Elizabeth Sutkowski - Academia.edu (original) (raw)

Papers by Elizabeth Sutkowski

Research paper thumbnail of Beta 1 subunits of sodium channels. Studies with subunit-specific antibodies

Journal of Biological Chemistry, 1990

The purified Na+ channel from rat brain consists of alpha (260 kDa), beta 1 (36 kDa), and beta 2 ... more The purified Na+ channel from rat brain consists of alpha (260 kDa), beta 1 (36 kDa), and beta 2 (33 kDa) subunits. Pure beta 1 subunits were prepared from purified rat brain Na+ channels which had been adsorbed to hydroxylapatite resin and used to prepare specific anti-beta 1 subunit antiserum. Antibodies purified from this antiserum by antigen affinity chromatography immunoprecipitate 125I-labeled, purified beta 1 subunits and purified Na+ channels (measured as high affinity [3H] saxitoxin binding sites) and recognize beta 1 subunits on immunoblots of solubilized rat brain membranes. The affinity-purified anti-beta 1 antibodies recognize beta 1 subunits in rat spinal cord, heart, skeletal muscle, and sciatic nerve, but do not detect immunoreactive beta 1 subunits in eel electroplax or eel brain. The developmental time course of expression of immunoreactive beta 1 subunits in rat forebrain was measured by immunoprecipitation followed by immunoblotting with affinity-purified anti-beta 1 antibodies. The amount of immunoreactive beta 1 subunits increased steadily to adult levels during the first 21 days of postnatal development.

Research paper thumbnail of Interaction of 7-bromoacetyl-7-desacetylforskolin, an alkylating derivative of forskolin, with bovine brain adenylyl cyclase and human erythrocyte glucose transporter

Biochemistry Usa, 1993

7-Bromoacetyl-7-desacetylforskolin (BrAcFsk), an alkylating derivative of forskolin, activated ad... more 7-Bromoacetyl-7-desacetylforskolin (BrAcFsk), an alkylating derivative of forskolin, activated adenylyl cyclase and irreversibly blocked high affinity forskolin binding sites in human platelet membranes and rat brain membranes (Laurenza et al., 1990). Photoincorporation of an iodinated arylazido derivative of forskolin, 1251-6-AIPP-Fsk, into adenylyl cyclase in bovine brain membranes was irreversibly inhibited by BrAcFsk but not by 1,9-dideoxy-BrAcFsk, suggesting that BrAcFsk was reacting specifically with a nucleophilic group(s) at the forskolin binding site of adenylyl cyclase. Immunoblotting with antiforskolin * To whom correspondence should be addressed: Food and Drug t FDA. NIH.

Research paper thumbnail of Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter

Journal of Biological Chemistry, Jul 15, 1991

Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6... more Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6-AIPP-Fsk) and N-(3-(4-azido-3-125I-phenyl)propionamide)-7-aminoethylcarbamyl-7- desacetylforskolin (125I-7-AIPP-Fsk) were synthesized with specific activities of 2200 Ci/mmol and used to label adenylyl cyclase and the glucose transporter. The affinities of the photolabels for adenylyl cyclase were determined by their inhibition of [3H]forskolin binding to bovine brain membranes. 6-AIPP-Fsk and 7-AIPP-Fsk inhibited [3H]forskolin binding with IC50 values of 15 nM and 200 nM, respectively. 125I-6-AIPP-Fsk labeled a 115-kDa protein in control and GTP gamma S-preactivated bovine brain membranes. This labeling was inhibited by forskolin but not by 1,9-dideoxyforskolin or cytochalasin B. 125I-6-AIPP-Fsk labeling of partially purified adenylyl cyclase was inhibited by forskolin but not by 1,9-dideoxyforskolin. 125I-7-AIPP-Fsk specifically labeled a 45-kDa protein and not a 115-kDa protein in control and GTP gamma S-preactivated brain membranes. This labeling was inhibited by forskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose but not cytochalasin E or L-glucose. Human erythrocyte membranes were photolyzed with 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk. 125I-7-AIPP-Fsk, but not 125I-6-AIPP-Fsk, strongly labeled a broad 45-70-kDa band. Forskolin, 7-bromoacetyl-7-desacetylforskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose, but not cytochalasin E or L-glucose, inhibited 125I-7-AIPP-Fsk labeling of the 45-70-kDa band. 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk are high affinity photolabels with specificity for adenylyl cyclase and the glucose transporter, respectively.

