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PubMed1986

Haloperidol binding to monoclonal antibodies: conformational analysis and relationships to D-2 receptor binding.

A library of 22 monoclonal antibodies to the D-2 dopaminergic receptor antagonist haloperidol has been developed by immunizing BALB/c mice with two conformationally distinct immunogens. The two immunogens were prepared by coupling haloperidol to bovine serum albumin through the tertiary alcohol at the 4-position of the piperidine ring with a succinic acid linkage and by coupling to bovine serum albumin through the ketone group of the butyrophenone with a carboxymethyl oxime linkage. Seventeen monoclonal antibodies displayed specific, saturable, high affinity binding of [3H]haloperidol which could be inhibited by a variety of neuroleptic drugs. Three monoclonal antibodies raised against the succinic acid conjugate and two monoclonal antibodies raised against the oxime conjugate were selected for detailed analysis of the molecular characteristics of binding specificity and for relationships to bovine striatal D-2 dopaminergic receptor binding. The monoclonal antibody with highest affinity for haloperidol, 185(2)-1 (raised against the succinic acid conjugate and herein referred to as mAb A), had a Kd of 3.3 (+/- 0.06) nM and primarily recognized chemical determinants in the butyrophenone ring (ring 1) and side chain. Inhibition of [3H]haloperidol binding to mAb A by 16 unlabeled haloperidol analogs displayed a good correlation [r = 0.82, n = 16, m = 1.06(+/- 0.38)] with D-2 receptor binding affinity, suggesting that the parts of the D-2 receptor combining site which recognize butyrophenone antagonists may have molecular characteristics which are similar to those of the monoclonal antibody. Other dopaminergic ligands such as dopamine and the D-1 antagonist SCH-23390 were not recognized by monoclonal antibodies raised against the succinic acid conjugate. Monoclonal antibodies raised against the oxime conjugate such as 258(2)-1 (herein referred to as mAb D) primarily recognized chemical determinants in ring 2 and the tertiary amino group of the piperidine ring. Although the D-2 receptor and mAb D both prefer electron-withdrawing substituents in the para position of ring 2, the antibody was more sensitive than the receptor to changes and displayed affinities that were much lower for substituents attached to the p-chlorophenyl ring (ring 2), which were electron donating. In addition, dopamine was able to completely displace 4 nM [3H]haloperidol from mAb D at a concentration of 6 mM.

Animals↗
PubMed1987

Solid-phase methods for sequencing nucleic acids. VII. Chemical degradation of synthetic DNA-RNA hybrid fragments using CCS and DE 81 anion-exchange papers.

Synthetic oligo(ribo-deoxyribo)nucleotides were analyzed and characterized by different solid-phase chemical degradation procedures, 5'- and 3'-end labelled mixed fragments were degraded by a slightly modified DNA cleavage procedure using 1 and 10% piperidine for the chain scission reaction and CCS anion-exchange paper. Besides the normal degradation products obtained by the usual modification and strand cleavage reactions of both deoxy- and ribonucleotide residues, additional bands were identified in the sequence patterns resulting from the hydrolysis of the RNA moiety induced by piperidine. Since both degradation reactions cleave the backbone of the mixed DNA-RNA fragments differently and produce nucleotide components with different charges, the degradation products do not interfere and can be resolved by gel electrophoresis on polyacrylamide. In addition, 3'-end labelled DNA-RNA oligomers were degraded by a RNA cleavage procedure using DE 81 anion-exchange paper as solid support. The combination of all three degradation methods allows to confirm the nucleotide sequence.

Base Sequence↗
PubMed1989

Hydroxyl free radical is not the main active species in site-specific DNA damage induced by copper (II) ion and hydrogen peroxide.

