The design, synthesis and biological data are presented for novel opioid peptides, which act as p... more The design, synthesis and biological data are presented for novel opioid peptides, which act as pure, delta receptor antagonists and contain an 'opioid' nitrogen which cannot exist in protonated form while interacting with the receptor. The K, values of Boc-Tyr-Pro-Gly-Phe-Leu, Boc-Tyr-Pro-Gly-Phe-Leu-Thr, Boc-Tyr-Pro-Gly-Phe-Leu-Thr (tBu) and phenylacetyl-or diphenylacetyl-Tyr-Pro-Gly-Phe-Lou-Thr determined against [Mets]enkephalin or DADLE in the mouse vas deferens bioassay fall into the l&'-4 x 10" M range, with a 50-350 fold delta over mu receptor selectivity. Displacement data in the receptor binding assay in rat brain membranes against 'H-DALE, 'H-DSLET, 3H-DAG0 and 3H-DHM matched the results of bioassay. Since not only the Nblocked derivatives but even the ones with a free N terminus were devoid of significant opioid agonist activity, it is suggested that these novel derivatives do not interact with the conventional 'opioid' nitrogen binding site although the hydrophobic binding site involved must be located in a reasonable vicinity. Conformational analyses of peptides appear to support this assumption.
ual challenge due to the complexity of the samples. A single chromatogram in which all the compon... more ual challenge due to the complexity of the samples. A single chromatogram in which all the components of such a complex mixture are represented by single peaks that are well resolved from each other would be ideal. The complexity of samples entails differences in size, charge, hydrophobicity, and chemical variety of the components. Tablet formulations generally contain three different groups of chemicals: small organic molecules, polymers that are controlled-release agents and binding materials, and solid particulates. Table 1 lists the most commonly used formulation materials, but space does not permit a listing of all possible components. The commercially available formulations comprise an astronomical variety of components and only a general categorization is possible here. One of the difficulties of the analytical chemist is the discretion on the part of the suppliers. The excipient supplier companies use only a few basic polymers, such as polyethylene glycol (PEG), polyvinylpyr...
Proceedings of The National Academy of Sciences, 1988
The aspartic residue (Asp-189) at the base of the substrate-binding pocket of trypsin was replace... more The aspartic residue (Asp-189) at the base of the substrate-binding pocket of trypsin was replaced by serine (present in a similar position in chymotrypsin) through site-directed mutagenesis. The wild-type (with Asp-189 in the mature trypsin sequence) and mutant (Ser-189) trypsinogens were expressed in Escherichia coli, purified to homogeneity, activated by enterokinase, and tested with a series of fluorogenic tetrapeptide substrates
The captopril-inhibited enzyme which forms [Met~]-enkephalin from [MetS]enkephalin-Arg6,Phe 7 in ... more The captopril-inhibited enzyme which forms [Met~]-enkephalin from [MetS]enkephalin-Arg6,Phe 7 in isolated rabbit ear artery was characterized further by using various natural substrate candidates/analogues ([MetS]-enkephalin-ArgS, Phe 7 and its amide, [MetS]enkephalin, angiotensin I and bradykinin), peptidase inhibitors such as captopril, enalaprilate and thiorphan and by endothel removal. 10 -~ and 10 4 M but not 10 -6 M captopril reduced the effectiveness of [MetS]-enkephalin-Arg%Phe 7 and potentiated the effect of bradykinin but did not affect markedly the action of the other peptides. Of the inhibitors, enalaprilate was less effective than captopril, and thiorphan had no effect. The [MetS]-enkephalin-Arg6,PheT~[MetS]enkephalin conversion was not affected by endothel removal. The substrate and inhibitor spectrum of this non-endothelial enzyme activity bears no relationship in other, hitherto characterized dipeptidylcarboxypeptidases/endopeptidases known to be involved in the metabolism of the tested peptides.