Research paper thumbnail of ChemInform Abstract: Synthesis and Evaluation of an 18F Analogue of Forskolin for Imaging Adenylyl Cyclase

Research paper thumbnail of Regulatory considerations in the nonclincal safety assessment of adjuvanted preventive vaccines

Immunopotentiators in Modern Vaccines, 2006

Research paper thumbnail of Photoaffinity labeling of adenylyl cyclase

Methods in enzymology, 2002

... of forskolin arid Ilion l~hotolabeled with 123~1 6 AIPP-Fsk. ShoV,'ll is the o,,elnight ... more ... of forskolin arid Ilion l~hotolabeled with 123~1 6 AIPP-Fsk. ShoV,'ll is the o,,elnight aulol-adiogram of the I()e/~ (w/v) SI)S-poryacrylamide gel The position of the cyclase is indicaled by Ihe arrow. [Reprinted from D. 1. Morris, ,1. D. Rohbins, AE Ruoho, EM Sulko\,~ski, :rod K. P ...

Research paper thumbnail of Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter

The Journal of biological chemistry, Jan 15, 1991

Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6... more Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6-AIPP-Fsk) and N-(3-(4-azido-3-125I-phenyl)propionamide)-7-aminoethylcarbamyl-7- desacetylforskolin (125I-7-AIPP-Fsk) were synthesized with specific activities of 2200 Ci/mmol and used to label adenylyl cyclase and the glucose transporter. The affinities of the photolabels for adenylyl cyclase were determined by their inhibition of [3H]forskolin binding to bovine brain membranes. 6-AIPP-Fsk and 7-AIPP-Fsk inhibited [3H]forskolin binding with IC50 values of 15 nM and 200 nM, respectively. 125I-6-AIPP-Fsk labeled a 115-kDa protein in control and GTP gamma S-preactivated bovine brain membranes. This labeling was inhibited by forskolin but not by 1,9-dideoxyforskolin or cytochalasin B. 125I-6-AIPP-Fsk labeling of partially purified adenylyl cyclase was inhibited by forskolin but not by 1,9-dideoxyforskolin. 125I-7-AIPP-Fsk specifically labeled a 45-kDa protein and not a 115-kDa protein in co...

Research paper thumbnail of Considerations in the development of live biotherapeutic products for clinical use

Current issues in molecular biology, 2008

Food products in the United States (U.S.), including dietary supplements, may contain live microo... more Food products in the United States (U.S.), including dietary supplements, may contain live microorganisms and can be promoted for general health, nutritional, or structure/function claims. In contrast, such preparations used with the intention of having a preventive or therapeutic effect in humans are regulated by the Food and Drug Administration (FDA) in the U.S. as biological products, specifically as live biotherapeutic products (LBPs). Discussion of considerations in the early development of LBPs may aid in preparation of an Investigational New Drug Application (IND) that is designed to collect clinical data to support marketing approval of a LBP in the U.S. for a specific clinical use. Product information is an important component of an IND to support a proposed clinical study.

Research paper thumbnail of Forskolin: a specific stimulator of adenylyl cyclase or a diterpene with multiple sites of action?

Trends in Pharmacological Sciences, 1989

Forskolin, a naturally occurring diterpene, directly stimulates adenylyl cyclase and has been use... more Forskolin, a naturally occurring diterpene, directly stimulates adenylyl cyclase and has been used extensively to increase cAMP and to elicit cAMP-dependent physiological responses. More recently, forskolin has been shown to inhibit a number of membrane transport proteins and channel proteins through a mechanism that does not involve the production of cAMP. Many of these channel proteins are predicted to have similar topographies in the membrane bilayer and it is tempting to speculate that forskolin may be binding at structurally homologous sites. Kenneth Seamon and colleagues discuss the cAMP-independent effects of forskolin and the structural similarity between forskolin and other physiologically important substances such as hexoses and steroids with respect to potential forskolin binding sites.

