Sep 13, 1978 - SUMMARY. The amino acid sequence of radioimmunoassayable neurotensin, isolated from bovine small intestinal ex- tracts, has been shown ...
THE JOURNAL OF BIOLOGICAL CHEM~~RY Vol. 253, No. 22, Issue of November 25, pp. 79967998, 1978 Printed m U.S.A.
Communication
MATERIALS
The Amino Acid Sequence of Radioimmunoassayable Neurotensin from Bovine Intestine IDENTITY TO NEUROTENSIN HYPOTHALAMUS* (Received
for publication,
August
FROM
14, 1978, and in revised form, September 13,1978)
Robert Carraway, Patrick Kitabgi,$ and Susan E. Leeman From the Department of Physiology and Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, Boston, Massachusetts 02115
SUMMARY
Neurotensin is a hypotensive, gut-contracting peptide originally discovered and isolated from acid/acetone extracts of bovine hypothalami (1). After its isolation, the structure of neurotensin was determined (2) and synthetic material was prepared and shown to be biologically and chemically indistinguishable from the native substance (3). Recently, a radioimmunoassay for neurotensin has been described which employs synthetic bovine neurotensin and rabbit antisera raised against various neurotensin-protein conjugates (4). An investigation of the distribution of radioimmunoassayable neurotensin in the rat suggested the presence of a neurotensinlike substance in small intestinal tissue (5). Since the concentration of this immunoreactive material was nearly the same when determined using these different antisera (with different specificities), it was assumed that this substance would be either very closely related to or identical with neurotensin. Further work led to the isolation of an immunoreactive tridecapeptide with the same amino acid composition as neurotensin from acid/acetone extracts of calf small intestine (6). In this communication, we report the determination of the complete amino acid sequence of this peptide and establish that it is identical to hypothalamic neurotensin. * This research was supported in part by National Institutes of Health Grants 5 ROI AM 19428 and ROI AM 16510. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Present address, Group I.N.S.E.R.M.U. 145, Faculte de Medintine (Pasteur), Chemin de Vallombrose, 06100 Nice, France.
7996
METHODS
The radioimmunoassayable neurotensin used here (approximately 80 nmol) is the same material, the isolation of which was previously reported from 45 kg of calf small intestinal fragments (6). The preparation was judged pure on the basis of its biological specific activity (approximately 1100 doses/mg of peptide determined by its hypotensive potency in rats) and its amino acid composition: Asp (l.l), Glu (2.1), Pro (2.0), Ile (0.8), Leu (1.8), Tyr (1.8), Lys (l.O), and Arg (2.0). Treatments with papain, chymotrypsin, and carboxypeptidase and aminopeptidase M were performed using enzyme preparations and procedures described previously (2). Digestions with L-pyroglutamyl peptide hydrolase (EC 3.4.11.8; Boehringer Mannheim) employed 100 ~1 of 100 mM NHdHCO:s buffer, pH 7.8,30 mM mercaptoethanol, 1 mM ethylenediaminetetraacetic acid with 10 pg of enzyme preparation, and 2 to 10 nmol of peptide. Digestions with leucine aminopeptidase (EC 3.4.11.1; Boehringer Mannheim) employed 100 ~1 of 50 mM TrisHCl buffer, pH 8.5, 2.5 mM MgC12 with 5 pg of enzyme, and 2 nmol of peptide. Treatments with carboxypeptidase Y (EC 3.4.12.1; Pierce) were done in 100 ~1 of 100 mM ammonium acetate buffer, pH 4.5, with 10 pg of enzyme and 4 nmol of peptide. High voltage paper electrophoresis was performed using a Michltype apparatus (Gilson) with Varsol as coolant. The relative mobilities of the peptides were calculated according to Offord (7). Amino acid analyses of acid hydrolysates and enzymic digests were performed as previously reported using a Durrum model D-500 automatic amino acid analyzer having a precision of +3% on a standard of 2.5 nmol (1). The results of the subtractive Edman procedure, performed manually as previously described (2), are presented in a format in which the value underlined denotes the residue removed at each step and the per cent recovery of the peptide is indicated in parentheses. Values less than 0.1 are considered to be 0. The symbols used in the diagrams are as follows: -, determined by subtractive Edman analysis or aminopeptidase treatment; -, determined by carboxypeptidases A and B. The fragments are lettered according to the enzyme used in their generation (P, papain; C, chymotrypsin) and numbered starting at the NH? terminus of the parent molecule. RESULTS
Studies
on the Intact
Peptide
Treatment of the intact peptide (2 nmol) with leucine aminopeptidase released less than 0.2 mol of any free amino acid per mol of peptide, indicating that the NH2 terminus was blocked. The COOH-terminal sequence of the intact peptide was established as -Tyr-Ile-Leu-OH by kinetic experiments using carboxypeptidase A. Digestions at 20°C using an enzyme/substrate molar ratio of 1:20 yielded the following moles of free amino acids per mole of peptide: 2 min, Leu (0.46) Ile (0.32); 6 min, Leu (0.65) Ile (0.50); 16 h, Leu (0.94) Ile (1.02) Tyr (0.95). Isolation
and Amino
Acid
Composition
of Fragments
The fragments obtained by treatments of the peptide with either papain or chymotrypsin were easily separated by high voltage paper electrophoresis during which they were found to migrate adjacent to the corresponding fragments of synthetic neurotensin (Fig. 1). The yields of each of the peptides obtained and their amino acid compositions as determined after acid hydrolysis are given in Table I. Sequence Peptide P-l (Residues Composition:
Studies
on Fragments A L1 to 4)