The distribution and properties of frog brain kappa-opioid receptor subtypes differ not only from... more The distribution and properties of frog brain kappa-opioid receptor subtypes differ not only from those of the guinea pig brain, but also from that of the rat brain. In guinea pig cerebellum the kappas is the dominant receptor subtype, frog brain contains mainly the kappa2 subtype, and the distribution of the rat brain subtypes is intermediate between the two others. In competition experiments it has been established that ethylketocyclazocine and N-cyclopropylmethyl-norazidomorphine, which are nonselective kappa-ligands, have relatively high affinities to frog brain membranes. The kappa2 ligands (MetS)enkephalin-Arg6-Phe v and etorphine also show high affinities to the frog brain. Kappa 1 binding sites measured in the presence of 5 txM/D-Ala2-LeuS/enkephalin represent 25-30% of [3H]ethylketocyclazocine binding in frog brain membranes. The kappa 2 subtype in frog brain resembles more to the mu subtype than the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and/D-AIa 2-(Me)Phe4-GlyS-ol/enkephalin (DAGO). From our data it is evident that the opioid receptor subtypes are already present in the amphibian brain but the differences among them are less pronounced than in mammalian brain.
The distribution and properties of frog brain kappa-opioid receptor subtypes differ not only from... more The distribution and properties of frog brain kappa-opioid receptor subtypes differ not only from those of the guinea pig brain, but also from that of the rat brain. In guinea pig cerebellum the kappa1 is the dominat receptor subtype, frog brain contains mainly the kappa2 subtype, and the distribution of the rat brain subtypes is intermediate between the two others. In competition experiments it has been established that ethylketocyclazocine and N-cyclopropylmethyl-norazidomorphine, which are nonselective kappa-ligands, have relatively high affinities to frog brain membranes. The kappa2 ligands (Met5)enkephalin-Arg6-Phe7 and etorphine also show high affinities to the frog brain. Kappa1 binding sites measured in the presence of 5 μM /D-Ala2-Leu5/enkephalin represent 25–30% of [3H]ethylketocyclazocine binding in frog brain membranes. The kappa2 subtype in frog brain resembles more to the mu subtype than the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and /D-Ala2-(Me)Phe4-Gly5-ol/enkephalin (DAGO). From our data it is evident that the opioid receptor subtypes are already present in the amphibian brain but the differences among them are less pronounced than in mammalian brain.
A kappa-opioid receptor subtype was purified from a digitonin extract of frog brain membranes, us... more A kappa-opioid receptor subtype was purified from a digitonin extract of frog brain membranes, using affinity chromatography. The affinity resin was prepared by coupling dynorphin (1–10) to AH Sepharose 4B. The purified receptor binds 4,750 pmol [3H]ethylketocyclazocine (EKC) per mg protein (5,600-fold purification over the membrane-bound receptor) with a Kd of 9.1 nM. The addition of cholesterol-phosphatidylethanolamine (2:1) enhanced 3.6-fold the binding activity of the purified material, which gives a purification very close to the theoretical. The purified receptor protein exhibits high affinity for kappa-selective ligands. The purified fraction shows one major band (65,000 Mr) in sodium dodecyl sulfate (SDS) gel electrophoresis.
The captopril-inhibited enzyme which forms [Met~]-enkephalin from [MetS]enkephalin-Arg6,Phe 7 in ... more The captopril-inhibited enzyme which forms [Met~]-enkephalin from [MetS]enkephalin-Arg6,Phe 7 in isolated rabbit ear artery was characterized further by using various natural substrate candidates/analogues ([MetS]-enkephalin-ArgS, Phe 7 and its amide, [MetS]enkephalin, angiotensin I and bradykinin), peptidase inhibitors such as captopril, enalaprilate and thiorphan and by endothel removal. 10 -~ and 10 4 M but not 10 -6 M captopril reduced the effectiveness of [MetS]-enkephalin-Arg%Phe 7 and potentiated the effect of bradykinin but did not affect markedly the action of the other peptides. Of the inhibitors, enalaprilate was less effective than captopril, and thiorphan had no effect. The [MetS]-enkephalin-Arg6,PheT~[MetS]enkephalin conversion was not affected by endothel removal. The substrate and inhibitor spectrum of this non-endothelial enzyme activity bears no relationship in other, hitherto characterized dipeptidylcarboxypeptidases/endopeptidases known to be involved in the metabolism of the tested peptides.
