Yasuyuki Shimohigashi

Receptor binding and biological activity of bivalent enkephalins

Two series of dimeric enkephalin analogues were assayed for opioid activity in two isolated smooth muscle preparations: the guinea pig ileum (GPI) and the mouse vas deferens (MVD). Dimers have the general structure: X-(CH2)n-X, where X is H-Tyr-D-Ala-Gly-Phe-Leu-NH-(n = 0, 2, 4, 6, 8, 10, 12), for the first series of dimeric pentapeptide enkephalins (DPEn), and H-Tyr-D-Ala-Gly-Phe-NH-(n = 2, 4, 6, 8, 12), for the series of dimeric tetrapeptide enkephalins (DTEn). Comparison of biological activities with binding affinities revealed that: (1) the DPE series with n = 2-8 showed increased potency in the MVD assay relative to monomeric [D-Ala2, Leu5]enkephalinamide (DALEA); (2) there was an associated increase affinity for the delta receptor of rat brain or neuroblastoma-glioma hybrid cells. (however, the relative potencies were higher in the MVD assay then predicted on the basis of binding affinities); (3) the DTE series also showed an increase in delta receptor affinities and MVD potencies relative to DALEA, for n = 2-12; (4) for the DTE series, the increase in MVD activities was less than that expected on the basis of delta binding affinity; (5) for both the DPE and DTE series, activities in the GPI assay and mu-receptor affinities were highly correlated: as the length of the methylene bridge increased from 2 to 12, there was a progressive loss of activity in both assays, with a similar pattern for DPE and DTE. Two selected dimers and their corresponding monomers were also assayed for antinociceptive activity in vivo: results were consistent with GPI and mu-binding but not with MVD and delta-binding. Two alkylamide analogs of penta- and tetrapeptide monomers, representing the monomer with the attached spacer of the most active dimers, were also assayed in biological and binding assays. Comparison of these compounds with the corresponding dimers suggest that the changes in activities and selectivities induced by dimerization are not a spurious effect of the presence of an akylamide derivative of the carboxy terminal of enkephalin but rather may represent a specific effect due to the bivalent nature of the ligands.

Carbohydrate structures in the β-subunit of human chorionic gonadotropin play a dominant role in hormonal activity

Human chorionic gonadotropin (hCG) is a glycoprotein hormone containing 30% carbohydrate. It consists of two non-identical polypeptide chains, designated as aand ,& subunits. The m-subunit is common among other glycoprotein hormones including LH, FSH and TSH, whereas the P-subunit dictates the specific hormonal activities. The assignment of P-subunit as the functional specific unit derives largely from the hybridization studies between the two subunits of interhormones and interspecies, in which the specific hormonal activity of such hybride always follows the P-subunit present. Studies based on chemical and enzymatical modifications of carbohydrate moieties in hCG have revealed that the terminal sialic acid residues are important in prolonging the plasma circulatory life of the hormone, and that successive exoglycosidase treatments of asialo-hCG reduce its ability to stimulate the production of CAMP and steroids in gonadal cells [l-5]. However, the role of the carbohydrate structures of each subunit in the essential hormonal activities of hCG has not been investigated. We therefore addressed the following questions in an attempt to clarify the role of carbohydate moieties in the subunits:

Importance of the stereo-orientation of aromatic groups in enkephalins to opiate receptor recognition

PO +Dehydrophenylalanine (delta Phe) having the E-configuration (delta EPhe ; phenyl and C = O cis) was incorporated into [Leu5]-enkephalin in order to restrict its conformation. Compared with the Z-isomer, in the radio-ligand receptor binding assays, [D-Ala2, delta EPhe4 , Leu5] enkephalin showed drastically decreased potency for the delta and mu opiate receptors, i.e., 260- and 150-fold loss of affinity, respectively. The results strongly indicate that the opiate receptors require the Z-configuration (phenyl and C = O, trans) of the delta Phe4 residue and may require a specific interrelationship between the aromatic rings of the Tyr1 and Phe4 residues in the molecule for binding. The conformation of [Leu5]-enkephalin specific for the delta receptors was analyzed and a comparison made with its crystal structure recently elucidated.