Research paper thumbnail of Synthesis and evaluation of an 18F analog of forskolin for imaging adenylyl cyclase

Journal of Fluorine Chemistry, 2000

We prepared two¯uorine containing carbamate analogs of forskolin as potential agents for imaging ... more We prepared two¯uorine containing carbamate analogs of forskolin as potential agents for imaging adenylyl cyclase in the living brain with positron emission tomography. Both compounds display high in vitro af®nity for adenylyl cyclase. The radiosynthesis of 7-(2-[ 18 F] uoroethyl) carbamoyl forskolin from an imidazolyl carbamate utilizes [ 18 F]¯uoroethyl amine, prepared by a novel method employing rapid deprotection and requiring no distillation. The brain uptake in rats and monkeys of 7-(2-[ 18 F]¯uoroethyl) carbamoyl forskolin is too low to be an effective imaging agent for this second messenger system.

Research paper thumbnail of Immunoprecipitation of adenylate cyclase with an antibody to a carboxyl-terminal peptide from Gs.alpha

Biochemistry, 1990

An antibody (RM) raised against the carboxyl-terminal decapeptide of the a subunit of the stimula... more An antibody (RM) raised against the carboxyl-terminal decapeptide of the a subunit of the stimulatory guanine nucleotide regulatory protein (Gsa) has been used to study the interaction of Gsa with bovine brain adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.11. R M antibody immunoprecipitated about 60% of the solubilized adenylate cyclase preactivated with either GTP-7-S or AlF4-. In contrast, RM antibody immunoprecipitated about 5% of the adenylate cyclase not preactivated (control) and 15% of the adenylate cyclase pretreated with forskolin. Adenylate cyclase solubilized from control membranes or GTP-y-S preactivated membranes was partially purified by using forskolin-agarose affinity chromatography. The amount of Gsa protein in the partially purified preparations was determined by immunoblotting with R M antibody. There was 3-fold more G s a detected in partially purified adenylate cyclase from preactivated membranes than in the partially purified adenylate cyclase from control membranes. Partially purified adenylate cyclase from preactivated membranes was immunoprecipitated with R M antibody and the amount of adenylate cyclase activity immunoprecipitated (65% of total) corresponded to the amount of Gscu protein immunoprecipitated. Only 15% of the partially purified adenylate cyclase from control membranes was immunoprecipitated. The presence of other G proteins in the partially purified preparations of adenylate cyclase was investigated by using specific antisera that detect Goa, Gia, and GO. The GO protein was the only subunit detected in the partially purified preparations of adenylate cyclase and the amount of GP was about the same in adenylate cyclase from preactivated membranes and from control membranes. Examination of the R M antibody immunoprecipitates from control and GTP-7-S preactivated solubilized membranes failed to detect any of these G proteins. These results indicate that the complex between the adenylate cyclase catalytic subunit and the activated Gsa protein can be isolated by immunoprecipitation with an anti-Gsa antibody. There does not appear to be a specific association of Goa, Gia, or GP with the preactivated complex of the catalytic subunit and the activated Gsa subunit. Hormone-sensit ive adenylate cyclase is regulated by the interaction of hormone receptors with specific guanine nucleotide binding proteins (G proteins), which transduce the hormone signal to the adenylate cyclase catalytic subunit (Gilman, 1987; Spiegel, 1987). G proteins are heterotrimeric proteins consisting of a, 0, and y subunits. The a subunits

Research paper thumbnail of Interaction of 7-bromoacetyl-7-desacetylforskolin, an alkylating derivative of forskolin, with bovine brain adenylyl cyclase and human erythrocyte glucose transporter

Biochemistry, 1993

7-Bromoacetyl-7-desacetylforskolin (BrAcFsk), an alkylating derivative of forskolin, activated ad... more 7-Bromoacetyl-7-desacetylforskolin (BrAcFsk), an alkylating derivative of forskolin, activated adenylyl cyclase and irreversibly blocked high affinity forskolin binding sites in human platelet membranes and rat brain membranes (Laurenza et al., 1990). Photoincorporation of an iodinated arylazido derivative of forskolin, 1251-6-AIPP-Fsk, into adenylyl cyclase in bovine brain membranes was irreversibly inhibited by BrAcFsk but not by 1,9-dideoxy-BrAcFsk, suggesting that BrAcFsk was reacting specifically with a nucleophilic group(s) at the forskolin binding site of adenylyl cyclase. Immunoblotting with antiforskolin * To whom correspondence should be addressed: Food and Drug t FDA. NIH.

