Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The characteristics of cholecystokinin (CCK) binding to its receptors in a particulate membrane fraction of mouse cerebral cortex were studied by employing biologically active radioiodinated CCK prepared by conjugation with 125I-Bolton-Hunter (125I-BH) reagent. At 24 degrees C binding was rapid, reversible, and linearly related to protein content. Binding was maximal at acidic pH (6.5) and reduced by the presence of monovalent cations. Under physiological conditions (pH 7.4, 118 mM-NaCL, 4.7 mM-KCl) Scatchard plots of CCK binding were linear with a KD value of 1.27 nM and binding capacity of 115 fmol/mg protein. Optimal binding required the presence of both Mg2+ and EGTA, and was inhibited by the addition of micromolar concentrations of Cu2+ (ID50 = 30 microM). The cortical receptor recognized all major forms of CCK, with an order of potency of: cholecystokinin octapeptide (CCK8) greater than CCK greater than cholecystokinin tetrapeptide (CCK4). Desulfated cholecystokinin octapeptide (dCCK8) had a 10-fold lower affinity than CCK8. Dibutyryl cyclic GMP, a potent competitive inhibitor of CCK binding to receptors in pancreas, was not a specific inhibitor of CCK binding to brain receptors. These present results support the concept that CCK may function as a regulatory peptide in brain, and that the cortical CCK receptor is different from the receptors mediating the peripheral action of CCK.
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PMID:Characterization of receptors for cholecystokinin and related peptides in mouse cerebral cortex. 626 5

The cholecystokinin (CCK) receptor in purified plasma membranes prepared from mouse pancreatic acini had a binding affinity of 1.8 nM, an acid pH optimum between 6.0 and 6.5, and an analog specificity of CCK8 greater than CCK33 greater than desulphated CCK8 greater than CCK4. Binding of CCK to its receptor was abolished by pretreatment of plasma membranes with trypsin. When [125I]CCK was cross-linked to its receptors with disuccinimidyl suberate, and the preparation solubilized and subjected to gel electrophoresis and autoradiography, the hormone was associated with Mr 80 000 protein in both the presence and absence of the reducing agent dithiothreitol.
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PMID:The CCK receptor on pancreatic plasma membranes: binding characteristics and covalent cross-linking. 629 90

Prior studies have shown that the cerebral cortex cholecystokinin (CCK) receptor can bind CCK and gastrin analogs with high affinity. In the present work the brain CCK receptor had approximately a three times greater affinity for CCK8 than its C-terminal tetrapeptide (CCK4) while the C-terminal tripeptide (CCK3) was 1000-fold less potent than CCK4. Thus the C-terminal tetrapeptide appears to be the minimal C-terminal CCK sequence required for high affinity binding. Since brain membranes degrade various peptides including CCK, we also evaluated the stability of CCK analogs under the conditions used to measure receptor binding by the following three methods: (1) Studies of degradation-resistant analogs in binding assays; (2) analysis of analog degradation by high performance liquid chromatography (HPLC); and (3) determination of the change in potency of CCK analogs in competitive binding studies subsequent to preincubation with brain membranes. These studies indicated that degradation of analogs by the brain membranes although significant did not account for the differences in potency of analogs in competitive binding studies. Therefore, the observed differences in potencies of the analogs tested are due to the receptor affinity and not sensitivity of the analog to degradation.
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PMID:Binding specificity of the mouse cerebral cortex receptor for small cholecystokinin peptides. 632 3

A precise and specific radioimmunoassay method for measuring plasma cholecystokinin (CCK) is described. The present assay system using a stable tracer iodinated by means of a modified Chloramine-T method followed by purification on a Sephadex G-15 and a SP Sephadex C-25 column, as well as careful corrections for non-specific plasma effects, allows measurements of fasting plasma CCK in the low pmol/l range; the significant rise in plasma CCK following duodenal infusion of fat; and the significant diurnal variation of plasma CCK. Apparent immunoreactive meal-stimulated plasma CCK was eluted from a Sephadex G-50 superfine column in four fractions. The first and largest peak probably represents plasma CCK bound to plasma proteins and non-specific plasma effects, the second and smaller peak big CCK with molecular weight between some 5,000 and some 30,000, the shoulders following the second peak ordinary CCK33 and CCK39 variant, and the final, and by far the smallest peak, may possibly represent COOH-terminal tetra- (CCK4) or octapeptides (CCK8) of CCK.
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PMID:Radioimmunoassay of plasma cholecystokinin (CCK), duodenal release of CCK, diurnal variation of plasma CCK, and immunoreactive plasma CCK components in man. 719 31

