216
C. Galoppini et al. / Il Farmaco 54 (1999) 213–217
triazole (HOBt)/N-methylmorpholine (NMM) and pro-
tected aminoacids or iminoacyl group [10] in four-fold
excess were recycled in the coupling step over a time of
50 min at a flow rate of 4 ml/min. Fmoc-deprotection
was performed with 20% (v/v) piperidine in DMF, 9
min at 4 ml/min.
(w/v) of ice-cold buffer containing 0.32 M sucrose, 20
mM Hepes-Tris (pH 7.4), 5 mM EDTA and protease
inhibitors (0.1 mM bacitracin, 0.1 mM phenylmethyl-
sulfonyl fluoride (PMSF), and 1 mg/ml leupeptin). The
homogenated was centrifuged at 48 000×g for 15 min
at 4°C. The pellet was resuspended in 10 volumes of 20
mM Hepes-Tris (pH 7.4), 5 mM EDTA (buffer HT)
containing protease inhibitors. The membrane homo-
genated was centrifuged at 48 000×g for 15 min at
4°C. The tissue preparation was used either immedi-
ately or stored in aliquots at −80°C.
Peptides were cleaved from the resin and deprotected
from the side-chain protecting groups with a solution of
trifluoroacetic acid (TFA)/phenol/anisole [14] (92/4/4
v/v) at room temperature for 1 h under magnetic
stirring, precipitated with cold ether and filtered. The
filtrate was dissolved in methanol and treated for 72 h
at room temperature to convert the carbamate on the
indole moiety of the tryptophan to the desired prod-
ucts; the total deprotection was observed by analytical
high performance liquid chromatography (HPLC). The
methanol was removed under vacuum and the residue
was suspended in water and lyophilized. Crude peptides
were analyzed by analytical RP-HPLC on a Beckman
System Gold apparatus in the following condition:
Vydac C18 column, 0.46×15 cm; eluant A: 0.1% TFA
in H2O, eluant B: 0.1% TFA in CH3CN; gradient from
20 to 95% B over 25 min; flow 1 ml/min; UV detection
at 210 nm.
3.3.1. Radioligand binding assay
Binding assays were performed as described by Cody
et al. [16], with some modifications. Briefly, cerebellar
(5 mg proteins) membranes were incubated in 0.25 ml of
T1 buffer (20 mM Tris–HCl, pH 7.4, 2 mM EDTA, 0.1
mM bacitracin, 0.1 mM PMSF, 1 mg/ml leupeptin, and
5 mg/ml aprotinin) with 15 pM [125I]ET-1 (2000 Ci/
mmol) for 2 h at 37°C. Reactions were terminated by
addition of 3 ml of ice-cold T2 buffer (50 mM Tris–
HCl, pH 7.3, 0.1 mM bacitracin) and rapid filtration of
samples through GF/C glass fiber filters which had
been soaked in T2 buffer containing 0.2% bovine serum
albumin (BSA) for 24 h. The filters were then washed
four times with 3 ml of ice-cold T2 buffer. Assays were
done in duplicate and non-specific binding was checked
in the presence of 100 nM ET-1. [125I]ET-1 was diluted
in T1 buffer plus 1 mg/ml BSA, while ET-1 and ET-3
were dissolved in the same buffer without BSA. Pep-
tides were diluted in T1 buffer plus 1 mg/ml BSA and
1% dimethyl sulfoxide (DMSO) to a concentration of 5
mM.
All peptides were purified by preparative RP-HPLC
on a Beckman System Gold apparatus; column Vydac
C18 (2.2×25 cm); eluants as above; gradient from 20 to
40% B over 100 min, flow 8 ml/min; UV detection 210
nm.
The characterization of peptides was performed by
electrospray ionization mass spectrometry (ES/MS)
[15], (see Table 1).
3.2. Synthesis of Fmoc-8-aminooctanoic acid
Saturation binding experiments (EDBA/LIGAND
Software, Cambridge, UK, and GraphPad Prism Soft-
ware, San Diego, CA) allowed the calculation of equi-
librium dissociation constant (KD) and binding capacity
(Bmax) values for the binding site in cerebellar mem-
brane that were 54 pM and 2.23 pM/mg proteins,
respectively.
The amino acid (1 equiv.) was suspended in water,
dissolved under reflux and pH adjusted to 9 with 10%
Na2CO3 solution. A solution of Fmoc–OSu (1 equiv.)
in dioxane was then added dropwise over 20 min and
the mixture was stirred under reflux for 24 h. The
reaction mixture was then diluted with water, acidified
to pH 3 with HCl and extracted twice with CH2Cl2.
The organic layer was washed, dried over Na2SO4, and
the solvent removed to dryness. Yield: 85%; m.p. 118–
119°C; Rf1=0.51, Rf2=0.92, Rf3=0.29, Rf4=0.05;
1H NMR (1×10−2 M CDCl3) l 7.80–7.26 (m, 8H,
Fmoc aromatic CH), 4.79 (s, 1H, NH), 4.41 (d, 2H,
Fmoc CH2), 4.22 (t, 1H, Fmoc 9-CH), 3.16 (m, 2H,
Aoc aCH2), 2.35 (t, 2H, Aoc bCH2), 1.68–1.24 (m,
10H, Aoc CH2).
References
[1] G.M. Rubany, M.A. Polokoff, Endothelins: molecular biology,
biochemistry, pharmacology, physiology, and phatophysiology,
Pharmacol. Rev. 46 (1994) 325–415.
[2] T. Masaki, M. Yanagisawa, K. Goto, Physiology and pharma-
cology of endothelins, Med. Res. Rev. 12 (1992) 391–421.
[3] M. Yanagisawa, K. Kurihara, S. Kimura, Y. Tomobe, M.
Kobayashi, Y. Mitsui, Y. Yazaki, K. Goto, T. Masaki, A novel
vosocostrictor peptide produced by vascular endothelial cells,
Nature 332 (1988) 411–415.
[4] J.P. Huggins, J.T. Pelton, R.C. Miller, The structure and specifi-
city of endothelin receptors—their importance in physiology and
medicine, Pharmacol. Ther. 59 (1993) 55–123.
[5] C.A. Maggi, S. Giuliani, R. Patacchini, P. Santicioli, P. Rovero,
A. Giachetti, A. Meli, The C-terminal hexapeptide, endothelin-
3.3. Pharmacology
Male Sprague-Dawley rats (160–250 g) were killed
by decapitation. The cerebellum was removed and ho-
mogenized using a Polytron homogenizer in 10 volumes