It has been previously found that chloromethyl ketone derivatives of enkephalins bind irreversibl... more It has been previously found that chloromethyl ketone derivatives of enkephalins bind irreversibly to the opioid receptors in vitro. Recently a novel affinity reagent, Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Gly chloromethyl ketone (Dynorphinil_10/-Glyll chloromethyl ketone, DynCMK) was synthesized, and its binding characteristics to frog (Rana esculenta) brain membranes were evaluated. In competition experiments, the product shows a relatively high affinity for the ~:-opioid binding sites labelled by [SH]ethylketocyclazocine (Ki = 200 nM), whereas its binding to the ([3H]dihydromorphine) and to the ~ sites ([3H]D-Ala2-Leu5]enkephalin) is weaker. Preincubation of the frog brain membranes with DynCMK at micromolar concentrations results in a washing-resistant and dose-dependent inhibition of the [3H]ethylketocyclazocine binding sites. Saturation binding analysis of the membranes preincubated with 50 ~M DynCMK reveals a significant decrease in the number of specific binding sites for [3H]ethylketocyclazocine compared to the control values. The K-preferring binding properties of the compound suggest that it could serve as an affinity label for the K-type of opioid receptors.
A radioactive enkephalin affinity reagent, selective for the b opioid receptor subtype, was synth... more A radioactive enkephalin affinity reagent, selective for the b opioid receptor subtype, was synthesized by a fragment condensation method. 3H-BOC-Tyr-D-Ala-Gly-OH was prepared by catalytic tritiation of the protected iodinated tripeptide. The protected tritiated tripeptide and N(Me)Phe-CH&I were condensed by the mixed anhydride method. The protecting group was removed by HCl/acetic acid. The tritiated tetrapeptide has a specific radioactivity of 56.8 Ci/mmole (2.1 TBq/mmole).
The design, synthesis and biological data are presented for novel opioid peptides, which act as p... more The design, synthesis and biological data are presented for novel opioid peptides, which act as pure, delta receptor antagonists and contain an 'opioid' nitrogen which cannot exist in protonated form while interacting with the receptor. The K, values of Boc-Tyr-Pro-Gly-Phe-Leu, Boc-Tyr-Pro-Gly-Phe-Leu-Thr, Boc-Tyr-Pro-Gly-Phe-Leu-Thr (tBu) and phenylacetyl-or diphenylacetyl-Tyr-Pro-Gly-Phe-Lou-Thr determined against [Mets]enkephalin or DADLE in the mouse vas deferens bioassay fall into the l&'-4 x 10" M range, with a 50-350 fold delta over mu receptor selectivity. Displacement data in the receptor binding assay in rat brain membranes against 'H-DALE, 'H-DSLET, 3H-DAG0 and 3H-DHM matched the results of bioassay. Since not only the Nblocked derivatives but even the ones with a free N terminus were devoid of significant opioid agonist activity, it is suggested that these novel derivatives do not interact with the conventional 'opioid' nitrogen binding site although the hydrophobic binding site involved must be located in a reasonable vicinity. Conformational analyses of peptides appear to support this assumption.
ual challenge due to the complexity of the samples. A single chromatogram in which all the compon... more ual challenge due to the complexity of the samples. A single chromatogram in which all the components of such a complex mixture are represented by single peaks that are well resolved from each other would be ideal. The complexity of samples entails differences in size, charge, hydrophobicity, and chemical variety of the components. Tablet formulations generally contain three different groups of chemicals: small organic molecules, polymers that are controlled-release agents and binding materials, and solid particulates. Table 1 lists the most commonly used formulation materials, but space does not permit a listing of all possible components. The commercially available formulations comprise an astronomical variety of components and only a general categorization is possible here. One of the difficulties of the analytical chemist is the discretion on the part of the suppliers. The excipient supplier companies use only a few basic polymers, such as polyethylene glycol (PEG), polyvinylpyr...
