PLGamide characterization and role in osmoregulation in leech brain.

Neurons immunoreactive to an antiserum specifically directed against the Prolyl-Leucyl-Glycinamide peptide (PLGamide: Melanocyte Inhibiting Factor: MIF) were detected in the brain of the leech Theromyzon tessulatum. Radioimmunoassay titrations of the PLGamide-like material at different physiological stages of the life cycle indicated a maximal amount at stage 3B, which is correlated to phase of both maximal water uptake and coelomic vitellogenin accumulation. In vivo experiments demonstrate that this cerebral PLGamide-like material is an anti-diuretic factor that would act at stage 3B in order to permit a water uptake leading to water retention allowing coelomic yolk protein accumulation. In brains of the Gnatobdellid leech Hirudo medicinalis and the Pharyngobdellid leech Erpobdella octoculata, anti-PLGamide material was also detected with an amount not differing with the degree of sex maturation of the animals, confirming the link between osmoregulation and ovogenesis in rhynchobdellid leeches. Using a combination of biochemical techniques including high-pressure gel permeation chromatography followed by reversed-phase HPLC on brain extracts and Edman degradation, we demonstrated the presence of an authentic MIF-1 peptide in leech brain. Finally, since in vertebrates MIF-1 belongs to the non-classical opioid peptide family, we studied its binding displacement, in contrast to morphine, on mu-receptors and on nitric oxide (NO) release experiments in leech brain. PLGamide did not bind to mu-alkaloid opioid receptors and did not stimulate NO release.

Isolation of a novel peptide with a unique binding profile from human brain cortex: Tyr-K-MIF-1 (Tyr-Pro-Lys-Gly-NH2).

Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2), and MIF-1 (Pro-Leu-Gly-NH2) are biologically active peptides previously isolated from brain tissue. We now have used size exclusion chromatography and several consecutive rp-HPLC steps monitored by RIA to isolate a structurally related peptide from human brain cortex with the sequence Tyr-Pro-Lys-Gly-NH2 (Tyr-K-MIF-1). Determination of the sequence, electrospray mass spectrometry, and comparison of its chromatographic behavior with synthetic Tyr-K-MIF-1 confirmed the structure. Unlike Tyr-MIF-1 and Tyr-W-MIF-1, Tyr-K-MIF-1 does not bind to the mu opiate site; unlike MIF-1, Tyr-K-MIF-1 can bind to the Tyr-MIF-1 site. Of these peptides, only Tyr-K-MIF-1 binds to its own site in brain tissue prepared in Tris buffer. Thus, a new member of the Tyr-MIF-1 family of peptides, with a unique profile of binding, has been isolated from human brain cortex.

Isolation of a novel tetrapeptide with opiate and antiopiate activity from human brain cortex: Tyr-Pro-Trp-Gly-NH2 (Tyr-W-MIF-1).

A novel tetrapeptide, Tyr-Pro-Trp-Gly-NH2 (Tyr-W-MIF-1), was purified from extracts of frontal cortex of human brain tissue by several consecutive reversed-phase high performance liquid chromatographic steps followed by a radioimmunoassay originally developed for Tyr-Pro-Leu-Gly-NH2 (Tyr-MIF-1). Sequencing, mass spectrometric analysis, and comparison of its chromatographic behavior with that of the synthetic peptide confirmed the structure. Like Tyr-MIF-1, which was previously isolated from human brain tissue, Tyr-W-MIF-1 can inhibit the binding of 3H-DAMGO (selective for mu opiate receptors) to rat brain and can act as an opiate agonist as well as antagonist. Tyr-W-MIF-1 was a more potent opiate agonist than Tyr-MIF-1, the free acid of Tyr-W-MIF-1, and the structurally related hemoglobin-derived opiate peptide hemorphin-4 (Tyr-Pro-Trp-Thr) in the guinea pig ileum. Each of these peptides acted as opiate antagonists on the ileum from morphine-tolerant guinea pigs; the free acid of Tyr-W-MIF-1 was the most potent antagonist in inhibiting the activity of DAMGO. The results demonstrate the presence in human brain of a new member of the Tyr-MIF-1 family of biologically active peptides.

Isolation of Tyr-W-MIF-1 from bovine hypothalami.

Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2) was recently isolated from human brain cortex. We have now isolated it from bovine hypothalami by solid phase extraction and several consecutive rpHPLC steps monitored by an RIA originally developed for the endogenous brain peptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2). Determination of the sequence of the purified material and comparison of its chromatographic behavior with synthetic Tyr-W-MIF-1 confirmed the structure. The synthetic peptide and the isolated material showed almost identical binding to mu opiate receptors.

