R. Glenn Hammonds

Synthetic insulin-like growth factor II

Human insulin-like growth factor II with 67 amino acid residues and three disulfide bridges has been synthesized by the solid-phase method. Homogeneity of the synthetic product is ascertained by chromatofocusing, high performance liquid chromatography and amino acid analysis. In both radioimmunoassay and radioreceptor assay, the synthetic product is indistinguishable from the natural hormone.

β-Endorphin: Thermodynamics of the binding reaction with rat brain membranes☆

Abstract The binding of human β-endorphin to rat brain membranes was studied at various temperatures in the absence and presence of sodium. Between 0 and 30 °C, 100 m m sodium depresses the binding of β h -endorphin by reducing its affinity for binding sites 7- to 10-fold but has no effect on the total number of binding sites present in the membranes preparation. Kinetic studies show that the decrease in affinity in sodium is largely accounted for by a 10-fold decrease in the association rate constant within the whole range of temperatures examined. High versus low temperatures markedly enhance the binding of β h -endorphin by increasing its affinity for receptors, both in the presence and absence of sodium, without change in the number of receptors. This enhancement in affinity is due to an increase in the association rate constant between 0 and 30 °C, coupled with a slight increase in the respective dissociation rate constants. Temperature variations do not interact with the influence of sodium on the β-endorphin receptor. The integrity of the tritiated peptide was assessed at each temperature used. No more than 6% of degraded material was found at the highest temperature of incubation examined. Large positive values of the standard enthalpy and entropy changes were observed both in the absence and presence of sodium indicating an entropically driven binding reaction in both cases. The dependence of binding affinity upon sodium and temperature strongly suggests that hydrophobic interactions play a major role in stabilizing the hormone-receptor complex while electrostatic and/or hydrogen-bonded interactions might play a more specific role in recognition processes.

Binding characteristics of dermorphin, and [dermorphin1–7]-βc-endorphin in rat brain membranes

Dermorphin and a camel beta-endorphin (beta c-EP) analog in which residues 1-7 correspond to the dermorphin sequence ([Dermorphin1-7]-beta c-EP) have been investigated with respect to their receptor binding characteristics using human and camel beta-EP as reference peptides. Tritiated dihydromorphine, [D-Ala2, D-Leu5]-enkephalin, ethylketocyclazocine and human beta-endorphin were used as primary ligands in the rat brain membrane preparation for radioreceptor assay. Camel beta-endorphin was the most potent peptide in all experiments. [Dermorphin1-7]-beta c-EP is significantly less potent towards 3H-ethylketocyclazocine and 3H-[D-Ala2, D-Leu5]-enkephalin but is as potent towards 3H-dihydromorphine and 3H-human beta-endorphin. Dermorphin itself weakly displaces tritiated dihydromorphine, [D-Ala2, D-Leu5]-enkephalin and ethylketocyclazocine (potency relative to camel beta-EP, 1-4%) but it is more potent (9%) in competition with tritiated human beta-endorphin. Dermorphin and the [Dermorphin-1-7]-beta c-EP appear to interact preferentially with mu opiate receptors.

β-Endorphin: Characteristics of binding sites in the rabbit cerebellar and brain membranes

Specific binding of human beta-endorphin to rabbit cerebellar and brain membranes was measured using [3H2-Tyr27]-beta h-endorphin as the primary ligand. In both tissues binding was time dependent and saturable, with apparent equilibrium dissociation constants of 0.275 nM and 0.449 nM in the cerebellum and brain, respectively. The binding capacity of cerebellum is greater than that of brain. Kinetic studies showed that the association rate constants were 2.7 X 10(7) M-1min-1 for cerebellum and 2.4 X 10(7) M-1min-1 for brain. Dissociation of tritiated beta h-endorphin from both cerebellum and brain is not consistent with a first order decay from a single site. In the cerebellum there is a time-dependent increase in slowly dissociating complex. The potency of several opioid peptides and opiates to inhibit the binding of tritiated beta h-endorphin was determined. Ligands with preference for mu, delta, and kappa opiate receptor (morphine, Metenkephalin and ethylketocyclazocine) all have similar affinities toward beta h-endorphin sites in both brain and cerebellar membranes.