Site-specific DNA damage by Cu(II) plus H2O2 was investigated by a DNA-sequencing technique. Cu(II) plus H2O2 induced strong DNA cleavage even without piperidine treatment. Piperidine-labile sites were induced frequently at thymine and guanine residues and rarely at adenine residue. A Cu(I)-specific chelating agent, bathocuproine, inhibited the DNA damage. Neither ethanol nor mannitol inhibited it. Of alcohols, tertbutyl alcohol, having relatively low reactivity to hydroxyl free radical, inhibited the DNA damage most strongly. Sodium azide and 1,4-diazobicyclo[2.2.2]octane completely inhibited cleavages at residues of the bases other than guanine. Tris inhibited the DNA damage. The enhancing effect of D2O on DNA damage was not observed. ESR studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) showed that the hydroxyl radical adduct of DMPO was formed during the reaction of Cu(II) with H2O2, and that the addition of sodium formate produced the CO2- radical adduct of DMPO more efficiently than expected. ESR studies showed that the nitroxide radical was formed from 2,2,6,6-tetramethyl-4-piperidone in the presence of Cu(II) plus H2O2, indicating the formation of singlet oxygen or its equivalent. The effects of scavengers on DNA damage have considerable correlation with the effects of scavengers on the nitroxide radical production and DMPO.OH formation. The results suggest that the main active species causing DNA damage are more likely copper-peroxide complexes, with similar reactivity to singlet oxygen and/or hydroxyl radical rather than hydroxyl free radical.

Alcohols↗
PubMed1989

The psychotomimetic drug phencyclidine labels two high affinity binding sites in guinea pig brain: evidence for N-methyl-D-aspartate-coupled and dopamine reuptake carrier-associated phencyclidine binding sites.

Numerous studies have now demonstrated that a binding site for the psychotomimetic drug phencyclidine (PCP) exists within the receptor channel complex for the excitatory amino acid neurotransmitter glutamate, specifically the glutamate receptor selectively activated by N-methyl-D-aspartate (NMDA). Several lines of evidence support the hypothesis that all PCP receptors in rat brain are associated with the NMDA receptor complex. In the present study, we reexamine this hypothesis. We report that the PCP analog [3H]1-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) labels two high affinity binding sites in membranes prepared from guinea pig brain site 1 (Kd = 14.1 nM, Bmax = 631 fmol/mg of protein) and site 2 (Kd = 46.5 nM, Bmax = 829 fmol/mg of protein). (+)-5-Methyl-10 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate bound to site 1 with high affinity (Kl = 3.2 nM) and to site 2 with low affinity (Kl = 5208 nM). The order of potency of drugs for inhibiting [3H]TCP binding to site 1 correlated with their ED50 values for inhibition of NMDA-mediated responses reported in the literature, whereas the order of potency of drugs for inhibiting [3H]TCP binding to site 2 correlated with their ED50 values for inhibition of [3H]dopamine reuptake reported in the literature. Kinetic experiments demonstrated that glutamate, 2-amino-7-phosphonoheptanoic acid, and Mg2+ modulated [3H]TCP binding to site 1 but not site 2. Preincubation of guinea pig striatal membranes with varying concentrations of the high affinity dopamine reuptake inhibitors N-[1-(2-benzo(b)thiophenyl)cyclohexyl]piperidine and 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-[3- phenylpropyl]piperazine caused a wash-resistant inhibition of [3H]TCP binding to site 2 but not site 1. Taken collectively, these data demonstrate the existence of a high affinity PCP binding site associated with the dopamine reuptake carrier and raise the possibility that the therapeutic and psychotomimetic effects of PCP in humans are separable and mediated via different binding sites.

Acetylcholine↗
PubMed1989

Anticonvulsant activities of 1-phenylcyclohexylamine and its conformationally restricted analog 1,1-pentamethylenetetrahydroisoquinoline.