Enhanced isolation of Tyr-MIF-1 from fresh human brain cortex.

Tyr-MIF-1 previously was isolated from tissue obtained after death. A possible role of autolysis could not be excluded. We report the isolation of Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) in increased amounts from fresh human brain cortex. This confirms the natural existence of this brain peptide.

Melanostatin, a new melanin synthesis inhibitor. Production, isolation, chemical properties, structure and biological activity.

Melanostatin, a new antibiotic with melanin synthesis inhibitor activity, was isolated from the fermentation broth of Streptomyces clavifer No. N924-2. Its structure was determined by spectral analysis and degradation experiments. Melanostatin strongly inhibited melanin formation in Streptomyces bikiniensis NRRL B-1049 and B16 melanoma cells.

H-Pro-[3H]Leu-Gly-NH2: metabolism in human and rat plasma investigated by high-pressure liquid chromatography.

H-Pro-Leu-Gly-NH2 (PLG) was labeled with 3H-leucine by catalytic tritiation of H-Pro-methylallylgylcyl-Gly-NH2 and purified by ion-exchange chromatography. Metabolism of 3H-PLG in rat and human plasma was investigated by reversed phase-paired ion high-pressure liquid chromatography. All possible metabolites could be completely separated within 25 min. Half-lives, based on disappearance of intact 3H-PLG, for in vitro metabolism were 26.4 min (rat) and 5.6 days (human). The only significant metabolite was 3H-leucine. A rate-limiting, species-specific enzyme seems responsible for the initial breakdown of PLG. Disappearance of 3H-PLG from rat plasma, following i.v. administration, proceeded with half-lives of 1.03 min (distribution) and 9.8 min (elimination).

Evidence for presence of Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) in human brain cortex.

Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) was previously isolated from bovine hypothalamus. We have now purified it from the parietal cortex of human brain tissue by gel filtration chromatography and four subsequent high performance liquid chromatographic steps. During isolation, the peptide content was followed by radioimmunoassay and compared with the elution of synthetic Tyr-MIF-1 in identical chromatographic systems. This extends evidence for the presence of Tyr-MIF-1 from bovine to human brain tissue and from hypothalamus to cortex.

Evidence that MIF plays a role in the development of pigmentation patterns in the frog.

A ventrally localized melanization-inhibiting factor (MIF) may play an important role in the expression of dorsal-ventral pigment patterns of amphibians. In efforts to purify this putative MIF, ventral skin conditioned medium (VCM) from Rana forreri was partially fractionated and used to immunize mice. A monoclonal antibody that has the ability to block the activity of MIF was isolated, and an immunoaffinity matrix was prepared by cross-linking the antibody to protein G-Sepharose. The fraction of VCM that bound to the affinity matrix decreased the number of melanized cells in the Xenopus laevis neural tube explant assay, but did not reduce significantly the number of cells that emigrated. The monoclonal antibody was used for immunohistochemical studies on R. pipiens skin. Strong staining with the antibody was observed beneath the basement membrane, in mucous glands, and in the subcutaneous tissue of the ventral skin. A weak staining was also observed in the ground substances of both ventral and dorsal skin. These results confirm that a monoclonal antibody has been secured against at least one of the MIF constituents and that it is useful as a probe in detecting the distribution of MIF in tissues. The results of its use in this study support the hypothesis that MIF plays a role in the expression, development, and maintenance of the dorsal-ventral pigmentation patterns of frogs.

Isolation of tyrosine-melanocyte-stimulating hormone release-inhibiting factor 1 from bovine brain tissue.

Although Tyr-melanocyte-stimulating hormone release-inhibiting factor 1 (MIF-1) (Tyr-Pro-Leu-Gly-NH2) can exert a number of biological actions in the brain and elsewhere, it has never been isolated from any tissue. Accordingly, we attempted to purify it from acetic acid extracts of bovine brain tissue by gel filtration chromatography and several different high performance liquid chromatographic systems. Peptide content was followed by a specific and sensitive radioimmunoassay with an antibody that was generated against synthetic Tyr-MIF-1. In each of the five applied high performance liquid chromatographic systems, the immunoreactive fractions coincided exactly with the elution time of synthetic Tyr-MIF-1 in the control runnings. The structure of the isolated peptide was identified by microsequence analysis as the tetrapeptide Tyr-Pro-Leu-Gly-NH2 and shown to be biologically active.