Manfred Westphal

β-Endorphin: Demonstration of binding sites in three human neuroblastoma cell lines specific for the COOH-terminal segment of the human hormone

This communication reports for the first time specific binding sites in human neuroblastoma cells for human beta-endorphin (beta h-EP). Three cell lines (IMR-32, NMB and Kelly) were investigated and found to bind tritiated beta h-EP with an apparent dissociation constant of 2.2-4.2 nM. Further characterization with camel beta-EP and synthetic analogs indicated that the binding is most likely mediated by the COOH-terminal segments. beta h-EP-(6-31) had significant potency (15-75%) and beta h-EP-(1-27) was without displacing activity. The camel beta-EP has below 1% of the human beta-EP activity.

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.

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: Evidence for the existence of opioid and non-opioid binding components for the tritiated human hormone in NG108-15 cells

Abstract Human β-endorphin (β h -EP) binding on neuroblastoma x glioma hybrid NG108-15 cells using tritiated human beta endorphin ( 3 H-β h -EP) as a primary ligand was found to have a component which was not displacable with [D-Ser 2 ]-Leu-enkephalin-Thr 6 (DSLET). The β h -EP binding on these cells after saturation of the δ opiate sites with 200 nM DSLET was further characterized with synthetic β h -EP analogs. the non-opioid binding site appears to recognize β h -EP-(6–31), β h -EP-(21–31) and β h -EP-(28–31). Under these conditions, these COOH-terminal segments fully displace the tritiated β h -EP. However, β h -EP-(1–27) does not further displace 3 H-β h -EP in the presence of DSLET. The fact that a combination of DSLET and β h -EP-(6–31) results in a full displacement of 3 H-β h -EP provides direct evidence for the existence of two binding sites for β h -EP in NG108-15 cells, one recognizing the NH 2 -terminal enkephalin sequence and the other the non-opioid COOH-terminal segment.

Epidermal growth factor receptors in the human glioblastoma cell line SF268 differ from those in epidermoid carcinoma cell line A431

Two established human tumor cell lines, epidermoid carcinoma line A431 and glioblastoma line SF268, were studied to compare the interaction of each with epidermal growth factor (EGF). SF268 cells bound [125I] EGF with 35-40 fold higher affinity than did the A431 cells. The EGF binding sites of both lines were photoaffinity labeled using 2,4-NAPS-[125I] EGF, a photoreactive derivative of EGF. Extracts of photolysed cells analyzed by SDS-PAGE showed a difference between the two cell lines in the high molecular weight component corresponding to the EGF receptor. EGF in a dose range from 0.3-200 nM had no effect on thymidine incorporation by SF268 cells, whereas thymidine incorporation by A431 cells was markedly inhibited by EGF.

Long-term culture of human corticotropin-secreting adenomas on extracellular matrix and evaluation of serum-free conditions

SummaryTissues from 12 human corticotropin-secreting adenomas, obtained during surgery for Cushings disease (CD, ten cases) or Nelsons Syndrome (NS, two cases), were exclusively mechanically dispersed. Single cells and cell aggregates were plated on extracellular matrix derived from bovine corneal endothelia. Functional responses to physiological stimuli were analyzed by measuring human β-endorphin (βh-EP) immunoreactivity (IR) by radioimmunoassay in the culture medium. All adenomas responded with stimulated secretory activity to arginine vasopressin (AVP), corticotropin-releasing factor (CRF), or both. Cortisol higher than 10−8 M suppressed basal secretion and CRF- or AVP-stimulated βh-EP-IR secretion. There was no consistent difference in response of the cells when cultured in medium containing 10% fetal calf serum (FCS) or in serum-free conditions. A change of cells from serum to serum-free conditions usually resulted in 10%–50% reduction in the basal secretion level that remained stable for at least 2 weeks and, in one case (NS), 10 weeks. In cells maintained in medium supplemented with 5% serum obtained from the respective patients 40 min after adenoma removal, basal secretion was suppressed to 60% of the baseline level in a 10% FCS control. Long-term incubation with CRF (10−9 M) showed sustained stimulation of hormone secretion. No remarkable cell proliferation was observed under basal conditions or during long-term, low-dose incubation with cholera toxin (10−12 M) in two cases (CD), or CRF (10−9 M) in two cases (NS, CD). Parallel β-EP-IR and adrenocorticotropin secretion was verified in selected cases.