1. Ionic conductances controlled by type A and type B cholecystokinin (CCK) receptors were studied in neurons of the rat nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMNV), using intracellular and whole-cell patch clamp recordings in current or voltage clamp configuration during bath application of agonists (CCK8, CCK4, BC 264) and antagonists. 2. CCKA receptor-related inhibition was associated with a membrane hyperpolarization and a decrease in input resistance that developed 2-6 min after the arrival of drug into the extracellular medium. These effects were induced by 5 nM CCK8 but not BC 264 and they were blocked by the CCKA antagonist, L-364,718, but not by the CCKB antagonist, L-365,260. 3. CCKA receptor-related inhibition was generated by a potassium current that reversed at a reversal potential E(rev) of -73 +/- 1 (mean +/- SE) mV with bathing potassium concentration [K+]o = 6 mM and at -88 +/- 1 with [K+]o = 3 mM, in agreement with the Nernst equation for potassium ions. 4. CCKB receptor-related excitation was associated with a membrane depolarization and an increase of the input resistance induced by the following agonists at threshold concentrations: CCK8 (0.2 nM) > or = BC 264 (0.4 nM) > CCK4 (10.9 nM). The increase of input resistance was abolished by L-365,260 and was maintained after blockade of the CCKA current by L-364,718. 5. CCKB receptor-related excitation, in the neurons (30% of cases) in which clear response reversal was observed, appeared to be generated by a decrease of a potassium conductance. Responses showed a reversal potential E(rev) of -68 +/- 4 mV with [K+]o = 6 mM and -89 +/- 1 mV with [K+]o = 3 mM, verifying predictions from the Nernst equation applied to potassium ions. However, in 70% of cases, clear reversal was not observed at membrane potentials negative to the theoretical potassium equilibrium potential EK. 6. In voltage clamp studies, CCK8 induced a 181 +/- 17 pA inward current associated with a 26 +/- 4% decrease in the instantaneous current (I(ins)) generated by hyperpolarizing voltage steps. This effect on I(ins) was demonstrated in the absence of effects on the outward noninactivating potassium current (IM) and on the inward noninactivating cationic current (IQ). 7. CCKB receptor-mediated excitation was not suppressed by cobalt, a blocker of calcium currents, and was not associated with a change of the calcium-dependent potassium current (IK(Ca)).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Cholecystokinin-gated currents in neurons of the rat solitary complex in vitro. 750 60

Cholecystokinin (CCK) is a gut hormone that regulates pancreatic endocrine functions via CCKA receptors. CCK4 (Trp-Met-Asp-Phe-NH2) has an insulinotropic effect, but is 1000-fold less potent than CCK8. The in vitro potencies and selectivity of newly synthesized CCK4 analogs were investigated. Exchanging various a amino acids, for example Met by Nle and modifying Phe and/or Trp, led to compounds that were much more effective than CCK4 itself and show insulinotropic effects comparable with those of CCK8. Compounds that possess electron withdrawing groups on the C-terminal phenylalanine were especially effective; compounds with electron-donating groups had no effect. In contrast to CCK8 the synthetic CCK4 compounds were selective for the endocrine pancreas: they had no agonistic or antagonistic effect on the contraction of the guinea pig ileum, amylase release from isolated acini, and no major effect on the feeding behavior of mice being supplied with either compound by an implantable AlzetR pump for 8 days. The data indicate that some of the synthetic tetrapeptides exhibit a high affinity for the CCK receptor of the endocrine pancreas and that they are highly selective for this (peripheral) CCKA receptor subtype. The beta-cell CCKA receptors are different from those in exocrine pancreas, smooth muscle, and those for regulating appetite; these peripheral receptor subtypes can be discriminated for the first time.
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PMID:Evidence for cholecystokinin receptor subtype in endocrine pancreas. 753 22

Given the high resistance of the cholecystokinin octapeptide (CCK8) to in vivo peptidase degradation, the possible existence of a reuptake system for this peptide was investigated. Efficient accumulation of intact, tritiated propionyl CCK8 ([3H]pCCK8) was observed following its incubation with rat cortical synaptosomes but not with cerebellar synaptosomes, where no cholecystokinin immunoreactivity was found. This uptake process appeared to be dependent on temperature, duration of incubation, concentration of radioligand, the presence of glucose and the integrity of the synaptosomes. A Lineweaver-Burk analysis indicated that the putative uptake process is characterized by a single Km value of 10.7 nM and a Vmax of 8.5 fmol/min/mg of protein. Carbonyl cyanide-m-chlorophenyl hydrazone, an uncoupler of oxidative phosphorylation, blocked accumulation of [3H]pCCK8, whereas ouabain did not. The uptake was found to be highly specific since, among all the cholecystokinin analogues tested, only CCK8 and, to a lesser extent, CCK7, were able to inhibit [3H]pCCK8 uptake. The rate of [3H]pCCK8 uptake was not affected by CCK4, CCK5, D-Trp CCK8, BC 264, a potent and radioactivity was observed using [3H]pBC 264, a result which is not in favour of a cholecystokinin receptor-induced internalization mechanism. The potent and selective uptake mechanism characterized in this study could participate, in conjunction with extra and intracellular degradation of CCK8 by peptidases, in the interruption of cholecystokinin-conveyed messages in the brain.
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PMID:Evidence for a high-affinity uptake system for cholecystokinin octapeptide (CCK8) in rat cortical synaptosomes. 761 12

