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M Wollemann

Publications and source records attributed to M Wollemann.

At least 37 records · Page 2Linked to original sources
PubMed1989

Effects of oxymorphazone in frogs: long lasting antinociception in vivo, and apparently irreversible binding in vitro.

Oxymorphazone (at doses of 50-200 mg/kg) was found to be a relatively weak antinociceptive drug in intact frog (Rana esculenta) when acetic acid was used as pain stimulus. Frogs remained analgesic for at least 48 hrs following oxymorphazone (200 mg/kg) administration. The ligand increased the latency of wiping reflex in spinal frogs too. These effects were blocked by naloxone. In equilibrium binding studies (3H)oxymorphazone had high affinity to the opioid receptors of frog brain and spinal cord as well (apparent Kd values were 8.9 and 10.6 nM, respectively). Kinetic experiments show that only 25% of the bound (3H)oxymorphazone is readily dissociable. Preincubation of the membranes with labeled oxymorphazone results in a washing resistant inhibition of the opioid binding sites. At least 70% of the (3H)oxymorphazone specific binding is apparently irreversible after reaction at 5 nM ligand concentration, and this can be enhanced by a higher concentration of tritiated ligand.

Analgesics↗
PubMed1989

Cation and guanine nucleotide effects on ligand binding properties of mu and delta opioid receptors in rat brain membranes.

Monovalent cations such as Na+, K+ and Li+ at 100 mM concentration inhibit the binding of mu and delta opioid agonists (e.g. (3H) dihydromorphine and (3H) D-Ala2-Leu5-enkephalin), enhance opioid antagonist [(3H) naloxone] binding in rat brain membranes. Divalent cations have an opposite effect: Mg2+ or Mn2+ (2mM) increase both mu and delta agonist binding, and decrease the antagonist binding. The effect of guanosine 5'-triphosphate and its non-hydrolysable analogue 5'-guanylyl-imidodiphosphate at micromolar concentrations is similar to that of sodium ion. In kinetic experiments, guanine nucleotides promote the dissociation of radiolabelled agonists from their binding sites by increasing the rate of dissociation. Equilibrium saturation binding studies show that only binding of the opioid agonist ligands are significantly inhibited by 5'-guanylyl-imidodiphosphate.

Animals↗
PubMed1988

Enkephalin photoaffinity probes: synthesis and binding properties.

The diazomethyl ketone derivative of D-Ala2-Leu-enkephalin and Leu-enkephalin were synthesized. Replacement of the C-terminal carboxyl group with CO-CHN2 resulted in a potency decrease, the new compounds display micromolar affinities to 3H-naloxone and 3H-DALE binding sites. Photolysis of the ligands bound to rat brain membranes resulted in an approximately 30% irreversible loss of the receptors. Photoinactivation was prevented by the opiate antagonist, naloxone, thus providing that the ligands are specific photoaffinity probes of the opioid receptors.

Affinity Labels↗
PubMed1987

Covalent labeling of opioid receptors with 3H-D-Ala2-Leu5-enkephalin chloromethyl ketone. I. Binding characteristics in rat brain membranes.

The chloromethyl ketone derivative of D-Ala2-Leu5-enkephalin was synthesized in a radioactive form, and the resulting compound (3H-DALECK) was used to label opioid receptors. 3H-DALECK binds with high affinity, specificity and saturability to rat brain membranes. The number of sites labeled is 130 fmoles/mg protein. Unlabeled opioids inhibited the binding of 3H-DALECK; etorphine and DAGO being most potent. A 10-fold preference for mu sites over delta was seen in site-specific competition experiments; while DALECK displayed low affinity for kappa sites of rat brain. DALECK irreversibly blocked a certain population of sites. Approximately 40% of 3H-DALECK binding at 15 min, and 60% at 60 min association time did not dissociate in the presence of a large excess of unlabeled DALECK and was resistant to washing. Autoradiography performed after SDS-PAGE revealed specific alkylation of proteins with molecular weight of 74, 65, 56, 43 and 34 kD. These results demonstrate the applicability of using 3H-DALECK to covalently label opioid receptors.

Amino Acid Chloromethyl Ketones↗
PubMed1987

Covalent labeling of opioid receptors with 3H-D-Ala2-Leu5-enkephalin chloromethyl ketone. II. Binding characteristics in frog brain membranes.