Peptide E: Opiate receptor binding profile in the rat brain membrane assay. Comparison with beta-endorphin

Beta-endorphin (beta-EP) and peptide E were compared in respect to their binding potency in the rat brain membrane by radioreceptor binding assay using tritiated human beta-EP, [D-Ala2,D-Leu5]-enkephalin (DADLE), dihydromorphine (DHM) and ethylketocyclazocine (EKC) as primary ligands. When the potency of beta h-EP was chosen to be 100%, peptide E was equipotent with beta-EP in displacing DHM (95%) and EKC (103%) less potent for competing with beta h-EP (60%) and least active (7%) for displacing DADLE. It may be concluded that peptide E binds preferentially with the opiate mu and kappa receptors in the rat brain membrane.

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.

Characterization of immunoreactive β-endorphin secreted from cultured human corticotropin-secreting adenomas

Seven human corticotropin-secreting adenomas causing Cushings disease or Nelsons syndrome were maintained in long-term culture. Pooled media from the individual adenomas were analyzed for the composition of their secretory products. From a radioimmunoassay (RIA) with 100% cross-reactivity for human beta-endorphin (beta h-EP) and beta-lipotropin (beta h-LPH), immunoreactive beta h-EP (IR X beta h-EP) was found to be the predominant secretory product after Sephadex G-50 analysis in 4 cases (40-80% of total IR), immunoreactive beta h-LPH (IR X beta h-LPH) predominated in 1 case, and both were equipresent in 2-cases. IR X beta h-EP was further purified by high-performance liquid chromatography (HPLC) and analyzed in 4 cases with ion-exchange chromatography on SP-Sephadex C-25 and a RIA which completely cross-reacts with beta h-EP, [N alpha-Ac]-beta h-EP, beta h-EP-(1-27) and [N alpha-Ac]-beta h-EP-(1-27). In all cases, the IR X beta h-EP was the main component (40-70%); the remaining IR material was attributable partially to [N alpha-Ac]-beta h-EP or other, less defined immunoreactive material. In 3 cases, enough IR X beta h-EP material was available for HPLC and to perform a radioreceptor assay using tritiated beta h-EP as primary ligand. The displacing potency of these preparations relative to synthetic beta h-EP was related to the content of the immunoreactive component eluting in the position of synthetic beta h-EP.

Synthesis of adrenal peptide E and some of its biological activities

A synthesis of peptide E, a highly potent, 25-amino acid adrenal opioid peptide containing both a [Met]enkephalin at the NH2-terminus and [Leu]enkephalin sequence at the COOH-terminus, originally isolated from bovine adrenal medulla [D. L. Kilpatrick, T. Taniguchi, B. N. Jones, A. S. Stern, J. E. Shively, J. Hullihan, S. Kimura, S. Stein, and S. Udenfriend (1981) Proc. Natl. Acad. Sci. USA 78, 3265-3268], is reported. The synthesis was accomplished by the solid-phase method employing the 4-(aminoacyloxymethyl)phenylacetamidomethyl(Pam)-copoly(styrene-1% divinylbenzene) resin. Two synthetic strategies (N-indole formyl protected vs unprotected tryptophan) were followed and results compared and evaluated. It was determined that peptide E prepared with protection of tryptophan (residues 13 and 14) was preferred and gave final product that was readily purified by HPLC. The biological activity of the synthetic material was found to be equivalent to the reported activity of the natural compound.