Research paper thumbnail of Regulation of forskolin interactions with type I, II, V, and VI adenylyl cyclases by Gs.alpha

Research paper thumbnail of Synthesis and Evaluation of NovelE-2-(2-Thienyl)- andZ-2-(3-Thienyl)-3-Arylacrylonitriles as Antifungal and Anticancer Agents

Archiv der Pharmazie, 2007

A series of 3-aryl-2-(2-thienyl)acrylonitriles 7 and 3-aryl-2-(3-thienyl)acrylonitriles 8 were sy... more A series of 3-aryl-2-(2-thienyl)acrylonitriles 7 and 3-aryl-2-(3-thienyl)acrylonitriles 8 were synthesized by the reaction of aromatic aldehydes 6 with 2- and 3-thienylacetonitriles 4 and 5, and evaluated for antifungal and cytotoxic activities against a panel of opportunistic and pathogenic fungi and three different cancer cell lines, respectively.

Research paper thumbnail of Forskolin: a specific stimulator of adenylyl cyclase or a diterpene with multiple sites of action?

Trends in Pharmacological Sciences, 1989

Forskolin, a naturally occurring diterpene, directly stimulates adenylyl cyclase and has been use... more Forskolin, a naturally occurring diterpene, directly stimulates adenylyl cyclase and has been used extensively to increase cAMP and to elicit cAMP-dependent physiological responses. More recently, forskolin has been shown to inhibit a number of membrane transport proteins and channel proteins through a mechanism that does not involve the production of cAMP. Many of these channel proteins are predicted to have similar topographies in the membrane bilayer and it is tempting to speculate that forskolin may be binding at structurally homologous sites. Kenneth Seamon and colleagues discuss the cAMP-independent effects of forskolin and the structural similarity between forskolin and other physiologically important substances such as hexoses and steroids with respect to potential forskolin binding sites.

Research paper thumbnail of Beta 1 subunits of sodium channels. Studies with subunit-specific antibodies

Journal of Biological Chemistry, 1990

The purified Na+ channel from rat brain consists of alpha (260 kDa), beta 1 (36 kDa), and beta 2 ... more The purified Na+ channel from rat brain consists of alpha (260 kDa), beta 1 (36 kDa), and beta 2 (33 kDa) subunits. Pure beta 1 subunits were prepared from purified rat brain Na+ channels which had been adsorbed to hydroxylapatite resin and used to prepare specific anti-beta 1 subunit antiserum. Antibodies purified from this antiserum by antigen affinity chromatography immunoprecipitate 125I-labeled, purified beta 1 subunits and purified Na+ channels (measured as high affinity [3H] saxitoxin binding sites) and recognize beta 1 subunits on immunoblots of solubilized rat brain membranes. The affinity-purified anti-beta 1 antibodies recognize beta 1 subunits in rat spinal cord, heart, skeletal muscle, and sciatic nerve, but do not detect immunoreactive beta 1 subunits in eel electroplax or eel brain. The developmental time course of expression of immunoreactive beta 1 subunits in rat forebrain was measured by immunoprecipitation followed by immunoblotting with affinity-purified anti-beta 1 antibodies. The amount of immunoreactive beta 1 subunits increased steadily to adult levels during the first 21 days of postnatal development.

Research paper thumbnail of Interaction of 7-bromoacetyl-7-desacetylforskolin, an alkylating derivative of forskolin, with bovine brain adenylyl cyclase and human erythrocyte glucose transporter

Biochemistry Usa, 1993

7-Bromoacetyl-7-desacetylforskolin (BrAcFsk), an alkylating derivative of forskolin, activated ad... more 7-Bromoacetyl-7-desacetylforskolin (BrAcFsk), an alkylating derivative of forskolin, activated adenylyl cyclase and irreversibly blocked high affinity forskolin binding sites in human platelet membranes and rat brain membranes (Laurenza et al., 1990). Photoincorporation of an iodinated arylazido derivative of forskolin, 1251-6-AIPP-Fsk, into adenylyl cyclase in bovine brain membranes was irreversibly inhibited by BrAcFsk but not by 1,9-dideoxy-BrAcFsk, suggesting that BrAcFsk was reacting specifically with a nucleophilic group(s) at the forskolin binding site of adenylyl cyclase. Immunoblotting with antiforskolin * To whom correspondence should be addressed: Food and Drug t FDA. NIH.