Proceedings of The National Academy of Sciences, 1988
The aspartic residue (Asp-189) at the base of the substrate-binding pocket of trypsin was replace... more The aspartic residue (Asp-189) at the base of the substrate-binding pocket of trypsin was replaced by serine (present in a similar position in chymotrypsin) through site-directed mutagenesis. The wild-type (with Asp-189 in the mature trypsin sequence) and mutant (Ser-189) trypsinogens were expressed in Escherichia coli, purified to homogeneity, activated by enterokinase, and tested with a series of fluorogenic tetrapeptide substrates
The captopril-inhibited enzyme which forms [Met~]-enkephalin from [MetS]enkephalin-Arg6,Phe 7 in ... more The captopril-inhibited enzyme which forms [Met~]-enkephalin from [MetS]enkephalin-Arg6,Phe 7 in isolated rabbit ear artery was characterized further by using various natural substrate candidates/analogues ([MetS]-enkephalin-ArgS, Phe 7 and its amide, [MetS]enkephalin, angiotensin I and bradykinin), peptidase inhibitors such as captopril, enalaprilate and thiorphan and by endothel removal. 10 -~ and 10 4 M but not 10 -6 M captopril reduced the effectiveness of [MetS]-enkephalin-Arg%Phe 7 and potentiated the effect of bradykinin but did not affect markedly the action of the other peptides. Of the inhibitors, enalaprilate was less effective than captopril, and thiorphan had no effect. The [MetS]-enkephalin-Arg6,PheT~[MetS]enkephalin conversion was not affected by endothel removal. The substrate and inhibitor spectrum of this non-endothelial enzyme activity bears no relationship in other, hitherto characterized dipeptidylcarboxypeptidases/endopeptidases known to be involved in the metabolism of the tested peptides.
The distribution and properties of frog brain kappa-opioid receptor subtypes differ not only from... more The distribution and properties of frog brain kappa-opioid receptor subtypes differ not only from those of the guinea pig brain, but also from that of the rat brain. In guinea pig cerebellum the kappas is the dominant receptor subtype, frog brain contains mainly the kappa2 subtype, and the distribution of the rat brain subtypes is intermediate between the two others. In competition experiments it has been established that ethylketocyclazocine and N-cyclopropylmethyl-norazidomorphine, which are nonselective kappa-ligands, have relatively high affinities to frog brain membranes. The kappa2 ligands (MetS)enkephalin-Arg6-Phe v and etorphine also show high affinities to the frog brain. Kappa 1 binding sites measured in the presence of 5 txM/D-Ala2-LeuS/enkephalin represent 25-30% of [3H]ethylketocyclazocine binding in frog brain membranes. The kappa 2 subtype in frog brain resembles more to the mu subtype than the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and/D-AIa 2-(Me)Phe4-GlyS-ol/enkephalin (DAGO). From our data it is evident that the opioid receptor subtypes are already present in the amphibian brain but the differences among them are less pronounced than in mammalian brain.
The distribution and properties of frog brain kappa-opioid receptor subtypes differ not only from... more The distribution and properties of frog brain kappa-opioid receptor subtypes differ not only from those of the guinea pig brain, but also from that of the rat brain. In guinea pig cerebellum the kappa1 is the dominat receptor subtype, frog brain contains mainly the kappa2 subtype, and the distribution of the rat brain subtypes is intermediate between the two others. In competition experiments it has been established that ethylketocyclazocine and N-cyclopropylmethyl-norazidomorphine, which are nonselective kappa-ligands, have relatively high affinities to frog brain membranes. The kappa2 ligands (Met5)enkephalin-Arg6-Phe7 and etorphine also show high affinities to the frog brain. Kappa1 binding sites measured in the presence of 5 μM /D-Ala2-Leu5/enkephalin represent 25–30% of [3H]ethylketocyclazocine binding in frog brain membranes. The kappa2 subtype in frog brain resembles more to the mu subtype than the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and /D-Ala2-(Me)Phe4-Gly5-ol/enkephalin (DAGO). From our data it is evident that the opioid receptor subtypes are already present in the amphibian brain but the differences among them are less pronounced than in mammalian brain.
