphomutase are described. PEP phosphomutase was first fractionated from T.
J Biol Chem, 1997
Protein geranylgeranyltransferase-I (PGGT-I) and protein farnesyltransferase (PFT) attach geranyl... more Protein geranylgeranyltransferase-I (PGGT-I) and protein farnesyltransferase (PFT) attach geranylgeranyl and farnesyl groups, respectively, to the C termini of eukaryotic cell proteins. In vitro, PGGT-I and PFT can transfer both geranylgeranyl and farnesyl groups from geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP) to their protein or peptide prenyl acceptor substrates. In the present study it is shown that PGGT-I binds GGPP 330-fold tighter than FPP and that PFT binds FPP 15-fold tighter than GGPP. Therefore, in vivo, where both GGPP and FPP compete for the binding to prenyltransferases, PGGT-I and PFT will likely be bound predominantly to GGPP and FPP, respectively. Previous studies have shown that K-Ras4B and the Ras-related GTPase TC21 are substrates for both PGGT-I and PFT in vitro. It is shown that TC21 can compete with the C-terminal peptide of the gamma subunit of heterotrimeric G proteins and with the C-terminal peptide of lamin B for geranylgeranylation by PGGT-I and for farnesylation by PFT, respectively. K-Ras4B competes in both cases but is almost exclusively farnesylated by PFT in the presence of the lamin B peptide competitor. Rapid and single turnover kinetic studies indicate that the rate constant for the PGGT-I-catalyzed geranylgeranyl transfer step of the reaction cycle is 14-fold larger than the steady-state turnover number, which indicates that the rate of the overall reaction is limited by a step subsequent to prenyl transfer such as release of products from the enzyme. PGGT-I-catalyzed farnesylation is 37-fold slower than geranylgeranylation and is limited by the farnesyl transfer step. These results together with earlier studies provide a paradigm for the substrate specificity of PGGT-I and PFT and provide information that is critical for the design of prenyltransferase inhibitors as anti-cancer agents.
J Am Chem Soc, 1988
'All reactions performed in 50 mM KCI/DMSO (l: l), pH 8.8. Initial velocities de... more 'All reactions performed in 50 mM KCI/DMSO (l: l), pH 8.8. Initial velocities determined by Radiometer pH-Stat at four or more substrate concentrations. Enzyme concentration 29 pM. Parameters determined by Lineweaver-Burke plots, with correlation coefficients of 0.98 or ...
Methods in Enzymology, 1999
American Peptide Symposia, 2002
... Kevon R. Shuler, Daniele M. Leonard, Dennis J. McNamara, Jeffrey D. Scholten, Judith S. Sebol... more ... Kevon R. Shuler, Daniele M. Leonard, Dennis J. McNamara, Jeffrey D. Scholten, Judith S. Sebolt-Leopold, Annette M. Doherty Parke-Davis Pharmaceutical Research ... 4. Bolton, GL, Creswell, MW, Hodges, JC, Gowan, RC, Scholten, J., and Sebolt-Leopold, JS, (209th ACS Natl. ...
Science, 1991
Microbial enzyme systems may be used in the biodegradation of persistent environmental pollutants... more Microbial enzyme systems may be used in the biodegradation of persistent environmental pollutants. The three polypeptide components of one such system, the 4-chlorobenzoate dehalogenase system, have been isolated, and the chemical steps of the 4-hydroxybenzoate-forming reaction that they catalyze have been identified. The genes contained within a 4.5-kilobase Pseudomonas sp. strain CBS3 chromosomal DNA fragment that encode dehalogenase activity were selectively expressed in transformed Escherichia coli. Oligonucleotide sequencing revealed a stretch of homology between the 57-kilodalton (kD) polypeptide and several magnesium adenosine triphosphate (MgATP)-cleaving enzymes that allowed MgATP and coenzyme A (CoA) to be identified as the dehalogenase cosubstrate and cofactor, respectively. The dehalogenase activity arises from two components, a 4-chlorobenzoate:CoA ligase-dehalogenase (an alpha beta dimer of the 57- and 30-kD polypeptides) and a thioesterase (the 16-kD polypeptide).
Protein Expression and Purification, 1998
Farnesyl:protein transferase (FPTase) catalyzes the transfer of a 15-carbon farnesyl isoprenoid g... more Farnesyl:protein transferase (FPTase) catalyzes the transfer of a 15-carbon farnesyl isoprenoid group from farnesyl diphosphate to the CaaX cysteine of a variety of cellular proteins. Since FPTase is a large (95-kDa) heterodimeric protein and is inactive unless the ␣and -subunits are coexpressed, large-scale overexpression of active enzyme has been challenging. We report the design of a translationally coupled expression system that will produce FPTase at levels as high as 30 mg/L Escherichia coli. Heterodimeric expression of FPTase was achieved using a translationally coupled operon from the T7 promoter of the pET23a (Novagen) expression plasmid. The -subunit-coding sequence was placed upstream of the ␣-subunit coding sequence linked by overlapping -subunit stop and ␣-subunit start codons. Additionally, the initial 88 codons of the ␣-subunit gene were altered, removing rare codons and replacing them with codons used in highly expressed proteins in E. coli. Since previous attempts at recombinantly expressing FPTase in E. coli from a translationally coupled system have demonstrated that initiation of translation of the ␣subunit is poor, we propose that the optimization of the codons at the start of the ␣-subunit gene leads to the observed high level of recombinant expression.
Molecular Neurobiology, 1994
Inhibition of brain acetylcholinesterase (AChE) can provide relief from the cognitive loss associ... more Inhibition of brain acetylcholinesterase (AChE) can provide relief from the cognitive loss associated with Alzheimer's disease (AD). However, unwanted peripheral side effects often limit the usefulness of the available anticholinesterases. Recently, we identified a dihydroquinazoline compound, PD 142676 (CI 1002) that is a potent anticholinesterase and a functional muscarinic antagonist at higher concentrations. Peripherally, PD 142676, unlike other anticholinesterases, inhibits gastrointestinal motility in rats, an effect consistent with its muscarinic antagonist properties. Centrally, the compound acts as a cholinomimetic. In rats, PD 142676 decreases core body temperature. It also increases neocortical arousal, as measured by quantitative electroencephalography, and cortical acetylcholine levels, measured by in vivo microdialysis. The compound improves the performance of C57/B10j mice in a water maze task and of aged rhesus monkeys in a delayed match-to-sample task involving short-term memory. The combined effect of AChE inhibition and muscarinic antagonism distinguishes PD 142676 from other anticholinesterases, and may be useful in treating the cognitive dysfunction of AD and produce fewer peripheral side effects.
Journal of the American Chemical Society, 1988
'All reactions performed in 50 mM KCI/DMSO (l: l), pH 8.8. Initial velocities de... more 'All reactions performed in 50 mM KCI/DMSO (l: l), pH 8.8. Initial velocities determined by Radiometer pH-Stat at four or more substrate concentrations. Enzyme concentration 29 pM. Parameters determined by Lineweaver-Burke plots, with correlation coefficients of 0.98 or ...
Journal of the American Chemical Society, 1988
Journal of the American Chemical Society, 1995