It has been reported that substitution of the Met31 residue in Boc-CCK4 (Boc-Trp30-Met31-Asp32-Phe33-NH2, CCK33 numbering) by trans-3-propyl-L-proline yields a highly potent and selective CCK-B agonist. To further explore the structural requirements of the Met31 side chain in the receptor-bound conformation of CCK4, we have synthesized several Ac-CCK4 analogs containing substitution of Met31 by 3- and 4-(alkylthio)-substituted proline derivatives. To this end we have developed novel synthetic routes to enantiomerically pure N-Boc-4-cis- and -trans-(methylthio)prolines and racemic N-Boc-3-cis and -trans-[(4-methylbenzyl)thio]prolines. The protected mercaptoprolines were incorporated into Ac-CCK4 analogs using SPPS and were alkylated using various electrophiles following cleavage from the solid support. Binding assays reveal that 3-(alkylthio)prolines analogs have higher affinities at the CCK-B receptor than the corresponding 4-(alkylthio)proline analogs, and that trans-3-(alkylthio)proline analogs had higher affinities than corresponding cis-3-(alkylthio)proline analogs. Within both the cis- and trans-3-(alkylthio)proline series, the order of potency was found to be Me < Et < n-Pr. The trans-3-(n-propylthio)-L-proline analog demonstrates a higher affinity than that reported for Boc-CCK4[trans-3-propyl-L-Pro31]. Comparison of the low-energy structures calculated for several high-affinity Ac-CCK4 analogs reveal a common geometry which we propose to be the CCK-B receptor-bound conformation. This model shows grouping of the hydrophobic side chains of Trp, Met, and Phe at one side of the molecule and the hydrophilic side chain of Asp and the C-terminal carboxamide at the other side.
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PMID:Ac-[3- and 4-alkylthioproline31]-CCK4 analogs: synthesis and implications for the CCK-B receptor-bound conformation. 783 25

Extracellular K+ activities (aKe) and neuronal and glial membrane potentials were recorded in the nucleus tractus solitarius (NTS) and in the dorsal vagal motor nucleus (DVMN) of rat brainstem slices after orthodromic stimulation of the tractus solitarius (TS). In glial cells, repetitive stimulation of the TS induced depolarizations of up to 30 mV followed by repolarizations which were fitted by exponential curves with a time constant of 1.6-5 s. Similar stimulations induced elevations of aKe of up to 8 mM, the recovery of which was fitted by single exponential curves with a time constant ranging between 1.6 and 4 s. These elevations in aKe were reduced by 75% during blockage of synaptic transmission in low Ca2+, high Mg2+ solution, and by 24% with application of 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX, 50 microM). Perfusion with a low Mg2+ solution increased the aKe response to stimulation of the TS, an effect that was reduced by the addition of 2-amino-5-phosphono-valeric acid (AP7, 50 microM) to the bath. No significant change in aKe and glial potential was seen when superfusing high concentrations of the C-terminal octapeptide of cholecystokinin (CCK8, 1-5 microM) and C-terminal tetrapeptide (CCK4, 50-100 microM). The effect of TS stimulations on solitary complex neurons suggests that extracellular K+ concentration is increased during synaptic activation of non-NMDA or NMDA ionotropic receptors. Conversely, slow depolarizations elicited by repetitive afferent activity or excitation by CCK agonists develop in neurons in the absence of measurable extracellular K+ fluctuations or glial depolarization.
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PMID:Extracellular potassium, glial and neuronal potentials in the solitary complex of rat brainstem slices. 809 69

1. Sulphated cholecystokinin octapeptide (CCK8S, 0.03-1.00 microM), pentapeptide (CCK5) and tetrapeptide (CCK4) elicited concentration dependent depolarizations of neonate rat ventral roots in vitro. 2. CCK5 was equipotent with CCK8S although CCK4 was weaker (equipotent molar ratio 17.5). 3. CCK8S-induced depolarizations were depressed by tetrodotoxin (0.1 microM), Mg2+ ions (0.75 mM) and the NMDA receptor antagonist 2-amino-5-phosphonopentanoate (AP5, 10 microM). These results suggest that CCK8S-induced depolarizations were predominantly mediated through the release of an excitatory amino acid from interneuronal sites. 4. The selective CCKA and CCKB receptor antagonists, L-364,718 and L-365,260 both depressed CCK8S-induced depolarizations. CCK8S dose ratios in the presence of 1 microM L-364,718 or L-365,260 were 4.5 and 11.2 respectively, suggesting the response was mediated predominantly through stimulation of CCKB receptors. 5. These results suggest that the neonate rat hemicord preparation is a suitable tissue for functional CCK receptor assays.
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PMID:Cholecystokinin-induced ventral root depolarization of neonate rat hemicord in vitro. 809 38


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