A kappa-opioid receptor subtype was purified from a digitonin extract of frog brain membranes, us... more A kappa-opioid receptor subtype was purified from a digitonin extract of frog brain membranes, using affinity chromatography. The affinity resin was prepared by coupling dynorphin (1–10) to AH Sepharose 4B. The purified receptor binds 4,750 pmol [3H]ethylketocyclazocine (EKC) per mg protein (5,600-fold purification over the membrane-bound receptor) with a Kd of 9.1 nM. The addition of cholesterol-phosphatidylethanolamine (2:1) enhanced 3.6-fold the binding activity of the purified material, which gives a purification very close to the theoretical. The purified receptor protein exhibits high affinity for kappa-selective ligands. The purified fraction shows one major band (65,000 Mr) in sodium dodecyl sulfate (SDS) gel electrophoresis.
The captopril-inhibited enzyme which forms [Met~]-enkephalin from [MetS]enkephalin-Arg6,Phe 7 in ... more The captopril-inhibited enzyme which forms [Met~]-enkephalin from [MetS]enkephalin-Arg6,Phe 7 in isolated rabbit ear artery was characterized further by using various natural substrate candidates/analogues ([MetS]-enkephalin-ArgS, Phe 7 and its amide, [MetS]enkephalin, angiotensin I and bradykinin), peptidase inhibitors such as captopril, enalaprilate and thiorphan and by endothel removal. 10 -~ and 10 4 M but not 10 -6 M captopril reduced the effectiveness of [MetS]-enkephalin-Arg%Phe 7 and potentiated the effect of bradykinin but did not affect markedly the action of the other peptides. Of the inhibitors, enalaprilate was less effective than captopril, and thiorphan had no effect. The [MetS]-enkephalin-Arg6,PheT~[MetS]enkephalin conversion was not affected by endothel removal. The substrate and inhibitor spectrum of this non-endothelial enzyme activity bears no relationship in other, hitherto characterized dipeptidylcarboxypeptidases/endopeptidases known to be involved in the metabolism of the tested peptides.
It has been previously found that chloromethyl ketone derivatives of enkephalins bind irreversibl... more It has been previously found that chloromethyl ketone derivatives of enkephalins bind irreversibly to the opioid receptors in vitro. Recently a novel affinity reagent, Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Gly chloromethyl ketone (Dynorphinil_10/-Glyll chloromethyl ketone, DynCMK) was synthesized, and its binding characteristics to frog (Rana esculenta) brain membranes were evaluated. In competition experiments, the product shows a relatively high affinity for the ~:-opioid binding sites labelled by [SH]ethylketocyclazocine (Ki = 200 nM), whereas its binding to the ([3H]dihydromorphine) and to the ~ sites ([3H]D-Ala2-Leu5]enkephalin) is weaker. Preincubation of the frog brain membranes with DynCMK at micromolar concentrations results in a washing-resistant and dose-dependent inhibition of the [3H]ethylketocyclazocine binding sites. Saturation binding analysis of the membranes preincubated with 50 ~M DynCMK reveals a significant decrease in the number of specific binding sites for [3H]ethylketocyclazocine compared to the control values. The K-preferring binding properties of the compound suggest that it could serve as an affinity label for the K-type of opioid receptors.
A radioactive enkephalin affinity reagent, selective for the b opioid receptor subtype, was synth... more A radioactive enkephalin affinity reagent, selective for the b opioid receptor subtype, was synthesized by a fragment condensation method. 3H-BOC-Tyr-D-Ala-Gly-OH was prepared by catalytic tritiation of the protected iodinated tripeptide. The protected tritiated tripeptide and N(Me)Phe-CH&I were condensed by the mixed anhydride method. The protecting group was removed by HCl/acetic acid. The tritiated tetrapeptide has a specific radioactivity of 56.8 Ci/mmole (2.1 TBq/mmole).
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