Abstract
Using a radioimmunoassay toward the COOH-terminal region of neurotensin, an immunoreactive and biologically active neurotensin-related peptide (NRP) has been isolated from pepsin-treated fractions of bovine, canine, human, and rat plasma. Bovine NRP was identified as H-Ile-Ala-Arg-Arg-His-Pro-Tyr-Phe-Leu-OH, which is similar in structure to both neurotensin and angiotensin I. Canine and human NRP also had the above amino acid composition, whereas that obtained from rat plasma had valine substituted for isoleucine. At their concentrations in pepsin-treated plasmas (2-6 microM) rat, human and canine NRP were shown to increase vascular permeability when injected intradermally into rats and to release histamine from rat mast cells in vitro. The pure peptides also cross-reacted very effectively at nanomolar concentrations in a radioreceptor assay for neurotensin. The protein(s) which liberated NRP upon pepsin treatment were purified about 7-fold and shown to behave like albumin during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and high pressure liquid chromatography on muBondapak C4. In addition, the purified preparations were found to react with anti-albumin antisera during immunodiffusion. Although the amino acid sequence of NRP was not found in albumin, a partial sequence homology was noted for NRP and various segments of bovine albumin. Using V8 protease, glutamyl residues were shown to lie within 3-4 amino acids of each end of NRP, as also occurs for the related segments in albumin. These results suggest that a subset of albumin-related protein(s) could serve as precursor(s) to biologically active neurotensin-related peptide(s).
Highlights
With the recognition that angiotensin is both stored inits reacted very effectively a t nanomolar concentrations processed forms within brain anadlso circulating in precursor in a radioreceptorassayforneurotensin.Theproforms within blood, we recently formulated the hypothesis tein(s) which liberatedNRP upon pepsin treatment that other currently known neuroendocrine peptides may be were purified about 7-fold and shown to behave like represented in thestewo signaling designs(11).In preliminary albumin during sodium dodecyl sulfate-polyacrylam- studies to address this hypothesis we demonstrated the foride gel electrophoresis, isoelectric focusing, and high mation of micromolar concentrations of immunoreactive neupressure liquid chromatography on pBondapak C4
UsingV8 protease, glutamyl residues were shown Here we report the isolation and identityof plasma neurotensin-related peptide (NRP) to lie within 3-4 amino acids of each end of NRP, as obtained from pepsin-treated human, bovine, canine, and rat occurs for the related segmenitns albumin
The isolated peptides show an amino acid sequence results suggest that a subset of albumin-related pro- homology to neurotensin and an ability to register in both tein(s) could serve as precursor(s) tobiologically active radioreceptor and bioassays
Summary
Peptides (1).Messenger peptides can be broadly categorized into ( a )those which are pre-processed, stored withinvesicles, RESULTS. Inthefinalstep, each Biological Properties of NRP-NRP was found to release preparation gave a single peak of absorbance co-elutingwith histamine from isolated rat mast cells in a dose-related manthe immunoreactivity (e.g. Fig. 4, Miniprint). Comparison of the dose-response relationships for NRP, compositions of theisolatedpeptides gave integral molar neurotensin, and bradykinin indicated that NRP was more ratios of the constituent amino acids (Table I). His-Pro-Tyr-Phe-Leu-OH.Comparison of theamino acid summarized in the Miniprint (Table VI) show that the subsequences given in TableI1 indicates that the COOH-terminalstrate(s) in human plasma which liberated NRP upon pepsin region of plasma NRP is partially homologous to both neu- treatment was purified about ?'-fold using the procedure derotensinandangiotensin I. 5 and 6, Miniprint), This relationship is consistent with other data presented in except during chromatography on blue-Sepharowseh,ere subthis paper, suggesting that plasma NRP originates from an strate activity eluted on the back side of the main peak of albumin-related protein(s). Molar ratios for Ser and Gly (common contaminants) were less than 0.3 and all other amino acids were less than 0.1
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