1-Phenylcyclohexylamine (PCA), an analog of phenyclidine (PCP) in which the piperidine ring is replaced by a primary amino group, and its conformationally restricted analog 1,1-pentamethylenetetrahydroisoquinoline (PM-THIQ) were potent anticonvulsants in the mouse maximal electroshock (MES) seizure test (ED50 values, 7.0 and 14.3 mg/kg, respectively). At higher doses, the drugs caused motor impairment (TD50 values, 16.3 and 43.0 mg/kg) and blocked the behavioral effects and lethality of i.p. injected N-methyl-D-aspartate (NMDA) (ED50 values, 36.3 and 127 mg/kg). The separation in potencies in the MES seizure and motor toxicity tests of PCA and PM-THIQ contrasts with PCP which was equally potent in both tests. Several compounds that were structurally related to PM-THIQ (N-ethyl-PCA, 2-methyl-PCA, N-methyl-PM-THIQ, tetrahydroisoquinoline and benzylamine) also blocked MES seizures and caused motor impairment, but failed to show as great a separation between MES anticonvulsant activity and motor toxicity. None of the compounds protected against seizures induced by the chemoconvulsant pentylenetetrazol at doses that lacked motor toxicity. The drugs were also evaluated for their ability to displace [3H]-1-[1-(2-thienyl)-cyclohexyl]piperidine from binding to high affinity acceptor sites (presumably on NMDA receptor-coupled channels) in rat brain homogenates. The rank order of potencies in the binding assay was similar to that in the behavioral tests, except for 2-methyl-PCA which was behaviorally more potent than expected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Hydroxyl radical production and human DNA damage induced by ferric nitrilotriacetate and hydrogen peroxide.

Reactivities of Fe3+ chelates of aminopolycarboxylic acids with DNA were investigated by the DNA-sequencing technique using 32P 5'-end-labeled DNA fragments obtained from the human c-Ha-ras-1 protooncogene, and the reaction mechanism was studied by electron spin resonance spectroscopy. Ferric nitrilotriacetate (Fe3+-NTA) plus hydrogen peroxide caused strong DNA cleavage in the presence of albumin. No or little DNA cleavage was observed with ferric chloride or Fe3+ chelates of other aminopolycarboxylic acids tested in the presence of hydrogen peroxide. The DNA cleavage by Fe3+-NTA plus hydrogen peroxide without piperidine treatment occurred at positions of every nucleotide although a specific cleavage was observed, whereas cleavages at the positions of guanine and thymine increased predominantly with piperidine treatment. Electron spin resonance studies using free radical traps demonstrated that of Fe3+ chelates of aminopolycarboxylic acids, Fe3+-NTA was the most effective catalyst in hydrogen peroxide-derived production of hydroxyl radicals under our conditions. The results suggest that Fe3+-NTA catalyzes the decomposition of hydrogen peroxide to produce hydroxyl radicals, which subsequently cause the strong base alterations of guanine and thymine, and deoxyribose-phosphate backbone breakages. The possibility that the Fe3+-NTA-induced DNA damage is the initiation and/or promotion of carcinogenesis by Fe3+-NTA is discussed.

DNA Damage↗
PubMed1987

Novel mu-selective Met-enkephalinamide analogs with antagonist activity. Synthesis, receptor binding, analgesic properties and conformational studies.

Four novel mu-selective peptide antagonists have been synthesized and examined for receptor binding, analgesic agonist and antagonist activity and energy conformational properties. These peptides were designed by analogy to results of molecular modeling of 3-phenyl piperidines which led to incorporating four modified tyrosine residues, m-Tyr, beta-methyl-m-Tyr, N-phenethyl-m-Tyr and alpha, beta-dimethyl-m-Tyr into D-Ala2-Met5-enkephalinamide. Peptides were synthesized by stepwise solution synthesis using an active ester coupling procedure. Receptor binding assays were performed on rat brain homogenates and data were analyzed by a modified version of the program LIGAND. Analgesic agonist and antagonist activity was evaluated by the mouse tail-flick test. Energy-optimized conformations were obtained using a program called Molecule-AIMS. The results demonstrate that relative ratios of in vivo agonist and antagonist potencies in D-Ala2-Met5-enkephalinamides can be modulated by chemical modification of the tyrosine residue. A shift in the phenolic-OH position from para to meta significantly enhances relative antagonist versus agonist activity; addition of a beta-CH3 group to the m-Tyr enhances mu-selectivity and leads to nearly equal agonist/antagonist activity. Energy conformational studies indicate that all analogs with high mu-receptor affinity examined have a common energy accessible B'II 2-3 turn conformation similar to that previously identified for high mu-affinity binding in peptides, lending further support to this candidate conformer. This conformer also has tyrosine side-chain angles which allowed total overlap with the amine and phenolic groups of a known structure of 3-(m-OH phenyl)-piperidine. This structural similarity together with the observation of mixed agonist antagonist activity in both types of opioids confirms the rationale upon which design of these peptides was based.