3H-D-Ala2-Leu5-enkephalin chloromethyl ketone (3H-DALECK) was used to label opioid receptors of frog brain membranes. We have previously shown (15) that 70% of the opioid receptors are of kappa type in this preparation. The binding of 3H-DALECK was of high affinity, half maximal binding being achieved by 0.9 nM of the radioligand. The number of sites labeled was calculated to be 108 fmol/mg protein. Opioid ligands, incubated with the membranes prior to the label, inhibited 3H-DALECK binding with the following rank order:etorphine greater than EKC greater than DAGO greater than DALECK greater than DADLE. Dissociation experiments showed that 70% of the binding is irreversible. Fluorography performed after SDS-PAGE revealed specific covalent labeling of protein subunits of 90, 58 and 20 kD molecular weights. Results will be compared to those obtained in rat brain (13). Our two studies demonstrate that 3H-DALECK is a useful probe for investigation the subunit structure of opioid receptors.

Amino Acid Chloromethyl Ketones↗
PubMed1987

The molecular structure of opiate receptors.

Examples are given which demonstrate that the kappa opiate receptor can be separated from mu and delta subtypes by their physical parameters. When the subunit composition of the subtypes are compared, no definite differences are encountered. The data from the literature are also contradictory. This may in part be explained by the fact that the different receptors appear to contain a structurally common high affinity binding site. A possible hypothesis would be that the subtypes differ from each other by the number of subunits.

Animals↗
PubMed1987

Tyr-D-Ala-Gly-(Me)Phe-chloromethyl ketone: a mu specific affinity label for the opioid receptor.

An alkylating tetrapeptide enkephalin derivative, Tyr-D-Ala-Gly-(Me)Phe-chloromethyl ketone (DAMK) was synthesized, and its binding characteristics on rat brain membranes were evaluated. In competition experiments, the product shows high affinity for the mu opioid binding site of the rat brain membranes, whereas its binding to the delta and kappa subtypes is weak. Micromolar concentrations of this ligand produce a dose-dependent, apparently irreversible inhibition of /3H/-naloxone binding, with apparent IC50 value of 1-5 uM. Neither reversibly binding opioids nor tosyl-amino acid chloromethyl ketones show these effects. Saturation binding analysis with /3H/-naloxone of membranes preincubated with Tyr-D-Ala-Gly-(Me)Phe-CH2Cl reveal a selective and irreversible inhibition of the high affinity /3H/-naloxone binding site. Irreversible blockade of mu-selective /3H/-ligand binding by Tyr-D-Ala-Gly-(Me)Phe-CH2Cl is much more effective than that of the binding of /3H/-enkephalin or /3H/-ethylketocyclazocine. The mu-selective binding properties of this new irreversible enkephalin analogue suggest that it could serve as an affinity label for the mu opioid receptor subtype.

Affinity Labels↗
PubMed1987

Purification of a kappa-opioid receptor subtype from frog brain.

A kappa-opioid receptor subtype was purified from a digitonin solubilized preparation of frog brain membranes using affinity chromatography. The affinity resin was prepared by coupling D-Ala2-Leu5-enkephalin to Sepharose-6B matrix. After elution of the receptor by 50 mumol naloxone, the kappa-subtype was separated from the mu- and delta-subtypes by gel permeation chromatography on Sepharose-6B. The purified receptor binds 3,900 pmol [3H]-ethylketocyclazocine per mg protein (a 4,300-fold purification over the membrane-bound receptor) with a KD of 8.3 nM. The purified receptor protein exhibits high affinity for kappa-selective ligands. The purified fraction shows two bands (Mr 65,000 and 58,000) in sodium dodecyl sulfate gel electrophoresis.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1986

Azidomorphine is an agonist of high-affinity opioid receptor binding sites.

Azidomorphine at low concentration (10(-9) M) inhibits the high-affinity binding site of labeled naloxone in rat brain membrane preparations. In the presence of Na+ and guanine nucleotides the displacement curves of azidomorphine are increased toward high concentrations, whereas Mg2+ ions decrease the IC50 values; This demonstrates the agonist behavior of azidomorphine in binding experiments. When compared with morphine, azidomorphine displayed five-fold lower IC50 values. Based on the presented results, azidomorphine appears to be a good candidate for photoaffinity labeling of opiate receptors.

Analgesics↗
PubMed1986

Hydrodynamic parameters of opioid receptors from frog brain.

Active opioid receptors were solubilized from frog (Rana esculenta) brain membranes using 1% digitonin. The solubilized preparation was sedimented in sucrose density gradient and applied to Sepharose-6B column. In the ultracentrifugation experiments, two distinct molecular forms of the opioid receptors were observed with apparent s20, w values of 15.7 and 10.8 S. The estimated molecular weights were 470 and 180 kD. The Stokes radii of the two separate forms were determined by gel filtration and found to be 71 and 42 A. The corresponding molecular weights were 500 and 140 kD indicating a good correlation with data obtained from the sedimentation experiments.