Research paper thumbnail of Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter

Journal of Biological Chemistry, Jul 15, 1991

Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6... more Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6-AIPP-Fsk) and N-(3-(4-azido-3-125I-phenyl)propionamide)-7-aminoethylcarbamyl-7- desacetylforskolin (125I-7-AIPP-Fsk) were synthesized with specific activities of 2200 Ci/mmol and used to label adenylyl cyclase and the glucose transporter. The affinities of the photolabels for adenylyl cyclase were determined by their inhibition of [3H]forskolin binding to bovine brain membranes. 6-AIPP-Fsk and 7-AIPP-Fsk inhibited [3H]forskolin binding with IC50 values of 15 nM and 200 nM, respectively. 125I-6-AIPP-Fsk labeled a 115-kDa protein in control and GTP gamma S-preactivated bovine brain membranes. This labeling was inhibited by forskolin but not by 1,9-dideoxyforskolin or cytochalasin B. 125I-6-AIPP-Fsk labeling of partially purified adenylyl cyclase was inhibited by forskolin but not by 1,9-dideoxyforskolin. 125I-7-AIPP-Fsk specifically labeled a 45-kDa protein and not a 115-kDa protein in control and GTP gamma S-preactivated brain membranes. This labeling was inhibited by forskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose but not cytochalasin E or L-glucose. Human erythrocyte membranes were photolyzed with 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk. 125I-7-AIPP-Fsk, but not 125I-6-AIPP-Fsk, strongly labeled a broad 45-70-kDa band. Forskolin, 7-bromoacetyl-7-desacetylforskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose, but not cytochalasin E or L-glucose, inhibited 125I-7-AIPP-Fsk labeling of the 45-70-kDa band. 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk are high affinity photolabels with specificity for adenylyl cyclase and the glucose transporter, respectively.

Research paper thumbnail of ChemInform Abstract: Synthesis and Evaluation of an 18F Analogue of Forskolin for Imaging Adenylyl Cyclase

Research paper thumbnail of Regulatory considerations in the nonclincal safety assessment of adjuvanted preventive vaccines

Immunopotentiators in Modern Vaccines, 2006

Research paper thumbnail of Photoaffinity labeling of adenylyl cyclase

Methods in enzymology, 2002

... of forskolin arid Ilion l~hotolabeled with 123~1 6 AIPP-Fsk. ShoV,'ll is the o,,elnight ... more ... of forskolin arid Ilion l~hotolabeled with 123~1 6 AIPP-Fsk. ShoV,'ll is the o,,elnight aulol-adiogram of the I()e/~ (w/v) SI)S-poryacrylamide gel The position of the cyclase is indicaled by Ihe arrow. [Reprinted from D. 1. Morris, ,1. D. Rohbins, AE Ruoho, EM Sulko\,~ski, :rod K. P ...

Research paper thumbnail of Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter

The Journal of biological chemistry, Jan 15, 1991

Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6... more Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6-AIPP-Fsk) and N-(3-(4-azido-3-125I-phenyl)propionamide)-7-aminoethylcarbamyl-7- desacetylforskolin (125I-7-AIPP-Fsk) were synthesized with specific activities of 2200 Ci/mmol and used to label adenylyl cyclase and the glucose transporter. The affinities of the photolabels for adenylyl cyclase were determined by their inhibition of [3H]forskolin binding to bovine brain membranes. 6-AIPP-Fsk and 7-AIPP-Fsk inhibited [3H]forskolin binding with IC50 values of 15 nM and 200 nM, respectively. 125I-6-AIPP-Fsk labeled a 115-kDa protein in control and GTP gamma S-preactivated bovine brain membranes. This labeling was inhibited by forskolin but not by 1,9-dideoxyforskolin or cytochalasin B. 125I-6-AIPP-Fsk labeling of partially purified adenylyl cyclase was inhibited by forskolin but not by 1,9-dideoxyforskolin. 125I-7-AIPP-Fsk specifically labeled a 45-kDa protein and not a 115-kDa protein in co...