Animals↗
PubMed1988

Electroencephalographic, behavioral and receptor binding correlates of phencyclinoids in the rat.

The pharmacology and structure-activity relationship of phencyclidine (PCP)-like drugs (phencyclinoids) were studied using electroencephalographic (EEG), behavioral and receptor binding techniques. The effects of PCP, 1-phenylcyclohexylamine HCl, N-methyl-1-phenycyclohexylamine HCl, N-ethyl-1-phenylcyclohexylamine HCl, N-(s-butyl)-1-phenylcyclohexylamine HCL, 1-(1-phenylcyclo-hexyl)-pyrrolidine HCl, 1-[1-(2-thienyl)cyclohexyl] piperidine HCl, 1-[1-(2-thienyl)cyclohexyl]-pyrrolidine HCl, ketamine and (+/-)-SKF 10047 were evaluated on the direct EEG and EEG spectra after acute i.v. injections (0.1-17.8 mg/kg). Similarities and differences were noted in the EEG dose-response curves. At lower doses of PCP and its analogs, low-amplitude theta waves predominated; however, at higher doses, high-amplitude, lower-frequency waves predominated. Qualitatively, the N-piperidine derivatives were similar to PCP and differed primarily in potency. The benzomorphan (+/-)-SKF 10047 produced only theta activity at doses up to 12.8 mg/kg. These EEG effects occurred in conjunction with overt behaviors including locomotion, stereotypy and ataxia, concurrently assessed via observer-based rating scales. A strong correlation (r = 0.98) was obtained between the EEG and behavioral effects and the IC50 values from [3H]PCP displacement experiments using crude rat brain homogenates.

Animals↗
PubMed1988

Characterization of opioid, sigma, and phencyclidine receptors in the neuroblastoma-brain hybrid cell line NCB-20.

Opioid, sigma, and phencyclidine (PCP) receptors were characterized in the mouse neuroblastoma--Chinese hamster brain hybrid cell line NCB-20. Quantitative receptor assays under equilibrium binding conditions with highly specific radioligands demonstrated the presence of delta, but not mu or kappa, opioid receptors on NCB-20 cell membranes. NCB-20 cells were shown to possess two distinct sites specific for sigma opioids and PCP derivatives. One site was labeled by (+)-[3H]N-allylnormetazocine [(+)-[3H]SKF-10,047] (Kd = 69 nM; Bmax = 4100 fmol/mg of protein). The rank order of potency of drugs at this site was (+)-3-(3-hydroxy-phenyl)-N-(1-propyl)piperidine [(+)-3-PPP] greater than haloperidol greater than (+)-SKF-10,047 greater than (+/-)-ethylketocyclazocine greater than (+/-)-bremazocine greater than N-[1-(2-thienyl) cyclohexyl]piperidine (TCP) greater than dexoxadrol. This site is similar in its ligand selectivity to the haloperidol-sensitive sigma receptor of rat brain. The other site was labeled by the potent phencyclidine derivative [3H]TCP (Kd = 335 nM; Bmax = 9300 fmol/mg of protein). This density is equivalent to approximately 60,000 sites/cell. The rank order of potency of drugs at this site was TCP greater than (+)-3-PPP greater than PCP greater than dexoxadrol greater than haloperidol greater than cyclazocine greater than levoxadrol greater than (+)-SKF-10,047; mu and delta ligands were inactive. This site is similar to the rat brain PCP receptor. The NCB-20 cell line is the only cultured cell line that has been demonstrated to have PCP receptors.

Animals↗
PubMed1988

Initial identification and characterization of sigma receptors on human peripheral blood leukocytes.