Animals↗
PubMed1986

Kinetics and physical parameters of rat brain opioid receptors solubilized by digitonin and CHAPS.

Rat brain opioid receptors were solubilized with digitonin and a zwitterionic detergent, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS). The yield of solubilization was 70-75% with digitonin and 30-35% with CHAPS. Kinetic and equilibrium studies performed from digitonin extracts resulted in KD values comparable with those of the membrane fractions. Two [3H]naloxone binding sites were obtained in the extracts similarly to membrane fractions. The rank order potency of drugs used in the competition experiments did not change during solubilization. The distributions of mu, delta, and kappa opioid receptor binding sites were similar in membrane and digitonin-solubilized fractions (48-50% mu, 35-37% kappa, and 13-17% delta subtypes). The hydrodynamic properties of digitonin- and CHAPS-solubilized preparations were studied by sucrose density gradient centrifugation and Sepharose-6B chromatography. In all cases, two receptor populations were identified with the following parameters: sedimentation coefficients for the digitonin extracts were 9.2S and 13.2S and for CHAPS extract 8S and 15.6S; the Stokes radii were 45 A and 65A for the digitonin extract and 31A and 76A for the CHAPS-solubilized preparation.

Animals↗
PubMed1985

Separation of kappa-opioid receptor subtype from frog brain.

Complete separation of the [3H]ethylketocyclazocine [( 3H]EKC) specific binding (kappa subtype) from tritiated Tyr-D-Ala2-Me-Phe4-Gly-ol5 enkephalin (DAGO) and Tyr-D-Ala2-L-Leu5-enkephalin (DALA) binding (mu-and delta-subtypes, respectively) was achieved by Sepharose-6B chromatography and sucrose density gradient centrifugation of digitonin solubilized frog brain membranes. The apparent sedimentation coefficient (s20.w) for the kappa receptor-detergent complex was 13.1 S and the corresponding Stokes radius 64 A. The isolated fractions exhibited high affinity for EKC and bremazocine, whereas mu- and delta-specific ligands were unable to compete for the [3H]EKC binding sites, indicating that the kappa subtype represents a separate molecular to compete for the [3H]EKC binding sites, indicating that the kappa subtype represents a separate molecular entity from the mu and delta receptor sites.

Animals↗
PubMed1985

Regulation of beta-adrenergic sensitivity by guanine nucleotides in rat brain.

Binding of beta-adrenergic receptor by labelled dihydroalprenolol was inhibited in the presence of GTP or GppNHp in rat brain particulate fractions. The inhibition was suspended when the particulate fraction was pretreated at 30 degrees C for 20 minutes and washed 3 times. It is concluded that the inhibition was due to endogenous bound catecholamines.

Animals↗
PubMed1984

Solubilization and characterization of opioid binding sites from frog (Rana esculenta) brain.

Active opioid receptors were solubilized from frog (Rana esculenta) brain membrane fractions by the use of 1% digitonin. It was found by kinetic as well as by equilibrium measurements that both the membrane and the solubilized fractions contain two binding sites. For the membrane preparations, KD values were 0.9 and 3.6 nM, and Bmax values were 293 and 734 fmol/mg protein. For the solubilized preparations, KD values were 0.4 and 2.6 nM, an Bmax values were 35 and 266 fmol/mg protein. The stereospecificity of the binding did not change during solubilization. Both the membrane-bound and the solubilized receptors showed weak binding of enkephalin and mu-specific drugs, suggesting that they are predominantly of the kappa-type. The membrane-bound and the soluble receptors showed the same distribution of subtypes, i.e., 70% kappa, 13% mu, and 17% delta for the membrane-bound and 71% kappa, 17% mu, and 12% delta for the soluble receptors.

Animals↗
PubMed1983

Binding characteristics and analgesic activity of D-ALA2-Leu5-enkephalin chloromethyl ketone.

The chloromethyl ketone derivative of D-Ala2-Leu5-enkephalin (DALECK) was synthesized and its potency was tested in competing for 3H-naloxone binding sites and inducing analgesia. It was established that the compound is a potent affinity reagent at alkaline pH, blocking selectively and irreversibly the high-affinity (KD less than 1 nM) binding site. Intracisternally given DALECK showed a long-lasting, dose-dependent antinociceptive effect in the rat tail-withdrawal test. This could be completely antagonized by naloxone administration showing the reversible nature of DALECK in this in vivo assay. It is suggested that DALECK binds reversibly to the morphine receptor which mediates analgesia but irreversibly to the enkephalin receptor, the function of which remains to be elucidated.

Amino Acid Chloromethyl Ketones↗