Research paper thumbnail of Considerations in the development of live biotherapeutic products for clinical use

Current issues in molecular biology, 2008

Food products in the United States (U.S.), including dietary supplements, may contain live microo... more Food products in the United States (U.S.), including dietary supplements, may contain live microorganisms and can be promoted for general health, nutritional, or structure/function claims. In contrast, such preparations used with the intention of having a preventive or therapeutic effect in humans are regulated by the Food and Drug Administration (FDA) in the U.S. as biological products, specifically as live biotherapeutic products (LBPs). Discussion of considerations in the early development of LBPs may aid in preparation of an Investigational New Drug Application (IND) that is designed to collect clinical data to support marketing approval of a LBP in the U.S. for a specific clinical use. Product information is an important component of an IND to support a proposed clinical study.

Research paper thumbnail of Forskolin: a specific stimulator of adenylyl cyclase or a diterpene with multiple sites of action?

Trends in Pharmacological Sciences, 1989

Forskolin, a naturally occurring diterpene, directly stimulates adenylyl cyclase and has been use... more Forskolin, a naturally occurring diterpene, directly stimulates adenylyl cyclase and has been used extensively to increase cAMP and to elicit cAMP-dependent physiological responses. More recently, forskolin has been shown to inhibit a number of membrane transport proteins and channel proteins through a mechanism that does not involve the production of cAMP. Many of these channel proteins are predicted to have similar topographies in the membrane bilayer and it is tempting to speculate that forskolin may be binding at structurally homologous sites. Kenneth Seamon and colleagues discuss the cAMP-independent effects of forskolin and the structural similarity between forskolin and other physiologically important substances such as hexoses and steroids with respect to potential forskolin binding sites.

Research paper thumbnail of Synthesis and evaluation of an 18F analog of forskolin for imaging adenylyl cyclase

Journal of Fluorine Chemistry, 2000

We prepared two¯uorine containing carbamate analogs of forskolin as potential agents for imaging ... more We prepared two¯uorine containing carbamate analogs of forskolin as potential agents for imaging adenylyl cyclase in the living brain with positron emission tomography. Both compounds display high in vitro af®nity for adenylyl cyclase. The radiosynthesis of 7-(2-[ 18 F] uoroethyl) carbamoyl forskolin from an imidazolyl carbamate utilizes [ 18 F]¯uoroethyl amine, prepared by a novel method employing rapid deprotection and requiring no distillation. The brain uptake in rats and monkeys of 7-(2-[ 18 F]¯uoroethyl) carbamoyl forskolin is too low to be an effective imaging agent for this second messenger system.

Research paper thumbnail of Immunoprecipitation of adenylate cyclase with an antibody to a carboxyl-terminal peptide from Gs.alpha