Phencyclidine (PCP) has been reported to suppress a variety of immune functions in vitro. Because PCP binds with high affinity to both PCP and sigma receptors, the identity of the receptor(s) mediating the immunological effects of PCP is unknown. The aim of the present study was to identify and characterize the sites of PCP action (sigma and/or PCP receptors) in human peripheral blood leukocytes (PBL) using [3H]haloperidol or 1,3 di(2-([5-3H]tolyl)guanidine ([3H]DTG) to specifically label sigma receptors and 3,4-[3H]-(N)-[1-(2-thienyl)-cyclohexyl]-piperidine ([3H]TCP) to specifically label PCP receptors. [3H]Haloperidol binding was saturable and of high affinity with comparable KD values in human PBL (0.44 +/- 0.10 nM) and rat cerebellum (0.51 +/- 0.09 nM). Similarly, [3H]DTG binding was saturable with comparable KD values of 29.5 +/- 3.5 and 26.4 +/- 3.6 nM in rat cerebellum and human PBL, respectively. In contrast, there was a notable absence of [3H]TCP-labeled PCP receptors in human PBL and rat cerebellum. In competition studies, the pharmacologic profile of [3H]haloperidol-labeled sigma receptors in human PBL was virtually identical with that in rat cerebellum (slope, 0.87; correlation coefficient, 0.96); the rank order of potency of competing drugs was haloperidol greater than l-butaclamol = pentazocine greater than d-3-(hydroxyphenyl)-N-(1-propyl)-piperidine greater than DTG = d-butaclamol = d-SKF 10,047 greater than levallorphan greater than or equal to PCP greater than or equal to l-SKF 10,047 greater than TCP greater than MK-801.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

L-pipecolic acid oxidation in the rabbit and cynomolgus monkey. Evidence for differing organellar locations and cofactor requirements in each species.

L-Pipecolic acid oxidation was studied in the rabbit and cynomolgus monkey. Tissue homogenates from both species incubated with L-[2,3,4,5,6-3H]pipecolic acid produced a single radioactive product identified as alpha-aminoadipic acid. In the rabbit, L-pipecolic acid oxidation was greatest in kidney cortex with progressively lesser specific activities in liver, heart, and brain. When rabbit kidney cortex was fractionated by differential centrifugation or on Percoll gradients, activity paralleled that of the mitochondrial marker, glutamate dehydrogenase. In sonicated mitochondria, 92% of the activity was in the soluble fraction. Activity was inhibited by both rotenone and antimycin A and was maximal when FAD, phenazine ethosulfate, and glycerol were included in the assay; Km,app was 0.74 +/- 0.16 mM. Nipecotic acid, piperidine, and cis-2,4-piperidine dicarboxylic acid did not inhibit L-pipecolic acid oxidation, while L-proline had a Ki greater than or equal to 10 mM. D-Alanine and kojic acid, substrate and inhibitor of D-amino acid oxidase, respectively, were also not inhibitory. When monkey kidney cortex was fractionated on Percoll gradients, L-pipecolic acid oxidation activity paralleled that of the peroxisomal marker, catalase. After organellar subfractionation, the activity was membrane-associated and maximal at pH 8.5; Km,app was 4.22 +/- 0.30 mM. L-Pipecolic acid oxidation produced hydrogen peroxide, suggesting involvement of an oxidase in alpha-aminoadipic acid formation. Antimycin A did not inhibit the reaction. No specific cofactor requirements were identified and phenazine ethosulfate inhibited the reaction. D-Pipecolic acid, L-proline, and the other compounds cited above did not significantly inhibit the activity.

Animals↗
PubMed1988

[Complementary addressed modification of single-stranded DNA using a [Fe-EDTA] derivative of oligonucleotide].

A one-tube synthesis scheme for coupling EDTA residue to the 5'-terminus of unprotected oligonucleotides via propylenediamine linker is described. The EDTA derivative of oligonucleotide d(pTGACCCTCTTCCC)A forms a kinetically stable complex with Fe2+ ion. In the presence of ascorbic acid with O2 limiting, this complex modifies single-stranded DNA (a 302-mer) in a site-specific way near the target complementary nucleotide sequence. The DNA fragment can be then cleaved predominantly at modified pyrimidine nucleotides (hidden breaks) by hot piperidine treatment, whereas few direct breaks (i.e. without piperidine) occurs at this site. No autocleavage of the [Fe.EDTA]-oligonucleotide derivative is observed under the experiment's conditions.

Chemical Phenomena↗
PubMed1988

Evaluation of antagonists of the discriminative stimulus and response rate effects of phencyclidine.

