Precursor forms of neurotensin (NT) in cat: processing with pepsin yields NT-(3-13) and NT-(4-13).
Basic proteins present in 0.1 N HCl extracts of feline CNS and intestine were found to liberate immunoreactive neurotensin (iNT) when treated with hog pepsin. These protein substrates were separated using Sephadex G-25, Sephadex G-75 and reverse-phase HPLC. In a calibrated SDS-polyacrylamide gel electrophoresis system, the major substrate from cat ileum exhibited a molecular weight of ca 16 kDa and minor substrates were observed at 30, 40 and 65 kDa. As shown previously for synthetic NT, pepsin-treatment of feline ileal NT converted it into the fully immunoreactive NT-(4-13) fragment (yield, 95%). When treated with pepsin, the partially purified ileal substrates gave rise to 4 immunoreactive peptides, one of which (ca 15% of total) eluted with the same retention time as NT-(4-13) while the major peptide formed (ca 40% of total) eluted near to the position of NT-(3-13). Both these products reacted equally well with two different antisera towards the C-terminal 5- and 8-residues of NT and were not recognized by an N-terminal antiserum. Experiments using various proteases demonstrated that the NT-related sequence(s) were located internally in each substrate and suggested that they were bounded by double basic residues. Substrate activity in isotonic homogenates of feline spinal cord, brain, adrenal and ileum cosedimented with iNT during equilibrium centrifugation, apparently in association with vesicle and/or synaptosomal particles. These findings indicate that basic proteins, colocalized with NT in vesicle-like particles of CNS, adrenals and ileum, could serve as precursors to this peptide, being liberated by pepsin-related enzyme(s).
D000311:Adrenal Glands / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism; D002415:Cats / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007082:Ileum / Q000378:metabolism; D007525:Isoelectric Focusing; D009496:Neurotensin / Q000096:biosynthesis / Q000378:metabolism*; D010434:Pepsin A / Q000494:pharmacology*; D010447:Peptide Hydrolases / Q000494:pharmacology*; D011498:Protein Precursors / Q000378:metabolism*; D011863:Radioimmunoassay; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution
D011498:Protein Precursors; D009496:Neurotensin; D010447:Peptide Hydrolases; D010434:Pepsin A