Biochemistry, 1990

An antibody (RM) raised against the carboxyl-terminal decapeptide of the a subunit of the stimula... more An antibody (RM) raised against the carboxyl-terminal decapeptide of the a subunit of the stimulatory guanine nucleotide regulatory protein (Gsa) has been used to study the interaction of Gsa with bovine brain adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.11. R M antibody immunoprecipitated about 60% of the solubilized adenylate cyclase preactivated with either GTP-7-S or AlF4-. In contrast, RM antibody immunoprecipitated about 5% of the adenylate cyclase not preactivated (control) and 15% of the adenylate cyclase pretreated with forskolin. Adenylate cyclase solubilized from control membranes or GTP-y-S preactivated membranes was partially purified by using forskolin-agarose affinity chromatography. The amount of Gsa protein in the partially purified preparations was determined by immunoblotting with R M antibody. There was 3-fold more G s a detected in partially purified adenylate cyclase from preactivated membranes than in the partially purified adenylate cyclase from control membranes. Partially purified adenylate cyclase from preactivated membranes was immunoprecipitated with R M antibody and the amount of adenylate cyclase activity immunoprecipitated (65% of total) corresponded to the amount of Gscu protein immunoprecipitated. Only 15% of the partially purified adenylate cyclase from control membranes was immunoprecipitated. The presence of other G proteins in the partially purified preparations of adenylate cyclase was investigated by using specific antisera that detect Goa, Gia, and GO. The GO protein was the only subunit detected in the partially purified preparations of adenylate cyclase and the amount of GP was about the same in adenylate cyclase from preactivated membranes and from control membranes. Examination of the R M antibody immunoprecipitates from control and GTP-7-S preactivated solubilized membranes failed to detect any of these G proteins. These results indicate that the complex between the adenylate cyclase catalytic subunit and the activated Gsa protein can be isolated by immunoprecipitation with an anti-Gsa antibody. There does not appear to be a specific association of Goa, Gia, or GP with the preactivated complex of the catalytic subunit and the activated Gsa subunit. Hormone-sensit ive adenylate cyclase is regulated by the interaction of hormone receptors with specific guanine nucleotide binding proteins (G proteins), which transduce the hormone signal to the adenylate cyclase catalytic subunit (Gilman, 1987; Spiegel, 1987). G proteins are heterotrimeric proteins consisting of a, 0, and y subunits. The a subunits

Research paper thumbnail of Interaction of 7-bromoacetyl-7-desacetylforskolin, an alkylating derivative of forskolin, with bovine brain adenylyl cyclase and human erythrocyte glucose transporter

Biochemistry, 1993

7-Bromoacetyl-7-desacetylforskolin (BrAcFsk), an alkylating derivative of forskolin, activated ad... more 7-Bromoacetyl-7-desacetylforskolin (BrAcFsk), an alkylating derivative of forskolin, activated adenylyl cyclase and irreversibly blocked high affinity forskolin binding sites in human platelet membranes and rat brain membranes (Laurenza et al., 1990). Photoincorporation of an iodinated arylazido derivative of forskolin, 1251-6-AIPP-Fsk, into adenylyl cyclase in bovine brain membranes was irreversibly inhibited by BrAcFsk but not by 1,9-dideoxy-BrAcFsk, suggesting that BrAcFsk was reacting specifically with a nucleophilic group(s) at the forskolin binding site of adenylyl cyclase. Immunoblotting with antiforskolin * To whom correspondence should be addressed: Food and Drug t FDA. NIH.

Research paper thumbnail of Regulation of forskolin interactions with type I, II, V, and VI adenylyl cyclases by Gs.alpha

Research paper thumbnail of Synthesis and Evaluation of NovelE-2-(2-Thienyl)- andZ-2-(3-Thienyl)-3-Arylacrylonitriles as Antifungal and Anticancer Agents

Archiv der Pharmazie, 2007

A series of 3-aryl-2-(2-thienyl)acrylonitriles 7 and 3-aryl-2-(3-thienyl)acrylonitriles 8 were sy... more A series of 3-aryl-2-(2-thienyl)acrylonitriles 7 and 3-aryl-2-(3-thienyl)acrylonitriles 8 were synthesized by the reaction of aromatic aldehydes 6 with 2- and 3-thienylacetonitriles 4 and 5, and evaluated for antifungal and cytotoxic activities against a panel of opportunistic and pathogenic fungi and three different cancer cell lines, respectively.

Research paper thumbnail of Forskolin: a specific stimulator of adenylyl cyclase or a diterpene with multiple sites of action?

Trends in Pharmacological Sciences, 1989

Forskolin, a naturally occurring diterpene, directly stimulates adenylyl cyclase and has been use... more Forskolin, a naturally occurring diterpene, directly stimulates adenylyl cyclase and has been used extensively to increase cAMP and to elicit cAMP-dependent physiological responses. More recently, forskolin has been shown to inhibit a number of membrane transport proteins and channel proteins through a mechanism that does not involve the production of cAMP. Many of these channel proteins are predicted to have similar topographies in the membrane bilayer and it is tempting to speculate that forskolin may be binding at structurally homologous sites. Kenneth Seamon and colleagues discuss the cAMP-independent effects of forskolin and the structural similarity between forskolin and other physiologically important substances such as hexoses and steroids with respect to potential forskolin binding sites.