Identification of the bioactive peptide PEC-60 in brain.

PEC-60 is a 60-residue peptide originally isolated from pig intestine. It inhibits glucose-induced insulin secretion from perfused pancreas in a hormonal manner and also has biological activity in the immune system. PEC-60-like immunoreactive material has been reported in catecholamine neurons of the central and peripheral nervous systems, but the peptide has not been identified from that material. We have now isolated PEC-60 from pig and rat brains with a method that combines column purification procedures with the specificity of a radioimmunoassay and the sensitivity of mass spectrometry to directly identify the peptide. The results show that PEC-60, like many other peptides, is expressed in the gastrointestinal tract and the central nervous system. The specific regional brain distribution and interaction with classical neurotransmitters raise the possibility that PEC-60 may play a role in the central nervous system disorders involving dopamine dysregulation.

Isolation and characterization of sulphated and nonsulphated forms of cholecystokinin-58 and their action on gallbladder contraction.

Cholecystokinin (CCK) exists in multiple molecular forms with different polypeptide lengths and the absence or presence of sulphation. We have isolated sulphated and nonsulphated forms of CCK-58 from porcine intestine and have determined their bioactivities in a guinea-pig gallbladder contraction assay. Both forms co-eluted in cation-exchange chromatography and in several rounds of reverse-phase (RP)-HPLC, but separated upon RP-HPLC using a water/acetonitrile system with heptafluorobutyric acid as counter ion. Nonsulphated CCK-58 was the form detected by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry because of desulphation in that process. The biological activity of CCK-58 and CCK-33 is equipotent, although the kinetics of the response differ. Sulphated CCK-58 was found to be 35 times more potent than nonsulphated CCK-58. In contrast, sulphated CCK-8 is 150 times more potent than nonsulphated CCK-8, and for sulphated and nonsulphated CCK-33, the activities differ by a factor of 100. This type of correlation indicates that the N-terminal end of CCK-58 partially compensates for the decrease in activity arising from the lack of sulphated tyrosine. Given its fairly high bioactivity, nonsulphated CCK-58 may have a physiological significance.

Micropurification and amino acid sequence of beta-casomorphin-8 in milk from a woman with postpartum psychosis.

Milk was obtained from a woman with acute postpartum psychosis and with ongoing lactation. Defatted samples were subjected to micropurification and collected fractions were analyzed by means of their beta-casomorphin-8 immunoreactivity. Immunoreactive material with the same chromatographic properties as synthetic human beta-casomorphin-8 was determined by amino acid sequence analysis to be Tyr-Pro-Phe-Val-Glu-Pro-Ile-Pro. Its molecular mass was determined by fast atom bombardment-mass spectrometry to be 962.3 Da. These determinations, which ultimately identify the immunoreactive material as human beta-casomorphin-8, represent the first structural identification of a beta-casomorphin peptide from a body fluid.

Peptide mapping on HIV polypeptides expressed in Escherichia coli. Quality control of different batches and identification of tryptic fragments containing residues of aromatic amino acids or cysteine.

Peptide mapping was used for the quality control of different batches of the recombinant HIV proteins p24 core and p24-gp41, expressed in Escherichia coli. These proteins comprise gag and env region polypeptides of the virus and may serve as suitable components in the diagnosis of HIV infections. The proteins were digested with trypsin and the mixtures were subjected to peptide mapping to prove batch equivalence of p24-gp41 and to isolate fragments of the p24-gp41 digest that differ from those of the p24 core digest. The proteins were reduced with dithiothreitol and the cysteine residues were derivatized by addition of 4-vinylpyridine. Peptide mapping was performed by means of reversed-phase high-performance liquid chromatography. Batch equivalence was proved by comparison of the maps. Peaks present in one map but not in the other were considered to be due to sequence differences or variability in digestion.

Characterization of a thermostable LH-immunoreactive material from bovine pituitary gland.

A thermostable peptide, immunologically similar to the beta-subunit of ovine lutropin, was isolated from a heated extract of bovine pituitary glands. The relative molecular mass of the peptide as well as its amino acid composition differed significantly from the lutropin subunits. Antibodies were raised to a preparation of this peptide.

Evaluation of several strategies for preparing a bovine gonadotropin-like peptide using isotachophoresis, isoelectrofocusing and high-performance liquid chromatography.

Several strategies for preparing a gonadotropin-like, thermostable peptide from the bovine adenohypophysis were compared. Initially, we extracted bovine pituitaries, in the form of an acetone powder, with acetic acid and water. Using one strategy, we isolated the peptide from the crude extract by gel filtration, isotachophoresis, and isoelectrofocusing. The yield was ca. 50 micrograms of peptide from 100 pituitaries. Using another strategy, we isolated the peptide from the crude extract by precipitating the active material with ethanol followed by high-performance liquid chromatography (HPLC). The yield was ca. 500 micrograms of peptide from 100 pituitaries. Using analytical HPLC, we found that material obtained using the different strategies gave similar results. The HPLC route was, however, more efficient and faster than the other routes, and we recommend it for preparing this gonadotropin-like peptide.

A gonadotropin-like thermostable peptide, prepared from bovine anterior pituitary, inducing spermiation in the frog Rana esculenta (L.) and binding to a rat ovarian FSH receptor.

A thermostable peptide, inducing sperm release in Rana esculenta L. and having immunological properties which resemble those of the gonadotropins but nonidentical with any one of the gonadotropins or their subunits, has been prepared from pars distalis of bovine anterior pituitaries by means of acetic acid extraction, heating, ethanol precipitation, and reverse-phase high-performance liquid chromatography (HPLC). During the purification, the biological activity was regularly followed by means of the potency of collected fractions to induce sperm release in frogs. Using HPLC (5 micron C18 silica), the activity was eluted with 38% of acetonitrile:water:trifluoroacetic acid (80:19.5:0.5) and 62% of 0.5% trifluoroacetic acid, pH 3.3. The active principle was also found to bind to a rat ovarian follitropin (FSH) receptor. In contrast, bovine FSH and LH treated identically to the peptide lost all such affinity, indicating that the peptide is not created from either of these during the purification procedure. Further, the peptide was, like FSH, also shown to stimulate aromatase activity in intact Sertoli cells from immature male rats in vitro.