The present studies examined whether a variety of drugs representative of diverse pharmacological classes were able to antagonize either the discriminative stimulus or response rate suppressing effects of phencyclidine (PCP). Rats were trained to lever press according to fixed-ratio 32 reinforcement schedules for food pellet delivery and to discriminate PCP from saline vehicle during daily (Monday-Friday) experimental sessions. Drugs combined with PCP to test for antagonism included an alpha-1 (prazosin) and an alpha-2 (yohimbine) adrenergic antagonist, the adenosine receptor agonist, I-phenylisopropyl adenosine, the gamma-aminobutyric acid agonist, imidazole acetic acid, the dopaminergic antagonist, haloperidol, the acylating agent of the PCP receptor, metaphit, and the dopaminergic drugs (+)- and (-)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine. All test drugs except metaphit were tested in rats trained to discriminate 2.5 mg/kg of PCP from saline. (+)- and (-)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine were additionally tested in rats trained to discriminate 1.0 mg/kg of PCP from saline. Metaphit was only tested in rats which had been trained to discriminate a 1.0-mg/kg PCP dose. None of the test drugs systematically reduced PCP level selection to vehicle levels. Yohimbine and prazosin, however, but not the other test drugs, partially reversed the response rate reducing effects of PCP. Evidence that alpha adrenergic antagonists have some PCP antagonistic effects were confirmed for the response rate effects of PCP, indicating a possible role of adrenergic mediation of these effects.

Animals↗
PubMed1987

Rational design of bis-intercalating drugs as antitumour agents: importance of rigidity in the linking chain.

Ditercalinium (NSC 366241), a dimer of 10-methoxy-7H-pyrido[4,3-c]carbazole quaternarized on the pyridine nitrogen by a rigid bis(1,1'-ethyl)-4,4'-bipiperidine linking chain, is endowed with antitumour properties and bis-intercalates with high affinity into DNA. New dimers have been designed in the same series to evaluate the importance of the rigidity of the linking chain for pharmacological activity. The dimers, characterized by one and two additional methylene groups between the two piperidine rings of the linking chain, remain as active as ditercalinium. However, a third additional CH2 group between the two piperidine rings leads to an inactive dimer. Relationships between the different pharmacological activities of the drugs and their intercalation complexes with DNA were investigated using viscosimetry, absorption spectroscopy and NMR analyses.

Animals↗
PubMedDate not supplied

Phencyclidine metabolism in vitro. The formation of a carbinolamine and its metabolites by rabbit liver preparations.

Four products of the in vitro oxidative metabolism of the piperidine ring of phencyclidine, 5-(1-phenylcyclohexylamino)valeraldehyde, V; N-(1-phenylcyclohexyl)-1,2,3,4-tetrahydropyridine, VIII; 5-(1-phenylcyclohexylamino)valeric acid, VII; and 1-phenylcyclohexylamine, IX, have been identified following derivatization, by GC/MS with stable isotope labeling and/or synthesis. The enamine, VIII, may be a work-up elimination product of a carbinolamine, alpha-hydroxy-N-(1-phenylcyclohexyl)piperidine, IV. The formation of all metabolites requires microsomal enzymes, but VII and the previously described N-(5-hydroxypentyl)-1-phenylcyclohexylamine, VI, also require soluble enzymes. Quantitative or semiquantitative data show that VI and VIII appear and disappear with time, whereas VII seems to be a terminal metabolite.

Animals↗
PubMedDate not supplied

Metabolism of aminoglutethimide in humans. Identification of four new urinary metabolites.