The enzyme stability of dehydro-enkephalins

Dehydro-enkephalins [delta Ala2]-, [delta Ala3]-, [delta Phe4]-, and [delta Leu5]enkephalins, were examined for their stability to enzymatic hydrolysis by carboxypeptidase Y [EC 3.4.16.1]. The successively liberated amino acids were determined quantitatively by amino acid analyses. The saturated leucine-enkephalin was rapidly hydrolyzed from the COOH-terminus. However, peptide linkages with alpha, beta-dehydroamino acid residues placed in the enkephalin molecule were strongly resistant to the enzyme at the carboxyl side and completely resistant at the amino side of the dehydro residue.

Gel high-performance liquid chromatographic studies on the elution behavior of chemically deglycosylated human chorionic gonadotropin and its subunits

The elution behavior of human chorionic gonadotropin and its subunits, and their desialylated and deglycosylated derivatives was studied on gel high-performance liquid chromatography. Using three TSK G-SW columns, excellent separations were achieved for eleven human chorionic gonadotropin derivatives. In a comparison of the native (30% carbohydrate content), asialo (24%) and HF-deglycosylated (8%) subunits, the estimated values of molecular weights on the basis of a series of reference globular proteins deviated in various degrees from the actual molecular weight. The extent of deviation depended on the carbohydrate content and possibly the location of carbohydrate chains. The data from recombination studies suggest that the beta-subunit is the dominant determinant for their expanded molecular size.

Differential effects of GTP and cations on binding of labeled dimeric and monomeric enkephalins to neuroblastoma-glioma cell delta opiate receptors

Abstract Binding of radio-labeled enkephalin monomers [D-Ala 2 ,Met 5 ]Enkephalin Amide (DAMEA) and [D-Ala 2 ,D-Leu 5 ]Enkephalin (DADLE) and a dimer of [D-Ala 2 ,Leu] Enkephalin Amide (DPE 2 ) to neuroblastoma-glioma (NG108-15) cells was examined in the presence and absence of GTP and/or cations. We found that: (1) binding occurs to a single class of homogeneous and non-interacting membane sites; (2) the affinity of the enkephalin dimer is reduced 50% in the presence of Mn 2+ and 65% in the presence of both Mn 2+ and GTP; (3) GTP alone either increases or does not change affinity of DPE 2 ; (4) Na + and GTP significantly decrease the affinities of monomers, but not that of the dimer; and (5) a higher concentration (0.1 mM) of GTP increases the binding of DPE 2 but significantly decreases binding of monomers. Conclusion: Changes in binding of a dimeric enkephalin by Na + , Mn 2+ and GTP are significantly and qualitatively different than those occurring for monomers.

DIMERIC PENTAPEPTIDE AND TETRAPEPTIDE ENKEPHALINS: NEW TOOLS FOR THE STUDY OF DELTA OPIOID RECEPTORS

AbstractWhen a dimeric ligand can react bivalently, one would expect an increase in affinity, selectivity, and possibly biological activity. On this premise, we have synthesized and characterized two series of dimers, viz.: Dimeric Pentapeptide Enkephalin (DPEn = (H-Tyr-D-Ala-Gly-Phe-Leu-NH-)2 · (CH2)n, and Dimeric Tetrapeptide Enkephalin (DTEn) = (H-Tyr-D-Ala-Gly-Phe-NH)2 · (CH2)n, with n = 2, 4, …, 12. These dimers display affinity, activity, and δ/μ selectivity which vary systematically with chain length (n). DPE2 shows a seven-fold increase in affinity for the δ receptor of whole brain and NG108-15 cells, relative to monomer, while its activity for the μ receptor is similar to enkephalin monomers. DTE12 shows a dramatic increase in δ selectivity relative to its monomer. The association rate constant for 3H-DPE2 is increased two-fold and its dissociation rate constant is significantly reduced, relative to monomer. DPE2 shows a loss of affinity in the presence of Na+ or Mn++, while GTP unexpectedly incr...