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Human beta-calcitonin gene-related peptide (beta-hCGRP) was isolated and purified from spinal cord. The complete characterization of this material was based on definitive mass analysis by fast atom bombardment mass spectrometry together with gas phase sequencing. Combining these data we have characterized the structure of beta-hCGRP including the C-terminal sequence, the presence of the S-S bridge and of phenylalanineamide as the C-terminal amino acid, and fully confirmed the amino acid sequence predicted from the nucleotide analysis.

The structure of beta-cell tropin, an insulin secretagogue released by the neuro-intermediate lobe of the obese (ob/ob) mouse, has recently been determined as the 22-39 moiety of ACTH. A method for the preparation of this octadecapeptide using mild solid-phase procedures followed by preparative high pressure liquid chromatography is described. The molecular weight of the synthetic peptide has been confirmed by Fast Atom Bombardment mass spectrometry. Synthetic beta-cell tropin is indistinguishable in its chromatographic, antigenic and biological properties from natural beta-cell tropin.

A new method is described for screening and establishing the primary structure of proteins. The procedure is both rapid and sensitive and allows study of the C-terminus of the protein with equal facility to the N-terminus. This new strategy which is illustrated here for the polypeptide hormone Insulin, has obvious applications in recombinant DNA biotechnology research, in post-translational modification and site-directed mutagenesis studies, or any other aspect of modern protein chemistry requiring accurate definition of primary structure.

The amino acid sequence of Sterol Carrier Protein2 (SCP2) isolated from rat has been investigated. Using a novel mass spectrometric mapping approach, the C-terminus was found to be extended beyond the previously published sequence. Carbohydrate analysis of SCP2 samples suggest the presence of tightly bound mannose oligosaccharide of 5-10 residues, although probably not in a glycoprotein linkage.

Fast atom bombardment mass spectrometry has been applied to the localization of phosphorylation sites in buffalo beta-casein. Two complementary strategies of identification are described. Phosphorylated residues in the tryptic peptide Tp 1 have been assigned by measuring the masses of peptide fragments obtained by enzymatic degradations. The phosphoserine residue in peptide Tp 2 has been identified by determining the intact molecular weight and confirmed by partial sequence information. This rapid and sensitive procedure appears of a great interest in structural studies of a wide range of post-translational modifications in proteins.

A radioimmunoassay for leukotriene D4 (LTD4) has been developed which exhibits sufficiently high sensitivity to be useful in conjunction with RP-HPLC in the detection of LTC4, LTD4 and LTE4 in physiological samples. The detection limit of the assay was approximately 240 amoles, using antiserum TG1 at a dilution of 6 X 10(3), with 50% displacement at 70 fmoles. Antiserum NW1, also at a dilution of 6 X 10(3), displayed a detection limit of 9 fmoles with 50% displacement at 100 fmoles. The two antisera have similiar crossreactivities, both manifesting useful affinities for LTE4 and LTC4, and low or negligible affinities for other arachidonic acid metabolites, or their derivatives. The radioimmunoassay was used to detect 1) LTC4, LTD4 and LTE4 released from perfused rat lung in response to platelet-activating factor (PAF) stimulation, 2) conversion of exogenous LTD4 to LTE4 in human blood, and 3) endogenous leukotrienes in human blood samples.

The biological activities of calcitonin gene-related peptide (CGRP) enzymic digest fragments, chemically modified products and beta-CGRP have been compared to that of intact alpha-CGRP on rat isolated paired atria. Tryptic and chymotryptic digests both produced inactive fragments. Acetylation of the N-terminal amino acid (Alanine) or either of Lys 24 or Lys 35, resulted in reduced, but measurable, biological activity. Destruction of the disulphide bridge between Cys 2 and Cys 7 abolished biological activity. Substitution of several amino acids, Asp 3, Val 22 and Asn 25, with Asn, Met and Ser respectively (beta-CGRP), produced a peptide with similar biological activity to alpha-CGRP.

Leukotriene (LT) release stimulated by platelet activating factor was inhibited by vasoactive intestinal polypeptide (VIP) in an in vitro rat lung preparation. This was detected by HPLC and radioimmunoassay. LTC4, although the major species in the stimulatory model used, was not detected in peptide-treated preparations and LTD4 and LTE4 levels were considerably reduced.

We have combined the push-pull perfusion of radiolabelled peptide, with reverse phase--HPLC procedures as a valuable method for monitoring peptide metabolism in the CNS. This model not only permits studies on metabolism of neuropeptides at specific sites in the CNS, but more importantly, allows (unlike in vitro methods) the study to be made under the appropriate physiological conditions. We have demonstrated unambiguously for the first time, the metabolism of CCK-8 in situ.

A new method for complementing existing protein chemical techniques for the assignment of S-S bridge positions in amino-acid sequences is described. The principle of the method is the direct examination of the masses of protein fragments, obtained by chemical or enzymatic degradation. Proteins are digested under conditions known to minimise disulphide reduction and reshuffling, and the unfractionated digest is examined directly by high field magnet (or other high mass) fast atom bombardment or Californium mass spectrometry. Disulphide linked peptides are identified from their unique masses, and by comparison with the spectrum of digested and reduced samples in which the signal corresponding to the S-S linked peptide(s) is replaced by two signals corresponding to the respective thiol peptide components, if INTER-bridged, or shifted by two mass units (dithiol) if INTRA-bridged. This rapid procedure has considerable potential in assisting with studies on the primary structure of proteins, in crystallographic studies and the monitoring of denaturation/renaturation of recombinant proteins.

The effect of prostaglandins E1 and F1 alpha on peptidoleukotriene biosynthesis/release from rat chopped lung stimulated with platelet activating factor was studied. Prostaglandin E1, known to stimulate adenylate cyclase in airways, inhibited the biosynthesis of leukotrienes C4, D4 and E4 and total peptidoleukotrienes whereas prostaglandin F1 alpha, which has no effect on adenylate cyclase, did not exert any effect on total peptidoleukotriene release, though a small inhibition was found for leukotriene D4. Cyclic AMP itself inhibited peptidoleukotriene release from platelet activating factor-stimulated lung, suggesting that the effect of prostaglandin E1 is mediated by cyclic AMP.