β-Endorphin-(1–27): Acetylation of alpha-amino groups enhances immunoreactivity but diminishes analgesic and receptor-binding activities with no changes in circular dichroism spectra

Four synthetic βh-endorphin analogs have been assessed for their immunoreactivity, receptor-binding activity and analgesic potency as well as their α-helical content by circular dichroism. These synthetic analogs are: βh-endorphin-(1–27), [Gln8]-βh-endorphin-(1–27), [Ac-Tyr1]-βh-endorphin-(1–27) and [Ac-Tyr1, Gln8]-βh-endorphin-(1–27). Results show that acetylation caused 3.8–8.8 X increase of immunoreactivity in comparison with the parent analog but abolished receptor-binding and analgesic activities. In addition, acetylation does not alter circular dichrois spectra in 90% methanol. Thus, there is no correlation of α-helix content with biological activities of these four synthetic βh-endorphin analogs.

β-endorphin: Replacement of glutamic acid in position 8 by glutamine increases analgesic potency and opiate receptor-binding activity

Summary A β-endorphin analog with replacement of glutamic acid in position 8 by glutamine has been synthesized by modified procedures of the solid-phase method. The analgesic potency of the synthetic analog was increased to nearly three-fold with a concomitant increase of opiate receptor-binding activity in neuroblastoma × glioma hybrid cells. This is the first instance in which a replacement of a single amino acid causes an increase of analgesic potency of β-endorphin.

Opiate receptor binding profile in the rabbit cerebellum and brain membranes

The equilibrium dissociation constants (Kd) and maximal binding capacities of tritiated dihydromorphine (DHM), [D-Ala2,D-Leu5]enkephalin (DADLE), ethylketocyclazocine (EKC) and human beta-endorphin (beta-EP) in rabbit cerebellum and brain membranes have been investigated. Binding of tritiated DHM and DADLE was adequately described by a single affinity class of binding sites, while that of EKC required two affinity sites. Binding of tritiated beta-EP was also consistent with a single affinity class of binding sites by Scatchard analysis, but inhibition of the binding with type selective opiate receptor ligands revealed multiple sites. Sequential displacement of a broad spectrum opiate ligand, diprenorphine (DIP), by type selective ligands showed that cerebellum membranes are relatively rich in mu (40%) and deficient in K (12%) binding sites, while brain membranes are relatively rich in K (32%) and deficient in mu (12%) binding sites. beta-EP displaces 88 and 73% of tritiated DIP from cerebellum and brain membranes, respectively, suggesting multiple sites of beta-EP binding.

β-Endorphin: Characteristics of binding sites in the mouse brain

Binding of human beta-endorphin to mouse brain membrane preparations has been characterized using the tritiated hormone as primary ligand. The binding was shown to be time and temperature dependent. The dissociation constant of the saturable binding was determined to be 0.5 nM. Both [Met5]enkephalin and naloxone were found to compete with tritiated beta-endorphin for the binding with a potency of 6.3 and 6.5% of the unlabeled hormone, respectively. Phospholipase A2 isolated from Formosan cobra venom was shown to be a potent inhibitor of the binding with a 50% inhibition concentration of 26 nM. Although Na+, K+, Ca2+, Mg2+ were found individually to exert a profound inhibition on the binding, the combined salt solution (Tyrode) did not abolish the specific binding of tritiated beta-endorphin to the mouse brain membrane preparations.

Detection of bovine pancreatic trypsin inhibitor (bPTI) in bovine and ovine pituitaries, bovine brain, and adrenal medulla. A specific radioimmunoassay for bPTI.

An antiserum against bovine pancreatic trypsin inhibitor (bPTI) has been raised in rabbits. The antiserum does not cross-react with known pituitary hormones and other proteins. A specific radioimmunoassay for bPTI has been developed. The ED50 is about 250 fmol per assay tube and the sensitivity is reliable to 15 fmol per tube. Immunoreactivity could be detected in bovine and sheep pituitaries, bovine brain, and adrenal medulla, but not in human, pig, and rat pituitaries. In the bovine pituitary the immunoreactivity is restricted to the posterior lobe.

β-Endorphin: Photoaffinity labelling of a non-opioid binding site in a human neuroblastoma

A photoaffinity reagent 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-Cl) and synthetic analogs of human beta-endorphin (beta h-EP) were employed to demonstrate the presence of receptor sites specific for beta h-EP but of non-opioid character in a human neuroblastoma cell line (IMR-32). The radioactive photoaffinity probe was carried out using [125I-Tyr1,2,4-NAPS-Trp27]-beta h-EP and IMR-32 cell membranes. After solubilization with sodium dodecyl sulfate (SDS) and SDS polyacrylamide gel electrophoresis, a single labelled protein band was identified with a molecular weight of 72,000. Labelling was blocked by beta h-EP or beta h-EP-(6-31) but remained in the presence of beta h-EP-(1-27). The specificity of this band is thus identical to that of the non-opioid site previously characterized. Various nonionic or zwitterionic detergents did not extract the labelled non-opioid site.