Four new metabolites of aminoglutethimide have been identified in the urine of patients being treated chronically with the drug. These were products of hydroxylation of the 3-ethylpiperidine-2,6-dione residue, namely 3-(4-aminophenyl)-3-ethyl-5-hydroxypiperidine-2,6-dione and its acetylamino analog, 3-(4-aminophenyl)-3-(1-hydroxyethyl)piperidine-2,6-dione, and 3-(4-aminophenyl)-3-(2-carboxamidoethyl)tetrahydrofuran-2-one, the lactone formed by rearrangement of 3-(4-aminophenyl)-3-(2-hydroxyethyl)piperidine-2,6-dione. The metabolites were isolated by reverse-phase thin layer chromatography and characterized by comparison of their mass spectra either with those of synthetic samples or with the mass spectra of analogous metabolites previously identified in the urine of rats. These new metabolites were minor constituents compared with aminoglutethimide and with the previously identified major metabolites 3-(4-acetylaminophenyl)-3-ethylpiperidine-2,6-dione and 3-(4-hydroxylaminophenyl)-3-ethylpiperidine-2,6-dione. There were marked species differences between rat and human inasmuch as almost all the metabolites in the urine of the rat were N-acetylated whereas most of the human metabolites were not. However, 5-hydroxylation of the piperidinedione residue was stereoselective in the same sense in both species, the cis isomer being formed exclusively. Synthetic cis-3-(4-aminophenyl)-3-ethyl-5-hydroxypiperidine-2,6-dione did not inhibit the activity of the target enzyme systems desmolase and aromatase in vitro, and therefore, like other metabolites so far described, is an inactivation product of the drug.

Acetylation↗
PubMed1980

[Conformational changes of spin-labeled native and modified phosphorylase B].

The phosphorylase B labelled with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-iodacetamide (phosphorylase I) and with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-ethylmaleinimide (phosphorylase II) was studied. It was shown that label I is characterized by a greater mobility with respect to the protein as compared to label II. In spin-labelled preparations of phosphorylase B the 1,5--2,0 SH-groups of the enzyme monomer having no effect on the enzyme activity were modified. The effects of AMP, glucose-1-phosphate and glucose-6-phosphate on the EPR spectrum of phosphorylase I were studied. The greatest changes in the spectrum (especially in the high field line) were found to occur in the presence of glucose-6-phosphate. These changes are due to the increase in the degree of anisotropic spin rotation. The experimental and theoretical spectra allowing to determine the correlation time for the protein moiety (tau b = 160 ns) were shown to be similar. The local conformation changes were found to occur in the vicinity of one of the two label-bound SH-groups of phosphorylase I. The EPR spectra demonstrate the S-shaped dependence of mobility of phosphorylase I label on concentration of glucose-6-phosphate (0,1--10 mM). In the presence of AMP no S-shaped dependence is observed. Reduced NaBH4 phosphorylase I does not reveal the S-shaped dependence of the label mobility on concentration of glucose-6-phosphate. The degree of the label immobilization in the apo-phosphorylase I--pyridoxal-5-chloromethylphosphonate complex in the presence of glucose-6-phosphate and AMP is the same as in cholophosphorylase I; however, in contrast to the choloenzyme it does not depend on glucose-6-phosphate (0,1--10,0 mM). The changes in the mobility of the spin label of apophosphorylase I and its complex with the AMP analog--adenosine-5'-chloromethylphosphonate--during the choloenzyme reconstruction by pyridoxalphosphate are indicative of participation of AMP and the phosphate group of AMP in the formation of the enzyme active center.

Adenosine Monophosphate↗
PubMed1978

Cyclic forms of the alpha-keto acid analogs of arginine, citrulline, homoarginine, and homocitrulline.

The alpha-keto acid analogs of arginine and citrulline (and of their next higher homologs, homoarginine and homocitrulline) were prepared enzymatically and shown to exist in equilibrium with cyclic forms which predominate in solution and in the solid state. The cyclic forms of the alpha-keto acid analogs of arginine and homoarginine have the structures pyrrolidine-1-amidino-2-hydroxy-2-carboxylic acid and piperidine-1-amidino-2-hydroxy-2-carboxylic acid, respectively. The cyclic forms of the alpha-keto acid analogs of citrulline and homocitrulline have the structures pyrrolidine-1-carbamyl-2-hydroxy-2-carboxylic acid and piperidine-1-carbamyl-2-carboxylic acid, respectively. The latter compound can undergo further cyclization and dehydration to form additional products, whose structures (2H, 5H, 7H-imidazo[1,5-a]pyridine-1,3-dione and 2H, 5H, 7H, 9H-9-hydroxy-imidazo[1,5-a]pyridine-1,3-dione) were deduced. The alpha-keto acids investigated here, which may be formed reversibly by enzymatic transamination of the corresponding amino acids, may be formed in vivo especially in the presence of metabolic abnormalities associated with inborn enzymatic defects.

Arginine↗