Marine mussels accumulate amino acids and glucose from seawater against considerable concentration gradients. The principal site for this uptake is the gill. Previous studies using intact, isolated gills from marine mussels have suggested that the transport mechanism involves coupling to Na⁺, similar to the mechanism of secondary active transport of amino acids and glucose in vertebrate epithelia, but until this dissertation there had been no rigorous test of this hypothesis. Brush border membrane vesicles (BBMV) were prepared from the gills of the marine mussel, Mytilus edulis, by differential and sucrose density centrifugation. The preparation procedure isolated a population of membranes enriched in brush border membrane markers. The transport of amino acids by two pathways, the alanine-lysine pathway (AK) and the alanine-proline pathway (AP), and the uptake of glucose was studied in the BBMV. The mechanism of transport through the three transport pathways is BBMV involved coupling to Na⁺. Concentrative uptake through the AK pathway, which transported alanine and lysine, also occurred in the presence of Li⁺ and K⁺ gradients. This pathway was the major route for alanine transport in BBMV. The AP pathway transported alanine and proline, and was strictly dependent on Na⁺. Glucose transport in gill BBMV resembled quite closely the Na⁺-coupled transport of glucose in vertebrate epithelia in such characteristics as Na⁺ and substrate specifically, and electrogenicity. Transport through the two amino acid uptake pathways (AK and AP) and through the glucose uptake pathway could be described by Michaelis-Menten kinetics, with high substrate affinities (K(t)'s below 10 μM). Furthermore, it is likely that multiple Na⁺ ions are involved in the transport of these amino acids and glucose in mussel gill BBMV. It appears that these transporters are adapted for function at low substrate concentrations and against large concentration gradients.

A number of α-melanotropin analogues have been prepared. Insight towards the development and understanding of the functional roles of each of the amino acid residues important for high melanotropic potency in a number of biological systems was studied. The melanotropin analogue Ac- α-MSH₄₋₁₂-NH₂ contains all of the structural requirements necessary for obtaining full biological potency on the lizard (Anolis carolinensis) melanophore and S-91 mouse melanoma. On the frog (Rana pipiens) melanophore, the melanotropin analogue Ac-[Nle⁴]-α-MSH₄₋₁₃-NH₂ possesses full melanotropic potency relative to the native hormone. The smallest melanotropin analogue capable of eliciting any biological response was Ac-α-MSH₆₋₉-NH₂ (Ac-His-Phe-Arg-Trp-NH₂) on all biological systems studied. The low biological activity found in previous studies for smaller melanotropin analogues may have been due to a trace contamination of a potent melanotropin. The importance of each of the thirteen amino acid residues of α-melanotropin in contributing to the melanotropic actions in a number of biological systems is discussed. We were unable to confirm the reported presence of a second independent active sequence in α-melanotropin. The structural requirements for prolongation of biological activity (following removal of exogenous hormone from the assay medium) and enzyme stability have been studied. Analysis suggests species-dependent differences in the structural relationships of the amino acid residues in the 4, 7 and 11 positions for prolonged melanotropic response. Analogues prepared with D-phenylalanine in place of its L-enantiomer in the seventh positions of α-melanotropin analogues generally results in an increased resistance to enzymatic degradation towards rat brain homogenate, rat serum, and to the purified enzymes, trypsin and chymotrypsin. Some of these analogues have been shown to be useful probes for the understanding of melanotropic actions in a number of biological systems. Two α-melanotropin analogues have been prepared which possess partial agonism on the mouse melanoma adenylate cyclase assay. A number of these analogues should prove useful in future studies directed towards a more detailed study of melanotropin receptor systems in a large variety of biological systems. The first known synthesis of melanin concentrating hormone (MCH) is reported. The overall synthetic yield of this cyclic heptadecapeptide was 14%. The chemical, physical and biological properties of synthetic MCH and naturally occurring telost MCH were in agreement. MCH was found to be a full agonist in stimulating melanosome dispersion in both the frog and lizard bioassay. Therefore MCH can stimulate melanosome dispersion or contraction depending upon the bioassay studied. Preliminary studies were done towards understanding the mechanisms of MCH action on telost fish.

No description available.

Thesis (M.S. in Molecular Physiology and Biophysics)--Vanderbilt University, Dec. 2007. / Title from title screen. Includes bibliographical references.

Dissertation (Ph.D.)--Auburn University, / Abstract. Includes bibliographic references (p.110-134)

Thesis (Ph. D.)-University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Thesis (doctoral)--University of Tartu, 2006. / This dissertation is based on 4 papers. Includes bibliographical references.

Several chemically-modified analogues of α-melanotropin (α-MSH, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂) were prepared by solid-phase peptide synthesis, including [Nle⁴]-α-MSH, Ac-[Nle⁴]-α-MSH₄₋₁₃-NH₂, Ac-[Nle⁴]-α-MSH₁₋₆-NH₂, Ac-α-MSH₇₋₁₀-NH₂, Ac-α-MSH₁₁₋₁₃-NH₂, Ac-[Nle⁴]-α-MSH(,4-10)-NH₂, Ac-[Nle⁴, D-Phe⁷]-α-MSH₄₋₁₀-NH₂, [Nle⁴, D-Phe⁷]-α-MSH, Ac-α-MSH₄₋₁₀-NH₂, Ac-[Tyr⁴]-α-MSH₄₋₁₀-NH₂ and [half-Cys⁴, half-Cys¹⁰]-α-MSH. The synthetic strategy involved: (1) p-methylbenzhydrylamine resin as a solid support, (2) N,N'-dicyclohexylcarbodiimide as a coupling reagent, (3) acetylation of the N-terminus and HF cleavage and deprotection (except for Nⁱ-For-Trp) of the fully assembled peptide-resin and (4) alkaline hydrolysis to deformylate Nⁱ-For-Trp. In the preparation of [half-Cys⁴, half-Cys¹⁰]-α-MSH, oxidative-cyclization provided formation of an intramolecular disulfide bridge. A comparative biological analysis in vitro of these above structural, stereoisomeric and conformationally-restricted analogues of α-MSH on several different vertebrate pigment cell systems provided the following results: (1) The [Nle⁴, D-Phe⁷]-α-MSH effected high melanotropic potency (> 60 times relative to α-MSH), ultralong biological activity and unprecedented metabolic stability. (2) Utilizing [Nle⁴, D-Phe⁷]-α-MSH as a molecular probe, two melanotropic receptor types were demonstrated which were mechanistically different in terms of calcium dependency and apparent hormone-receptor complex reversibility. (3) The Ac-[Nle⁴, D-Phe⁷]-α-MSH₄₋₁₀-NH₂ was a highly potent active site (Met-Glu-His-Phe-Arg-Trp-Gly) analogue of α-MSH (ranging from 0.2- to 10-times relative to α-MSH) without the ultralong melanotropic activity possessed by the parent stereostructural tridecapeptide. (4) The [half-Cys4, half-Cys10]-α-MSH exhibited superpotency on frog (Rana pipiens) melanophores (≥ 10,000 times relative to α-MSH), and provided experimental evidence that a pseudocyclic conformation of the native hormone containing a β-turn structural requirement at His-Phe-Arg-Trp might be related to its biological activity at the pigment cell receptor. The [Nle⁴, D-Phe⁷]-α-MSH may be suitable for use as a radio-labeled tracer or drug-delivery agent for the localization or treatment of human melanoma in vivo.

No description available.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy