Biomedical subjects

N W DeLapp

Publications and source records attributed to N W DeLapp.

At least 19 recordsLinked to original sources

PubMed2001

Potent antagonism of 5-HT(3) and 5-HT(6) receptors by olanzapine.

The interaction of the psychotropic agent olanzapine with serotonin 5-HT(3) and 5-HT(6) receptors was investigated. Olanzapine did not contract the isolated guinea pig ileum, but blocked contractions induced by the 5-HT(3) receptor agonist 2-methyl serotonin (2-CH(3) 5-HT) with a pK(B) value of 6.38+/-0.03, close to the affinity of the 5-HT(3) receptor antagonist ondansetron. The atypical antipsychotic risperidone (1 microM) did not significantly inhibit 2-CH(3) 5-HT-induced contractions. Olanzapine had high affinity (pK(i)=8.30+/-0.06) for human 5-HT(6) receptors in radioligand binding studies. Olanzapine did not stimulate [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding to the G protein G(s) in cells containing human 5-HT(6) receptors, but inhibited 5-HT-stimulated [35S]GTPgammaS binding (pK(B)=7.38+/-0.16). Among other antipsychotics investigated, clozapine antagonized 5-HT(6) receptors with a pK(B)=7.42+/-0.15, ziprasidone was three-fold less potent, and risperidone, quetiapine and haloperidol were weak antagonists. Thus, olanzapine was not an agonist, but was a potent antagonist at 5-HT(6) receptors and had marked antagonism at 5-HT(3) receptors.

Animals↗

PubMed2001

Receptor reserve of phosphoinositide-coupled muscarinic receptors in mouse hippocampus in vivo.

The ability of the partial muscarinic agonist pilocarpine to increase in vivo phosphoinositide (PI) hydrolysis in mouse brain was compared to two full agonists. Pilocarpine increased in vivo phosphoinositide (PI) hydrolysis in cortex, striatum, and to the greatest extent in the hippocampus. Pilocarpine injected either subcutaneously or intracerebroventricularly robustly increased in vivo PI hydrolysis in hippocampus up to 500% of control levels and the increases were blocked by the muscarinic antagonist scopolamine. The increases in vivo PI hydrolysis induced by pilocarpine were 60-75% of the magnitude of the full muscarinic agonists oxotremorine-M and cis-dioxolane. The muscarinic M(1) preferring antagonist pirenzepine potently blocked pilocarpine-induced increases in in vivo PI hydrolysis, consistent with the increase being mediated by M(1) receptors. Since pilocarpine is a relatively weak partial agonist, these data suggest a substantial level of receptor reserve for the PI response in mouse hippocampus.

Acetylcholine↗

PubMed2001

The muscarinic receptor agonist BuTAC, a novel potential antipsychotic, does not impair learning and memory in mouse passive avoidance.

(5R,6R)-6-(3-butylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]octane) (BuTAC) is a novel, selective muscarinic receptor ligand with partial agonist mode of action at muscarinic M2 and M4 and antagonist mode of action at M1, M3 and M5 receptor subtypes in cloned cell lines. BuTAC exhibits functional dopamine receptor antagonism despite its lack of affinity for dopamine receptors, and parasympathomimetic effects in mice are produced only at doses well beyond the doses exhibiting the antipsychotic-like effects. In the present study we investigated the effects of BuTAC and the antipsychotic compounds clozapine, sertindole and olanzapine using one trial passive avoidance with mice as a model of learning and memory. Pharmacologically relevant doses of BuTAC and reference antipsychotics were identified, based on inhibition of apomorphine-induced climbing in mice as an assay measuring antidopaminergic potency. When ratios between the minimum effective dose (MED) for impairment of retention in passive avoidance and the MED for inhibition of apomorphine-induced climbing were calculated, BuTAC displayed a high ratio of >10, compared with clozapine (0.3), sertindole (3) and olanzapine (3). These data suggest that BuTAC is a potential novel antipsychotic which may have favourable effects on aspects of learning and memory.

Animals↗

PubMed2000

Muscarinic receptor agonists decrease cocaine self-administration rates in drug-naive mice.

(5R,6R)-6-(3-Propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[ 3.2.1]octane (PTAC) is a selective muscarinic receptor ligand. The compound exhibits high affinity for central muscarinic receptors with partial agonist mode of action at muscarinic M(2) and M(4) and antagonist mode of action at muscarinic M(1), M(3) and M(5) receptor subtypes. The compound was earlier reported to exhibit functional dopamine receptor antagonism in rodents despite its lack of affinity for dopamine receptors. In the present study, we report that PTAC, as well as the muscarinic receptor agonists pilocarpine and oxotremorine, dose-dependently decreased rates of intravenous self-administration (fixed ratio 1) of the indirect dopamine receptor agonist cocaine in drug naive mice. Similar decreases in cocaine self-administration rates were obtained with the dopamine receptor antagonists olanzapine, clozapine, risperidone, fluphenazine and haloperidol. These findings suggest that compounds with partial muscarinic receptor agonist mode of action may be used in the medical treatment of cocaine abuse.

Animals↗

PubMed1999

Antagonism by olanzapine of dopamine D1, serotonin2, muscarinic, histamine H1 and alpha 1-adrenergic receptors in vitro.

The atypical antipsychotic olanzapine has relatively high affinity for a number of neuronal receptors in radioreceptor binding assays. The ability of olanzapine to activate or antagonize a number of neuronal receptors was investigated in vitro, in cell lines transfected selectively with receptor subtypes and in receptor-selective isolated tissue studies. Olanzapine had no agonist activity at any of the receptors examined. However, olanzapine was a potent antagonist of 5-HT-stimulated increases in IP3 in cell lines transfected with 5-HT2A or 5-HT2B receptors with IC50 values of 30-40 nM. Olanzapine weakly blocked 5-HT-induced formation of IP3 in cell lines transfected with 5-HT2c receptors, but in this cell line potently inhibited 5-HT-stimulated [35S]GTP gamma S binding with a Ki value of 15 nM. Olanzapine blocked dopamine-stimulated adenylyl cyclase in rat retina with modest potency (Ki = 69 nM), consistent with its relatively low affinity for dopamine D1 receptors. Olanzapine blocked agonist-induced activities at the muscarinic receptor subtypes M1, M2, M3, and M5 with Ki values of 70, 622, 126, and 82 nM, respectively. In studies using cell lines transfected with muscarinic M4 receptors, olanzapine and the atypical antipsychotic clozapine did not have agonist activities as determined with cAMP inhibition and stimulation assays, arachidonic acid release and [35S]GTP gamma S binding assays. However, olanzapine antagonized agonist-induced effects in muscarinic M4 cells with a Ki value of 350 nM. In isolated tissue studies, olanzapine potently blocked agonist-induced effects at alpha 1-adrenergic and histamine H1 receptors (KB = 9 and 19 nM, respectively). Thus, olanzapine was an antagonist at all receptors investigated and was a particularly potent antagonist at 5-HT2A, 5-HT2B, 5-HT2C, alpha 1-adrenergic and histamine H1 receptors. Olanzapine was a weaker antagonist at muscarinic and dopamine D1 receptors.

Adenylyl Cyclases↗

PubMed1999

Potential role of muscarinic receptors in schizophrenia.

The role of muscarinic receptors in schizophrenia was investigated using the muscarinic agonist PTAC. PTAC was highly selective for muscarinic receptors, was a partial agonist at muscarinic M2/M4 receptors and an antagonist at M1, M3 and M5 receptors. PTAC was highly active in animal models predictive of antipsychotic behavior including inhibition of conditioned avoidance responding in rats and blockade of apomorphine-induced climbing behavior in mice. d-Amphetamine-induced Fos expression in rat nucleus accumbens was inhibited by PTAC, thus directly demonstrating the ability of PTAC to modulate DA activity. In electrophysiological studies in rats, PTAC acutely inhibited the firing of A10 DA cells and after chronic administration decreased the number of spontaneously firing DA cells in the A10 brain area. However, PTAC did not appreciably alter the firing of A9 DA cells. Thus, PTAC appears to have novel antipsychotic-like activity and these data suggest that muscarinic compounds such as PTAC may represent a new class of antipsychotic agents.

Animals↗

PubMed1999

Determination of [35S]guanosine-5'-O-(3-thio)triphosphate binding mediated by cholinergic muscarinic receptors in membranes from Chinese hamster ovary cells and rat striatum using an anti-G protein scintillation proximity assay.

An assay for measuring agonist-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate (GTPgamma35S) binding to heterotrimeric GTP binding proteins was developed for use in 96-well format using commercially available anti-G protein antibodies captured by anti-IgG-coated scintillation proximity assay beads. Use of an anti-Galphaq/11 antibody to measure GTPgamma35S binding mediated by M1, M3, and M5 receptors stably expressed in Chinese hamster ovary (CHO) cells resulted in a marked increase in agonist-stimulated/basal binding ratio compared with whole membrane binding. Pertussis toxin (PTX) treatment of CHO M1 cells before membrane preparation resulted in a marked reduction in agonist-stimulated GTPgamma35S binding to whole membranes. Direct coupling of M1 receptors in CHO cells to inhibitory G proteins was demonstrated using an anti-Galphai(1-3) antibody, and this binding was inhibited by 76% following PTX treatment. However, PTX had no effect on M1-mediated binding determined using anti-Galphaq/11. CHO M2 receptors mediated robust agonist-stimulated GTPgamma35S binding measured with anti-Galphai(1-3), but coupled only weakly to Galphaq/11. Using membranes from rat striatum, GTPgamma35S binding stimulated by oxotremorine M was demonstrated using anti-Galphaq/11, anti-Galphai(1-3), and anti-Galphao antibodies. Agonist-stimulated binding to striatal membranes showed a marked antibody-dependent GDP requirement with robust signals obtained using 0.1 microM GDP for anti-Galphaq/11 compared with 50 microM GDP for anti-Galphai(1-3) and anti-Galphao. The potencies observed for pirenzepine and AFDX 116 blockade of agonist-stimulated GTPgamma35S binding to striatal membranes determined with anti-Galphaq/11 and anti-Galphao suggested mediation of these responses primarily by M1 and M4 receptors, respectively. Antibody capture GTPgamma35S binding using scintillation proximity assay technology provides a convenient, productive alternative to immunoprecipitation for exploration of receptor-G protein interaction in cells and tissues.

Algorithms↗

PubMed1999

Muscarinic receptor agonists, like dopamine receptor antagonist antipsychotics, inhibit conditioned avoidance response in rats.

The purpose of our studies was to determine the effects of muscarinic receptor agonists on conditioned avoidance responding in the rat. Rats were trained to avoid or escape an electric shock delivered to the feet in a discrete trial procedure. The muscarinic receptor agonists pilocarpine and [2-ethyl-8-methyl-2,8-diazaspiro(4. 5)decane-1,3-dione] hydrochloride (RS86) and the cholinesterase inhibitor physostigmine all decreased the percentage of avoidance responses at doses that produced less than approximately 30% response failures. Similar results were obtained with the antipsychotic drugs haloperidol, trifluoperazine, chlorpromazine, and clozapine. However, the benzodiazepine anxiolytic diazepam did not decrease avoidance responding up to doses that produced ataxia. On the other hand, oxotremorine and arecoline decreased avoidance responding only by producing response failures, whereas aceclidine produced intermediate changes. The muscarinic receptor antagonists scopolamine, trihexyphenidyl, and benztropine were without effect when administered alone but antagonized the decreases in avoidance responding produced by pilocarpine and RS86. Scopolamine had little effect on the decreases in avoidance responding produced by haloperidol. The newer muscarinic receptor partial agonists or agonist/antagonists [R-(Z)-(+)-alpha-(methoxyimino)-1-azabicyclo[2.2. 2]octane-3-acetonitrile] hydrochloride, talsaclidine, milameline, and xanomeline also produced dose-related decreases in avoidance responding. Our results demonstrate that muscarinic receptor agonists can decrease avoidance responding in a manner similar to dopamine-receptor antipsychotic drugs, suggesting that muscarinic receptor agonists may provide an alternative approach to the treatment of psychosis.

Animals↗

PubMed1998

Muscarinic agonists with antipsychotic-like activity: structure-activity relationships of 1,2,5-thiadiazole analogues with functional dopamine antagonist activity.

Muscarinic agonists were tested in two models indicative of clinical antipsychotic activity: conditioned avoidance responding (CAR) in rats and inhibition of apomorphine-induced climbing in mice. The standard muscarinic agonists oxotremorine and pilocarpine were both active in these tests but showed little separation between efficacy and cholinergic side effects. Structure-activity relationships of the alkylthio-1,2,5-thiadiazole azacyclic type muscarinic partial agonists are shown, revealing the exo-6-(3-propyl/butylthio-1,2, 5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]octane analogues (4a,b and 9a, b) to be the most potent antipsychotic agents with large separation between efficacy and cholinergic side effects. The lack of enantiomeric selectivity suggests the pharmacophoric elements are in the mirror plane of the compounds. A model explaining the potency differences of closely related compounds is offered. The data suggest that muscarinic agonists act as functional dopamine antagonists and that they could become a novel treatment of psychotic patients.

Animals↗

PubMed1998

Identification of side chains on 1,2,5-thiadiazole-azacycles optimal for muscarinic m1 receptor activation.

Series of analogs to the functional m1 selective agonist, xanomeline (hexyloxy-TZTP), were evaluated for their in vitro m1 efficacy in cell lines transfected with the human m1 receptor. Systematic variation of the side chain and the azacyclic ring led to the discovery of potent muscarinic agonists with robust m1 efficacy, all having the phenylpropargyloxy/thio as the side chain. The most selective compound was the phenylpropargylthio-[3.2.1] endo analog 28, which is a potent and efficacious m1 agonist with no m2 activity.

Animals↗

PubMed1998

Conformationally constrained analogues of the muscarinic agonist 3-(4-(methylthio)-1,2,5-thiadiazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyr idine. Synthesis, receptor affinity, and antinociceptive activity.

Conformationally constrained analogues of the potent muscarinic agonist 3-(4-methylthio)-1,2,5-thiadiazol-3-yl)-1,2,5,6-tetrahydro-1-methy lpyridine (methylthio-TZTP, 17) were designed and synthesized with the aim of (a) improving the antinociceptive selectivity over salivation and tremor and (b) predicting the active conformation of 17 with respect to the dihedral angle C4-C3-C3'-N2'. Using MOPAC 6.0 tricyclic analogues (7, 15, 16) with C4-C3-C3'-N2' dihedral angles close to 180 degrees and a rotation hindered analogue (9) with a C4-C3-C3'-N2' dihedral angle close to 274 degrees were designed, as these conformations had previously been suggested as being the active conformations. The analogues were tested for central muscarinic receptor binding affinity, for their antinociceptive activity in the mouse grid shock test, and, in the same assay, for their ability to induce tremor and salivation. The data showed that the tricyclic analogues (7, 15, 16) were equipotent with 17 as analgesics, but with no improved side effect profiles. The rotation-hindered analogue 9 had neither muscarinic receptor binding affinity nor antinociceptive activity. These results suggest that the active conformation of 17 has a C3-C4-C3'-N2' dihedral angle close to 180 degrees.

Analgesics↗

PubMed1997

Intrathecal pertussis toxin produces hyperalgesia and allodynia in mice.

Pertussis toxin (PTX), which causes the ADP-ribosylation and thereby inactivation of Gi-proteins, has been employed in analgesia testing to elucidate receptors that are coupled to inhibitory G-proteins, such as the mu-opioid receptor. Consistent with previous findings, the antinociceptive effects of morphine (1-10 microg) as measured by tail-flick latency using a 55 degrees C water bath, were blocked by a single intrathecal injection of 0.5 microg PTX 6 days prior to intrathecal morphine administration. In addition, mice treated intrathecally with 0.5 microg of PTX had significantly shorter baseline tail-flick latencies compared with vehicle treated mice using a 55 degrees C water bath when tested 6 days after PTX or vehicle administration. Morphine-induced antinociception was blocked in a dose-dependent manner by PTX with complete blockade of morphine following a 0.3-microg dose of PTX. Further, mice administered 0.1 microg or 0.3 microg PTX intrathecally had significantly shorter tail-flick latencies compared with vehicle injected mice using a 40, 45 or 50 degrees C water bath when tested 7 days after intrathecal injection. Shorter tail-flick latencies were observed at 45 degrees C as early as 48 h after intrathecal administration of 0.03, 0.1 or 0.3 microg PTX and these shorter tail-flick latencies were observed up to 90 days after intrathecal PTX administration. The intrathecal administration of PTX caused hyperalgesia and allodynia that appears similar to the symptoms reported by patients suffering from neuropathic pain, and suggests that deficiencies in inhibitory systems, as compared with increases in excitatory systems, may play a role in the pathophysiology of at least some central or neuropathic pain states.

Analgesics, Opioid↗

PubMed1997

In vitro and in vivo biochemistry of olanzapine: a novel, atypical antipsychotic drug.

BACKGROUND: Classical (typical) antipsychotic drugs are in wide use clinically, but some patients do not respond at all to treatment, while in others, negative symptoms and cognitive deficits fail to respond. Also, these drugs often cause serious motor disturbances. Clozapine, an atypical antipsychotic, appears to correct many of these deficiencies, but has a significant incidence of potentially fatal agranulocytosis. Accordingly, we attempted to develop a prototype of a new generation of antipsychotics that is both more efficacious and safe. Our strategy was to create a compound that is not only active in behavioral tests that predict antipsychotic action but also shares the rich, multifaceted receptor pharmacology of clozapine without its side effects. To this end, Eli Lilly and Co. developed olanzapine. In this article we characterize the in vitro and in vivo receptor pharmacology of olanzapine. METHOD: We evaluated olanzapine interactions with neuronal receptors using standard assays of radioreceptor binding in vitro and well-established in vivo (functional) assays. RESULTS: Binding studies showed that olanzapine interacts with key receptors of interest in schizophrenia, having a nanomolar affinity for dopaminergic, serotonergic, alpha 1-adrenergic, and muscarinic receptors. In vivo olanzapine is a potent antagonist at DA receptors (DOPAC levels; pergolide-stimulated increases in plasma corticosterone) and 5-HT receptors (quipazine-stimulated increases in corticosterone), but is weaker at alpha-adrenergic and muscarinic receptors. Olanzapine has little or no effect at other receptors, enzymes, or key proteins in neuronal function. Olanzapine has a receptor profile that is similar to that of clozapine: it is relatively nonselective at dopamine receptor subtypes and it shows selectivity for mesolimbic and mesocortical over striatal dopamine tracts (electrophysiology; Fos). CONCLUSION: The binding and functional profile of olanzapine (1) is similar to that of clozapine, (2) indicates that olanzapine is an atypical antipsychotic drug, and (3) is consistent with clinical efficacy. If olanzapine also proves to be safe, then it will have high potential to become a more ideal antipsychotic drug.

Animals↗

PubMed1996

Muscarinic agonist inhibition of rat striatal adenylate cyclase is enhanced by dopamine stimulation.

Rat striatal adenylate cyclase stimulated with combinations of 100 microM dopamine plus 40-100 nM forskolin was inhibited to a significantly greater extent by the muscarinic agonist carbachol than was forskolin-stimulated activity in the absence of dopamine. In the presence of Ro 201724 as phosphodiesterase inhibitor, a 100 microM concentration of the adenosine agonist 2-chloroadenosine stimulated adenylate cyclase activity 6.6 -fold over basal activity. In contrast to dopamine-stimulated activity, carbachol did not significantly inhibit adenylate cyclase activity elevated by the adenosine agonist, indicating specificity of the muscarinic response for dopamine stimulation. The effects of muscarinic antagonists on striatal versus heart adenylate cyclase indicated that the striatal response was mediated primarily through M4 receptors. The present results suggest that muscarinic M4 and dopamine D1 receptors are co-localized and functionally coupled in rat striatum.

Adenylyl Cyclase Inhibitors↗

PubMed1995

The muscarinic M1 agonist xanomeline increases soluble amyloid precursor protein release from Chinese hamster ovary-m1 cells.

The functionally selective M1 agonist xanomeline, which is currently undergoing clinical trials as a therapy for Alzheimer's disease, was compared to the muscarinic agonist carbachol for effects on secretion of soluble amyloid precursor protein (APPs) from Chinese hamster ovary cells transfected with the human m1 receptor (CHO-m1). Release of APPs from CHO-m1 cells was increased maximally (4-10 fold) by 100 microM carbachol (EC50 = 11 microM) and by 100 nM xanomeline (EC50 = 10 nM). Stimulation of APPs secretion by xanomeline and carbachol was blocked by preincubation with 1 microM atropine. Carbachol did not stimulate APPs secretion from non-transfected CHO cells. Pilocarpine at 1 mM also increased APPs release. The efficacy of carbachol, xanomeline and pilocarpine for stimulating APPs secretion did not differ significantly. Activation of protein kinase C (PKC) in m1 transfected cell lines by 1 microM phorbol dibutyrate (PDBu) increased APPs release, and this was inhibited 97% by the PKC inhibitor bisindolemalemide. The PKC inhibitor decreased xanomeline and carbachol-stimulated APPs secretion by only 25-30%. These results demonstrate that xanomeline increased APPs release by activation of m1 muscarinic receptors and support the possibility that cholinergic replacement therapy for Alzheimer's Disease may reduce amyloid deposition.

Amyloid beta-Protein Precursor↗

PubMed1992

Forskolin stimulation of cyclic AMP accumulation in rat brain cortex slices is markedly enhanced by endogenous adenosine.

