alpha-chymotrypsin and peroxyformic-acid

A study on the effect of cysteic acid position on the types of fragment ions formed by collision-induced dissociation (CID) of [M - H](-) ions is presented. Of particular note is the observation of d-type fragment ions for peptides that contain an N-terminal cysteic acid (fixed negative charge) and cleavable amino acid side chains possessing a β-γ carbon-carbon bond. For example, the CID mass spectrum of oxidized cys-kemptide (C(ox)LRRASLG) [M - H + O(3)](-) ions contains abundant series of d-type fragment ions, and similar results are observed for oxidized cysteine-containing ribonuclease A proteolytic peptides. The d(i) fragment ions are assumed to arise by a charge-remote and/or charge-assisted fragmentation mechanism, which both occur at high collision energies and involve consecutive reactions (i.e., the formation of a(i) ions followed by the elimination of the side chain to form d(i) ions).

Topics: Amino Acid Sequence; Chymotrypsin; Cysteic Acid; Formates; Molecular Sequence Data; Oxidation-Reduction; Peptide Fragments; Ribonuclease, Pancreatic; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure-Activity Relationship; Tandem Mass Spectrometry; Trypsin

Carboxymethylation of bovine skimmed milk with 14C-labelled iodoacetic acid followed by purification of the alpha s2-casein dimer showed that all four cysteine residues in the protein are engaged in disulfide linkages. Mass spectrometry and sequence analysis of cystine-containing tryptic peptides revealed the presence of two interchain disulfide bridges in the protein. Sequence analysis of disulfide-linked peptides resulting from an enzymatic cleavage between the bridges demonstrated that the individual chains in the dimers are either aligned in an antiparallel or a parallel orientation. The identity of some of the disulfide-linked peptides was further verified by performic acid oxidation followed by sequence analysis of the resulting peptides.

Topics: Amino Acid Sequence; Animals; Caseins; Cattle; Chromatography, High Pressure Liquid; Chymotrypsin; Disulfides; Formates; Mass Spectrometry; Molecular Sequence Data; Oxidation-Reduction; Peptides; Trypsin

We previously reported that the rat posterior pituitary contains a potent PRL-releasing factor (PRF) which is distinct from oxytocin (OT), TRH, and angiotensin II (AII). The objectives of this study were 1) to examine whether posterior pituitary extracts stimulate PRL release in the presence of dopamine (DA), 2) to determine the chemical nature of PRF, and 3) to estimate its mol wt. Perifused anterior pituitary cells were used to assess PRF activity. Posterior pituitaries and medial basal hypothalamus (MBH) fragments were extracted with perchloric acid and lyophilized. Subsequent to various treatments, samples were reconstituted in the perifusion medium and introduced to the cells in short pulses. Fractions were collected and analyzed for hormone content by RIA. During a constant infusion of DA (50 nM), PRL secretion was inhibited by 75%, yet the posterior pituitary extract retained its ability to rapidly stimulate PRL release. Studies using proteolytic enzymes showed that posterior pituitary PRF was resistant to inactivation by trypsin, whereas the PRF activity of AII was abolished. Both chymotrypsin and proline-specific endopeptidase significantly reduced the PRF activity in the posterior pituitary. The PRL-releasing activity of TRH was not affected by chymotrypsin. Immunoreactive vasoactive intestinal polypeptide was undetectable in posterior pituitary extracts. Oxidation of posterior pituitary extracts with performic acid caused only a modest reduction of their PRF activity, while the ability of OT to stimulate PRL release as well as immunoreactive OT was abolished. Studies using ultrafiltration membranes showed that the PRF activity in the posterior pituitary was less than 5,000 mol wt. Furthermore, posterior pituitary PRF partitioned in nearly equal amounts across 1K membranes, as did AII and OT. In contrast, about 80% of the PRF activity in the MBH and all of the synthetic TRH passed through the 1K membranes. We conclude that 1) posterior pituitary PRF can stimulate PRL secretion from perifused anterior pituitary cells in the presence of physiological concentrations of DA; 2) PRF is a small peptide(s) of less than 5,000, and perhaps closer to 1,000, mol wt; 3) PRF is resistant to inactivation by trypsin and to oxidation by performic acid, but is hydrolyzed by both chymotrypsin and proline-specific endopeptidase; and 4) these data further distinguish posterior pituitary PRF from known PRL secretagogues.

Topics: Animals; Chymotrypsin; Dopamine; Female; Formates; Hypothalamus, Middle; Molecular Weight; Oxidation-Reduction; Pituitary Gland, Anterior; Pituitary Gland, Posterior; Prolactin; Prolyl Oligopeptidases; Radioimmunoassay; Rats; Rats, Inbred Strains; Serine Endopeptidases; Thyrotropin-Releasing Hormone; Tissue Extracts; Trypsin; Vasoactive Intestinal Peptide