Stimulation of cyclic AMP (cAMP) accumulation in rat cortex slices by 1 microM forskolin (F) was markedly reduced (96%) by treatment with adenosine deaminase (ADA). The effect of ADA was progressively less at higher concentrations of F, but still inhibited the response by 50% at 100 microM F. ADA-mediated inhibition of the cAMP response to 1 microM F was completely reversed by 5 microM 2-chloroadenosine (CA), an ADA-resistant analogue. Stimulation by F (controls) and F plus CA (ADA treated) in cortex slices was significantly inhibited by 200 microM caffeine (CAF) and by 10 microM 8-phenyltheophylline. cAMP accumulation in ADA-treated cortex slices stimulated with CA at concentrations from 5 to 100 microM was markedly enhanced by 1 microM F. Neither ADA treatment nor 200 microM CAF significantly affected cAMP accumulation in slices stimulated by 1 microM vasoactive intestinal polypeptide or adenylate cyclase in membranes stimulated by 1 microM F. CAF (1 mM) did not significantly increase basal cAMP levels in cortex slices, whereas 1 mM 3-isobutyl-1-methylxanthine caused a significant 80% increase and 100 microM rolipram enhanced cAMP levels by 4.5-fold. F-stimulated cAMP accumulation (1 microM) in cortex slices was inhibited 98% by 1 mM CAF and 49% by 1 mM 3-isobutyl-1-methylxanthine, and was enhanced 2.5-fold by 100 microM rolipram. These data have been interpreted to indicate that the stimulation of cAMP accumulation in rat cortex slices by 1 microM F is predominantly due to synergistic interaction with endogenous adenosine and that the inhibition of this response by CAF is largely due to blockade of adenosine receptors.

2-Chloroadenosine↗

PubMed1990

Effect of basic fibroblast growth factor (bFGF) and insulin-like growth factors type I (IGF-I) and type II (IGF-II) on adult human keratinocyte growth and fibronectin secretion.

Effects of growth factors on keratinocyte migration and proliferation are of interest as an indication of their potential use in acceleration of wound re-epithelialization. Various growth factors were examined for effects on normal adult human keratinocyte growth and fibronectin (Fn) secretion for cells cultured in serum-free medium. Accumulation of Fn in the medium of cells growth with H + I + EGF + BPE paralleled growth during the exponential phase and declined as the cells approached confluence. Cells maintained in low Ca++ (0.15 mM) post-confluence and fed daily to prevent cornification continued to accumulate Fn in the medium, while those grown continuously in 1.2 mM Ca++ ceased Fn secretion at confluence. EGF, bFGF, and transforming growth factor-beta (TGF beta) stimulated keratinocyte Fn secretion in correlation with literature reports on the ability of these factors to stimulate the migration of these cells. In contrast, despite its marked effects on cell growth, BPE was found to consistently reduce the amount of Fn found in the medium when added to cultures containing either EGF or bFGF. Addition of BPE to cultures containing EGF or bFGF stimulated growth to the same extent, indicating that the effects of BPE on keratinocyte growth are not solely due to its content of bFGF.

Adult↗

PubMed1988

Gamma glutamyl peptidase: a novel enzyme from hairless mouse epidermis.

Recent literature has suggested that pyroglutamate (PCA) formation in stratum corneum occurs by spontaneous cyclization of glutamine residues derived from filaggrin breakdown. This paper describes an enzymatic alternative. Epidermal homogenates from hairless mice were found to catalyze the formation of PCA from both glutamine and glutamic acid at pH 6.2. Enzyme activity responsible for the first step in this reaction, gamma-glutamyl peptide formation, was partially purified using ammonium sulfate precipitation followed by ion exchange, gel filtration, and hydroxylapatite chromatography. Enzyme preparations free of gamma-glutamyl cyclotransferase activity (which forms PCA from certain gamma-glutamyl peptides) catalyzed formation of gamma-glutamyl-glutamine from glutamine and gamma-glutamyl-glutamate from glutamic acid. Enzyme preparations catalyzed hydrolysis of a variety of gamma-glutamyl peptides but did not split non-gamma-glutamyl peptides or the transpeptidase substrate gamma-glutamyl-rho-nitroanilide. Ammonium sulfate fractions containing both gamma-glutamyl peptidase and gamma-glutamyl cyclotransferase activity catalyzed linear formation of PCA from glutamic acid for periods of up to 19 h. Using gamma-glutamyl-leucine as a substrate, gamma-glutamyl peptidase activity was found to be much higher in crude extracts from epidermis than in preparations from liver, kidney, spleen, intestine, lung, brain, or heart. This activity has not, to our knowledge, been previously described in mammalian tissues.

Ammonium Sulfate↗